Adsorption of insulin peptide on charged single-walled carbon nanotubes: significant role of ordered water molecules.

PubMed

Shen, Jia-Wei; Wu, Tao; Wang, Qi; Kang, Yu; Chen, Xin

2009-06-02

Ordered hydration shells: The more ordered hydration shells outside the charged CNT surfaces prevent more compact adsorption of the peptide in the charged CNT systems [picture: see text], but peptide binding strengths on the charged CNT surfaces are stronger due to the electrostatic interaction.Studies of adsorption dynamics and stability for peptides/proteins on single-walled carbon nanotubes (SWNTs) are of great importance for a better understanding of the properties and nature of nanotube-based biosystems. Herein, the dynamics and mechanism of the adsorption of the insulin chain B peptide on different charged SWNTs are investigated by explicit solvent molecular dynamics simulations. The results show that all types of surfaces effectively attract the model peptide. Water molecules play a significant role in peptide adsorption on the surfaces of charged carbon nanotubes (CNTs). Compared to peptide adsorption on neutral CNT surfaces, the more ordered hydration shells outside the tube prevent more compact adsorption of the peptide in charged CNT systems. This shield effect leads to a smaller conformational change and van der Waals interaction between the peptide and surfaces, but peptide binding strengths on charged CNT surfaces are stronger than those on the neutral CNT surface due to the strong electrostatic interaction. The result of these simulations implies the possibility of improving the binding strength of peptides/proteins on CNT surfaces, as well as keeping the integrity of the peptide/protein conformation in peptide/protein-CNT complexes by charging the CNTs.

Role of Surface Charge Density in Nanoparticle-templated Assembly of Bromovirus Protein Cages

PubMed Central

Daniel, Marie-Christine; Tsvetkova, Irina B.; Quinkert, Zachary T.; Murali, Ayaluru; De, Mrinmoy; Rotello, Vincent M.; Kao, C. Cheng; Dragnea, Bogdan

2010-01-01

Self-assembling icosahedral protein cages have potencially useful physical and chemical characteristics for a variety of nanotechnology applications, ranging from therapeutic or diagnostic vectors to building blocks for hierarchical materials. For application-specific functional control of protein cage assemblies, a deeper understanding of the interaction between the protein cage and its payload is necessary. Protein-cage encapsulated nanoparticles, with their well-defined surface chemistry, allow for systematic control over key parameters of encapsulation such as the surface charge, hydrophobicity, and size. Independent control over these variables allows experimental testing of different assembly mechanism models. Previous studies done with Brome mosaic virus capsids and negatively-charged gold nanoparticles indicated that the result of the self-assembly process depends on the diameter of the particle. However, in these experiments, the surface-ligand density was maintained at saturation levels, while the total charge and the radius of curvature remained coupled variables, making the interpretation of the observed dependence on the core size difficult. The current work furnishes evidence of a critical surface charge density for assembly through an analysis aimed at decoupling the surface charge the core size. PMID:20575505

Interaction of gold and silver nanoparticles with human plasma: Analysis of protein corona reveals specific binding patterns.

PubMed

Lai, Wenjia; Wang, Qingsong; Li, Lumeng; Hu, Zhiyuan; Chen, Jiankui; Fang, Qiaojun

2017-04-01

Determining how nanomaterials interact with plasma will assist in understanding their effects on the biological system. This work presents a systematic study of the protein corona formed from human plasma on 20nm silver and gold nanoparticles with three different surface modifications, including positive and negative surface charges. The results show that all nanoparticles, even those with positive surface modifications, acquire negative charges after interacting with plasma. Approximately 300 proteins are identified on the coronas, while 99 are commonly found on each nanomaterial. The 20 most abundant proteins account for over 80% of the total proteins abundance. Remarkably, the surface charge and core of the nanoparticles, as well as the isoelectric point of the plasma proteins, are found to play significant roles in determining the nanoparticle coronas. Albumin and globulins are present at levels of less than 2% on these nanoparticle coronas. Fibrinogen, which presents in the plasma but not in the serum, preferably binds to negatively charged gold nanoparticles. These observations demonstrate the specific plasma protein binding pattern of silver and gold nanoparticles, as well as the importance of the surface charge and core in determining the protein corona compositions. The potential downstream biological impacts of the corona proteins were also investigated. Copyright © 2017 Elsevier B.V. All rights reserved.

Model simulations of the adsorption of statherin to solid surfaces: Effects of surface charge and hydrophobicity

NASA Astrophysics Data System (ADS)

Skepö, M.

2008-11-01

The structural properties of the salivary protein statherin upon adsorption have been examined using a coarse-grained model and Monte Carlo simulation. A simple model system with focus on electrostatic interactions and short-ranged attractions among the uncharged amino acids has been used. To mimic hydrophobically modified surfaces, an extra short-ranged interaction was implemented between the amino acids and the surface. It has been shown that the adsorption and the thickness of the adsorbed layer are determined by (i) the affinity for the surface, i.e., denser layer with an extrashort-ranged potential, and (ii) the distribution of the charges along the chain. If all the amino acids have a high affinity for the surface, the protein adsorbs in a train conformation, if the surface is negatively charged the protein adsorbs in a tail-train conformation, whereas if the surface is positively charged the protein adsorbs in a loop conformation. The latter gives rise to a more confined adsorbed layer.

Fibronectin module FN(III)9 adsorption at contrasting solid model surfaces studied by atomistic molecular dynamics.

PubMed

Kubiak-Ossowska, Karina; Mulheran, Paul A; Nowak, Wieslaw

2014-08-21

The mechanism of human fibronectin adhesion synergy region (known as integrin binding region) in repeat 9 (FN(III)9) domain adsorption at pH 7 onto various and contrasting model surfaces has been studied using atomistic molecular dynamics simulations. We use an ionic model to mimic mica surface charge density but without a long-range electric field above the surface, a silica model with a long-range electric field similar to that found experimentally, and an Au {111} model with no partial charges or electric field. A detailed description of the adsorption processes and the contrasts between the various model surfaces is provided. In the case of our model silica surface with a long-range electrostatic field, the adsorption is rapid and primarily driven by electrostatics. Because it is negatively charged (-1e), FN(III)9 readily adsorbs to a positively charged surface. However, due to its partial charge distribution, FN(III)9 can also adsorb to the negatively charged mica model because of the absence of a long-range repulsive electric field. The protein dipole moment dictates its contrasting orientation at these surfaces, and the anchoring residues have opposite charges to the surface. Adsorption on the model Au {111} surface is possible, but less specific, and various protein regions might be involved in the interactions with the surface. Despite strongly influencing the protein mobility, adsorption at these model surfaces does not require wholesale FN(III)9 conformational changes, which suggests that the biological activity of the adsorbed protein might be preserved.

Quantitative study of protein-protein interactions by quartz nanopipettes

NASA Astrophysics Data System (ADS)

Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

2014-08-01

In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions. Electronic supplementary information (ESI) available: Determination of nanopipette diameter; surface modification scheme; numerical simulation; noise analysis; SPR experiments. See DOI: 10.1039/c4nr02964j

Carbodiimide-mediated immobilization of acidic biomolecules on reversed-charge zwitterionic sensor chip surfaces.

PubMed

Risse, Fabian; Gedig, Erk T; Gutmann, Jochen S

2018-04-30

The carbodiimide-mediated amine coupling of protein ligands to sensor chips coated with anionic polycarboxylate hydrogels, such as carboxymethyl dextran, is the predominant covalent immobilization procedure utilized in optical biosensors, namely surface plasmon resonance (SPR) biosensors. Usually, electrostatic interactions at a slightly acidic pH and low ionic strength are employed to efficiently accumulate neutral and basic ligands on the chip surface, which are then covalently coupled by surface-bound active N-hydroxysuccinimide (NHS) esters. Unfortunately, this approach is not suitable for acidic proteins or other ligands with low isoelectric points (IEPs), such as nucleic acids, because the charge density of the polycarboxylates is greatly reduced at acidic pH or because electrostatic attraction cannot be achieved. To overcome these drawbacks, we have established a charge-reversal approach that allows the preconcentration of acidic proteins above their IEPs. A precisely controlled amount of tertiary amines is applied to reverse the previous anionic surface charge while maintaining carbodiimide compatibility with future protein immobilization. The mechanism of this reversed-charge immobilization approach was demonstrated employing protein A as a model protein and using attenuated total reflectance Fourier transform infrared spectroscopy, dynamic contact angle measurements, colorimetric quantification, and SPR analysis to characterize surface derivatization. Furthermore, even though it had previously proven impossible to preconcentrate DNA electrostatically and to covalently couple it to polyanionic chip surfaces, we demonstrated that our approach allowed DNA to be preconcentrated and immobilized in good yields. Graphical abstract Principle of the covalent immobilization of acidic ligands on reversed-charge zwitterionic sensor chip surfaces.

Biochemical Study of Anti-Inflammatory Proteins vCCI and vMIP-II

DTIC Science & Technology

2014-07-17

protein ), where we showed that vCCI is able to bind so many different chemokines due to its general negatively charged surface , allowing it to bind...sample of these competition curves. Our conclusion from the data in Table 1 and Figure 1 is that the negatively charged surface of vCCI allows it to...Similar to our mutagenesis results, the overall data indicate that vCCI uses a negatively charged surface to bind positive charges on the chemokine

Influence of the Amino Acid Sequence on Protein-Mineral Interactions in Soil

NASA Astrophysics Data System (ADS)

Chacon, S. S.; Reardon, P. N.; Purvine, S.; Lipton, M. S.; Washton, N.; Kleber, M.

2017-12-01

The intimate associations between protein and mineral surfaces have profound impacts on nutrient cycling in soil. Proteins are an important source of organic C and N, and a subset of proteins, extracellular enzymes (EE), can catalyze the depolymerization of soil organic matter (SOM). Our goal was to determine how variation in the amino acid sequence could influence a protein's susceptibility to become chemically altered by mineral surfaces to infer the fate of adsorbed EE function in soil. We hypothesized that (1) addition of charged amino acids would enhance the adsorption onto oppositely charged mineral surfaces (2) addition of aromatic amino acids would increase adsorption onto zero charged surfaces (3) Increase adsorption of modified proteins would enhance their susceptibility to alterations by redox active minerals. To test these hypotheses, we generated three engineered proxies of a model protein Gb1 (IEP 4.0, 6.2 kDA) by inserting either negatively charged, positively charged or aromatic amino acids in the second loop. These modified proteins were allowed to interact with functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnessite) at pH 5 and 7. We used LC-MS/MS and solution-state Heteronuclear Single Quantum Coherence Spectroscopy NMR to observe modifications on engineered proteins as a consequence to mineral interactions. Preliminary results indicate that addition of any amino acids to a protein increase its susceptibility to fragmentation and oxidation by redox active mineral surfaces, and alter adsorption to the other mineral surfaces. This suggest that not all mineral surfaces in soil may act as sorbents for EEs and chemical modification of their structure should also be considered as an explanation for decrease in EE activity. Fragmentation of proteins by minerals can bypass the need to produce proteases, but microbial acquisition of other nutrients that require enzymes such as cellulases, ligninases or phosphatases may be hampered by mineral association.

Nanoparticles Penetrate into the Multicellular Spheroid-on-Chip: Effect of Surface Charge, Protein Corona, and Exterior Flow.

PubMed

Huang, Ke; Boerhan, Rena; Liu, Changming; Jiang, Guoqiang

2017-12-04

Nanoparticles (NPs) are widely studied as tumor targeted vehicles. The penetration of NPs into the tumor is considered as a major barrier for delivery of NPs into tumor cell and a big challenge to translate NPs from lab to the clinic. The objective of this study is to know how the surface charge of NPs, the protein corona surrounding the NPs, and the fluid flow around the tumor surface affect the penetration and accumulation of NPs into the tumor, through in vitro penetration study based on a spheroid-on-chip system. Surface decorated polystyrene (PS) NPs (100 nm) carrying positive and negative surface charge were loaded to the multicellular spheroids under static and flow conditions, in the presence or absence of serum proteins. NP penetration was investigated by confocal laser microscopy scanning followed with quantitative image analysis. The results reveal that negatively charged NPs are attached more on the spheroid surface and easier to penetrate into the spheroids. Protein corona, which is formed surrounding the NPs in the presence of serum protein, changes the surface properties of the NPs, weakens the NP-cell affinity, and, therefore, results in lower NP concentration on the spheroid surface but might facilitate deeper penetration. The exterior fluid flow enhances the interstitial flow into the spheroid, which benefits the penetration but also strips the NPs (especially the NPs with protein corona) on the spheroid surface, which decreases the penetration flux significantly. The maximal penetration was obtained by applying negatively charged NPs without protein corona under the flow condition. We hope the present study will help to understand the spatiotemporal performance of drug delivery NPs and inform the rational design of NPs with highly defined drug accumulation localized at a target site.

Modulation of Silica Nanoparticle Uptake into Human Osteoblast Cells by Variation of the Ratio of Amino and Sulfonate Surface Groups: Effects of Serum

PubMed Central

2015-01-01

To study the importance of the surface charge for cellular uptake of silica nanoparticles (NPs), we synthesized five different single- or multifunctionalized fluorescent silica NPs (FFSNPs) by introducing various ratios of amino and sulfonate groups into their surface. The zeta potential values of these FFSNPs were customized from highly positive to highly negative, while other physicochemical properties remained almost constant. Irrespective of the original surface charge, serum proteins adsorbed onto the surface, neutralized the zeta potential values, and prevented the aggregation of the tailor-made FFSNPs. Depending on the surface charge and on the absence or presence of serum, two opposite trends were found concerning the cellular uptake of FFSNPs. In the absence of serum, positively charged NPs were more strongly accumulated by human osteoblast (HOB) cells than negatively charged NPs. In contrast, in serum-containing medium, anionic FFSNPs were internalized by HOB cells more strongly, despite the similar size and surface charge of all types of protein-covered FFSNPs. Thus, at physiological condition, when the presence of proteins is inevitable, sulfonate-functionalized silica NPs are the favorite choice to achieve a desired high rate of NP internalization. PMID:26030456

Probing protein orientation near charged nanosurfaces for simulation-assisted biosensor design.

PubMed

Cooper, Christopher D; Clementi, Natalia C; Barba, Lorena A

2015-09-28

Protein-surface interactions are ubiquitous in biological processes and bioengineering, yet are not fully understood. In biosensors, a key factor determining the sensitivity and thus the performance of the device is the orientation of the ligand molecules on the bioactive device surface. Adsorption studies thus seek to determine how orientation can be influenced by surface preparation, varying surface charge, and ambient salt concentration. In this work, protein orientation near charged nanosurfaces is obtained under electrostatic effects using the Poisson-Boltzmann equation, in an implicit-solvent model. Sampling the free energy for protein G B1 D4' at a range of tilt and rotation angles with respect to the charged surface, we calculated the probability of the protein orientations and observed a dipolar behavior. This result is consistent with published experimental studies and combined Monte Carlo and molecular dynamics simulations using this small protein, validating our method. More relevant to biosensor technology, antibodies such as immunoglobulin G are still a formidable challenge to molecular simulation, due to their large size. With the Poisson-Boltzmann model, we obtained the probability distribution of orientations for the iso-type IgG2a at varying surface charge and salt concentration. This iso-type was not found to have a preferred orientation in previous studies, unlike the iso-type IgG1 whose larger dipole moment was assumed to make it easier to control. Our results show that the preferred orientation of IgG2a can be favorable for biosensing with positive charge on the surface of 0.05 C/m(2) or higher and 37 mM salt concentration. The results also show that local interactions dominate over dipole moment for this protein. Improving immunoassay sensitivity may thus be assisted by numerical studies using our method (and open-source code), guiding changes to fabrication protocols or protein engineering of ligand molecules to obtain more favorable orientations.

Probing protein orientation near charged nanosurfaces for simulation-assisted biosensor design

NASA Astrophysics Data System (ADS)

Cooper, Christopher D.; Clementi, Natalia C.; Barba, Lorena A.

2015-09-01

Protein-surface interactions are ubiquitous in biological processes and bioengineering, yet are not fully understood. In biosensors, a key factor determining the sensitivity and thus the performance of the device is the orientation of the ligand molecules on the bioactive device surface. Adsorption studies thus seek to determine how orientation can be influenced by surface preparation, varying surface charge, and ambient salt concentration. In this work, protein orientation near charged nanosurfaces is obtained under electrostatic effects using the Poisson-Boltzmann equation, in an implicit-solvent model. Sampling the free energy for protein G B1 D4' at a range of tilt and rotation angles with respect to the charged surface, we calculated the probability of the protein orientations and observed a dipolar behavior. This result is consistent with published experimental studies and combined Monte Carlo and molecular dynamics simulations using this small protein, validating our method. More relevant to biosensor technology, antibodies such as immunoglobulin G are still a formidable challenge to molecular simulation, due to their large size. With the Poisson-Boltzmann model, we obtained the probability distribution of orientations for the iso-type IgG2a at varying surface charge and salt concentration. This iso-type was not found to have a preferred orientation in previous studies, unlike the iso-type IgG1 whose larger dipole moment was assumed to make it easier to control. Our results show that the preferred orientation of IgG2a can be favorable for biosensing with positive charge on the surface of 0.05 C/m2 or higher and 37 mM salt concentration. The results also show that local interactions dominate over dipole moment for this protein. Improving immunoassay sensitivity may thus be assisted by numerical studies using our method (and open-source code), guiding changes to fabrication protocols or protein engineering of ligand molecules to obtain more favorable orientations.

Effect of Surface Hydration on Antifouling Properties of Mixed Charged Polymers.

PubMed

Leng, Chuan; Huang, Hao; Zhang, Kexin; Hung, Hsiang-Chieh; Xu, Yao; Li, Yaoxin; Jiang, Shaoyi; Chen, Zhan

2018-05-07

Interfacial water structure on a polymer surface in water (or surface hydration) is related to the antifouling activity of the polymer. Zwitterionic polymer materials exhibit excellent antifouling activity due to their strong surface hydration. It was proposed to replace zwitterionic polymers using mixed charged polymers because it is much easier to prepare mixed charged polymer samples with much lower costs. In this study, using sum frequency generation (SFG) vibrational spectroscopy, we investigated interfacial water structures on mixed charged polymer surfaces in water, and how such structures change while exposing to salt solutions and protein solutions. The 1:1 mixed charged polymer exhibits excellent antifouling property while other mixed charged polymers with different ratios of the positive/negative charges do not. It was found that on the 1:1 mixed charged polymer surface, SFG water signal is dominated by the contribution of the strongly hydrogen bonded water molecules, indicating strong hydration of the polymer surface. The responses of the 1:1 mixed charged polymer surface to salt solutions are similar to those of zwitterionic polymers. Interestingly, exposure to high concentrations of salt solutions leads to stronger hydration of the 1:1 mixed charged polymer surface after replacing the salt solution with water. Protein molecules do not substantially perturb the interfacial water structure on the 1:1 mixed charged polymer surface and do not adsorb to the surface, showing that this mixed charged polymer is an excellent antifouling material.

Protein Corona Composition of Superparamagnetic Iron Oxide Nanoparticles with Various Physico-Chemical Properties and Coatings

NASA Astrophysics Data System (ADS)

Sakulkhu, Usawadee; Mahmoudi, Morteza; Maurizi, Lionel; Salaklang, Jatuporn; Hofmann, Heinrich

2014-05-01

Because of their biocompatibility and unique magnetic properties, superparamagnetic iron oxide nanoparticles NPs (SPIONs) are recognized as some of the most prominent agents for theranostic applications. Thus, understanding the interaction of SPIONs with biological systems is important for their safe design and efficient applications. In this study, SPIONs were coated with 2 different polymers: polyvinyl alcohol polymer (PVA) and dextran. The obtained NPs with different surface charges (positive, neutral, and negative) were used as a model study of the effect of surface charges and surface polymer materials on protein adsorption using a magnetic separator. We found that the PVA-coated SPIONs with negative and neutral surface charge adsorbed more serum proteins than the dextran-coated SPIONs, which resulted in higher blood circulation time for PVA-coated NPs than the dextran-coated ones. Highly abundant proteins such as serum albumin, serotransferrin, prothrombin, alpha-fetoprotein, and kininogen-1 were commonly found on both PVA- and dextran-coated SPIONs. By increasing the ionic strength, soft- and hard-corona proteins were observed on 3 types of PVA-SPIONs. However, the tightly bound proteins were observed only on negatively charged PVA-coated SPIONs after the strong protein elution.

Quantitative study of protein-protein interactions by quartz nanopipettes.

PubMed

Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

2014-09-07

In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.

Electrostatic complementarity at protein/protein interfaces.

PubMed

McCoy, A J; Chandana Epa, V; Colman, P M

1997-05-02

Calculation of the electrostatic potential of protein-protein complexes has led to the general assertion that protein-protein interfaces display "charge complementarity" and "electrostatic complementarity". In this study, quantitative measures for these two terms are developed and used to investigate protein-protein interfaces in a rigorous manner. Charge complementarity (CC) was defined using the correlation of charges on nearest neighbour atoms at the interface. All 12 protein-protein interfaces studied had insignificantly small CC values. Therefore, the term charge complementarity is not appropriate for the description of protein-protein interfaces when used in the sense measured by CC. Electrostatic complementarity (EC) was defined using the correlation of surface electrostatic potential at protein-protein interfaces. All twelve protein-protein interfaces studied had significant EC values, and thus the assertion that protein-protein association involves surfaces with complementary electrostatic potential was substantially confirmed. The term electrostatic complementarity can therefore be used to describe protein-protein interfaces when used in the sense measured by EC. Taken together, the results for CC and EC demonstrate the relevance of the long-range effects of charges, as described by the electrostatic potential at the binding interface. The EC value did not partition the complexes by type such as antigen-antibody and proteinase-inhibitor, as measures of the geometrical complementarity at protein-protein interfaces have done. The EC value was also not directly related to the number of salt bridges in the interface, and neutralisation of these salt bridges showed that other charges also contributed significantly to electrostatic complementarity and electrostatic interactions between the proteins. Electrostatic complementarity as defined by EC was extended to investigate the electrostatic similarity at the surface of influenza virus neuraminidase where the epitopes of two monoclonal antibodies, NC10 and NC41, overlap. Although NC10 and NC41 both have quite high values of EC for their interaction with neuraminidase, the similarity in electrostatic potential generated by the two on the overlapping region of the epitopes is insignificant. Thus, it is possible for two antibodies to recognise the electrostatic surface of a protein in dissimilar ways.

Protein-Nanoparticle Interactions: Improving Immobilized Lytic Enzyme Activity and Surface Energy Effects

NASA Astrophysics Data System (ADS)

Downs, Emily Elizabeth

Protein-nanostructure conjugates, particularly particles, are a subject of significant interest due to changes in their fundamental behavior compared to bulk surfaces. As the size scale of nano-structured materials and proteins are on the same order of magnitude, nanomaterial properties can heavily influence how proteins adsorb and conform to the surface. Previous work has demonstrated the ability of nanoscale surfaces to modulate protein activity, conformation, and retention by modifying the particle surface curvature, morphology, and surface charge. This work has improved our understanding of the protein material interactions, but a complete understanding is still lacking. The goal of this thesis is to investigate two missing areas of understanding using two distinct systems. The first system utilizes a particle with controlled surface energy to observe the impact of surface energy on protein-particle interactions, while the second system uses a modified Listeria-specific protein to determine how protein structure and flexibility affects protein adsorption and activity on particles. Spherical, amorphous, and uniformly doped Zn-silica particles with tailored surface energies were synthesized to understand the impact of surface energy on protein adsorption behavior. Particle surface energy increased with a decrease in particle size and greater dopant concentrations. Protein adsorption and structural loss increased with both particle size and particle surface energy. Higher surface energies promoted protein-particle association and increased protein unfolding. Particle curvature and protein steric hindrance effects limited adsorption and structural loss on smaller particles. Protein surface charge heterogeneity was also found to be linked to both protein adsorption and unfolding behavior on larger particles. Greater surface charge heterogeneity led to higher adsorption concentrations and multilayer formation. These multilayers transitioned from protein-particle interactions to protein-protein interactions and were thicker with greater surface energy, which resulted in the recovery of secondary structure in the outermost layer. To help understand the impact of protein structure on nano-bio conjugate interactions, a listeria specific protein was used. This system was chosen as it has applications in the food industry in preventing bacterial contamination. The insertion of an amino acid linker between the enzymatic and binding domain of the protein improved the flexibility between domains, leading to increased adsorption, and improved activity in both cell-wall and plating assays. Additionally, linker modified protein incorporated into the silica-polymer nanocomposite showed significant activity in a real-world example of contaminated lettuce. This thesis study has isolated the impact of surface energy and protein flexibility on protein adsorption and structure. Particle surface energy affects adsorbed protein concentration and conformation. Coupled with protein surface charge, surface energy was also found to dictate multilayer thickness. The conformational flexibility of the protein was shown to help in controlling not only protein adsorption concentration but also in retaining protein activity after immobilization. Also, a controllable synthesis method for particles with adjustable surface energy, an ideal platform for studying protein-particle interactions, has been established.

Determination of the surface charge density and temperature dependence of purple membrane by electric force microscopy.

PubMed

Du, Huiwen; Li, Denghua; Wang, Yibing; Wang, Chenxuan; Zhang, Dongdong; Yang, Yan-lian; Wang, Chen

2013-08-29

We report here the measurement of the temperature-dependent surface charge density of purple membrane (PM) by using electrostatic force microscopy (EFM). The surface charge density was measured to be 3.4 × 10(5) e/cm(2) at room temperature and reaches the minimum at around 52 °C. The initial decrease of the surface charge density could be attributed to the reduced dipole alignment because of the thermally induced protein mobility in PM. The increase of charge density at higher temperature could be ascribed to the weakened interaction between proteins and the lipids, which leads to the exposure of the charged amino acids. This work could be a benefit to the direct assessment of the structural stability and electric properties of biological membranes at the nanoscale.

Plasma protein adsorption to zwitterionic poly (carboxybetaine methacrylate) modified surfaces: chain chemistry and end-group effects on protein adsorption kinetics, adsorbed amounts and immunoblots.

PubMed

Abraham, Sinoj; Bahniuk, Markian S; Unsworth, Larry D

2012-12-01

Protein-surface interactions are crucial to the overall biocompatability of biomaterials, and are thought to be the impetus towards the adverse host responses such as blood coagulation and complement activation. Only a few studies hint at the ultra-low fouling potential of zwitterionic poly(carboxybetaine methacrylate) (PCBMA) grafted surfaces and, of those, very few systematically investigate their non-fouling behavior. In this work, single protein adsorption studies as well as protein adsorption from complex solutions (i.e. human plasma) were used to evaluate the non-fouling potential of PCBMA grafted silica wafers prepared by nitroxide-mediated free radical polymerization. PCBMAs used for surface grafting varied in charge separating spacer groups that influence the overall surface charges, and chain end-groups that influence the overall hydrophilicity, thereby, allows a better understanding of these effects towards the protein adsorption for these materials. In situ ellipsometry was used to quantify the adsorbed layer thickness and adsorption kinetics for the adsorption of four proteins from single protein buffer solutions, viz, lysozyme, α-lactalbumin, human serum albumin and fibrinogen. Total amount of protein adsorbed on surfaces differed as a function of surface properties and protein characteristics. Finally, immunoblots results showed that human plasma protein adsorption to these surfaces resulted, primarily, in the adsorption of human serum albumin, with total protein adsorbed amounts being the lowest for PCBMA-3 (TEMPO). It was apparent that surface charge and chain hydrophilicity directly influenced protein adsorption behavior of PCBMA systems and are promising materials for biomedical applications.

Influence of surface charge on the rate, extent, and structure of adsorbed Bovine Serum Albumin to gold electrodes.

PubMed

Beykal, Burcu; Herzberg, Moshe; Oren, Yoram; Mauter, Meagan S

2015-12-15

The objective of this work is to investigate the rate, extent, and structure of amphoteric proteins with charged solid surfaces over a range of applied potentials and surface charges. We use Electrochemical Quartz Crystal Microbalance with Dissipation Monitoring (E-QCM-D) to investigate the adsorption of amphoteric Bovine Serum Albumin (BSA) to a gold electrode while systematically varying the surface charge on the adsorbate and adsorbent by manipulating pH and applied potential, respectively. We also perform cyclic voltammetry-E-QCM-D on an adsorbed layer of BSA to elucidate conformational changes in response to varied applied potentials. We confirm previous results demonstrating that increasing magnitude of applied potential on the gold electrode is positively correlated with increasing mass adsorption when the protein and the surface are oppositely charged. On the other hand, we find that the rate of BSA adsorption is not governed by simple electrostatics, but instead depends on solution pH, an observation not well documented in the literature. Cyclic voltammetry with simultaneous E-QCM-D measurements suggest that BSA protein undergoes a conformational change as the surface potential varies. Copyright © 2015 Elsevier Inc. All rights reserved.

Hydrophilic crosslinked-polymeric surface capable of effective suppression of protein adsorption

NASA Astrophysics Data System (ADS)

Kamon, Yuri; Inoue, Naoko; Mihara, Erika; Kitayama, Yukiya; Ooya, Tooru; Takeuchi, Toshifumi

2016-08-01

We investigated the nonspecific adsorption of proteins towards three hydrophilic crosslinked-polymeric thin layers prepared by surface-initiated atom transfer radical polymerization using N,N‧-methylenebisacrylamide, 2-(methacryloyloxy)ethyl-[N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC), or 6,6‧-diacryloyl-trehalose crosslinkers. Protein binding experiments were performed by surface plasmon resonance with six proteins of different pI values including α-lactalbumin, bovine serum albumin (BSA), myoglobin, ribonuclease A, cytochrome C, and lysozyme in buffer solution at pH 7.4. All of the obtained crosslinked-polymeric thin layers showed low nonspecific adsorption of negatively charged proteins at pH 7.4 such as α-lactalbumin, BSA, and myoglobin. Nonspecific adsorption of positively charged proteins including ribonuclease A, cytochrome C, and lysozyme was the lowest for poly(MMPC). These results suggest poly(MMPC) can effectively reduce nonspecific adsorption of a wide range of proteins that are negatively or positively charged at pH 7.4. MMPC is a promising crosslinker for a wide range of polymeric materials requiring low nonspecific protein binding.

Surface functionalization of dopamine coated iron oxide nanoparticles for various surface functionalities

NASA Astrophysics Data System (ADS)

Sherwood, Jennifer; Xu, Yaolin; Lovas, Kira; Qin, Ying; Bao, Yuping

2017-04-01

We present effective conjugation of four small molecules (glutathione, cysteine, lysine, and Tris(hydroxymethyl)aminomethane) onto dopamine-coated iron oxide nanoparticles. Conjugation of these molecules could improve the surface functionality of nanoparticles for more neutral surface charge at physiological pH and potentially reduce non-specific adsorption of proteins to nanoparticles surfaces. The success of conjugation was evaluated with dynamic light scattering by measuring the surface charge changes and Fourier transform infrared spectroscopy for surface chemistry analysis. The stability of dopamine-coated nanoparticles and the ability of conjugated nanoparticles to reduce the formation of protein corona were evaluated by measuring the size and charge of the nanoparticles in biological medium. This facile conjugation method opens up possibilities for attaching various surface functionalities onto iron oxide nanoparticle surfaces for biomedical applications.

Hydrolysis-controlled protein adsorption and antifouling behaviors of mixed charged self-assembled monolayer: A molecular simulation study.

PubMed

Liu, Jie; Zhou, Jian

2016-08-01

Understanding the mechanism of the antimicrobial and antifouling properties of mixed charged materials is of great significance. The interactions between human gamma fibrinogen (γFg) and mixed carboxylic methyl ether-terminated (COOCH3-) and trimethylamino-terminated (N(CH3)3(+)-) SAMs and the influence of hydrolysis were studied by molecular simulations. After hydrolysis, the mixed SAMs exhibit behaviors from antimicrobial to antifouling, since the COOCH3-thiols were translated into carboxylic acid (COO(-)-) terminated thiols, which carried a net charge of -1 e. Simulation results showed that the main differences between COOCH3-/N(CH3)3(+)-SAM and COO(-)-/N(CH3)3(+)-SAM are the charged property and the hydration layer above the surface. γFg could stably adsorb on the positively-charged COOCH3-/N(CH3)3(+)-SAM. The adsorption behavior is mainly induced by the strong electrostatic attraction. There is a single hydration layer bound to the surface, which is related to the N(CH3)3(+) groups. The van der Waals repulsion between γFg and the single hydration layer are not strong enough to compensate the strong electrostatic attraction. After hydrolysis, the positively-charged SAM was transferred to a neutral mixed charged surface, the electrostatic attraction between γFg and the surface disappears. Meanwhile, the SAM surface is covered by double hydration layers, which is induced by the N(CH3)3(+) and COO(-) groups; water molecules around COO(-) groups are obviously denser than that around N(CH3)3(+) groups. With the combined contribution from double hydration layers and the vanishment of electrostatic attraction, γFg is forced to desorb from the surface. After hydrolysis, the internal structure of mixed SAM appears more ordered due to the electrostatic interactions between charged groups on the top of SAMs. The antimicrobial and antifouling materials are of great importance in many biological applications. The strong hydration property of surfaces and the interactions between proteins and surfaces play a key role in resisting protein adsorption. The mixed SAMs, constructed from a 1:1 combination of COOCH3- and N(CH3)3(+)-terminated thiols, can induce protein adsorption mainly through the electrostatic interaction. When the COOCH3-terminated thiols were hydrolyzed to negatively charged COO(-)-terminated thiols, the mixed-charged SAMs switched from antimicrobial to antifouling. Due to the strong hydration property of the mixed charged SAMs, the adsorbed γFg moved away from the surface. Understanding the interactions between protein and mixed-charged SAMs in the atomistic level is important for the practical design and development of new antimicrobial and antifouling materials. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Label-free biosensing with functionalized nanopipette probes.

PubMed

Umehara, Senkei; Karhanek, Miloslav; Davis, Ronald W; Pourmand, Nader

2009-03-24

Nanopipette technology can uniquely identify biomolecules such as proteins based on differences in size, shape, and electrical charge. These differences are determined by the detection of changes in ionic current as the proteins interact with the nanopipette tip coated with probe molecules. Here we show that electrostatic, biotin-streptavidin, and antibody-antigen interactions on the nanopipette tip surface affect ionic current flowing through a 50-nm pore. Highly charged polymers interacting with the glass surface modulated the rectification property of the nanopipette electrode. Affinity-based binding between the probes tethered to the surface and their target proteins caused a change in the ionic current due to a partial blockade or an altered surface charge. These findings suggest that nanopipettes functionalized with appropriate molecular recognition elements can be used as nanosensors in biomedical and biological research.

Suppression of protein adsorption on a charged phospholipid polymer interface.

PubMed

Xu, Yan; Takai, Madoka; Ishihara, Kazuhiko

2009-02-09

High capability of a charged interface to suppress adsorption of both anionic and cationic proteins was reported. The interface was covalently constructed on quartz by modifying with an anionic phospholipid copolymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-potassium 3-methacryloyloxypropyl sulfonate (PMPS)-co-3-methacryloxypropyl trimethoxysilane (MPTMSi)) (PMBSSi). The PMBSSi interfaces were very hydrophilic and homogeneous and could function effectively for a long time even under long-term fluidic working conditions. The PMBSSi density on the interface, which was controllable by adjusting the PMBSSi concentration of the modification solution, affected the surface properties, including the surface contact angle, the surface roughness, and the surface zeta-potential. When a PMBSSi modification was applied, the adsorption of various proteins (isoelectric point varying from 1.0 to 11.0) on quartz was reduced to at least 87% in amount, despite the various electrical natures these proteins have. The protein adsorption behavior on the PMBSSi interface depended more on the PMBSSi density than on the surface charge. The PMBSSi modification had a stable impact on the surface, not only at the physiologic ionic strength, but also over a range of the ionic strength, suggesting that electrostatic interactions do not dominate the behavior of protein adsorption to the PMBSSi surface.

Single helically folded aromatic oligoamides that mimic the charge surface of double-stranded B-DNA

NASA Astrophysics Data System (ADS)

Ziach, Krzysztof; Chollet, Céline; Parissi, Vincent; Prabhakaran, Panchami; Marchivie, Mathieu; Corvaglia, Valentina; Bose, Partha Pratim; Laxmi-Reddy, Katta; Godde, Frédéric; Schmitter, Jean-Marie; Chaignepain, Stéphane; Pourquier, Philippe; Huc, Ivan

2018-05-01

Numerous essential biomolecular processes require the recognition of DNA surface features by proteins. Molecules mimicking these features could potentially act as decoys and interfere with pharmacologically or therapeutically relevant protein-DNA interactions. Although naturally occurring DNA-mimicking proteins have been described, synthetic tunable molecules that mimic the charge surface of double-stranded DNA are not known. Here, we report the design, synthesis and structural characterization of aromatic oligoamides that fold into single helical conformations and display a double helical array of negatively charged residues in positions that match the phosphate moieties in B-DNA. These molecules were able to inhibit several enzymes possessing non-sequence-selective DNA-binding properties, including topoisomerase 1 and HIV-1 integrase, presumably through specific foldamer-protein interactions, whereas sequence-selective enzymes were not inhibited. Such modular and synthetically accessible DNA mimics provide a versatile platform to design novel inhibitors of protein-DNA interactions.

The influence of PAMAM dendrimers surface groups on their interaction with porcine pepsin.

PubMed

Ciolkowski, Michal; Rozanek, Monika; Bryszewska, Maria; Klajnert, Barbara

2013-10-01

In this study the ability of three polyamidoamine (PAMAM) dendrimers with different surface charge (positive, neutral and negative) to interact with a negatively charged protein (porcine pepsin) was examined. It was shown that the dendrimer with a positively charged surface (G4 PAMAM-NH2), as well as the dendrimer with a neutral surface (G4 PAMAM-OH), were able to inhibit enzymatic activity of pepsin. It was also found that these dendrimers act as mixed partially non-competitive pepsin inhibitors. The negatively charged dendrimer (G3.5 PAMAM-COOH) was not able to inhibit the enzymatic activity of pepsin, probably due to the electrostatic repulsion between this dendrimer and the protein. No correlation between changes in enzymatic activity of pepsin and alterations in CD spectrum of the protein was observed. It indicates that the interactions between dendrimers and porcine pepsin are complex, multidirectional and not dependent only on disturbances of the secondary structure. © 2013.

Increasing binding density of yeast cells by control of surface charge with allylamine grafting to ion modified polymer surfaces.

PubMed

Tran, Clara T H; Kondyurin, Alexey; Chrzanowski, Wojciech; Bilek, Marcela M M; McKenzie, David R

2014-10-01

Plasma immersion ion implantation (PIII) treatment of polymers creates a biointerface capable of direct covalent immobilization of biomolecules. The immobilization of protein molecules is achieved by covalent bonds formed between embedded radicals on the treated surface and amino acid side chains and cells can be immobilized through cell-wall proteins. The attachment density of negatively charged entities on a PIII treated surface is inhibited by its negative surface charge at neutral pH. To reduce the negative charge of PIII treated surfaces in phosphate buffer (pH 7.4, 11mM), we develop an effective approach of grafting allylamine monomers onto the treated surface. The results reveal reactions between allylamine and radicals on the PIII treated surface. One of these triggers polymerization, increasing the number of amine groups grafted. As a consequence, the PIII treated polystyrene surface after allylamine exposure becomes more hydrophobic and less negatively charged in phosphate buffer. Using yeast cells as an example, we have shown a significant improvement (6-15 times) of cell density immobilized on the PIII treated surface after exposure to allylamine. Copyright © 2014 Elsevier B.V. All rights reserved.

Label-free biosensing with functionalized nanopipette probes

PubMed Central

Umehara, Senkei; Karhanek, Miloslav; Davis, Ronald W.; Pourmand, Nader

2009-01-01

Nanopipette technology can uniquely identify biomolecules such as proteins based on differences in size, shape, and electrical charge. These differences are determined by the detection of changes in ionic current as the proteins interact with the nanopipette tip coated with probe molecules. Here we show that electrostatic, biotin-streptavidin, and antibody-antigen interactions on the nanopipette tip surface affect ionic current flowing through a 50-nm pore. Highly charged polymers interacting with the glass surface modulated the rectification property of the nanopipette electrode. Affinity-based binding between the probes tethered to the surface and their target proteins caused a change in the ionic current due to a partial blockade or an altered surface charge. These findings suggest that nanopipettes functionalized with appropriate molecular recognition elements can be used as nanosensors in biomedical and biological research. PMID:19264962

Domain Formation Induced by the Adsorption of Charged Proteins on Mixed Lipid Membranes

PubMed Central

Mbamala, Emmanuel C.; Ben-Shaul, Avinoam; May, Sylvio

2005-01-01

Peripheral proteins can trigger the formation of domains in mixed fluid-like lipid membranes. We analyze the mechanism underlying this process for proteins that bind electrostatically onto a flat two-component membrane, composed of charged and neutral lipid species. Of particular interest are membranes in which the hydrocarbon lipid tails tend to segregate owing to nonideal chain mixing, but the (protein-free) lipid membrane is nevertheless stable due to the electrostatic repulsion between the charged lipid headgroups. The adsorption of charged, say basic, proteins onto a membrane containing anionic lipids induces local lipid demixing, whereby charged lipids migrate toward (or away from) the adsorption site, so as to minimize the electrostatic binding free energy. Apart from reducing lipid headgroup repulsion, this process creates a gradient in lipid composition around the adsorption zone, and hence a line energy whose magnitude depends on the protein's size and charge and the extent of lipid chain nonideality. Above a certain critical lipid nonideality, the line energy is large enough to induce domain formation, i.e., protein aggregation and, concomitantly, macroscopic lipid phase separation. We quantitatively analyze the thermodynamic stability of the dressed membrane based on nonlinear Poisson-Boltzmann theory, accounting for both the microscopic characteristics of the proteins and lipid composition modulations at and around the adsorption zone. Spinodal surfaces and critical points of the dressed membranes are calculated for several different model proteins of spherical and disk-like shapes. Among the models studied we find the most substantial protein-induced membrane destabilization for disk-like proteins whose charges are concentrated in the membrane-facing surface. If additional charges reside on the side faces of the proteins, direct protein-protein repulsion diminishes considerably the propensity for domain formation. Generally, a highly charged flat face of a macroion appears most efficient in inducing large compositional gradients, hence a large and unfavorable line energy and consequently lateral macroion aggregation and, concomitantly, macroscopic lipid phase separation. PMID:15626713

Mapping the surface charge distribution of amyloid fibril

NASA Astrophysics Data System (ADS)

Lee, Gyudo; Lee, Wonseok; Lee, Hyungbeen; Woo Lee, Sang; Sung Yoon, Dae; Eom, Kilho; Kwon, Taeyun

2012-07-01

It is of high importance to measure and map the surface charge distribution of amyloids, since electrostatic interaction between amyloidogenic proteins and biomolecules plays a vital role in amyloidogenesis. In this work, we have measured and mapped the surface charge distributions of amyloids (i.e., β-lactoglobulin fibril) using Kelvin probe force microscopy. It is shown that the surface charge distribution is highly dependent on the conformation of amyloids (e.g., the helical pitch of amyloid fibrils) as well as the pH of a solvent.

Adhesion of osteoblasts to a nanorough titanium implant surface

PubMed Central

Gongadze, Ekaterina; Kabaso, Doron; Bauer, Sebastian; Slivnik, Tomaž; Schmuki, Patrik; van Rienen, Ursula; Iglič, Aleš

2011-01-01

This work considers the adhesion of cells to a nanorough titanium implant surface with sharp edges. The basic assumption was that the attraction between the negatively charged titanium surface and a negatively charged osteoblast is mediated by charged proteins with a distinctive quadrupolar internal charge distribution. Similarly, cation-mediated attraction between fibronectin molecules and the titanium surface is expected to be more efficient for a high surface charge density, resulting in facilitated integrin mediated osteoblast adhesion. We suggest that osteoblasts are most strongly bound along the sharp convex edges or spikes of nanorough titanium surfaces where the magnitude of the negative surface charge density is the highest. It is therefore plausible that nanorough regions of titanium surfaces with sharp edges and spikes promote the adhesion of osteoblasts. PMID:21931478

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

PubMed

Karkov, Hanne Sophie; Krogh, Berit Olsen; Woo, James; Parimal, Siddharth; Ahmadian, Haleh; Cramer, Steven M

2015-11-01

In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules. © 2015 Wiley Periodicals, Inc.

Improved mucoadhesion and cell uptake of chitosan and chitosan oligosaccharide surface-modified polymer nanoparticles for mucosal delivery of proteins.

PubMed

Dyawanapelly, Sathish; Koli, Uday; Dharamdasani, Vimisha; Jain, Ratnesh; Dandekar, Prajakta

2016-08-01

The main aim of the present study was to compare mucoadhesion and cellular uptake efficiency of chitosan (CS) and chitosan oligosaccharide (COS) surface-modified polymer nanoparticles (NPs) for mucosal delivery of proteins. We have developed poly (D, L-lactide-co-glycolide) (PLGA) NPs, surface-modified COS-PLGA NPs and CS-PLGA NPs, by using double emulsion solvent evaporation method, for encapsulating bovine serum albumin (BSA) as a model protein. Surface modification of NPs was confirmed using physicochemical characterization methods such as particle size and zeta potential, SEM, TEM and FTIR analysis. Both surface-modified PLGA NPs displayed a slow release of protein compared to PLGA NPs. Furthermore, we have explored the mucoadhesive property of COS as a material for modifying the surface of polymeric NPs. During in vitro mucoadhesion test, positively charged COS-PLGA NPs and CS-PLGA NPs exhibited enhanced mucoadhesion, compared to negatively charged PLGA NPs. This interaction was anticipated to improve the cell interaction and uptake of NPs, which is an important requirement for mucosal delivery of proteins. All nanoformulations were found to be safe for cellular delivery when evaluated in A549 cells. Moreover, intracellular uptake behaviour of FITC-BSA loaded NPs was extensively investigated by confocal laser scanning microscopy and flow cytometry. As we hypothesized, positively charged COS-PLGA NPs and CS-PLGA NPs displayed enhanced intracellular uptake compared to negatively charged PLGA NPs. Our results demonstrated that CS- and COS-modified polymer NPs could be promising carriers for proteins, drugs and nucleic acids via nasal, oral, buccal, ocular and vaginal mucosal routes.

Orienting proteins by nanostructured surfaces: evidence of a curvature-driven geometrical resonance.

PubMed

Messina, Grazia M L; Bocchinfuso, Gianfranco; Giamblanco, Nicoletta; Mazzuca, Claudia; Palleschi, Antonio; Marletta, Giovanni

2018-04-26

Experimental and theoretical reports have shown that nanostructured surfaces have a dramatic effect on the amount of protein adsorbed and the conformational state and, in turn, on the performances of the related devices in tissue engineering strategies. Here we report an innovative method to prepare silica-based nanostructured surfaces with a reproducible, well-defined local curvature, consisting of ordered hexagonally packed arrays of curved hemispheres, from nanoparticles of different diameters (respectively 147 nm, 235 nm and 403 nm). The nanostructured surfaces have been made chemically homogeneous by partially embedding silica nanoparticles in poly(hydroxymethylsiloxane) films, further modified by means of UV-O3 treatments. This paper has been focused on the experimental and theoretical study of laminin, taken as a model protein, to study the nanocurvature effects on the protein configuration at nanostructured surfaces. A simple model, based on the interplay of electrostatic interactions between the charged terminal domains of laminin and the nanocurved charged surfaces, closely reproduces the experimental findings. In particular, the model suggests that nanocurvature drives the orientation of rigid proteins by means of a "geometrical resonance" effect, involving the matching of dimensions, charge distribution and spatial arrangement of both adsorbed molecules and adsorbent nanostructures. Overall, the results pave the way to unravel the nanostructured surface effects on the intra- and inter-molecular organization processes of proteins.

Parallel tempering Monte Carlo simulations of lysozyme orientation on charged surfaces

NASA Astrophysics Data System (ADS)

Xie, Yun; Zhou, Jian; Jiang, Shaoyi

2010-02-01

In this work, the parallel tempering Monte Carlo (PTMC) algorithm is applied to accurately and efficiently identify the global-minimum-energy orientation of a protein adsorbed on a surface in a single simulation. When applying the PTMC method to simulate lysozyme orientation on charged surfaces, it is found that lysozyme could easily be adsorbed on negatively charged surfaces with "side-on" and "back-on" orientations. When driven by dominant electrostatic interactions, lysozyme tends to be adsorbed on negatively charged surfaces with the side-on orientation for which the active site of lysozyme faces sideways. The side-on orientation agrees well with the experimental results where the adsorbed orientation of lysozyme is determined by electrostatic interactions. As the contribution from van der Waals interactions gradually dominates, the back-on orientation becomes the preferred one. For this orientation, the active site of lysozyme faces outward, which conforms to the experimental results where the orientation of adsorbed lysozyme is co-determined by electrostatic interactions and van der Waals interactions. It is also found that despite of its net positive charge, lysozyme could be adsorbed on positively charged surfaces with both "end-on" and back-on orientations owing to the nonuniform charge distribution over lysozyme surface and the screening effect from ions in solution. The PTMC simulation method provides a way to determine the preferred orientation of proteins on surfaces for biosensor and biomaterial applications.

Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haarmeyer, Carolyn N.; Smith, Matthew D.; Chundawat, Shishir P. S.

Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterizedmore » 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Altogether, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases.« less

Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein

DOE PAGES

Haarmeyer, Carolyn N.; Smith, Matthew D.; Chundawat, Shishir P. S.; ...

2016-10-17

Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterizedmore » 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Altogether, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases.« less

Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein.

PubMed

Haarmeyer, Carolyn N; Smith, Matthew D; Chundawat, Shishir P S; Sammond, Deanne; Whitehead, Timothy A

2017-04-01

Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue toward energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28-0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases. Biotechnol. Bioeng. 2017;114: 740-750. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Strong Electrostatic Interactions Lead to Entropically Favorable Binding of Peptides to Charged Surfaces.

PubMed

Sprenger, K G; Pfaendtner, Jim

2016-06-07

Thermodynamic analyses can provide key insights into the origins of protein self-assembly on surfaces, protein function, and protein stability. However, obtaining quantitative measurements of thermodynamic observables from unbiased classical simulations of peptide or protein adsorption is challenging because of sampling limitations brought on by strong biomolecule/surface binding forces as well as time scale limitations. We used the parallel tempering metadynamics in the well-tempered ensemble (PTMetaD-WTE) enhanced sampling method to study the adsorption behavior and thermodynamics of several explicitly solvated model peptide adsorption systems, providing new molecular-level insight into the biomolecule adsorption process. Specifically studied were peptides LKα14 and LKβ15 and trpcage miniprotein adsorbing onto a charged, hydrophilic self-assembled monolayer surface functionalized with a carboxylic acid/carboxylate headgroup and a neutral, hydrophobic methyl-terminated self-assembled monolayer surface. Binding free energies were calculated as a function of temperature for each system and decomposed into their respective energetic and entropic contributions. We investigated how specific interfacial features such as peptide/surface electrostatic interactions and surface-bound ion content affect the thermodynamic landscape of adsorption and lead to differences in surface-bound conformations of the peptides. Results show that upon adsorption to the charged surface, configurational entropy gains of the released solvent molecules dominate the configurational entropy losses of the bound peptide. This behavior leads to an apparent increase in overall system entropy upon binding and therefore to the surprising and seemingly nonphysical result of an apparent increased binding free energy at elevated temperatures. Opposite effects and conclusions are found for the neutral surface. Additional simulations demonstrate that by adjusting the ionic strength of the solution, results that show the expected physical behavior, i.e., peptide binding strength that decreases with increasing temperature or is independent of temperature altogether, can be recovered on the charged surface. On the basis of this analysis, an overall free energy for the entire thermodynamic cycle for peptide adsorption on charged surfaces is constructed and validated with independent simulations.

Unfolding and inactivation of proteins by counterions in protein-nanoparticles interaction.

PubMed

Ghosh, Goutam; Gaikwad, Pallavi S; Panicker, Lata; Nath, Bimalendu B; Mukhopadhyaya, Rita

2016-09-01

In this work, the structure and activity of proteins; such as, hen egg lysozyme (HEWL) and calf intestine alkaline phosphatase (CIAP); have been investigated after incubation with surface coated iron oxide nanoparticles (IONPs) in water. IONPs were coated with counterions bound charge-ligands and were named as the charge-ligand counterions iron oxide nanoparticles (CLC-IONPs). The coating was done with tri-lithium citrate (TLC) and tri-potassium citrate (TKC) to have negative surface charge of CLC-IONPs and Li(+) and K(+), respectively, as counterions. To have positive surface charge, IONPs were coated with cetylpyridinium chloride (CPC) and cetylpyridinium iodide (CPI) having Cl(-) and I(-), respectively, as counterions. The secondary structure of proteins was measured using far ultraviolet circular dichroism (CD) spectroscopy which showed that both proteins were irreversibly unfolded after incubation with CLC-IONPs. The unfolded proteins were seen to be functionally inactive, as confirmed through their activity assays, i.e., HEWL with Escherichia coli (E. coli) and CIAP with para-nitrophenyl phosphate (pNPP). Additionally, we have observed that monomeric hemoglobin (Hb) from radio-resistant insect Chironomus ramosus (ChHb) was also partially unfolded upon interaction with CLC-IONPs. This work clearly shows the role of counterions in protein inactivation via protein-nanoparticles interaction and, therefore, CLC-IONPs could be used for therapeutic purpose. Copyright © 2016 Elsevier B.V. All rights reserved.

Zeta Potential Measurements on Solid Surfaces for in Vitro Biomaterials Testing: Surface Charge, Reactivity Upon Contact With Fluids and Protein Absorption

PubMed Central

Ferraris, Sara; Cazzola, Martina; Peretti, Veronica; Stella, Barbara; Spriano, Silvia

2018-01-01

Surface properties of biomaterials (e.g., roughness, chemical composition, charge, wettability, and hydroxylation degree) are key features to understand and control the complex interface phenomena that happens upon contact with physiological fluids. Numerous physico-chemical techniques can be used in order to investigate in depth these crucial material features. Among them, zeta potential measurements are widely used for the characterization of colloidal suspensions, but actually poorly explored in the study of solid surfaces, even if they can give significant information about surface charge in function of pH and indirectly about surface functional groups and reactivity. The aim of the present research is application of zeta potential measurements of solid surfaces for the in vitro testing of biomaterials. In particular, bare and surface modified Ti6Al4V samples have been compared in order to evaluate their isoelectric points (IEPs), surface charge at physiological pH, in vitro bioactivity [in simulated body fluid (SBF)] and protein absorption. Zeta potential titration was demonstrated as a suitable technique for the surface characterization of surface treated Ti6Al4V substrates. Significant shift of the isoelectric point was recorded after a chemical surface treatment (because of the exposition of hydroxyl groups), SBF soaking (because of apatite precipitation IEP moves close to apatite one) and protein absorption (IEP moves close to protein ones). Moreover, the shape of the curve gives information about exposed functional groups (e.g., a plateau in the basic range appears due to the exposition of acidic OH groups and in the acidic range due to exposition of basic NH2 groups). PMID:29868575

Lipids and topological rules of membrane protein assembly: balance between long and short range lipid-protein interactions.

PubMed

Vitrac, Heidi; Bogdanov, Mikhail; Heacock, Phil; Dowhan, William

2011-04-29

The N-terminal six-transmembrane domain (TM) bundle of lactose permease of Escherichia coli is uniformly inverted when assembled in membranes lacking phosphatidylethanolamine (PE). Inversion is dependent on the net charge of cytoplasmically exposed protein domains containing positive and negative residues, net charge of the membrane surface, and low hydrophobicity of TM VII acting as a molecular hinge between the two halves of lactose permease (Bogdanov, M., Xie, J., Heacock, P., and Dowhan, W. (2008) J. Cell Biol. 182, 925-935). Net neutral lipids suppress the membrane translocation potential of negatively charged amino acids, thus increasing the cytoplasmic retention potential of positively charged amino acids. Herein, TM organization of sucrose permease (CscB) and phenylalanine permease (PheP) as a function of membrane lipid composition was investigated to extend these principles to other proteins. For CscB, topological dependence on PE only becomes evident after a significant increase in the net negative charge of the cytoplasmic surface of the N-terminal TM bundle. High negative charge is required to overcome the thermodynamic block to inversion due to the high hydrophobicity of TM VII. Increasing the positive charge of the cytoplasmic surface of the N-terminal TM hairpin of PheP, which is misoriented in PE-lacking cells, favors native orientation in the absence of PE. PheP and CscB also display co-existing dual topologies dependent on changes in the charge balance between protein domains and the membrane lipids. Therefore, the topology of both permeases is dependent on PE. However, CscB topology is governed by thermodynamic balance between opposing lipid-dependent electrostatic and hydrophobic interactions.

Alternative Computational Protocols for Supercharging Protein Surfaces for Reversible Unfolding and Retention of Stability

PubMed Central

Der, Bryan S.; Kluwe, Christien; Miklos, Aleksandr E.; Jacak, Ron; Lyskov, Sergey; Gray, Jeffrey J.; Georgiou, George; Ellington, Andrew D.; Kuhlman, Brian

2013-01-01

Reengineering protein surfaces to exhibit high net charge, referred to as “supercharging”, can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from −11 to −61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and −30 net charge. Mid-charge variants demonstrated ∼3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding. PMID:23741319

Nano-functionalization of protein microspheres

NASA Astrophysics Data System (ADS)

Yoon, Sungkwon; Nichols, William T.

2014-08-01

Protein microspheres are promising building blocks for the assembly of complex functional materials. Here we demonstrate a set of three techniques that add functionality to the surface of protein microspheres. In the first technique, a positive surface charge on the protein spheres is deposited by electrostatic adsorption. Negatively charged silica and gold nanoparticle colloids can then electrostatically bind reversibly to the microsphere surface. In the second technique, nanoparticles are covalently anchored to the protein shell using a simple one-pot process. The strong covalent bond between sulfur groups in cysteine in the protein shell irreversibly binds to the gold nanoparticles. In the third technique, surface morphology of the protein microsphere is tuned through hydrodynamic instability at the water-oil interface. This is accomplished through the degree of solubility of the oil phase in water. Taken together these three techniques form a platform to create nano-functionalized protein microspheres, which can then be used as building blocks for the assembly of more complex macroscopic materials.

Competitive adsorption in model charged protein mixtures: Equilibrium isotherms and kinetics behavior

NASA Astrophysics Data System (ADS)

Fang, F.; Szleifer, I.

2003-07-01

The competitive adsorption of proteins of different sizes and charges is studied using a molecular theory. The theory enables the study of charged systems explicitly including the size, shape, and charge distributions in all the molecular species in the mixture. Thus, this approach goes beyond the commonly used Poisson-Boltzmann approximation. The adsorption isotherms of the protein mixtures are studied for mixtures of two proteins of different size and charge. The amount of proteins adsorbed and the fraction of each protein is calculated as a function of the bulk composition of the solution and the amount of salt in the system. It is found that the total amount of proteins adsorbed is a monotonically decreasing function of the fraction of large proteins on the bulk solution and for fixed protein composition of the salt concentration. However, the composition of the adsorbed layer is a complicated function of the bulk composition and solution ionic strength. The structure of the adsorb layer depends upon the bulk composition and salt concentration. In general, there are multilayers adsorbed due to the long-range character of the electrostatic interactions. When the composition of large proteins in bulk is in very large excess it is found that the structure of the adsorb multilayer is such that the layer in contact with the surface is composed by a mixture of large and small proteins. However, the second and third layers are almost exclusively composed of large proteins. The theory is also generalized to study the time-dependent adsorption. The approach is based on separation of time scales into fast modes for the ions from the salt and the solvent and slow for the proteins. The dynamic equations are written for the slow modes, while the fast ones are obtained from the condition of equilibrium constrained to the distribution of proteins given by the slow modes. Two different processes are presented: the adsorption from a homogeneous solution to a charged surface at low salt concentration, and large excess of the large proteins in bulk. The second process is the kinetics of structural and adsorption change by changing the salt concentration of the bulk solution from low to high. The first process shows a large overshoot of the large proteins on the surface due to their excess in solution, followed by a surface replacement by the smaller molecules. The second process shows a very fast desorption of the large proteins followed by adsorption at latter stages. This process is found to be driven by large electrostatic repulsions induced by the fast ions from the salt approaching the surface. The relevance of the theoretical predictions to experimental system and possible directions for improvements of the theory are discussed.

Examination and Manipulation of Protein Surface Charge in Solution with Electrospray Ionization Mass Spectrometry

ERIC Educational Resources Information Center

Gross, Deborah S.; Van Ryswyk, Hal

2014-01-01

Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool for examining the charge of proteins in solution. The charge can be manipulated through choice of solvent and pH. Furthermore, solution-accessible, protonated lysine side chains can be specifically tagged with 18-crown-6 ether to form noncovalent adducts. Chemical derivatization…

Interaction of proteins with weak amphoteric charged membrane surfaces: effect of pH.

PubMed

Matsumoto, Hidetoshi; Koyama, Yoshiyuki; Tanioka, Akihiko

2003-08-01

Weak amphoteric charged membranes were prepared by the graft copolymerization of poly(ethylene glycol) (PEG) derivatives with pendant ionizable groups onto polyethylene (PE) porous membranes. Two types of weak amphoteric charged membranes and two types of weak single charged membranes were prepared. The pH dependence of the protein (fluorescein isothiocyanate-labeled bovine serum albumin, FITC-BSA) adsorption onto the membranes was investigated by fluorescence spectroscopy. The interfacial charge properties of the membranes and protein were also characterized at different pH values by streaming potential and electrophoretic light scattering (ELS) measurements, respectively. The adsorbed amount onto each ionic PEG chain grafted membrane showed a uniform maximum value near the isoelectric point (IEP) of the protein (pH 4.1). On both sides of the IEP (pHs 3.3 and 7.2), the adsorption experiments and zeta (zeta) potential measurements were well correlated: the contribution of electrostatic interaction was dominant for the protein adsorption behavior. In the alkaline condition (pH 10.2), the adsorption experiments contradict the zeta potential measurements. It suggested that the conformational change of protein molecule influenced the adsorption behavior. Finally, these results indicated the potential of controlling the protein-ionic PEG chain interaction on the membrane surfaces by the pH adjustment of the outer solution.

Zwitterionic sulfobetaine-grafted poly(vinylidene fluoride) membrane with highly effective blood compatibility via atmospheric plasma-induced surface copolymerization.

PubMed

Chang, Yung; Chang, Wan-Ju; Shih, Yu-Ju; Wei, Ta-Chin; Hsiue, Ging-Ho

2011-04-01

Development of nonfouling membranes to prevent nonspecific protein adsorption and platelet adhesion is critical for many biomedical applications. It is always a challenge to control the surface graft copolymerization of a highly polar monomer from the highly hydrophobic surface of a fluoropolymer membrane. In this work, the blood compatibility of poly(vinylidene fluoride) (PVDF) membranes with surface-grafted electrically neutral zwitterionic poly(sulfobetaine methacrylate) (PSBMA), from atmospheric plasma-induced surface copolymerization, was studied. The effect of surface composition and graft morphology, electrical neutrality, hydrophilicity and hydration capability on blood compatibility of the membranes were determined. Blood compatibility of the zwitterionic PVDF membranes was systematically evaluated by plasma protein adsorption, platelet adhesion, plasma-clotting time, and blood cell hemolysis. It was found that the nonfouling nature and hydration capability of grafted PSBMA polymers can be effectively controlled by regulating the grafting coverage and charge balance of the PSBMA layer on the PVDF membrane surface. Even a slight charge bias in the grafted zwitterionic PSBMA layer can induce electrostatic interactions between proteins and the membrane surfaces, leading to surface protein adsorption, platelet activation, plasma clotting and blood cell hemolysis. Thus, the optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and the best antifouling, anticoagulant, and antihemolytic activities when comes into contact with human blood. © 2011 American Chemical Society

Dendrimer-protein interactions versus dendrimer-based nanomedicine.

PubMed

Shcharbin, Dzmitry; Shcharbina, Natallia; Dzmitruk, Volha; Pedziwiatr-Werbicka, Elzbieta; Ionov, Maksim; Mignani, Serge; de la Mata, F Javier; Gómez, Rafael; Muñoz-Fernández, Maria Angeles; Majoral, Jean-Pierre; Bryszewska, Maria

2017-04-01

Dendrimers are hyperbranched polymers belonging to the huge class of nanomedical devices. Their wide application in biology and medicine requires understanding of the fundamental mechanisms of their interactions with biological systems. Summarizing, electrostatic force plays the predominant role in dendrimer-protein interactions, especially with charged dendrimers. Other kinds of interactions have been proven, such as H-bonding, van der Waals forces, and even hydrophobic interactions. These interactions depend on the characteristics of both participants: flexibility and surface charge of a dendrimer, rigidity of protein structure and the localization of charged amino acids at its surface. pH and ionic strength of solutions can significantly modulate interactions. Ligands and cofactors attached to a protein can also change dendrimer-protein interactions. Binding of dendrimers to a protein can change its secondary structure, conformation, intramolecular mobility and functional activity. However, this strongly depends on rigidity versus flexibility of a protein's structure. In addition, the potential applications of dendrimers to nanomedicine are reviwed related to dendrimer-protein interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

NASA Astrophysics Data System (ADS)

Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

2015-01-01

Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein-particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

Modulating protein behaviors on responsive surface by external electric fields: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Xie, Yun; Pan, Yufang; Zhang, Rong; Liang, Ying; Li, Zhanchao

2015-01-01

Molecular dynamics simulations were employed to investigate the modulation of protein behaviors on the electrically responsive zwitterionic phosphorylcholine self-assembled monolayers (PC-SAMs). Results show that PC-SAMs could sensitively respond to the applied electric fields and exhibit three states with different charge distributions, namely both the negatively charged phosphate groups and the positively charged choline groups are exposed to the solution in the absence of electric fields (state 1), phosphate groups exposed in the presence of positive electric fields (state 2), and choline groups exposed in the presence of negative electric fields (state 3). Under state 1, the adsorption of Cyt c on the PC-SAM is reversible and the orientations of Cyt c are randomly distributed. Under state 2, the adsorption of Cyt c is enhanced due to the electrostatic attractions between the exposed phosphate groups and the positively charged protein; when adsorbed on the PC-SAMs, Cyt c tends to adopt the orientation with the heme plane perpendicular to the surface plane, and the percentage of this orientation increases as the field strength rises up. Under state 3, the adsorption of Cyt c is retarded because of the electrostatic repulsions between the exposed choline groups and the protein; however, if the gaps between PC chains are large enough, Cyt c could insert into the PC-SAM and access the phosphate groups after overcoming a slight energy barrier. Under three states, the basic backbone structures of Cyt c are well kept within the simulation time since the conformation of Cyt c is mainly affected by the surface-generated electric fields, whose strengths are modulated by the external electric fields and are not strong enough to deform protein. The results indicate the possibility of regulating protein behaviors, including promoting or retarding protein adsorption and regulating protein orientations, on responsive surfaces by applying electric fields on the surfaces without worrying protein deformation, which may be helpful in the applications of protein separation and controlled drug delivery.

Adsorption of transgenic insecticidal Cry1Ab protein to silica particles. Effects on transport and bioactivity.

PubMed

Madliger, Michael; Gasser, Christoph A; Schwarzenbach, René P; Sander, Michael

2011-05-15

Bt crops are genetically modified to be resistant against insect pests by expressing insecticidal Cry proteins. The processes governing the fate and bioavailability of the expressed transgenic Cry proteins in soils are poorly understood. We studied adsorption of Cry1Ab to negatively charged silica (SiO(2)) particles, a major soil constituent and a model for negatively charged mineral surfaces, at pH 5 to 10 and ionic strengths I = 10 mM to 250 mM, both in solution depletion and saturated column transport experiments. Cry1Ab-SiO(2) interactions were dominated by patch-controlled electrostatic attraction (PCEA), as evident from increasing Cry1Ab attraction to SiO(2) with decreasing I at pH at which both Cry1Ab and SiO(2) were net negatively charged. Experimental and modeling evidence is provided that the surface heterogeneity of SiO(2) particles modulated PCEA, leading to a fraction of adsorption sites with slow Cry1Ab desorption kinetics. Desorption rates from these sites increased upon increasing the solution pH. In toxicity bioassays, we demonstrated that Cry1Ab retained insecticidal activity when adsorbed to SiO(2), suggesting high protein conformational stability during adsorption-desorption cycles. Models predicting Cry1A protein adsorption in soils therefore need to account for combined effects of the nonuniform protein surface charge distribution and of sorbent surface heterogeneity.

Adsorption of hydrophobin on different self-assembled monolayers: the role of the hydrophobic dipole and the electric dipole.

PubMed

Peng, Chunwang; Liu, Jie; Zhao, Daohui; Zhou, Jian

2014-09-30

In this work, the adsorptions of hydrophobin (HFBI) on four different self-assembled monolayers (SAMs) (i.e., CH3-SAM, OH-SAM, COOH-SAM, and NH2-SAM) were investigated by parallel tempering Monte Carlo and molecular dynamics simulations. Simulation results indicate that the orientation of HFBI adsorbed on neutral surfaces is dominated by a hydrophobic dipole. HFBI adsorbs on the hydrophobic CH3-SAM through its hydrophobic patch and adopts a nearly vertical hydrophobic dipole relative to the surface, while it is nearly horizontal when adsorbed on the hydrophilic OH-SAM. For charged SAM surfaces, HFBI adopts a nearly vertical electric dipole relative to the surface. HFBI has the narrowest orientation distribution on the CH3-SAM, and thus can form an ordered monolayer and reverse the wettability of the surface. For HFBI adsorption on charged SAMs, the adsorption strength weakens as the surface charge density increases. Compared with those on other SAMs, a larger area of the hydrophobic patch is exposed to the solution when HFBI adsorbs on the NH2-SAM. This leads to an increase of the hydrophobicity of the surface, which is consistent with the experimental results. The binding of HFBI to the CH3-SAM is mainly through hydrophobic interactions, while it is mediated through a hydration water layer near the surface for the OH-SAM. For the charged SAM surfaces, the adsorption is mainly induced by electrostatic interactions between the charged surfaces and the oppositely charged residues. The effect of a hydrophobic dipole on protein adsorption onto hydrophobic surfaces is similar to that of an electric dipole for charged surfaces. Therefore, the hydrophobic dipole may be applied to predict the probable orientations of protein adsorbed on hydrophobic surfaces.

On the design of composite protein-quantum dot biomaterials via self-assembly.

PubMed

Majithia, Ravish; Patterson, Jan; Bondos, Sarah E; Meissner, Kenith E

2011-10-10

Incorporation of nanoparticles during the hierarchical self-assembly of protein-based materials can impart function to the resulting composite materials. Herein we demonstrate that the structure and nanoparticle distribution of composite fibers are sensitive to the method of nanoparticle addition and the physicochemical properties of both the nanoparticle and the protein. Our model system consists of a recombinant enhanced green fluorescent protein-Ultrabithorax (EGFP-Ubx) fusion protein and luminescent CdSe-ZnS core-shell quantum dots (QDs), allowing us to optically assess the distribution of both the protein and nanoparticle components within the composite material. Although QDs favorably interact with EGFP-Ubx monomers, the relatively rough surface morphology of composite fibers suggests EGFP-Ubx-QD conjugates impact self-assembly. Indeed, QDs templated onto EGFP-Ubx film post-self-assembly can be subsequently drawn into smooth composite fibers. Additionally, the QD surface charge impacts QD distribution within the composite material, indicating that surface charge plays an important role in self-assembly. QDs with either positively or negatively charged coatings significantly enhance fiber extensibility. Conversely, QDs coated with hydrophobic moieties and suspended in toluene produce composite fibers with a heterogeneous distribution of QDs and severely altered fiber morphology, indicating that toluene severely disrupts Ubx self-assembly. Understanding factors that impact the protein-nanoparticle interaction enables manipulation of the structure and mechanical properties of composite materials. Since proteins interact with nanoparticle surface coatings, these results should be applicable to other types of nanoparticles with similar chemical groups on the surface.

Miscibility of binary monolayers at the air-water interface and interaction of protein with immobilized monolayers by surface plasmon resonance technique.

PubMed

Wang, Yuchun; Du, Xuezhong

2006-07-04

The miscibility and stability of the binary monolayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DOMA) at the air-water interface and the interaction of ferritin with the immobilized monolayers have been studied in detail using surface pressure-area isotherms and surface plasmon resonance technique, respectively. The surface pressure-area isotherms indicated that the binary monolayers of DPPC and DOMA at the air-water interface were miscible and more stable than the monolayers of the two individual components. The surface plasmon resonance studies indicated that ferritin binding to the immobilized monolayers was primarily driven by the electrostatic interaction and that the amount of adsorbed protein at saturation was closely related not only to the number of positive charges in the monolayers but also to the pattern of positive charges at a given mole fraction of DOMA. The protein adsorption kinetics was determined by the properties of the monolayers (i.e., the protein-monolayer interaction) and the structure of preadsorbed protein molecules (i.e., the protein-protein interaction).

Glutamate Induced Thermal Equilibrium Intermediate and Counteracting Effect on Chemical Denaturation of Proteins.

PubMed

Anumalla, Bramhini; Prabhu, N Prakash

2018-01-25

When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two-state transitions during chemical denaturation. The extent of stability exerted by Glu is higher for RNase A at higher temperature, whereas it provides more stability for α-LA at lower temperature. Thus, the experiments indicate that Glu induces a thermal equilibrium intermediate and increases the thermodynamic stability of proteins irrespective of their surface charges. The extent of stability varies between the proteins in a temperature-dependent manner.

Modelling charge interactions in the prion protein: predictions for pathogenesis.

PubMed

Warwicker, J

1999-04-30

Calculations are presented for the pH-dependence of stability and membrane charge complementarity of prion protein fragments. The theoretical results are compared with reported characterisations of prion protein folding in vitro. Discussion of models for conformational change and pathogenesis in vivo leads to the prediction of amino acids that could mediate sensitivity to the endosomal pH and to a design strategy for recombinant prion proteins with an increased susceptibility to prion proteinSc-like properties in vitro. In this model, the protective effect of certain basic polymorphisms can be interpreted in terms of oligomerisation on a negatively-charged surface.

The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces

PubMed Central

Arbely, Eyal; Neuweiler, Hannes; Sharpe, Timothy D; Johnson, Christopher M; Fersht, Alan R

2010-01-01

Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Hückel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution. PMID:20662005

Charge Segregation and Low Hydrophobicity Are Key Features of Ribosomal Proteins from Different Organisms*

PubMed Central

Fedyukina, Daria V.; Jennaro, Theodore S.; Cavagnero, Silvia

2014-01-01

Ribosomes are large and highly charged macromolecular complexes consisting of RNA and proteins. Here, we address the electrostatic and nonpolar properties of ribosomal proteins that are important for ribosome assembly and interaction with other cellular components and may influence protein folding on the ribosome. We examined 50 S ribosomal subunits from 10 species and found a clear distinction between the net charge of ribosomal proteins from halophilic and non-halophilic organisms. We found that ∼67% ribosomal proteins from halophiles are negatively charged, whereas only up to ∼15% of ribosomal proteins from non-halophiles share this property. Conversely, hydrophobicity tends to be lower for ribosomal proteins from halophiles than for the corresponding proteins from non-halophiles. Importantly, the surface electrostatic potential of ribosomal proteins from all organisms, especially halophiles, has distinct positive and negative regions across all the examined species. Positively and negatively charged residues of ribosomal proteins tend to be clustered in buried and solvent-exposed regions, respectively. Hence, the majority of ribosomal proteins is characterized by a significant degree of intramolecular charge segregation, regardless of the organism of origin. This key property enables the ribosome to accommodate proteins within its complex scaffold regardless of their overall net charge. PMID:24398678

Adsorption of transgenic insecticidal Cry1Ab protein to SiO2. 2. Patch-controlled electrostatic attraction.

PubMed

Madliger, Michael; Sander, Michael; Schwarzenbach, René P

2010-12-01

Adsorption governs the fate of Cry proteins from genetically modified Bt crops in soils. The effect of ionic strength (I) on the adsorption of Cry1Ab (isoelectric point IEP(Cry1Ab) ≈ 6) to negatively charged quartz (SiO(2)) and positively charged poly-L-lysine (PLL) was investigated at pH 5 to 8, using quartz crystal microbalance with dissipation monitoring and optical waveguide lightmode spectroscopy. Cry1Ab adsorbed via positively and negatively charged surface patches to SiO(2) and PLL, respectively. This patch controlled electrostatic attraction (PCEA) explains the observed increase in Cry1Ab adsorption to sorbents that carried the same net charge as the protein (SiO(2) at pH > IEP(Cry1Ab) and PLL at pH < IEP(Cry1Ab)) with decreasing I. In contrast, the adsorption of two reference proteins, BSA and HEWL, with different adsorption mechanism, were little affected by similar changes of I. Consistent with PCEA, Cry1Ab desorption from SiO(2) at pH > IEP(Cry1Ab) increased with increasing I and pH. Weak Cry1Ab-SiO(2) PCEA above pH 7 resulted in reversible, concentration dependent adsorption. Solution depletion experiments showed that PCEA also governed Cry1Ab adsorption to SiO(2) particles at environmentally relevant concentrations (a few ng mL(-1)). These results imply that models describing Cry1Ab adsorption to charged surfaces in soils need to account for the nonuniform surface charge distribution of the protein.

Modeling of protein-anion exchange resin interaction for the human growth hormone charge variants.

PubMed

Lapelosa, Mauro; Patapoff, Thomas W; Zarraga, Isidro E

2015-12-01

Modeling ion exchange chromatography (IEC) behavior has generated significant interest because of the wide use of IEC as an analytical technique as well as a preparative protein purification process; indeed there is a need for better understanding of what drives the unique behavior of protein charge variants. We hypothesize that a complex protein molecule, which contains both hydrophobic and charged moieties, would interact strongly with an in silico designed resin through charged electrostatic patches on the surface of the protein. In the present work, variants of recombinant human growth hormone that mimic naturally-occurring deamidation products were produced and characterized in silico. The study included these four variants: rhGH, N149D, N152D, and N149D/N152D. Poisson-Boltzmann calculations were used to determine surface electrostatic potential. Metropolis Monte Carlo simulations were carried out with the resulting variants to simulate IEC systems, examining the free energy of the interaction of the protein with an in silico anion exchange column represented by polylysine polypeptide. The results show that the charge variants have different average binding energies and the free energy of interaction can be used to predict the retention time for the different variants. Copyright © 2015 Elsevier B.V. All rights reserved.

Kinesin motor protein as an electrostatic ratchet machine

NASA Astrophysics Data System (ADS)

Tsironis, George; Ciudad, Aleix; Sancho, Jose Maria

2008-03-01

Kinesin and related motor proteins utilize ATP fuel to propel themselves along the external surface of microtubules in a processive and directional fashion. We show that the observed step-like motion is possible through time varying charge distributions furnished by the ATP hydrolysis circle while the static charge configuration on the microtuble provides the guide for motion. Thus, while the chemical hydrolysis energy induces appropriate local conformational changes, the motor translational energy is fundamentally electrostatic. Numerical simulations of the mechanical equations of motion show that processivity and directionality are direct consequences of the ATP-dependent electrostatic interaction between the different charge distributions of kinesin and microtubule. Treating proterins as continuous dielectric media and using a Green's function formalism we find analytical expressions for the electrostatic energy in the vicinity of the protein surfaces. We calculate the Bjerrum length in the interior of the protein and analyze its dependence on the charge proximity to the protein interface. We apply these results to kinesin and estimate the pure electrostatic ATP-ADP interaction to be larger than 2k T.

Biosafety evaluations of well-dispersed mesoporous silica nanoparticles: towards in vivo-relevant conditions

NASA Astrophysics Data System (ADS)

Liu, Tsang-Pai; Wu, Si-Han; Chen, Yi-Ping; Chou, Chih-Ming; Chen, Chien-Tsu

2015-04-01

This study aimed to investigate how mesoporous silica nanoparticles (MSNs), especially focussing on their surface functional groups, interacted with Raw 264.7 macrophages, as well as with zebrafish embryos. Upon introducing nanoparticles into a biological milieu, adsorption of proteins and biomolecules onto the nanoparticle surface usually progresses rapidly. Nanoparticles bound with proteins can result in physiological and pathological changes, but the mechanisms remain to be elucidated. In order to evaluate how protein corona affected MSNs and the subsequent cellular immune responses, we experimented in both serum and serum-deprived conditions. Our findings indicated that the level of p-p38 was significantly elevated by the positively charged MSNs, whereas negatively charged MSNs resulted in marked ROS production. Most significantly, our experiments demonstrated that the presence of protein efficiently mitigated the potential nano-hazard. On the other hand, strongly positively charged MSNs caused 94% of the zebrafish embryos to die. In that case, the toxicity caused by the quaternary ammonium ligands on the surface of those nanoparticles was exerted in a dose-dependent manner. In summary, these fundamental studies here provide valuable insights into the design of better biocompatible nanomaterials in the future.This study aimed to investigate how mesoporous silica nanoparticles (MSNs), especially focussing on their surface functional groups, interacted with Raw 264.7 macrophages, as well as with zebrafish embryos. Upon introducing nanoparticles into a biological milieu, adsorption of proteins and biomolecules onto the nanoparticle surface usually progresses rapidly. Nanoparticles bound with proteins can result in physiological and pathological changes, but the mechanisms remain to be elucidated. In order to evaluate how protein corona affected MSNs and the subsequent cellular immune responses, we experimented in both serum and serum-deprived conditions. Our findings indicated that the level of p-p38 was significantly elevated by the positively charged MSNs, whereas negatively charged MSNs resulted in marked ROS production. Most significantly, our experiments demonstrated that the presence of protein efficiently mitigated the potential nano-hazard. On the other hand, strongly positively charged MSNs caused 94% of the zebrafish embryos to die. In that case, the toxicity caused by the quaternary ammonium ligands on the surface of those nanoparticles was exerted in a dose-dependent manner. In summary, these fundamental studies here provide valuable insights into the design of better biocompatible nanomaterials in the future. Electronic supplementary information (ESI) available: TEM image, size distribution histogram of as-synthesized wn-R-MSN@PEG. TGA measurements. Cellular uptake efficiency. WST-1 analysis. Western blot assays. Confocal images and zebrafish mortality. See DOI: 10.1039/c4nr07421a

In-cell thermodynamics and a new role for protein surfaces.

PubMed

Smith, Austin E; Zhou, Larry Z; Gorensek, Annelise H; Senske, Michael; Pielak, Gary J

2016-02-16

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

Organophosphonate biofunctionalization of diamond electrodes.

PubMed

Caterino, R; Csiki, R; Wiesinger, M; Sachsenhauser, M; Stutzmann, M; Garrido, J A; Cattani-Scholz, A; Speranza, Giorgio; Janssens, S D; Haenen, K

2014-08-27

The modification of the diamond surface with organic molecules is a crucial aspect to be considered for any bioapplication of this material. There is great interest in broadening the range of linker molecules that can be covalently bound to the diamond surface. In the case of protein immobilization, the hydropathicity of the surface has a major influence on the protein conformation and, thus, on the functionality of proteins immobilized at surfaces. For electrochemical applications, particular attention has to be devoted to avoid that the charge transfer between the electrode and the redox center embedded in the protein is hindered by a thick insulating linker layer. This paper reports on the grafting of 6-phosphonohexanoic acid on OH-terminated diamond surfaces, serving as linkers to tether electroactive proteins onto diamond surfaces. X-ray photoelectron spectroscopy (XPS) confirms the formation of a stable layer on the surface. The charge transfer between electroactive molecules and the substrate is studied by electrochemical characterization of the redox activity of aminomethylferrocene and cytochrome c covalently bound to the substrate through this linker. Our work demonstrates that OH-terminated diamond functionalized with 6-phosphonohexanoic acid is a suitable platform to interface redox-proteins, which are fundamental building blocks for many bioelectronics applications.

Adsorption of polyelectrolyte-like proteins to silica surfaces and the impact of pH on the response to ionic strength. A Monte Carlo simulation and ellipsometry study.

PubMed

Hyltegren, Kristin; Skepö, Marie

2017-05-15

The adsorbed amount of the polyelectrolyte-like protein histatin 5 on a silica surface depends on the pH and the ionic strength of the solution. Interestingly, an increase in ionic strength affects the adsorbed amount differently depending on the pH of the solution, as shown by ellipsometry measurements (Hyltegren, 2016). We have tested the hypothesis that the same (qualitative) trends can be found also from a coarse-grained model that takes all charge-charge interactions into account within the frameworks of Gouy-Chapman and Debye-Hückel theories. Using the same coarse-grained model as in our previous Monte Carlo study of single protein adsorption (Hyltegren, 2016), simulations of systems with many histatin 5 molecules were performed and then compared with ellipsometry measurements. The strength of the short-ranged attractive interaction between the protein and the surface was varied. The coarse-grained model does not qualitatively reproduce the pH-dependence of the experimentally observed trends in adsorbed amount as a function of ionic strength. However, the simulations cast light on the balance between electrostatic attraction between protein and surface and electrostatic repulsion between adsorbed proteins, the deficiencies of the Langmuir isotherm, and the implications of protein charge regulation in concentrated systems. Copyright © 2017 Elsevier Inc. All rights reserved.

Rational modification of protein stability by targeting surface sites leads to complicated results

PubMed Central

Xiao, Shifeng; Patsalo, Vadim; Shan, Bing; Bi, Yuan; Green, David F.; Raleigh, Daniel P.

2013-01-01

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions. PMID:23798426

Non-interacting surface solvation and dynamics in protein-protein interactions.

PubMed

Visscher, Koen M; Kastritis, Panagiotis L; Bonvin, Alexandre M J J

2015-03-01

Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein-protein binding, even for simple lock-and-key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein-protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein-protein surfaces. We compare properties of the interface, rim, and non-interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non-interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non-interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein-protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein-protein complexes from unintended protein-protein interactions. © 2014 Wiley Periodicals, Inc.

Overcharging below the nanoscale: Multivalent cations reverse the ion selectivity of a biological channel

NASA Astrophysics Data System (ADS)

García-Giménez, Elena; Alcaraz, Antonio; Aguilella, Vicente M.

2010-02-01

We report charge inversion within a nanoscopic biological protein ion channel in salts of multivalent ions. The presence of positive divalent and trivalent counterions reverses the cationic selectivity of the OmpF channel, a general diffusion porin located in the outer membrane of E. coli. We discuss the conditions under which charge inversion can be inferred from the change in sign of the measured quantity, the channel zero current potential. By comparing experimental results in protein channels whose charge has been modified after site-directed mutagenesis, the predictions of current theories of charge inversion are critically examined. It is emphasized that charge inversion does not necessarily increase with the bare surface charge density of the interface and that even this concept of surface charge density may become meaningless in some biological ion channels. Thus, any theory based on electrostatic correlations or chemical binding should explicitly take into account the particular structure of the charged interface.

Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy

PubMed Central

Klausen, Lasse Hyldgaard; Fuhs, Thomas; Dong, Mingdong

2016-01-01

Local surface charge density of lipid membranes influences membrane–protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values. PMID:27561322

Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy

NASA Astrophysics Data System (ADS)

Klausen, Lasse Hyldgaard; Fuhs, Thomas; Dong, Mingdong

2016-08-01

Local surface charge density of lipid membranes influences membrane-protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values.

Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy.

PubMed

Klausen, Lasse Hyldgaard; Fuhs, Thomas; Dong, Mingdong

2016-08-26

Local surface charge density of lipid membranes influences membrane-protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values.

Studies on surface morphology and electrical conductivity of PEDOT:PSS thin films in presence of gold nanoparticles

NASA Astrophysics Data System (ADS)

Bhowal, Ashim Chandra; Kundu, Sarathi

2018-04-01

PEDOT:PSS is a water soluble conducting polymer consists of positively charged PEDOT and negatively charged PSS. However, this polymer suffers low conductivity problem which restrict its use. In this paper, electrical conductivity of PEDOT:PSS thin films is improved by using charged gold nanoparticles. The nanoparticles used are synthesized using lysozyme protein. The nanoparticles coated with lysozyme protein possess positive zeta potential. In the presence of gold nanoparticles due to electrostatic interaction between positively charged nanoparticles and negatively charged PSS chains, modification takes place in the surface morphology and electrical behaviors of PEDOT:PSS thin films. The changes in the polymer matrix conformations in the presence of nanoparticles are studied by Fourier transformed Infra-red (FTIR) spectroscopy, whereas the surface morphology of prepared thin films before and after interaction with nanoparticles is investigated through atomic force microscopy (AFM). Four probe method is used to measure the variation of electrical conductivity from I-V characteristics curves.

Pulsed laser deposited metal oxide thin films mediated controlled adsorption of proteins

NASA Astrophysics Data System (ADS)

Kim, Se Jin

Several metal oxide thin films were grown on Si substrate by pulsed laser deposition for controlling adsorption of proteins. No intentional heating of substrate and introduction of oxygen gas during growth were employed. Additionally, fibrinogen, bovine serum albumin (BSA), and lysozyme were used as model protein in this study. The film properties such as cyratllinity, surface roughness, surface electrical charge and chemistry were investigated by many techniques in order to obtain the relationship with protein adsorption. Firstly, as grown Ta2O5 and ZnO thin film were used to study the effects of surface charge on the behaviors of BSA and lysozyme adsorption. The protein thickness results by ellipsometry showed that negatively charged Ta2O5 had a stronger affinity to positively charged lysozyme, while positively charged ZnO had a stronger affinity to negatively charged BSA. The results confirmed electrostatic interaction due to surface charge is one of main factors for determining adsorption of proteins. Furthermore, annealing studies were performed by heat treatment of as grown Ta2O5 and ZnO at 800°C in air ambience. Annealed Ta2O5 thin film had almost wetting property (from 10.02° to less than 1˜2°) and the change of cystallinity (from amorphous to cyrsalline) while annealed ZnO thin film had a reduced contact angle (from 75.65° to 39.41°) and remained to crystalline structure. The fibrinogen thickness on annealed Ta2O5 film was increased compared with as grown sample, while heat treated ZnO film showed much reduction of fibrinogen adsorption. Binary Ta-Zn oxide thin films (TZ) were grown by preparing PLD target composed of 50 wt% Ta2O5 and 50 wt% ZnO. This binary film had IEP pH 7.1 indicating nearly neutral charge in pH 7.4 PBS solution, and hydrophilic property. Ellipsometrical results showed that TZ film had the lowest fibrinogen, BSA and lysozyme thickness after 120 min adsorption compared with Ta2O5 and ZnO. Other samples, bilayer oxide films in which Ta2O5 and ZnO coexist were also employed to study adsorption behaviors. Especially, Ta2O 5-based bilayer films revealed zero adsorption of lysozyme.

Theoretical models for electrochemical impedance spectroscopy and local ζ-potential of unfolded proteins in nanopores

NASA Astrophysics Data System (ADS)

Vitarelli, Michael J.; Talaga, David S.

2013-09-01

Single solid-state nanopores find increasing use for electrical detection and/or manipulation of macromolecules. These applications exploit the changes in signals due to the geometry and electrical properties of the molecular species found within the nanopore. The sensitivity and resolution of such measurements are also influenced by the geometric and electrical properties of the nanopore. This paper continues the development of an analytical theory to predict the electrochemical impedance spectra of nanopores by including the influence of the presence of an unfolded protein using the variable topology finite Warburg impedance model previously published by the authors. The local excluded volume of, and charges present on, the segment of protein sampled by the nanopore are shown to influence the shape and peak frequency of the electrochemical impedance spectrum. An analytical theory is used to relate the capacitive response of the electrical double layer at the surface of the protein to both the charge density at the protein surface and the more commonly measured zeta potential. Illustrative examples show how the theory predicts that the varying sequential regions of surface charge density and excluded volume dictated by the protein primary structure may allow for an impedance-based approach to identifying unfolded proteins.

Effects of polymer graft properties on protein adsorption and transport in ion exchange chromatography: a multiscale modeling study.

PubMed

Basconi, Joseph E; Carta, Giorgio; Shirts, Michael R

2015-04-14

Multiscale simulation is used to study the adsorption of lysozyme onto ion exchangers obtained by grafting charged polymers into a porous matrix, in systems with various polymer properties and strengths of electrostatic interaction. Molecular dynamics simulations show that protein partitioning into the polymer-filled pore space increases with the overall charge content of the polymers, while the diffusivity in the pore space decreases. However, the combination of greatly increased partitioning and modestly decreased diffusion results in macroscopic transport rates that increase as a function of charge content, as the large concentration driving force due to enhanced pore space partitioning outweighs the reduction in the pore space diffusivity. Matrices having greater charge associated with the grafted polymers also exhibit more diffuse intraparticle concentration profiles during transient adsorption. In systems with a high charge content per polymer and a low protein loading, the polymers preferentially partition toward the surface due to favorable interactions with the surface-bound protein. These results demonstrate the potential of multiscale modeling to illuminate qualitative trends between molecular properties and the adsorption equilibria and kinetic properties observable on macroscopic scales.

Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport.

PubMed

McCauliff, Leslie A; Xu, Zhi; Li, Ran; Kodukula, Sarala; Ko, Dennis C; Scott, Matthew P; Kahn, Peter C; Storch, Judith

2015-11-06

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2(-/-) fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Counterion effects in protein nanoparticle electrostatic binding: a theoretical study.

PubMed

Ghosh, Goutam

2015-04-01

Effects of counterions on the folding conformation of proteins, bound electrostatically on the surface of charge-ligand functionalized nanoparticles, have been investigated based on the protein folding energy calculation. The folding energy of a protein has been taken as a sum of the short range interaction energies, like, the van der Waals attraction and the hydrogen bond energies, and the long range coulomb interaction energy. On electrostatic binding, counterions associated with surface ligands of nanoparticles diffuse into bound proteins through the medium of dispersion. As a result, bound proteins partially unfold, as observed in circular dichroism experiments, which has been realized using the "charge-dipole" and the "charge-induced dipole" interactions of counterions with polar and non-polar residues, respectively. The effect of counterions solvation in the dispersing medium, e.g., water, which causes water molecules to polarize around the counterions, has also been considered. The folding energy of bound proteins has been seen to decrease proportionally with the increasing number of diffusion of counterions and their polarizability. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of Acids, Bases, and Heteroatoms on Proximal Radial Distribution Functions for Proteins.

PubMed

Nguyen, Bao Linh; Pettitt, B Montgomery

2015-04-14

The proximal distribution of water around proteins is a convenient method of quantifying solvation. We consider the effect of charged and sulfur-containing amino acid side-chain atoms on the proximal radial distribution function (pRDF) of water molecules around proteins using side-chain analogs. The pRDF represents the relative probability of finding any solvent molecule at a distance from the closest or surface perpendicular protein atom. We consider the near-neighbor distribution. Previously, pRDFs were shown to be universal descriptors of the water molecules around C, N, and O atom types across hundreds of globular proteins. Using averaged pRDFs, a solvent density around any globular protein can be reconstructed with controllable relative error. Solvent reconstruction using the additional information from charged amino acid side-chain atom types from both small models and protein averages reveals the effects of surface charge distribution on solvent density and improves the reconstruction errors relative to simulation. Solvent density reconstructions from the small-molecule models are as effective and less computationally demanding than reconstructions from full macromolecular models in reproducing preferred hydration sites and solvent density fluctuations.

Surface Induced Dissociation Yields Quaternary Substructure of Refractory Noncovalent Phosphorylase B and Glutamate Dehydrogenase Complexes

NASA Astrophysics Data System (ADS)

Ma, Xin; Zhou, Mowei; Wysocki, Vicki H.

2014-03-01

Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.

Surface charge- and space-dependent transport of proteins in crowded environments of nanotailored posts.

PubMed

Choi, Chang Kyoung; Fowlkes, Jason D; Retterer, Scott T; Siuti, Piro; Iyer, Sukanya; Doktycz, Mitchel J

2010-06-22

The reaction and diffusion of molecules across barriers and through crowded environments is integral to biological system function and to separation technologies. Ordered, microfabricated post arrays are a promising route to creating synthetic barriers with controlled chemical and physical characteristics. They can be used to create crowded environments, to mimic aspects of cellular membranes, and to serve as engineered replacements of polymer-based separation media. Here, the translational diffusion of fluorescein isothiocyante and various forms of green fluorescent protein (GFP), including "supercharged" variants, are examined in a silicon-based post array environment. The technique of fluorescence recovery after photobleaching (FRAP) is combined with analytical approximations and numerical simulations to assess the relative effects of reaction and diffusion on molecular transport, respectively. FRAP experiments were conducted for 64 different cases where the molecular species, the density of the posts, and the chemical surface charge of the posts were varied. In all cases, the dense packing of the posts hindered the diffusive transport of the fluorescent species. The supercharged GFPs strongly interacted with oppositely charged surfaces. With similar molecular and surface charges, transport is primarily limited by hindered diffusion. For conventional, enhanced GFP in a positively charged surface environment, transport was limited by the coupled action of hindered diffusion and surface interaction with the posts. Quantification of the size-, space-, time-, and charge-dependent translational diffusion in the post array environments can provide insight into natural processes and guide the design and development of selective membrane systems.

Spin relaxation measurements of electrostatic bias in intermolecular exploration

NASA Astrophysics Data System (ADS)

Teng, Ching-Ling; Bryant, Robert G.

2006-04-01

We utilize the paramagnetic contribution to proton spin-lattice relaxation rate constants induced by freely diffusing charged paramagnetic centers to investigate the effect of charge on the intermolecular exploration of a protein by the small molecule. The proton NMR spectrum provided 255 resolved resonances that report how the explorer molecule local concentration varies with position on the surface. The measurements integrate over local dielectric constant variations, and, in principle, provide an experimental characterization of the surface free energy sampling biases introduced by the charge distribution on the protein. The experimental results for ribonuclease A obtained using positive, neutral, and negatively charged small nitroxide radicals are qualitatively similar to those expected from electrostatic calculations. However, while systematic electrostatic trends are apparent, the three different combinations of the data sets do not yield internally consistent values for the electrostatic contribution to the intermolecular free energy. We attribute this failure to the weakness of the electrostatic sampling bias for charged nitroxides in water and local variations in effective translational diffusion constant at the water-protein interface, which enters the nuclear spin relaxation equations for the nitroxide-proton dipolar coupling.

Contribution of Charged Groups to the Enthalpic Stabilization of the Folded States of Globular Proteins

PubMed Central

Dadarlat, Voichita M.; Post, Carol Beth

2016-01-01

In this paper we use the results from all atom MD simulations of proteins and peptides to assess individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the “unfolded state”) and charged amino acids in globular proteins (the “folded state”). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability. The strongest enthalpic contributions favoring the folded state come from the carboxylate (COO−) groups of either Glu or Asp. The contributions from Arg guanidinium groups are generally somewhat stabilizing, while NH3+ groups from Lys contribute little toward stabilizing the folded state. The average enthalpic gain due to the transfer of a methyl group in an apolar amino acid from solution to the protein interior is described for comparison. Notably, charged groups that are less exposed to solvent contribute more favorably to protein native-state enthalpic stability than charged groups that are solvent exposed. While solvent reorganization/release has favorable contributions to folding for all charged atomic groups, the variation in folded state stability among proteins comes mainly from the change in the nonbonded interaction energy of charged groups between the unfolded and folded states. A key outcome is that the calculated enthalpic stabilization is found to be inversely proportional to the excess charge density on the surface, in support of an hypothesis proposed previously. PMID:18303881

Modeling the surface of Campylobacter fetus: protein surface layer stability and resistance to cationic antimicrobial peptides.

PubMed

Roberts, James M D; Graham, Lori L; Quinn, Bonnie; Pink, David A

2013-03-01

Campylobacter fetus is a Gram negative bacterium recognized for its virulence in animals and humans. This bacterium possesses a paracrystalline array of high molecular weight proteins known as surface-layer proteins covering its cell surface. A mathematical model has been made of the outer membrane of this bacterium, both with its surface-layer proteins (S+) and without (S-). Monte Carlo computer simulation was used to understand the stability of the surface-layer protein structure as a function of ionic concentration. The interactions of an electrically-charged antimicrobial agent, the cationic antimicrobial peptide protamine, with surface-layer proteins and with the lipopolysaccharides of the outer membrane were modeled and analyzed. We found that (1) divalent ions stabilize the surface-layer protein array by reducing the fluctuations perpendicular and parallel to the membrane plane thereby promoting adhesion to the LPS region. This was achieved via (2) divalent ions bridging the negatively-charged LPS Core. The effect of this bridging is to bring individual Core regions closer together so that the O-antigens can (3) increase their attractive van der Waals interactions and "collapse" to form a surface with reduced perpendicular fluctuations. These findings provide support for the proposal of Yang et al. [1]. (4) No evidence for a significant increase in Ca(2+) concentration in the region of the surface-layer protein subunits was observed in S+ simulations compared to S- simulations. (5) We predicted the trends of protamine MIC tests performed on C. fetus and these were in good agreement with our experimental results. Copyright © 2012 Elsevier B.V. All rights reserved.

Protein corona affects the relaxivity and MRI contrast efficiency of magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Amiri, Houshang; Bordonali, Lorenzo; Lascialfari, Alessandro; Wan, Sha; Monopoli, Marco P.; Lynch, Iseult; Laurent, Sophie; Mahmoudi, Morteza

2013-08-01

Magnetic nanoparticles (NPs) are increasingly being considered for use in biomedical applications such as biosensors, imaging contrast agents and drug delivery vehicles. In a biological fluid, proteins associate in a preferential manner with NPs. The small sizes and high curvature angles of NPs influence the types and amounts of proteins present on their surfaces. This differential display of proteins bound to the surface of NPs can influence the tissue distribution, cellular uptake and biological effects of NPs. To date, the effects of adsorption of a protein corona (PC) on the magnetic properties of NPs have not been considered, despite the fact that some of their potential applications require their use in human blood. Here, to investigate the effects of a PC (using fetal bovine serum) on the MRI contrast efficiency of superparamagnetic iron oxide NPs (SPIONs), we have synthesized two series of SPIONs with variation in the thickness and functional groups (i.e. surface charges) of the dextran surface coating. We have observed that different physico-chemical characteristics of the dextran coatings on the SPIONs lead to the formation of PCs of different compositions. 1H relaxometry was used to obtain the longitudinal, r1, and transverse, r2, relaxivities of the SPIONs without and with a PC, as a function of the Larmor frequency. The transverse relaxivity, which determines the efficiency of negative contrast agents (CAs), is very much dependent on the functional group and the surface charge of the SPIONs' coating. The presence of the PC did not alter the relaxivity of plain SPIONs, while it slightly increased the relaxivity of the negatively charged SPIONs and dramatically decreased the relaxivity of the positively charged ones, which was coupled with particle agglomeration in the presence of the proteins. To confirm the effect of the PC on the MRI contrast efficiency, in vitro MRI experiments at ν = 8.5 MHz were performed using a low-field MRI scanner. The MRI contrasts, produced by different samples, were fully in agreement with the relaxometry findings.Magnetic nanoparticles (NPs) are increasingly being considered for use in biomedical applications such as biosensors, imaging contrast agents and drug delivery vehicles. In a biological fluid, proteins associate in a preferential manner with NPs. The small sizes and high curvature angles of NPs influence the types and amounts of proteins present on their surfaces. This differential display of proteins bound to the surface of NPs can influence the tissue distribution, cellular uptake and biological effects of NPs. To date, the effects of adsorption of a protein corona (PC) on the magnetic properties of NPs have not been considered, despite the fact that some of their potential applications require their use in human blood. Here, to investigate the effects of a PC (using fetal bovine serum) on the MRI contrast efficiency of superparamagnetic iron oxide NPs (SPIONs), we have synthesized two series of SPIONs with variation in the thickness and functional groups (i.e. surface charges) of the dextran surface coating. We have observed that different physico-chemical characteristics of the dextran coatings on the SPIONs lead to the formation of PCs of different compositions. 1H relaxometry was used to obtain the longitudinal, r1, and transverse, r2, relaxivities of the SPIONs without and with a PC, as a function of the Larmor frequency. The transverse relaxivity, which determines the efficiency of negative contrast agents (CAs), is very much dependent on the functional group and the surface charge of the SPIONs' coating. The presence of the PC did not alter the relaxivity of plain SPIONs, while it slightly increased the relaxivity of the negatively charged SPIONs and dramatically decreased the relaxivity of the positively charged ones, which was coupled with particle agglomeration in the presence of the proteins. To confirm the effect of the PC on the MRI contrast efficiency, in vitro MRI experiments at ν = 8.5 MHz were performed using a low-field MRI scanner. The MRI contrasts, produced by different samples, were fully in agreement with the relaxometry findings. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr00345k

Amine-Rich Organic Thin Films for Cell Culture: Possible Electrostatic Effects in Cell-Surface Interactions

NASA Astrophysics Data System (ADS)

Wertheimer, Michael R.; St-Georges-Robillard, Amélie; Lerouge, Sophie; Mwale, Fackson; Elkin, Bentsian; Oehr, Christian; Wirges, Werner; Gerhard, Reimund

2012-11-01

In recent communications from these laboratories, we observed that amine-rich thin organic layers are very efficient surfaces for the adhesion of mammalian cells. We prepare such deposits by plasma polymerization at low pressure, atmospheric pressure, or by vacuum-ultraviolet photo-polymerization. More recently, we have also investigated a commercially available material, Parylene diX AM. In this article we first briefly introduce literature relating to electrostatic interactions between cells, proteins, and charged surfaces. We then present certain selected cell-response results that pertain to applications in orthopedic and cardiovascular medicine: we discuss the influence of surface properties on the observed behaviors of two particular cell lines, human U937 monocytes, and Chinese hamster ovary cells. Particular emphasis is placed on possible electrostatic attractive forces due to positively charged R-NH3+ groups and negatively charged proteins and cells, respectively. Experiments carried out with electrets, polymers with high positive or negative surface potentials are added for comparison.

Regular Nanoscale Protein Patterns via Directed Adsorption through Self-Assembled DNA Origami Masks.

PubMed

Ramakrishnan, Saminathan; Subramaniam, Sivaraman; Stewart, A Francis; Grundmeier, Guido; Keller, Adrian

2016-11-16

DNA origami has become a widely used method for synthesizing well-defined nanostructures with promising applications in various areas of nanotechnology, biophysics, and medicine. Recently, the possibility to transfer the shape of single DNA origami nanostructures into different materials via molecular lithography approaches has received growing interest due to the great structural control provided by the DNA origami technique. Here, we use ordered monolayers of DNA origami nanostructures with internal cavities on mica surfaces as molecular lithography masks for the fabrication of regular protein patterns over large surface areas. Exposure of the masked sample surface to negatively charged proteins results in the directed adsorption of the proteins onto the exposed surface areas in the holes of the mask. By controlling the buffer and adsorption conditions, the protein coverage of the exposed areas can be varied from single proteins to densely packed monolayers. To demonstrate the versatility of this approach, regular nanopatterns of four different proteins are fabricated: the single-strand annealing proteins Redβ and Sak, the iron-storage protein ferritin, and the blood protein bovine serum albumin (BSA). We furthermore demonstrate the desorption of the DNA origami mask after directed protein adsorption, which may enable the fabrication of hierarchical patterns composed of different protein species. Because selectivity in adsorption is achieved by electrostatic interactions between the proteins and the exposed surface areas, this approach may enable also the large-scale patterning of other charged molecular species or even nanoparticles.

Adsorption of intrinsically disordered barnacle adhesive proteins on silica surface

NASA Astrophysics Data System (ADS)

Wang, Xiaoqiang; Wang, Chao; Xu, Baomei; Wei, Junting; Xiao, Yang; Huang, Fang

2018-01-01

The adsorption of recombinant barnacle proteins Bacp19k and Mrcp19k on hydrophilic silica surface was characterized by spectroscopic ellipsometry in artificial seawater (pH = 8.2). They are homologous adhesive proteins destined for underwater adhesion but bear opposite net charges in seawater. As assessed with their primary and secondary structures, both proteins are intrinsically disordered and thus distinct from globular proteins that have dominated research in the field. Different from Mrcp19k, higher initial rate and adsorbed amount were obtained via curve fitting for Bacp19k in kinetic studies, due to favorable charge interactions with silica surface. The good fitting with the same dynamic model also indicates the formation of monolayer coverage in both cases. The two adsorption isotherms of Bacp19k and Mrcp19k are different in the initial change and maximum adsorption level, indicating different protein-surface affinities and charge interactions. Each isotherm fits the Langmuir model well, which is commonly used to describe monolayer adsorption, thus consistent with the predication from kinetic fitting. To further examine the effect of electrostatic interaction on the adsorption, the isotherm of the 1:1 mixture of Bacp19k and Mrcp19k was also constructed, which showed a higher correlation fit for Jovanovic than for Langmuir model. The presence of electrostatic attraction between Bacp19k and Mrcp19k deviated from one of the required conditions for Langmuir behavior, which may also result in the highest coadsorption level but slowest initial change among the three isotherms. The surface state of the adhesive proteins and the change with adsorption time were also examined by atomic force microscopy. The results thus obtained are in good agreement with the corresponding ellipsometric measurement.

Regulation of Macrophage Recognition through the Interplay of Nanoparticle Surface Functionality and Protein Corona.

PubMed

Saha, Krishnendu; Rahimi, Mehran; Yazdani, Mahdieh; Kim, Sung Tae; Moyano, Daniel F; Hou, Singyuk; Das, Ridhha; Mout, Rubul; Rezaee, Farhad; Mahmoudi, Morteza; Rotello, Vincent M

2016-04-26

Using a family of cationic gold nanoparticles (NPs) with similar size and charge, we demonstrate that proper surface engineering can control the nature and identity of protein corona in physiological serum conditions. The protein coronas were highly dependent on the hydrophobicity and arrangement of chemical motifs on NP surface. The NPs were uptaken in macrophages in a corona-dependent manner, predominantly through recognition of specific complement proteins in the NP corona. Taken together, this study shows that surface functionality can be used to tune the protein corona formed on NP surface, dictating the interaction of NPs with macrophages.

Protein Adsorption to In-Line Filters of Intravenous Administration Sets.

PubMed

Besheer, Ahmed

2017-10-01

Ensuring compatibility of administered therapeutic proteins with intravenous administration sets is an important regulatory requirement. A low-dose recovery during administration of low protein concentrations is among the commonly observed incompatibilities, and it is mainly due to adsorption to in-line filters. To better understand this phenomenon, we studied the adsorption of 4 different therapeutic proteins (2 IgG1s, 1 IgG4, and 1 Fc fusion protein) diluted to 0.01 mg/mL in 5% glucose (B. Braun EcoFlac; B. Braun Melsungen AG, Melsungen, Germany) or 0.9% sodium chloride (NaCl; Freeflex; Fresenius Kabi, Friedberg, Germany) solutions to 8 in-line filters (5 positively charged and 3 neutral filters made of different polymers and by different suppliers). The results show certain patterns of protein adsorption, which depend to a large extent on the dilution solution and filter material, and to a much lower extent on the proteins' biophysical properties. Investigation of the filter membranes' zeta potential showed a correlation between the observed adsorption pattern in 5% glucose solution and the filter's surface charge, with higher protein adsorption for the strongly negatively charged membranes. In 0.9% NaCl solution, the surface charges are masked, leading to different adsorption patterns. These results contribute to the general understanding of the protein adsorption to IV infusion filters and allow the design of more efficient compatibility studies. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Influence of charge and flexibility on smectic phase formation in filamentous virus suspensions

NASA Astrophysics Data System (ADS)

Purdy, Kirstin R.; Fraden, Seth

2007-07-01

We present experimental measurements of the cholesteric-smectic phase transition of suspensions of charged semiflexible rods as a function of rod flexibility and surface charge. The rod particles consist of the bacteriophage M13 and closely related mutants, which are structurally identical to M13, but vary either in contour length and therefore ratio of persistence length to contour length, or surface charge. Surface charge is altered in two ways; by changing solution pH and by comparing M13 with fd virus, a virus which differs from M13 only by the substitution of a single charged amino acid for a neutral one per viral coat protein. Phase diagrams are measured as a function of particle length, particle charge, and ionic strength. The experimental results are compared with existing theoretical predictions for the phase behavior of flexible rods and charged rods.

Structure of the human DNA-repair protein RAD52 containing surface mutations.

PubMed

Saotome, Mika; Saito, Kengo; Onodera, Keiichi; Kurumizaka, Hitoshi; Kagawa, Wataru

2016-08-01

The Rad52 protein is a eukaryotic single-strand DNA-annealing protein that is involved in the homologous recombinational repair of DNA double-strand breaks. The isolated N-terminal half of the human RAD52 protein (RAD52(1-212)) forms an undecameric ring structure with a surface that is mostly positively charged. In the present study, it was found that RAD52(1-212) containing alanine mutations of the charged surface residues (Lys102, Lys133 and Glu202) is highly amenable to crystallization. The structure of the mutant RAD52(1-212) was solved at 2.4 Å resolution. The structure revealed an association between the symmetry-related RAD52(1-212) rings, in which a partially unfolded, C-terminal region of RAD52 extended into the DNA-binding groove of the neighbouring ring in the crystal. The alanine mutations probably reduced the surface entropy of the RAD52(1-212) ring and stabilized the ring-ring association observed in the crystal.

Plantaricin A, a cationic peptide produced by Lactobacillus plantarum, permeabilizes eukaryotic cell membranes by a mechanism dependent on negative surface charge linked to glycosylated membrane proteins.

PubMed

Sand, Sverre L; Nissen-Meyer, Jon; Sand, Olav; Haug, Trude M

2013-02-01

Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH(4) cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH(4) cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts. Copyright © 2012 Elsevier B.V. All rights reserved.

Effects of Acids, Bases, and Heteroatoms on Proximal Radial Distribution Functions for Proteins

PubMed Central

Nguyen, Bao Linh; Pettitt, B. Montgomery

2015-01-01

The proximal distribution of water around proteins is a convenient method of quantifying solvation. We consider the effect of charged and sulfur-containing amino acid side-chain atoms on the proximal radial distribution function (pRDF) of water molecules around proteins using side-chain analogs. The pRDF represents the relative probability of finding any solvent molecule at a distance from the closest or surface perpendicular protein atom. We consider the near-neighbor distribution. Previously, pRDFs were shown to be universal descriptors of the water molecules around C, N, and O atom types across hundreds of globular proteins. Using averaged pRDFs, a solvent density around any globular protein can be reconstructed with controllable relative error. Solvent reconstruction using the additional information from charged amino acid side-chain atom types from both small models and protein averages reveals the effects of surface charge distribution on solvent density and improves the reconstruction errors relative to simulation. Solvent density reconstructions from the small-molecule models are as effective and less computationally demanding than reconstructions from full macromolecular models in reproducing preferred hydration sites and solvent density fluctuations. PMID:26388706

Effect of surface charge alteration on stability of L-asparaginase II from Escherichia sp.

PubMed

Vidya, Jalaja; Ushasree, Mrudula Vasudevan; Pandey, Ashok

2014-03-05

Escherichia coli L-asparaginases have great significance in the treatment of leukemia. Consequently, there is considerable interest in engineering this enzyme for improving its stability. In this work, the effect of surface charge on the stability of the enzyme l-asparaginase II was studied by site-directed mutagenesis of the cloned ansB gene from Escherichia sp. Replacement of two positively charged residues (K139 and K207) on the surface loops with neutral and reverse charges resulted in altered thermo stability in designed variants. Neutral charge substitutions (K139A and K207A) retained greater tolerance and stability followed by negative charge substitutions (K139D and K207D) compared to control mutant K139R and wild enzyme. From the results, it was concluded that the optimization of surface charge contributed much to the thermal properties of proteins without affecting the structure. Copyright © 2013 Elsevier Inc. All rights reserved.

The Limitations of an Exclusively Colloidal View of Protein Solution Hydrodynamics and Rheology

PubMed Central

Sarangapani, Prasad S.; Hudson, Steven D.; Migler, Kalman B.; Pathak, Jai A.

2013-01-01

Proteins are complex macromolecules with dynamic conformations. They are charged like colloids, but unlike colloids, charge is heterogeneously distributed on their surfaces. Here we overturn entrenched doctrine that uncritically treats bovine serum albumin (BSA) as a colloidal hard sphere by elucidating the complex pH and surface hydration-dependence of solution viscosity. We measure the infinite shear viscosity of buffered BSA solutions in a parameter space chosen to tune competing long-range repulsions and short-range attractions (2 mg/mL ≤ [BSA] ≤ 500 mg/mL and 3.0 ≤ pH ≤ 7.4). We account for surface hydration through partial specific volume to define volume fraction and determine that the pH-dependent BSA intrinsic viscosity never equals the classical hard sphere result (2.5). We attempt to fit our data to the colloidal rheology models of Russel, Saville, and Schowalter (RSS) and Krieger-Dougherty (KD), which are each routinely and successfully applied to uniformly charged suspensions and to hard-sphere suspensions, respectively. We discover that the RSS model accurately describes our data at pH 3.0, 4.0, and 5.0, but fails at pH 6.0 and 7.4, due to steeply rising solution viscosity at high concentration. When we implement the KD model with the maximum packing volume fraction as the sole floating parameter while holding the intrinsic viscosity constant, we conclude that the model only succeeds at pH 6.0 and 7.4. These findings lead us to define a minimal framework for models of crowded protein solution viscosity wherein critical protein-specific attributes (namely, conformation, surface hydration, and surface charge distribution) are addressed. PMID:24268154

Adsorption of charged protein residues on an inorganic nanosheet: Computer simulation of LDH interaction with ion channel

NASA Astrophysics Data System (ADS)

Tsukanov, Alexey A.; Psakhie, Sergey G.

2016-08-01

Quasi-two-dimensional and hybrid nanomaterials based on layered double hydroxides (LDH), cationic clays, layered oxyhydroxides and hydroxides of metals possess large specific surface area and strong electrostatic properties with permanent or pH-dependent electric charge. Such nanomaterials may impact cellular electrostatics, changing the ion balance, pH and membrane potential. Selective ion adsorption/exchange may alter the transmembrane electrochemical gradient, disrupting potential-dependent cellular processes. Cellular proteins as a rule have charged residues which can be effectively adsorbed on the surface of layered hydroxide based nanomaterials. The aim of this study is to attempt to shed some light on the possibility and mechanisms of protein "adhesion" an LDH nanosheet and to propose a new direction in anticancer medicine, based on physical impact and strong electrostatics. An unbiased molecular dynamics simulation was performed and the combined process free energy estimation (COPFEE) approach was used.

The Efficiency of Different Salts to Screen Charge Interactions in Proteins: A Hofmeister Effect?

PubMed Central

Perez-Jimenez, Raul; Godoy-Ruiz, Raquel; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

2004-01-01

Understanding the screening by salts of charge-charge interactions in proteins is important for at least two reasons: a), screening by intracellular salt concentration may modulate the stability and interactions of proteins in vivo; and b), the in vitro experimental estimation of the contributions from charge-charge interactions to molecular processes involving proteins is generally carried out on the basis of the salt effect on process energetics, under the assumption that these interactions are screened out by moderate salt concentrations. Here, we explore experimentally the extent to which the screening efficiency depends on the nature of the salt. To this end, we have carried out an energetic characterization of the effect of NaCl (a nondenaturing salt), guanidinium chloride (a denaturing salt), and guanidinium thiocyanate (a stronger denaturant) on the stability of the wild-type form and a T14K variant of Escherichia coli thioredoxin. Our results suggest that the efficiency of different salts to screen charge-charge interactions correlates with their denaturing strength and with the position of the constituent ions in the Hofmeister rankings. This result appears consistent with the plausible relation of the Hofmeister rankings with the extent of solute accumulation/exclusion from protein surfaces. PMID:15041679

Use of a mixture of n-dodecyl-beta-D-maltoside and sodium dodecyl sulfate in poly(dimethylsiloxane) microchips to suppress adhesion and promote separation of proteins.

PubMed

Huang, Bo; Kim, Samuel; Wu, Hongkai; Zare, Richard N

2007-12-01

Dynamic modification of poly(dimethylsiloxane) channels using a mixture of n-dodecyl-beta-D-maltoside (DDM) and sodium dodecyl sulfate (SDS) is able to suppress analyte adsorption and control electroosmotic flow (EOF). In this mixed surfactant system, the nonionic surfactant DDM functions as a surface blocking reagent, whereas the anionic surfactant SDS introduces negative charges to the channel walls. Changing the DDM/SDS mixing ratio tunes the surface charge density and the strength of EOF. Using 0.1% (w/v) DDM and 0.03% (w/v) SDS, Alexa Fluor 647 labeled streptavidin can be analyzed according to the charges added by the fluorophores. Protein molecules with different numbers of fluorophores are well resolved. DDM and SDS also form negatively charged mixed micelles, which act as a separation medium. The low critical micellar concentration of DDM/SDS mixed micelles also allows the use of SDS at a nondenaturing concentration, which enables the analysis of proteins in their native state. The immunocomplex between a membrane protein, beta2 adrenergic receptor, and anti-FLAG antibody has been fully separated using 0.1% (w/v) DDM and 0.03% (w/v) SDS. We have also analyzed the composition of light-harvesting protein-chromophore complexes in cyanobacteria.

Spectroscopic studies of conformational changes of β-lactoglobulin adsorbed on gold nanoparticle surfaces.

PubMed

Winuprasith, Thunnalin; Suphantharika, Manop; McClements, David Julian; He, Lili

2014-02-15

In this work, we investigated the conformational changes of a globular protein (β-lactoglobulin, β-lg) coated on the surface of 200 nm gold nanoparticles (GNPs) using a number of analytical techniques: dynamic light scattering (DLS); particle electrophoresis (ζ-potential); localized surface plasmon resonance (LSPR) spectroscopy; transmission electron microscopy (TEM); and surface-enhanced Raman scattering (SERS). The β-lg (pH 3) concentration had a pronounced effect on the aggregation and surface charge of β-lg-coated GNPs. The surface charge of GNPs changed from negative to positive as increasing amounts of β-lg molecule were added, indicating that the globular protein molecules adsorbed to the surfaces of the particles. Extensive particle aggregation occurred when β-lg did not saturate the GNP surfaces, which was attributed to electrostatic bridging flocculation. Modifications in LSPR and SERS spectra after addition of β-lg to the GNP suspensions supported the adsorption of β-lg to the particle surfaces. Moreover, SERS highlighted the importance of a number of specific molecular groups in the binding interaction, and suggested conformational changes of the globular protein after adsorption. This research provides useful information for characterizing and understanding the interactions between globular proteins and colloidal particles. Copyright © 2013 Elsevier Inc. All rights reserved.

Interactions of long DNA chains with charged surfaces: Entropy, Conformations and Applications

NASA Astrophysics Data System (ADS)

Rondelez, Francis

2004-03-01

The adsorption of long DNA chains on positively charged surfaces is controlled by electrostatics. We demonstrate experimentally on two different systems that the driving force for adsorption is the release of the small counterions surrounding the DNA chains and the charged surface. We then proceed to the study of the conformation of the adsorbed DNA chains. In the first series of experiments, the DNA is in contact with a Langmuir monolayer of cationic amphiphiles. The advantage is that the surface charge density can be varied over a factor of 10 and also that the immobilized DNA chains can be mechanically manipulated. We observe by neutron reflectometry that the chains are essentially flat on the interface, with a few dangling loops. In the second series of experiments the DNA chains are in contact with a solution of cationic polystyrene microspheres. Due to the small size of the particles, the DNA chains adsorb only partially. The fraction of nucleotides localized around the beads can be measured by fluorescence spectroscopy and we compare it to the total number of charges on the particle. We also study the conditions to maximize the wrapping. Such experiments should be useful to better understand the compaction of DNA by histone proteins and formation of nucleosomes. The immobilization of DNA by surfaces also provides a way to control the interactions of DNA with proteins like DNases, exonucleases or RNA polymerases.

Brownian dynamics simulations of simplified cytochrome c molecules in the presence of a charged surface

NASA Astrophysics Data System (ADS)

Gorba, C.; Geyer, T.; Helms, V.

2004-07-01

Simulations were performed for up to 150 simplified spherical horse heart cytochrome c molecules in the presence of a charged surface, which serves as an approximate model for a lipid membrane. Screened electrostatic and short-ranged attractive as well as repulsive van der Waals forces for interparticle and particle-membrane interactions are utilized in the simulations. At a distance from the membrane, where particle-membrane interactions are negligible, the simulation is coupled to a noninteraction continuum analogous to a heat bath [Geyer et al., J. Chem. Phys. 120, 4573 (2004)]. From the particles' density profiles perpendicular to the planar surface binding isotherms are derived and compared to experimental results [Heimburg et al. (1999)]. Using a negatively charged structureless membrane surface a saturation effect was found for relatively large particle concentrations. Since biological membranes often contain membrane proteins, we also studied the influence of additional charges on our model membrane mimicking bacterial reaction centers. We find that the onset of the saturation occurs for much lower concentrations and is sensitive to the detailed implementation. Therefore we suggest that local distortion of membrane planarity (undulation), or lipid demixing, or the presence of charged integral membrane proteins create preferential binding sites on the membrane. Only then do we observe saturation at physiological concentrations.

Subtle charge balance controls surface-nucleated self-assembly of designed biopolymers.

PubMed

Charbonneau, Céline; Kleijn, J Mieke; Cohen Stuart, Martien A

2014-03-25

We report the surface-nucleated self-assembly into fibrils of a biosynthetic amino acid polymer synthesized by the yeast Pichia pastoris. This polymer has a block-like architecture, with a central silk-like block labeled SH, responsible for the self-assembly into fibrils, and two collagen-like random coil end blocks (C) that colloidally stabilize the fibers in aqueous solution. The silk-like block contains histidine residues (pKa≈6) that are positively charged in the low pH region, which hinders self-assembly. In aqueous solution, CSHC self-assembles into fibers above a pH-dependent critical nucleation concentration Ccb. Below Ccb, where no self-assembly occurs in solution, fibril formation can be induced by a negatively charged surface (silica) in the pH range of 3.5-7. The density of the fibers at the surface and their length are controlled by a subtle balance in charge between the protein polymer and the silica surface, which is evidenced from the dependence on pH. With increasing number density of the fibers at the surface, their average length decreases. The results can be explained on the basis of a nucleation-and-growth mechanism: the surface density of fibers depends on the rate of nucleation, while their growth rate is limited by transport of proteins from solution. Screening of the charges on the surface and histidine units by adding NaCl influences the nucleation-and-growth process in a complicated fashion: at low pH, the growth is improved, while at high pH, the nucleation is limited. Under conditions where nucleation in the bulk solution is not possible, growth of the surface-nucleated fibers into the solution--away from the surface--can still occur.

Molecular Dynamics Simulations on Gas-Phase Proteins with Mobile Protons: Inclusion of All-Atom Charge Solvation.

PubMed

Konermann, Lars

2017-08-31

Molecular dynamics (MD) simulations have become a key tool for examining the properties of electrosprayed protein ions. Traditional force fields employ static charges on titratable sites, whereas in reality, protons are highly mobile in gas-phase proteins. Earlier studies tackled this problem by adjusting charge patterns during MD runs. Within those algorithms, proton redistribution was subject to energy minimization, taking into account electrostatic and proton affinity contributions. However, those earlier approaches described (de)protonated moieties as point charges, neglecting charge solvation, which is highly prevalent in the gas phase. Here, we describe a mobile proton algorithm that considers the electrostatic contributions from all atoms, such that charge solvation is explicitly included. MD runs were broken down into 50 ps fixed-charge segments. After each segment, the electrostatics was reanalyzed and protons were redistributed. Challenges associated with computational cost were overcome by devising a streamlined method for electrostatic calculations. Avidin (a 504-residue protein complex) maintained a nativelike fold over 200 ns. Proton transfer and side chain rearrangements produced extensive salt bridge networks at the protein surface. The mobile proton technique introduced here should pave the way toward future studies on protein folding, unfolding, collapse, and subunit dissociation in the gas phase.

In vivo encapsulation of nucleic acids using an engineered nonviral protein capsid.

PubMed

Lilavivat, Seth; Sardar, Debosmita; Jana, Subrata; Thomas, Geoffrey C; Woycechowsky, Kenneth J

2012-08-15

In Nature, protein capsids function as molecular containers for a wide variety of molecular cargoes. Such containers have great potential for applications in nanotechnology, which often require encapsulation of non-native guest molecules. Charge complementarity represents a potentially powerful strategy for engineering novel encapsulation systems. In an effort to explore the generality of this approach, we engineered a nonviral, 60-subunit capsid, lumazine synthase from Aquifex aeolicus (AaLS), to act as a container for nucleic acid. Four mutations were introduced per subunit to increase the positive charge at the inner surface of the capsid. Characterization of the mutant (AaLS-pos) revealed that the positive charges lead to the uptake of cellular RNA during production and assembly of the capsid in vivo. Surprisingly, AaLS-pos capsids were found to be enriched with RNA molecules approximately 200-350 bases in length, suggesting that this simple charge complementarity approach to RNA encapsulation leads to both high affinity and a degree of selectivity. The ability to control loading of RNA by tuning the charge at the inner surface of a protein capsid could illuminate aspects of genome recognition by viruses and pave the way for the development of improved RNA delivery systems.

Increasing the Detection Limit of the Parkinson Disorder through a Specific Surface Chemistry Applied onto Inner Surface of the Titration Well.

PubMed

Mille, Caroline; Debarnot, Dominique; Zorzi, Willy; El Moualij, Benaïssa; Coudreuse, Arnaud; Legeay, Gilbert; Quadrio, Isabelle; Perret-Liaudet, Armand; Poncin-Epaillard, Fabienne

2012-04-18

The main objective of this paper was to illustrate the enhancement of the sensitivity of ELISA titration for neurodegenerative proteins by reducing nonspecific adsorptions that could lead to false positives. This goal was obtained thanks to the association of plasma and wet chemistries applied to the inner surface of the titration well. The polypropylene surface was plasma-activated and then, dip-coated with different amphiphilic molecules. These molecules have more or less long hydrocarbon chains and may be charged. The modified surfaces were characterized in terms of hydrophilic-phobic character, surface chemical groups and topography. Finally, the coated wells were tested during the ELISA titration of the specific antibody capture of the α-synuclein protein. The highest sensitivity is obtained with polar (Θ = 35°), negatively charged and smooth inner surface.

Increasing the Detection Limit of the Parkinson Disorder through a Specific Surface Chemistry Applied onto Inner Surface of the Titration Well

PubMed Central

Mille, Caroline; Debarnot, Dominique; Zorzi, Willy; Moualij, Benaïssa El; Coudreuse, Arnaud; Legeay, Gilbert; Quadrio, Isabelle; Perret-Liaudet, Armand; Poncin-Epaillard, Fabienne

2012-01-01

The main objective of this paper was to illustrate the enhancement of the sensitivity of ELISA titration for neurodegenerative proteins by reducing nonspecific adsorptions that could lead to false positives. This goal was obtained thanks to the association of plasma and wet chemistries applied to the inner surface of the titration well. The polypropylene surface was plasma-activated and then, dip-coated with different amphiphilic molecules. These molecules have more or less long hydrocarbon chains and may be charged. The modified surfaces were characterized in terms of hydrophilic—phobic character, surface chemical groups and topography. Finally, the coated wells were tested during the ELISA titration of the specific antibody capture of the α-synuclein protein. The highest sensitivity is obtained with polar (Θ = 35°), negatively charged and smooth inner surface. PMID:24955533

Probing the nanoscale interaction forces and elastic properties of organic and inorganic materials using force-distance (F-D) spectroscopy

NASA Astrophysics Data System (ADS)

Vincent, Abhilash

Due to their therapeutic applications such as radical scavenging, MRI contrast imaging, Photoluminescence imaging, drug delivery, etc., nanoparticles (NPs) have a significant importance in bio-nanotechnology. The reason that prevents the utilizing NPs for drug delivery in medical field is mostly due to their biocompatibility issues (incompatibility can lead to toxicity and cell death). Changes in the surface conditions of NPs often lead to NP cytotoxicity. Investigating the role of NP surface properties (surface charges and surface chemistry) on their interactions with biomolecules (Cells, protein and DNA) could enhance the current understanding of NP cytotoxicity. Hence, it is highly beneficial to the nanotechnology community to bring more attention towards the enhancement of surface properties of NPs to make them more biocompatible and less toxic to biological systems. Surface functionalization of NPs using specific ligand biomolecules have shown to enhance the protein adsorption and cellular uptake through more favorable interaction pathways. Cerium oxide NPs (CNPs also known as nanoceria) are potential antioxidants in cell culture models and understanding the nature of interaction between cerium oxide NPs and biological proteins and cells are important due to their therapeutic application (especially in site specific drug delivery systems). The surface charges and surface chemistry of CNPs play a major role in protein adsorption and cellular uptake. Hence, by tuning the surface charges and by selecting proper functional molecules on the surface, CNPs exhibiting strong adhesion to biological materials can be prepared. By probing the nanoscale interaction forces acting between CNPs and protein molecules using Atomic Force Microscopy (AFM) based force-distance (F-D) spectroscopy, the mechanism of CNP-protein adsorption and CNP cellular uptake can be understood more quantitatively. The work presented in this dissertation is based on the application of AFM in studying the interaction forces as well as the mechanical properties of nanobiomaterials. The research protocol employed in the earlier part of the dissertation is specifically aimed to understand the operation of F-D spectroscopy technique. The elastic properties of thin films of silicon dioxide NPs were investigated using F-D spectroscopy in the high force regime of few 100 nN to 1 microN. Here, sol-gel derived porous nanosilica thin films of varying surface morphology, particle size and porosity were prepared through acid and base catalyzed process. AFM nanoindentation experiments were conducted on these films using the F-D spectroscopy mode and the nanoscale elastic properties of these films were evaluated. The major contribution of this dissertation is a study exploring the interaction forces acting between CNPs and transferrin proteins in picoNewton scale regime using the force-distance spectroscopy technique. This study projects the importance of obtaining appropriate surface charges and surface chemistry so that the NP can exhibit enhanced protein adsorption and NP cellular uptake.

Anions in Electrothermal Supercharging of Proteins with Electrospray Ionization Follow a Reverse Hofmeister Series

PubMed Central

2015-01-01

The effects of different anions on the extent of electrothermal supercharging of proteins from aqueous ammonium and sodium salt solutions were investigated. Sulfate and hydrogen phosphate are the most effective anions at producing high charge state protein ions from buffered aqueous solution, whereas iodide and perchlorate are ineffective with electrothermal supercharging. The propensity for these anions to produce high charge state protein ions follows the following trend: sulfate > hydrogen phosphate > thiocyanate > bicarbonate > chloride > formate ≈ bromide > acetate > iodide > perchlorate. This trend correlates with the reverse Hofmeister series over a wide range of salt concentrations (1 mM to 2 M) and with several physical properties, including solvent surface tension, anion viscosity B-coefficient, and anion surface/bulk partitioning coefficient, all of which are related to the Hofmeister series. The effectiveness of electrothermal supercharging does not depend on bubble formation, either from thermal degradation of the buffer or from coalescence of dissolved gas. These results provide evidence that the effect of different ions in the formation of high charge state ions by electrothermal supercharging is largely a result of Hofmeister effects on protein stability leading to protein unfolding in the heated ESI droplet. PMID:24410546

Measurements and theoretical interpretation of points of zero charge/potential of BSA protein.

PubMed

Salis, Andrea; Boström, Mathias; Medda, Luca; Cugia, Francesca; Barse, Brajesh; Parsons, Drew F; Ninham, Barry W; Monduzzi, Maura

2011-09-20

The points of zero charge/potential of proteins depend not only on pH but also on how they are measured. They depend also on background salt solution type and concentration. The protein isoelectric point (IEP) is determined by electrokinetical measurements, whereas the isoionic point (IIP) is determined by potentiometric titrations. Here we use potentiometric titration and zeta potential (ζ) measurements at different NaCl concentrations to study systematically the effect of ionic strength on the IEP and IIP of bovine serum albumin (BSA) aqueous solutions. It is found that high ionic strengths produce a shift of both points toward lower (IEP) and higher (IIP) pH values. This result was already reported more than 60 years ago. At that time, the only available theory was the purely electrostatic Debye-Hückel theory. It was not able to predict the opposite trends of IIP and IEP with ionic strength increase. Here, we extend that theory to admit both electrostatic and nonelectrostatic (NES) dispersion interactions. The use of a modified Poisson-Boltzmann equation for a simple model system (a charge regulated spherical colloidal particle in NaCl salt solutions), that includes these ion specific interactions, allows us to explain the opposite trends observed for isoelectric point (zero zeta potential) and isoionic point (zero protein charge) of BSA. At higher concentrations, an excess of the anion (with stronger NES interactions than the cation) is adsorbed at the surface due to an attractive ionic NES potential. This makes the potential relatively more negative. Consequently, the IEP is pushed toward lower pH. But the charge regulation condition means that the surface charge becomes relatively more positive as the surface potential becomes more negative. Consequently, the IIP (measuring charge) shifts toward higher pH as concentration increases, in the opposite direction from the IEP (measuring potential). © 2011 American Chemical Society

Analysis of SiO2 nanoparticles binding proteins in rat blood and brain homogenate.

PubMed

Shim, Kyu Hwan; Hulme, John; Maeng, Eun Ho; Kim, Meyoung-Kon; An, Seong Soo A

2014-01-01

A multitude of nanoparticles, such as titanium oxide (TiO2), zinc oxide, aluminum oxide, gold oxide, silver oxide, iron oxide, and silica oxide, are found in many chemical, cosmetic, pharmaceutical, and electronic products. Recently, SiO2 nanoparticles were shown to have an inert toxicity profile and no association with an irreversible toxicological change in animal models. Hence, exposure to SiO2 nanoparticles is on the increase. SiO2 nanoparticles are routinely used in numerous materials, from strengthening filler for concrete and other construction composites, to nontoxic platforms for biomedical application, such as drug delivery and theragnostics. On the other hand, recent in vitro experiments indicated that SiO2 nanoparticles were cytotoxic. Therefore, we investigated these nanoparticles to identify potentially toxic pathways by analyzing the adsorbed protein corona on the surface of SiO2 nanoparticles in the blood and brain of the rat. Four types of SiO2 nanoparticles were chosen for investigation, and the protein corona of each type was analyzed using liquid chromatography-tandem mass spectrometry technology. In total, 115 and 48 plasma proteins from the rat were identified as being bound to negatively charged 20 nm and 100 nm SiO2 nanoparticles, respectively, and 50 and 36 proteins were found for 20 nm and 100 nm arginine-coated SiO2 nanoparticles, respectively. Higher numbers of proteins were adsorbed onto the 20 nm sized SiO2 nanoparticles than onto the 100 nm sized nanoparticles regardless of charge. When proteins were compared between the two charges, higher numbers of proteins were found for arginine-coated positively charged SiO2 nanoparticles than for the negatively charged nanoparticles. The proteins identified as bound in the corona from SiO2 nanoparticles were further analyzed with ClueGO, a Cytoscape plugin used in protein ontology and for identifying biological interaction pathways. Proteins bound on the surface of nanoparticles may affect functional and conformational properties and distributions in complicated biological processes.

Unprecedented Ionization Processes in Mass Spectrometry Provide Missing Link between ESI and MALDI.

PubMed

Trimpin, Sarah; Lee, Chuping; Weidner, Steffen M; El-Baba, Tarick J; Lutomski, Corinne A; Inutan, Ellen D; Foley, Casey D; Ni, Chi-Kung; McEwen, Charles N

2018-03-05

In the field of mass spectrometry, producing intact, highly-charged protein ions from surfaces is a conundrum with significant potential payoff in application areas ranging from biomedical to clinical research. Here, we report on the ability to form intact, highly-charged protein ions on high vacuum time-of-flight mass spectrometers in the linear and reflectron modes achievable using experimental conditions that allow effective matrix removal from both the sample surfaces and from the charged clusters formed by the laser ablation event. The charge states are the highest reported on high vacuum mass spectrometers, yet they remain at only around a third of the highest charge obtained using laser ablation with a suitable matrix at atmospheric pressure. Other than physical instrument modifications, the key to forming abundant and stable highly-charged ions appears to be the volatility of the matrix used. Cumulative results suggest mechanistic links between the ionization process reported here and traditional ionization methods of electrospray ionization and matrix-assisted laser desorption/ionization. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Neisseria conserved protein DMP19 is a DNA mimic protein that prevents DNA binding to a hypothetical nitrogen-response transcription factor

PubMed Central

Wang, Hao-Ching; Ko, Tzu-Ping; Wu, Mao-Lun; Ku, Shan-Chi; Wu, Hsing-Ju; Wang, Andrew H.-J.

2012-01-01

DNA mimic proteins occupy the DNA binding sites of DNA-binding proteins, and prevent these sites from being accessed by DNA. We show here that the Neisseria conserved hypothetical protein DMP19 acts as a DNA mimic. The crystal structure of DMP19 shows a dsDNA-like negative charge distribution on the surface, suggesting that this protein should be added to the short list of known DNA mimic proteins. The crystal structure of another related protein, NHTF (Neisseria hypothetical transcription factor), provides evidence that it is a member of the xenobiotic-response element (XRE) family of transcriptional factors. NHTF binds to a palindromic DNA sequence containing a 5′-TGTNAN11TNACA-3′ recognition box that controls the expression of an NHTF-related operon in which the conserved nitrogen-response protein [i.e. (Protein-PII) uridylyltransferase] is encoded. The complementary surface charges between DMP19 and NHTF suggest specific charge–charge interaction. In a DNA-binding assay, we found that DMP19 can prevent NHTF from binding to its DNA-binding sites. Finally, we used an in situ gene regulation assay to provide evidence that NHTF is a repressor of its down-stream genes and that DMP19 can neutralize this effect. We therefore conclude that the interaction of DMP19 and NHTF provides a novel gene regulation mechanism in Neisseria spps. PMID:22373915

Ordered adsorption of coagulation factor XII on negatively charged polymer surfaces probed by sum frequency generation vibrational spectroscopy.

PubMed

Chen, Xiaoyun; Wang, Jie; Paszti, Zoltan; Wang, Fulin; Schrauben, Joel N; Tarabara, Volodymyr V; Schmaier, Alvin H; Chen, Zhan

2007-05-01

Electrostatic interactions between negatively charged polymer surfaces and factor XII (FXII), a blood coagulation factor, were investigated by sum frequency generation (SFG) vibrational spectroscopy, supplemented by several analytical techniques including attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), quartz crystal microbalance (QCM), zeta-potential measurement, and chromogenic assay. A series of sulfonated polystyrenes (sPS) with different sulfonation levels were synthesized as model surfaces with different surface charge densities. SFG spectra collected from FXII adsorbed onto PS and sPS surfaces with different surface charge densities showed remarkable differences in spectral features and especially in spectral intensity. Chromogenic assay experiments showed that highly charged sPS surfaces induced FXII autoactivation. ATR-FTIR and QCM results indicated that adsorption amounts on the PS and sPS surfaces were similar even though the surface charge densities were different. No significant conformational change was observed from FXII adsorbed onto surfaces studied. Using theoretical calculations, the possible contribution from the third-order nonlinear optical effect induced by the surface electric field was evaluated, and it was found to be unable to yield the SFG signal enhancement observed. Therefore it was concluded that the adsorbed FXII orientation and ordering were the main reasons for the remarkable SFG amide I signal increase on sPS surfaces. These investigations indicate that negatively charged surfaces facilitate or induce FXII autoactivation on the molecular level by imposing specific orientation and ordering on the adsorbed protein molecules.

Dipolar response of hydrated proteins

NASA Astrophysics Data System (ADS)

Matyushov, Dmitry V.

2012-02-01

The paper presents an analytical theory and numerical simulations of the dipolar response of hydrated proteins in solution. We calculate the effective dielectric constant representing the average dipole moment induced at the protein by a uniform external field. The dielectric constant shows a remarkable variation among the proteins, changing from 0.5 for ubiquitin to 640 for cytochrome c. The former value implies a negative dipolar susceptibility, that is a dia-electric dipolar response and negative dielectrophoresis. It means that ubiquitin, carrying an average dipole of ≃240 D, is expected to repel from the region of a stronger electric field. This outcome is the result of a negative cross-correlation between the protein and water dipoles, compensating for the positive variance of the intrinsic protein dipole in the overall dipolar susceptibility. In contrast to the neutral ubiquitin, charged proteins studied here show para-electric dipolar response and positive dielectrophoresis. The study suggests that the dipolar response of proteins in solution is strongly affected by the coupling of the protein surface charge to the hydration water. The protein-water dipolar cross-correlations are long-ranged, extending ˜2 nm from the protein surface into the bulk. A similar correlation length of about 1 nm is seen for the electrostatic potential produced by the hydration water inside the protein. The analysis of numerical simulations suggests that the polarization of the protein-water interface is highly heterogeneous and does not follow the standard dielectric results for cavities carved in dielectrics. The polarization of the water shell gains in importance, relative to the intrinsic protein dipole, at high frequencies, above the protein Debye peak. The induced interfacial dipole can be either parallel or antiparallel to the protein dipole, depending on the distribution of the protein surface charge. As a result, the high-frequency absorption of the protein solution can be either higher or lower than the absorption of water. Both scenarios have been experimentally observed in the THz window of radiation.

Dipolar response of hydrated proteins.

PubMed

Matyushov, Dmitry V

2012-02-28

The paper presents an analytical theory and numerical simulations of the dipolar response of hydrated proteins in solution. We calculate the effective dielectric constant representing the average dipole moment induced at the protein by a uniform external field. The dielectric constant shows a remarkable variation among the proteins, changing from 0.5 for ubiquitin to 640 for cytochrome c. The former value implies a negative dipolar susceptibility, that is a dia-electric dipolar response and negative dielectrophoresis. It means that ubiquitin, carrying an average dipole of ≃240 D, is expected to repel from the region of a stronger electric field. This outcome is the result of a negative cross-correlation between the protein and water dipoles, compensating for the positive variance of the intrinsic protein dipole in the overall dipolar susceptibility. In contrast to the neutral ubiquitin, charged proteins studied here show para-electric dipolar response and positive dielectrophoresis. The study suggests that the dipolar response of proteins in solution is strongly affected by the coupling of the protein surface charge to the hydration water. The protein-water dipolar cross-correlations are long-ranged, extending ~2 nm from the protein surface into the bulk. A similar correlation length of about 1 nm is seen for the electrostatic potential produced by the hydration water inside the protein. The analysis of numerical simulations suggests that the polarization of the protein-water interface is highly heterogeneous and does not follow the standard dielectric results for cavities carved in dielectrics. The polarization of the water shell gains in importance, relative to the intrinsic protein dipole, at high frequencies, above the protein Debye peak. The induced interfacial dipole can be either parallel or antiparallel to the protein dipole, depending on the distribution of the protein surface charge. As a result, the high-frequency absorption of the protein solution can be either higher or lower than the absorption of water. Both scenarios have been experimentally observed in the THz window of radiation.

Top-down mass spectrometry imaging of intact proteins by laser ablation ESI FT-ICR MS.

PubMed

Kiss, András; Smith, Donald F; Reschke, Brent R; Powell, Matthew J; Heeren, Ron M A

2014-05-01

Laser ablation ESI (LAESI) is a recent development in MS imaging. It has been shown that lipids and small metabolites can be imaged in various samples such as plant material, tissue sections or bacterial colonies without any sample pretreatment. Further, LAESI has been shown to produce multiply charged protein ions from liquids or solid surfaces. This presents a means to address one of the biggest challenges in MS imaging; the identification of proteins directly from biological tissue surfaces. Such identification is hindered by the lack of multiply charged proteins in common MALDI ion sources and the difficulty of performing tandem MS on such large, singly charged ions. We present here top-down identification of intact proteins from tissue with a LAESI ion source combined with a hybrid ion-trap FT-ICR mass spectrometer. The performance of the system was first tested with a standard protein with electron capture dissociation and infrared multiphoton dissociation fragmentation to prove the viability of LAESI FT-ICR for top-down proteomics. Finally, the imaging of a tissue section was performed, where a number of intact proteins were measured and the hemoglobin α chain was identified directly from tissue using CID and infrared multiphoton dissociation fragmentation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prediction of purification of biopharmeceuticals with molecular dynamics

NASA Astrophysics Data System (ADS)

Ustach, Vincent; Faller, Roland

Purification of biopharmeceuticals remains the most expensive part of protein-based drug production. In ion exchange chromatography (IEX), prediction of the elution ionic strength of host cell and target proteins has the potential to reduce the parameter space for scale-up of protein production. The complex shape and charge distribution of proteins and pores complicates predictions of the interactions in these systems. All-atom molecular dynamics methods are beyond the scope of computational limits for mass transport regimes. We present a coarse-grained model for proteins for prediction of elution pH and ionic strength. By extending the raspberry model for colloid particles to surface shapes and charge distributions of proteins, we can reproduce the behavior of proteins in IEX. The average charge states of titratatable amino acid residues at relevant pH values are determined by extrapolation from all-atom molecular dynamics at pH 7. The pH specific all-atom electrostatic field is then mapped onto the coarse-grained surface beads of the raspberry particle. The hydrodynamics are reproduced with the lattice-Boltzmann scheme. This combination of methods allows very long simulation times. The model is being validated for known elution procedures by comparing the data with experiments. Defense Threat Reduction Agency (Grant Number HDTRA1-15-1-0054).

Bovine serum albumin surface imprinted polymer fabricated by surface grafting copolymerization on zinc oxide rods and its application for protein recognition.

PubMed

Li, Xiangjie; Zhou, Jingjing; Tian, Lei; Li, Wei; Zhang, Baoliang; Zhang, Hepeng; Zhang, Qiuyu

2015-10-01

A novel bovine serum albumin (BSA) surface imprinted polymer based on ZnO rods was synthesized by surface grafting copolymerization. It exhibited an excellent recognition performance to bovine serum albumin. The adsorption capacity and imprinting factor of bovine serum albumin could reach 89.27 mg/g and 2.35, respectively. Furthermore, the fluorescence property of ZnO was used for tracing the process of protein imprinting and it implied the excellent optical sensing property of this material. More importantly, the hypothesis that the surface charge of carrier could affect the imprinting process was confirmed. That is, ZnO with positive surface charge could not only improve the recognition specificity of binding sites to template proteins (pI < 7), but also deteriorate the bindings between sites and non-template proteins (pI > 7). It was also important that the reusability of ZnO@BSA molecularly imprinted polymers was satisfactory. This implied that the poor mechanical/chemical stability of traditional zinc oxide sensors could be solved by the introduction of surface grafting copolymerization. These results revealed that the ZnO@BSA molecularly imprinted polymers are a promising optical/electrochemical sensor element. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative examination of adsorption of serum proteins on HSA- and PLGA-based nanoparticles using SDS-PAGE and LC-MS.

PubMed

Gossmann, R; Fahrländer, E; Hummel, M; Mulac, D; Brockmeyer, J; Langer, K

2015-06-01

The behavior of nanosized drug carrier systems under cell culture conditions and therefore also the destiny in the body are highly influenced by the protein corona, which is formed upon entering a biological environment. Some of the adsorbed proteins, named opsonins, lead to a shortened plasma circulation half-life of the nanoparticles. Others are attributed to promote the transport of nanoparticles into other compartments of the body, just to mention two examples. Hence, detailed knowledge concerning the composition of the protein corona is of great importance. The aim of this work was to investigate the influence of the nanoparticle starting material and the surface modification on the composition of the adsorbed serum proteins in a cell culture environment. Therefore, positively charged nanoparticles based on the biodegradable polymer poly(dl-lactide-co-glycolide) (PLGA) stabilized with didodecyldimethylammonium bromide (DMAB) and negatively charged nanoparticles based on human serum albumin (HSA) were prepared and modified with hydrophilic polymers. By incubating the nanoparticles with fetal bovine serum (FBS) the adsorption of serum proteins on the colloidal system was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) a semi-quantitative analysis of the protein corona was performed and after enzymatic in-solution-digestion the adsorbed proteins were identified using high resolution LC-MS. Our study accentuates the influence of the core material, surface charge, and surface modification on the amount and nature of the adsorbed proteins. The combination of SDS-PAGE and LC-MS turns out to be a simple and reliable method to investigate the protein corona of nanoparticles. Copyright © 2015 Elsevier B.V. All rights reserved.

Positively charged mini-protein Zbasic2 as a highly efficient silica binding module: opportunities for enzyme immobilization on unmodified silica supports.

PubMed

Bolivar, Juan M; Nidetzky, Bernd

2012-07-03

Silica is a highly attractive support material for protein immobilization in a wide range of biotechnological and biomedical-analytical applications. Without suitable derivatization, however, the silica surface is not generally usable for attachment of proteins. We show here that Z(basic2) (a three α-helix bundle mini-protein of 7 kDa size that exposes clustered positive charges from multiple arginine residues on one side) functions as highly efficient silica binding module (SBM), allowing chimeras of target protein with SBM to become very tightly attached to underivatized glass at physiological pH conditions. We used two enzymes, d-amino acid oxidase and sucrose phosphorylase, to demonstrate direct immobilization of Z(basic2) protein from complex biological samples with extremely high selectivity. Immobilized enzymes displayed full biological activity, suggesting that their binding to the glass surface had occurred in a preferred orientation via the SBM. We also show that charge complementarity was the main principle of affinity between SBM and glass surface, and Z(basic2) proteins were bound in a very strong, yet fully reversible manner, presumably through multipoint noncovalent interactions. Z(basic2) proteins were immobilized on porous glass in a loading of 30 mg protein/g support or higher, showing that attachment via the SBM combines excellent binding selectivity with a technically useful binding capacity. Therefore, Z(basic2) and silica constitute a fully orthogonal pair of binding module and insoluble support for oriented protein immobilization, and this opens up new opportunities for the application of silica-based materials in the development of supported heterogeneous biocatalysts.

Salt induced reduction of lysozyme adsorption at charged interfaces

NASA Astrophysics Data System (ADS)

Göhring, Holger; Paulus, Michael; Salmen, Paul; Wirkert, Florian; Kruse, Theresa; Degen, Patrick; Stuhr, Susan; Rehage, Heinz; Tolan, Metin

2015-06-01

A study of lysozyme adsorption below a behenic acid membrane and at the solid-liquid interface between aqueous lysozyme solution and a silicon wafer in the presence of sodium chloride is presented. The salt concentration was varied between 1 mmol L-1 and 1000 mmol L-1. X-ray reflectivity data show a clear dependence of the protein adsorption on the salt concentration. Increasing salt concentrations result in a decreased protein adsorption at the interface until a complete suppression at high concentrations is reached. This effect can be attributed to a reduced attractive electrostatic interaction between the positively charged proteins and negatively charged surfaces by charge screening. The measurements at the solid-liquid interfaces show a transition from unoriented order of lysozyme in the adsorbed film to an oriented order with the short protein axis perpendicular to the solid-liquid interface with rising salt concentration.

The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation

PubMed Central

Winter, Jody A; Christofi, Panayiotis; Morroll, Shaun; Bunting, Karen A

2009-01-01

Background The high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA) to a resolution of 2.0 Å. Results The overall architecture of HvPCNA is very similar to other known PCNAs, which are highly structurally conserved. Three commonly observed adaptations in halophilic proteins are higher surface acidity, bound ions and increased numbers of intermolecular ion pairs (in oligomeric proteins). HvPCNA possesses the former two adaptations but not the latter, despite functioning as a homotrimer. Strikingly, the positive surface charge considered key to PCNA's role as a sliding clamp is dramatically reduced in the halophilic protein. Instead, bound cations within the solvation shell of HvPCNA may permit sliding along negatively charged DNA by reducing electrostatic repulsion effects. Conclusion The extent to which individual proteins adapt to halophilic conditions varies, presumably due to their diverse characteristics and roles within the cell. The number of ion pairs observed in the HvPCNA monomer-monomer interface was unexpectedly low. This may reflect the fact that the trimer is intrinsically stable over a wide range of salt concentrations and therefore additional modifications for trimer maintenance in high salt conditions are not required. Halophilic proteins frequently bind anions and cations and in HvPCNA cation binding may compensate for the remarkable reduction in positive charge in the pore region, to facilitate functional interactions with DNA. In this way, HvPCNA may harness its environment as opposed to simply surviving in extreme halophilic conditions. PMID:19698123

Macroscopic modeling and simulations of supercoiled DNA with bound proteins

NASA Astrophysics Data System (ADS)

Huang, Jing; Schlick, Tamar

2002-11-01

General methods are presented for modeling and simulating DNA molecules with bound proteins on the macromolecular level. These new approaches are motivated by the need for accurate and affordable methods to simulate slow processes (on the millisecond time scale) in DNA/protein systems, such as the large-scale motions involved in the Hin-mediated inversion process. Our approaches, based on the wormlike chain model of long DNA molecules, introduce inhomogeneous potentials for DNA/protein complexes based on available atomic-level structures. Electrostatically, treat those DNA/protein complexes as sets of effective charges, optimized by our discrete surface charge optimization package, in which the charges are distributed on an excluded-volume surface that represents the macromolecular complex. We also introduce directional bending potentials as well as non-identical bead hydrodynamics algorithm to further mimic the inhomogeneous effects caused by protein binding. These models thus account for basic elements of protein binding effects on DNA local structure but remain computational tractable. To validate these models and methods, we reproduce various properties measured by both Monte Carlo methods and experiments. We then apply the developed models to study the Hin-mediated inversion system in long DNA. By simulating supercoiled, circular DNA with or without bound proteins, we observe significant effects of protein binding on global conformations and long-time dynamics of the DNA on the kilo basepair length.

A study on the nature of interactions of mixed-mode ligands HEA and PPA HyperCel using phenylglyoxal modified lysozyme.

PubMed

Pezzini, J; Cabanne, C; Dupuy, J-W; Gantier, R; Santarelli, X

2014-06-01

Mixed mode chromatography, or multimodal chromatography, involves the exploitation of combinations of several interactions in a controlled manner, to facilitate the rapid capture of proteins. Mixed-mode ligands like HEA and PPA HyperCel™ facilitate different kinds of interactions (hydrophobic, ionic, etc.) under different conditions. In order to better characterize the nature of this multi-modal interaction, we sought to study a protein, lysozyme, which is normally not retained by these mixed mode resins under normal binding conditions. Lysozyme was modified specifically at Arginine residues by the action of phenylglyoxal, and was extensively studied in this work to better characterize the mixed-mode interactions of HEA HyperCel™ and PPA HyperCel™ chromatographic supports. We show here that the adsorption behaviour of lysozyme on HEA and PPA HyperCel™ mixed mode sorbents varies depending on the degree of charge modification at the surface of the protein. Experiments using conventional cation exchange and hydrophobic interaction chromatography confirm that both charge and hydrophobicity modification occurs at the surface of the protein after lysozyme reaction with phenylglyoxal. The results emanating from this work using HEA and PPA HyperCel sorbents strongly suggest that mixed mode chromatography can efficiently separate closely related proteins of only minor surface charge and/or hydrophobicity differences. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein sensing by nanofluidic crystal and its signal enhancement

PubMed Central

Sang, Jianming; Du, Hongtan; Wang, Wei; Chu, Ming; Wang, Yuedan; Li, Haichao; Alice Zhang, Haixia; Wu, Wengang; Li, Zhihong

2013-01-01

Nanofluidics has a unique property that ionic conductance across a nanometer-sized confined space is strongly affected by the space surface charge density, which can be utilized to construct electrical read-out biosensor. Based on this principle, this work demonstrated a novel protein sensor along with a sandwich signal enhancement approach. Nanoparticles with designed aptamer onside are assembled in a suspended micropore to form a 3-dimensional network of nanometer-sized interstices, named as nanofluidic crystal hereafter, as the basic sensing unit. Proteins captured by aptamers will change the surface charge density of nanoparticles and thereby can be detected by monitoring the ionic conductance across this nanofluidic crystal. Another aptamer can further enlarge the variations of the surface charge density by forming a sandwich structure (capturing aptamer/protein/signal enhancement aptamer) and the read-out conductance as well. The preliminary experimental results indicated that human α-thrombin was successfully detected by the corresponding aptamer modified nanofluidic crystal with the limit of detection of 5 nM (0.18 μg/ml) and the read-out signal was enhanced up to 3 folds by using another thrombin aptamer. Being easy to graft probe, facile and low-cost to prepare the nano-device, and having an electrical read-out, the present nanofluidic crystal scheme is a promising and universal strategy for protein sensing. PMID:24404017

Crystal structure of human proteasome assembly chaperone PAC4 involved in proteasome formation.

PubMed

Kurimoto, Eiji; Satoh, Tadashi; Ito, Yuri; Ishihara, Eri; Okamoto, Kenta; Yagi-Utsumi, Maho; Tanaka, Keiji; Kato, Koichi

2017-05-01

The 26S proteasome is a large protein complex, responsible for degradation of ubiquinated proteins in eukaryotic cells. Eukaryotic proteasome formation is a highly ordered process that is assisted by several assembly chaperones. The assembly of its catalytic 20S core particle depends on at least five proteasome-specific chaperones, i.e., proteasome-assembling chaperons 1-4 (PAC1-4) and proteasome maturation protein (POMP). The orthologues of yeast assembly chaperones have been structurally characterized, whereas most mammalian assembly chaperones are not. In the present study, we determined a crystal structure of human PAC4 at 1.90-Å resolution. Our crystallographic data identify a hydrophobic surface that is surrounded by charged residues. The hydrophobic surface is complementary to that of its binding partner, PAC3. The surface also exhibits charge complementarity with the proteasomal α4-5 subunits. This will provide insights into human proteasome-assembling chaperones as potential anticancer drug targets. © 2017 The Protein Society.

Exploring the Relationship between Structural and Air-Water Interfacial Properties of Wheat (Triticum aestivum L.) Gluten Hydrolysates in a Food System Relevant pH Range.

PubMed

Wouters, Arno G B; Fierens, Ellen; Rombouts, Ine; Brijs, Kristof; Joye, Iris J; Delcour, Jan A

2017-02-15

The relationship between structural and foaming properties of two tryptic and two peptic wheat gluten hydrolysates was studied at different pH conditions. The impact of pH on foam stability (FS) of the samples heavily depended on the peptidase used and the degree of hydrolysis reached. Surface dilatational moduli were in most, but not all, instances related to FS, implying that, although the formation of a viscoelastic protein hydrolysate film is certainly important, this is not the only phenomenon that determines FS. In contrast to what might be expected, surface charge was not a major factor contributing to FS, except when close to the point-of-zero-charge. Surface hydrophobicity and intrinsic fluorescence measurements suggested that changes in protein conformation take place when the pH is varied, which can in turn influence foaming. Finally, hydrolyzed gluten proteins formed relatively large particles, suggesting that protein hydrolysate aggregation probably influences its foaming properties.

Interaction of Charged Patchy Protein Models with Like-Charged Polyelectrolyte Brushes.

PubMed

Yigit, Cemil; Kanduč, Matej; Ballauff, Matthias; Dzubiella, Joachim

2017-01-10

We study the adsorption of charged patchy particle models (CPPMs) on a thin film of a like-charged and dense polyelectrolyte (PE) brush (of 50 monomers per chain) by means of implicit-solvent, explicit-salt Langevin dynamics computer simulations. Our previously introduced set of CPPMs embraces well-defined one- and two-patched spherical globules, each of the same net charge and (nanometer) size, with mono- and multipole moments comparable to those of small globular proteins. We focus on electrostatic effects on the adsorption far away from the isoelectric point of typical proteins, i.e., where charge regulation plays no role. Despite the same net charge of the brush and globule, we observe large binding affinities up to tens of the thermal energy, k B T, which are enhanced by decreasing salt concentration and increasing charge of the patch(es). Our analysis of the distance-resolved potentials of mean force together with a phenomenological description of all leading interaction contributions shows that the attraction is strongest at the brush surface, driven by multipolar, Born (self-energy), and counterion-release contributions, dominating locally over the monopolar and steric repulsions.

Crystal structure of the DNA polymerase III β subunit (β-clamp) from the extremophile Deinococcus radiodurans.

PubMed

Niiranen, Laila; Lian, Kjersti; Johnson, Kenneth A; Moe, Elin

2015-02-27

Deinococcus radiodurans is an extremely radiation and desiccation resistant bacterium which can tolerate radiation doses up to 5,000 Grays without losing viability. We are studying the role of DNA repair and replication proteins for this unusual phenotype by a structural biology approach. The DNA polymerase III β subunit (β-clamp) acts as a sliding clamp on DNA, promoting the binding and processivity of many DNA-acting proteins, and here we report the crystal structure of D. radiodurans β-clamp (Drβ-clamp) at 2.0 Å resolution. The sequence verification process revealed that at the time of the study the gene encoding Drβ-clamp was wrongly annotated in the genome database, encoding a protein of 393 instead of 362 amino acids. The short protein was successfully expressed, purified and used for crystallisation purposes in complex with Cy5-labeled DNA. The structure, which was obtained from blue crystals, shows a typical ring-shaped bacterial β-clamp formed of two monomers, each with three domains of identical topology, but with no visible DNA in electron density. A visualisation of the electrostatic surface potential reveals a highly negatively charged outer surface while the inner surface and the dimer forming interface have a more even charge distribution. The structure of Drβ-clamp was determined to 2.0 Å resolution and shows an evenly distributed electrostatic surface charge on the DNA interacting side. We hypothesise that this charge distribution may facilitate efficient movement on encircled DNA and help ensure efficient DNA metabolism in D. radiodurans upon exposure to high doses of ionizing irradiation or desiccation.

Complexation of lysozyme with adsorbed PtBS-b-SCPI block polyelectrolyte micelles on silver surface.

PubMed

Papagiannopoulos, Aristeidis; Christoulaki, Anastasia; Spiliopoulos, Nikolaos; Vradis, Alexandros; Toprakcioglu, Chris; Pispas, Stergios

2015-01-20

We present a study of the interaction of the positively charged model protein lysozyme with the negatively charged amphiphilic diblock polyelectrolyte micelles of poly(tert-butylstyrene-b-sodium (sulfamate/carboxylate)isoprene) (PtBS-b-SCPI) on the silver/water interface. The adsorption kinetics are monitored by surface plasmon resonance, and the surface morphology is probed by atomic force microscopy. The micellar adsorption is described by stretched-exponential kinetics, and the micellar layer morphology shows that the micelles do not lose their integrity upon adsorption. The complexation of lysozyme with the adsorbed micellar layers depends on the micelles arrangement and density in the underlying layer, and lysozyme follows the local morphology of the underlying roughness. When the micellar adsorbed amount is small, the layers show low capacity in protein complexation and low resistance in loading. When the micellar adsorbed amount is high, the situation is reversed. The adsorbed layers both with or without added protein are found to be irreversibly adsorbed on the Ag surface.

Fabrication and characteristics of MOSFET protein chip for detection of ribosomal protein.

PubMed

Park, Keun-Yong; Kim, Min-Suk; Choi, Sie-Young

2005-04-15

A metal oxide silicon field effect transistor (MOSFET) protein chip for the easy detection of protein was fabricated and its characteristics were investigated. Generally, the drain current of the MOSFET is varied by the gate potential. It is expected that the formation of an antibody-antigen complex on the gate of MOSFET would lead to a detectable change in the charge distribution and thus, directly modulate the drain current of MOSFET. As such, the drain current of the MOSFET protein chip can be varied by ribosomal proteins absorbed by the self-assembled monolayer (SAM) immobilized on the gate (Au) surface, as ribosomal protein has positive charge, and these current variations then used as the response of the protein chip. The gate of MOSFET protein chip is not directly biased by an external voltage source, so called open gate or floating gate MOSFET, but rather chemically modified by immobilized molecular receptors called self-assembled monolayer (SAM). In our experiments, the current variation in the proposed protein chip was about 8% with a protein concentration of 0.7 mM. As the protein concentration increased, the drain current also gradually increased. In addition, there were some drift of the drain current in the device. It is considered that these drift might be caused by the drift from the MOSFET itself or protein absorption procedures that are relied on the facile attachment of thiol (-S) ligands to the gate (Au) surface. We verified the formation of SAM on the gold surface and the absorption of protein through the surface plasmon resonance (SPR) measurement.

Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa.

PubMed

Trevino, Saul R; Scholtz, J Martin; Pace, C Nick

2007-02-16

Poor protein solubility is a common problem in high-resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all 20 amino acids to protein solubility has not been done. Here, 20 variants at the completely solvent-exposed position 76 of ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II beta-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine.

Plasma membrane translocation of a protein needle based on a triple-stranded β-helix motif.

PubMed

Sanghamitra, Nusrat J M; Inaba, Hiroshi; Arisaka, Fumio; Ohtan Wang, Dan; Kanamaru, Shuji; Kitagawa, Susumu; Ueno, Takafumi

2014-10-01

Plasma membrane translocation is challenging due to the barrier of the cell membrane. Contrary to the synthetic cell-penetrating materials, tailed bacteriophages use cell-puncturing protein needles to puncture the cell membranes as an initial step of the DNA injection process. Cell-puncturing protein needles are thought to remain functional in the native phages. In this paper, we found that a bacteriophage T4 derived protein needle of 16 nm length spontaneously translocates through the living cell membrane. The β-helical protein needle (β-PN) internalizes into human red blood cells that lack endocytic machinery. By comparing the cellular uptake of β-PNs with modified surface charge, it is shown that the uptake efficiency is maximum when it has a negative charge corresponding to a zeta potential value of -16 mV. In HeLa cells, uptake of β-PN incorporates endocytosis independent mechanisms with partial macropinocytosis dependence. The endocytosis dependence of the uptake increases when the surface charges of β-PNs are modified to positive or negative. Thus, these results suggest that natural DNA injecting machinery can serve as an inspiration to design new class of cell-penetrating materials with a tailored mechanism.

A mobile precursor determines protein resistance on nanostructured surfaces.

PubMed

Wang, Kang; Chen, Ye; Gong, Xiangjun; Xia, Jianlong; Zhao, Junpeng; Shen, Lei

2018-05-09

Biomaterials are often engineered with nanostructured surfaces to control interactions with proteins and thus regulate their biofunctions. However, the mechanism of how nanostructured surfaces resist or attract proteins together with the underlying design rules remains poorly understood at a molecular level, greatly limiting attempts to develop high-performance biomaterials and devices through the rational design of nanostructures. Here, we study the dynamics of nonspecific protein adsorption on block copolymer nanostructures of varying adhesive domain areas in a resistant matrix. Using surface plasmon resonance and single molecule tracking techniques, we show that weakly adsorbed proteins with two-dimensional diffusivity are critical precursors to protein resistance on nanostructured surfaces. The adhesive domain areas must be more than tens or hundreds of times those of the protein footprints to slow down the 2D-mobility of the precursor proteins for their irreversible adsorption. This precursor model can be used to quantitatively analyze the kinetics of nonspecific protein adsorption on nanostructured surfaces. Our method is applicable to precisely manipulate protein adsorption and resistance on various nanostructured surfaces, e.g., amphiphilic, low-surface-energy, and charged nanostructures, for the design of protein-compatible materials.

Negatively Charged Lipid Membranes Promote a Disorder-Order Transition in the Yersinia YscU Protein

PubMed Central

Weise, Christoph F.; Login, Frédéric H.; Ho, Oanh; Gröbner, Gerhard; Wolf-Watz, Hans; Wolf-Watz, Magnus

2014-01-01

The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscUC variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscUC that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia. PMID:25418176

Negatively charged lipid membranes promote a disorder-order transition in the Yersinia YscU protein.

PubMed

Weise, Christoph F; Login, Frédéric H; Ho, Oanh; Gröbner, Gerhard; Wolf-Watz, Hans; Wolf-Watz, Magnus

2014-10-21

The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscUC variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscUC that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia.

Properties of an αs-casein-rich casein fraction: influence of dialysis on surface properties, miscibility, and micelle formation.

PubMed

Kessler, Anne; Menéndez-Aguirre, Orquidéa; Hinrichs, Jörg; Stubenrauch, Cosima; Weiss, Jochen

2013-09-01

In this study, the surface tension, miscibility, and particle size distribution of a solution containing an αs-casein (CN)-rich CN fraction (54 wt % αs-CN, 32 wt % β-CN, and 15 wt % κ-CN) were determined at pH 6.6. The nondialyzed CN fraction was compared with a dialyzed one. In the nondialyzed sample, every charge on the protein was compensated by 0.3 charges coming from counterions, whereas in the dialyzed sample, only 0.2 charges could be assigned to each charge on the protein. This relation was determined by calculating the charges at the proteins, taking the measured mineral content into account. The surface tension was measured as a function of the protein concentration by the du Noüy ring method at room temperature. Results indicated alterations in the surface properties after reduction of counterions. The equilibrium surface tension above the critical micelle concentration increased from 40.1×10(-3) to 45×10(-3) N/m, the critical micelle concentration increased from 0.9×10(-4) to 2×10(-3) mol/L, and the minimal area occupied per molecule at the surface increased from 2.4×10(-18) to 4.6×10(-18) m(2). Cloud points were determined by measuring the absorbance of CN solutions as a function of the temperature. The cloud points were found to be concentration dependent and had a minimum at 0.2 wt % at 34°C for nondialyzed CN and at 0.25 wt % at 28°C for dialyzed CN, again demonstrating the influence of counterion reduction. Below the cloud point, a micellar phase was found to exist. The hydrodynamic diameter of the micelles were characterized by dynamic light scattering in both auto- and cross-correlation mode. However, no influence of reduction in counterions could be observed, possibly due to the fact that dynamic light scattering is not a suitable method for this type of system. The presence of self-assembled structures was verified by freeze-fracture electron microscopy. The observed differences between dialyzed and nondialyzed samples were explained by changes in the counterion cloud surrounding the proteins. Consequently, the electrostatic interactions between as well as within the CN are altered by dialysis, which, in turn, affects the behavior at the surface as well as the properties in the solution. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

[Interaction of protein with charged colloidal particles].

PubMed

Durdenko, E V; Kuznetsova, S M; Basova, L V; Tikhonenko, S A; Saburova, E A

2011-01-01

The functional state of three proteins of different molecular weight (urease, lactate dehydrogenase, and hemoglobin) in the presence of the linear polyelectrolytes poly(allylamine hydrochloride) (PAA) and sodium poly(styrenesulfonate) (PSS) in the dissolved state and of the same polyelectrolytes bound to the surface of microspheres has been investigated. Microspheres were prepared by consecutive absorption of oppositely charged polyelectrolytes so that the outer layer of the shell was PAA for the acidic protein urease, and PSS for the alkaline proteins LDH and hemoglobin. It was shown that the dissolved polyelectrolyte completely inactivates all three proteins within one minute with a slight difference in the time constant. (By Hb inactivation are conventionally meant changes in the heme environment observed from the spectrum in the Soret band.) In the presence of microspheres, the proteins were adsorbed on their surface; in this case, more than 95% of the activity was retained within two hours. The proportion of the protein adsorbed on microspheres accounted for about 98% for urease, 72% for Hb, and 35% for LDH, as determined from the tryptophan fluorescence data. The interaction of hemoglobin with another type of charged colloidal particles, phospholipid vesicles, leads to the destruction of the tertiary structure of the protein, which made itself evident in the optical absorption spectra in the Soret band, as well as the spectra of tryptophan fluorescence and circular dichroism. In this case, according to circular dichroism, the percentage of alpha-helical structure of Hb was maintained. The differences in the physical and chemical mechanisms of interaction of proteins with these two types of charged colloidal particles that leads to differences in the degree of denaturing effects are discussed.

Toward a Molecular Understanding of Protein Solubility: Increased Negative Surface Charge Correlates with Increased Solubility

PubMed Central

Kramer, Ryan M.; Shende, Varad R.; Motl, Nicole; Pace, C. Nick; Scholtz, J. Martin

2012-01-01

Protein solubility is a problem for many protein chemists, including structural biologists and developers of protein pharmaceuticals. Knowledge about how intrinsic factors influence solubility is limited due to the difficulty of obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, because gels or supersaturated solutions often form, making it impossible to determine solubility values for many proteins. Protein precipitants can be used to obtain comparative solubility measurements and, in some cases, estimations of solubility in buffer alone. Protein precipitants fall into three broad classes: salts, long-chain polymers, and organic solvents. Here, we compare the use of representatives from two classes of precipitants, ammonium sulfate and polyethylene glycol 8000, by measuring the solubility of seven proteins. We find that increased negative surface charge correlates strongly with increased protein solubility and may be due to strong binding of water by the acidic amino acids. We also find that the solubility results obtained for the two different precipitants agree closely with each other, suggesting that the two precipitants probe similar properties that are relevant to solubility in buffer alone. PMID:22768947

Lentil and chickpea protein-stabilized emulsions: optimization of emulsion formulation.

PubMed

Can Karaca, Asli; Nickerson, Michael T; Low, Nicholas H

2011-12-28

Chickpea and lentil protein-stabilized emulsions were optimized with regard to pH (3.0-8.0), protein concentration (1.1-4.1% w/w), and oil content (20-40%) for their ability to form and stabilize oil-in-water emulsions using response surface methodology. Specifically, creaming stability, droplet size, and droplet charge were assessed. Optimum conditions for minimal creaming (no serum separation after 24 h), small droplet size (<2 μm), and high net droplet charge (absolute value of ZP > 40 mV) were identified as 4.1% protein, 40% oil, and pH 3.0 or 8.0, regardless of the plant protein used for emulsion preparation.

Nanoscale observation of local bound charges of patterned protein arrays by scanning force microscopy

NASA Astrophysics Data System (ADS)

Oh, Y. J.; Jo, W.; Kim, S.; Park, S.; Kim, Y. S.

2008-09-01

A protein patterned surface using micro-contact printing methods has been investigated by scanning force microscopy. Electrostatic force microscopy (EFM) was utilized for imaging the topography and detecting the electrical properties such as the local bound charge distribution of the patterned proteins. It was found that the patterned IgG proteins are arranged down to 1 µm, and the 90° rotation of patterned anti-IgG proteins was successfully undertaken. Through the estimation of the effective areas, it was possible to determine the local bound charges of patterned proteins which have opposite electrostatic force behaviors. Moreover, we studied the binding probability between IgG and anti-IgG in a 1 µm2 MIMIC system by topographic and electrostatic signals for applicable label-free detections. We showed that the patterned proteins can be used for immunoassay of proteins on the functional substrate, and that they can also be used for bioelectronics device application, indicating distinct advantages with regard to accuracy and a label-free detection.

The simulation study of protein-protein interfaces based on the 4-helix bundle structure

NASA Astrophysics Data System (ADS)

Fukuda, Masaki; Komatsu, Yu; Morikawa, Ryota; Miyakawa, Takeshi; Takasu, Masako; Akanuma, Satoshi; Yamagishi, Akihiko

2013-02-01

Docking of two protein molecules is induced by intermolecular interactions. Our purposes in this study are: designing binding interfaces on the two proteins, which specifically interact to each other; and inducing intermolecular interactions between the two proteins by mixing them. A 4-helix bundle structure was chosen as a scaffold on which binding interfaces were created. Based on this scaffold, we designed binding interfaces involving charged and nonpolar amino acid residues. We performed molecular dynamics (MD) simulation to identify suitable amino acid residues for the interfaces. We chose YciF protein as the scaffold for the protein-protein docking simulation. We observed the structure of two YciF protein molecules (I and II), and we calculated the distance between centroids (center of gravity) of the interfaces' surface planes of the molecules I and II. We found that the docking of the two protein molecules can be controlled by the number of hydrophobic and charged amino acid residues involved in the interfaces. Existence of six hydrophobic and five charged amino acid residues within an interface were most suitable for the protein-protein docking.

1-anilino-8-naphthalene sulfonate as a protein conformational tightening agent.

PubMed

Matulis, D; Baumann, C G; Bloomfield, V A; Lovrien, R E

1999-05-01

1-Anilino-8-naphthalene sulfonate (ANS) anion is conventionally considered to bind to preexisting hydrophobic (nonpolar) surfaces of proteins, primarily through its nonpolar anilino-naphthalene group. Such binding is followed by an increase in ANS fluorescence intensity, similar to that occurring when ANS is dissolved in organic solvents. It is generally assumed that neither the negative sulfonate charge on the ANS, nor charges on the protein, participate significantly in ANS-protein interaction. However, titration calorimetry has demonstrated that most ANS binding to a number of proteins occurs through electrostatic forces, in which ion pairs are formed between ANS sulfonate groups and cationic groups on the proteins (D. Matulis and R. E. Lovrien, Biophys. J., 1998, Vol. 74, pp. 1-8). Here we show by viscometry and diffusion coefficient measurements that bovine serum albumin and gamma-globulin, starting from their acid-expanded, most hydrated conformations, undergo extensive molecular compaction upon ANS binding. As the cationic protein binds negatively charged ANS anion it also takes up positively charged protons from water to compensate the effect of the negative charge, and leaves the free hydroxide anions in solution thus shifting pH upward (the Scatchard-Black effect). These results indicate that ANS is not always a definitive reporter of protein molecular conformation that existed before ANS binding. Instead, ANS reports on a conformationally tightened state produced by the interplay of ionic and hydrophobic characters of both protein and ligand.

Surface charge dependent separation of modified and hybrid ferritin in native PAGE: Impact of lysine 104.

PubMed

Subhadarshanee, Biswamaitree; Mohanty, Abhinav; Jagdev, Manas Kumar; Vasudevan, Dileep; Behera, Rabindra K

2017-10-01

Preparation of modified and hybrid ferritin provides a great opportunity to understand the mechanisms of iron loading/unloading, protein self-assembly, size constrained nanomaterial synthesis and targeted drug delivery. However, the large size (M.W.=490kDa) has been limiting the separation of different modified and/or hybrid ferritin nanocages from each other in their intact assembled form and further characterization. Native polyacrylamide gel electrophoresis (PAGE) separates proteins on the basis of both charge and mass, while maintaining their overall native structure and activity. Altering surface charge distribution by substitution of amino acid residues located at the external surface of ferritin (K104E & D40A) affected the migration rate in native PAGE while internal modification had little effect. Crystal structures confirmed that ferritin nanocages made up of subunits with single amino acid substitutions retain the overall structure of ferritin nanocage. Taking advantage of K104E migration behavior, formation of hybrid ferritins with subunits of wild type (WT) and K104E were confirmed and separated in native PAGE. Cage integrity and iron loading ability (ferritin activity) were also tested. The migration pattern of hybrid ferritins in native PAGE depends on the subunit ratio (WT: K104E) in the ferritin cage. Our work shows that native PAGE can be exploited in nanobiotechnology, by analyzing modifications of large proteins like ferritin. Native PAGE, a simple, straight-forward technique, can be used to analyze small modification (by altering external surface charge) in large proteins like ferritin, without disintegrating its self-assembled nanocage structure. In doing so, native PAGE can complement the information obtained from mass spectrometry. The confirmation and separation of modified and hybrid ferritin protein nanocages in native PAGE, opens up various prospects of bio-conjugation, which can be useful in targeted drug delivery, nanobiotechnology and in understanding nature's idea of synthesizing hybrid ferritins in different human tissues. Copyright © 2017 Elsevier B.V. All rights reserved.

Substantial conformational change mediated by charge-triad residues of the death effector domain in protein-protein interactions.

PubMed

Twomey, Edward C; Cordasco, Dana F; Kozuch, Stephen D; Wei, Yufeng

2013-01-01

Protein conformational changes are commonly associated with the formation of protein complexes. The non-catalytic death effector domains (DEDs) mediate protein-protein interactions in a variety of cellular processes, including apoptosis, proliferation and migration, and glucose metabolism. Here, using NMR residual dipolar coupling (RDC) data, we report a conformational change in the DED of the phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) protein in the complex with a mitogen-activated protein (MAP) kinase, extracellular regulated kinase 2 (ERK2), which is essential in regulating ERK2 cellular distribution and function in cell proliferation and migration. The most significant conformational change in PEA-15 happens at helices α2, α3, and α4, which also possess the highest flexibility among the six-helix bundle of the DED. This crucial conformational change is modulated by the D/E-RxDL charge-triad motif, one of the prominent structural features of DEDs, together with a number of other electrostatic and hydrogen bonding interactions on the protein surface. Charge-triad motif promotes the optimal orientation of key residues and expands the binding interface to accommodate protein-protein interactions. However, the charge-triad residues are not directly involved in the binding interface between PEA-15 and ERK2.

Albumin adsorption on CoCrMo alloy surfaces

NASA Astrophysics Data System (ADS)

Yan, Yu; Yang, Hongjuan; Su, Yanjing; Qiao, Lijie

2015-12-01

Proteins can adsorb on the surface of artificial joints immediately after being implanted. Although research studying protein adsorption on medical material surfaces has been carried out, the mechanism of the proteins’ adsorption which affects the corrosion behaviour of such materials still lacks in situ observation at the micro level. The adsorption of bovine serum albumin (BSA) on CoCrMo alloy surfaces was studied in situ by AFM and SKPFM as a function of pH and the charge of CoCrMo alloy surfaces. Results showed that when the specimens were uncharged, hydrophobic interaction could govern the process of the adsorption rather than electrostatic interaction, and BSA molecules tended to adsorb on the surfaces forming a monolayer in the side-on model. Results also showed that adsorbed BSA molecules could promote the corrosion process for CoCrMo alloys. When the surface was positively charged, the electrostatic interaction played a leading role in the adsorption process. The maximum adsorption occurred at the isoelectric point (pH 4.7) of BSA.

Anisotropy of the Coulomb Interaction between Folded Proteins: Consequences for Mesoscopic Aggregation of Lysozyme

PubMed Central

Chan, Ho Yin; Lankevich, Vladimir; Vekilov, Peter G.; Lubchenko, Vassiliy

2012-01-01

Toward quantitative description of protein aggregation, we develop a computationally efficient method to evaluate the potential of mean force between two folded protein molecules that allows for complete sampling of their mutual orientation. Our model is valid at moderate ionic strengths and accounts for the actual charge distribution on the surface of the molecules, the dielectric discontinuity at the protein-solvent interface, and the possibility of protonation or deprotonation of surface residues induced by the electric field due to the other protein molecule. We apply the model to the protein lysozyme, whose solutions exhibit both mesoscopic clusters of protein-rich liquid and liquid-liquid separation; the former requires that protein form complexes with typical lifetimes of approximately milliseconds. We find the electrostatic repulsion is typically lower than the prediction of the Derjaguin-Landau-Verwey-Overbeek theory. The Coulomb interaction in the lowest-energy docking configuration is nonrepulsive, despite the high positive charge on the molecules. Typical docking configurations barely involve protonation or deprotonation of surface residues. The obtained potential of mean force between folded lysozyme molecules is consistent with the location of the liquid-liquid coexistence, but produces dimers that are too short-lived for clusters to exist, suggesting lysozyme undergoes conformational changes during cluster formation. PMID:22768950

Electrostatic Interactions Influence Protein Adsorption (but Not Desorption) at the Silica-Aqueous Interface.

PubMed

McUmber, Aaron C; Randolph, Theodore W; Schwartz, Daniel K

2015-07-02

High-throughput single-molecule total internal reflection fluorescence microscopy was used to investigate the effects of pH and ionic strength on bovine serum albumin (BSA) adsorption, desorption, and interfacial diffusion at the aqueous-fused silica interface. At high pH and low ionic strength, negatively charged BSA adsorbed slowly to the negatively charged fused silica surface. At low pH and low ionic strength, where BSA was positively charged, or in solutions at higher ionic strength, adsorption was approximately 1000 times faster. Interestingly, neither surface residence times nor the interfacial diffusion coefficients of BSA were influenced by pH or ionic strength. These findings suggested that adsorption kinetics were dominated by energy barriers associated with electrostatic interactions, but once adsorbed, protein-surface interactions were dominated by short-range nonelectrostatic interactions. These results highlight the ability of single-molecule techniques to isolate elementary processes (e.g., adsorption and desorption) under steady-state conditions, which would be impossible to measure using ensemble-averaging methods.

Surface charge sensing by altering the phase transition in VO2

NASA Astrophysics Data System (ADS)

Kumar, S.; Esfandyarpour, R.; Davis, R.; Nishi, Y.

2014-08-01

Detection of surface charges has various applications in medicine, electronics, biotechnology, etc. The source of surface charge induction may range from simple charge-polarized molecules like water to complicated proteins. It was recently discovered that surface charge accumulation can alter the temperature at which VO2 undergoes a Mott transition. Here, we deposited polar molecules onto the surface of two-terminal thin-film VO2 lateral devices and monitored the joule-heating-driven Mott transition, or conductance switching. We observed that the power required to induce the conductance switching reduced upon treatment with polar molecules and, using in-situ blackbody-emission direct measurement of local temperature, we show that this reduction in power was accompanied by reduction in the Mott transition temperature. Further evidence suggested that this effect has specificity to the nature of the species used to induce surface charges. Using x-ray absorption spectroscopy, we also show that there is no detectable change in oxidation state of vanadium or structural phase in the bulk of the 40 nm VO2 thin-film even as the phase transition temperature is reduced by up to 20 K by the polar molecules. The ability to alter the phase transition parameters by depositing polar molecules suggests a potential application in sensing surface charges of different origins and this set of results also highlights interesting aspects of the phase transition in VO2.

Surface-mediated molecular events in material-induced blood-plasma coagulation

NASA Astrophysics Data System (ADS)

Chatterjee, Kaushik

Coagulation and thrombosis persist as major impediments associated with the use of blood-contacting medical devices. We are investigating the molecular mechanism underlying material-induced blood-plasma coagulation focusing on the role of the surface as a step towards prospective development of improved hemocompatible biomaterials. A classic observation in hematology is that blood/blood-plasma in contact with clean glass surface clots faster than when in contact with many plastic surfaces. The traditional biochemical theory explaining the underlying molecular mechanism suggests that hydrophilic surfaces, like that of glass, are specific activators of the coagulation cascade because of the negatively-charged groups on the surface. Hydrophobic surfaces are poor procoagulants or essentially "benign" because they lack anionic groups. Further, these negatively-charged surfaces are believed to not only activate blood factor XII (FXII), the key protein in contact activation, but also play a cofactor role in the amplification and propagation reactions that ultimately lead to clot formation. In sharp contrast to the traditional theory, our investigations indicate a need for a paradigm shift in the proposed sequence of contact activation events to incorporate the role of protein adsorption at the material surfaces. These studies have lead to the central hypothesis for this work proposing that protein adsorption to hydrophobic surfaces attenuates the contact activation reactions so that poorly-adsorbent hydrophilic surfaces appear to be stronger procoagulants relative to hydrophobic surfaces. Our preliminary studies measuring the plasma coagulation response of activated FXII (FXIIa) on different model surfaces suggested that the material did not play a cofactor role in the processing of this enzyme dose through the coagulation pathway. Therefore, we focused our efforts on studying the mechanism of initial production of enzyme at the procoagulant surface. Calculations for the amounts of FXIIa generated at material surfaces in plasma using a mathematical model for measured coagulation responses indicate that the relative contributions of the individual pathways of enzyme generation are similar at both hydrophilic and hydrophobic surfaces, only the amounts of enzyme generated scale with surface energy and area of the activating surface. Further, from direct measurement of enzyme activation at test surfaces we observed that contact activation reactions are not specific to negatively-charged hydrophilic surfaces. Rather, the molecular interactions are attenuated at hydrophobic surfaces due to protein adsorption so that poorly-adsorbent hydrophilic surfaces exhibit an apparent specificity for contact activation reactions. Preliminary studies were preformed to assay the plasma coagulation response to low-fouling surfaces prepared by either grafting poly(ethylene glycol) chains or using zwitterions. Results indicate that poly(ethylene glycol)-modified surfaces are significantly weaker procoagulants than surfaces containing zwitterions underscoring a need to specifically evaluate the coagulation response despite similarities in observed protein adsorption to both surfaces. In summary, our studies demonstrate a need to incorporate protein-adsorption competition at procoagulant surfaces into the mechanism of contact activation to account for the observed moderation of FXII activation by blood proteins unrelated to the plasma coagulation cascade.

Protein Adaptations in Archaeal Extremophiles

PubMed Central

Reed, Christopher J.; Lewis, Hunter; Trejo, Eric; Winston, Vern; Evilia, Caryn

2013-01-01

Extremophiles, especially those in Archaea, have a myriad of adaptations that keep their cellular proteins stable and active under the extreme conditions in which they live. Rather than having one basic set of adaptations that works for all environments, Archaea have evolved separate protein features that are customized for each environment. We categorized the Archaea into three general groups to describe what is known about their protein adaptations: thermophilic, psychrophilic, and halophilic. Thermophilic proteins tend to have a prominent hydrophobic core and increased electrostatic interactions to maintain activity at high temperatures. Psychrophilic proteins have a reduced hydrophobic core and a less charged protein surface to maintain flexibility and activity under cold temperatures. Halophilic proteins are characterized by increased negative surface charge due to increased acidic amino acid content and peptide insertions, which compensates for the extreme ionic conditions. While acidophiles, alkaliphiles, and piezophiles are their own class of Archaea, their protein adaptations toward pH and pressure are less discernible. By understanding the protein adaptations used by archaeal extremophiles, we hope to be able to engineer and utilize proteins for industrial, environmental, and biotechnological applications where function in extreme conditions is required for activity. PMID:24151449

Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa

PubMed Central

Trevino, Saul R.; Scholtz, J. Martin; Pace, C. Nick

2009-01-01

SUMMARY Poor protein solubility is a common problem in high resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all twenty amino acids to protein solubility has not been done. Here, twenty variants at the completely solvent-exposed position 76 of Ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II β-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine. PMID:17174328

Spatial variation of charge and sulfur oxidation state in a surface gradient affects plasma proteins adsorption

PubMed Central

Ding, Yong-Xue; Streitmatter, Seth; Wright, Bryon E.; Hlady, Vladimir

2010-01-01

A gradient of negative surface charge based on 1-D spatial variation from surface sulfhydryl to mixed sulfhydryl-sulfonate moities was prepared by controlled UV oxidation of 3-mercaptopropylsilane monolayer on fused silica. Adsorption of three human plasma proteins, albumin (HSA), immunoglobulin G (IgG), and fibrinogen (Fgn) onto such surface gradient was studied using spatially-resolved total internal reflection fluorescence (TIRF) and autoradiography. Adsorption was measured from dilute solutions equivalent to 1/100 (TIRF, autoradiography), and 1/500 and 1/1000 (autoradiography) of protein’s physiological concentrations in plasma. All three proteins adsorbed more to the non-oxidized sulfhydryl region than to the oxidized, mixed sulfhydryl-sulfonate region of the gradient. In the case of HSA the adsorption contrast along the gradient was largest when the adsorption took place from more dilute protein solutions. Increasing the concentration to 1/100 of protein plasma concentration eliminated the effect of the gradient on HSA adsorption and to the lesser extent on IgG adsorption. In the case of Fgn the greatest adsorption contrast was observed at the highest concentration used. Based on adsorption kinetics, the estimated binding affinity of HSA for the sulfhydryl region what twice the affinity for the mixed sulfhydryl-sulfonate region of the gradient. For IgG and Fgn the initial adsorption was transport-limited and the initial adsorption rates approached the computed flux of the protein to the surface. PMID:20568822

Protein adsorption on electrospun zinc doped hydroxyapatite containing nylon 6 membrane: kinetics and isotherm.

PubMed

Esfahani, Hamid; Prabhakaran, Molamma P; Salahi, Esmaeil; Tayebifard, Ali; Keyanpour-Rad, Mansour; Rahimipour, Mohamad Reza; Ramakrishna, Seeram

2015-04-01

Surface modification of electrospun polymeric membrane surfaces is a critical step towards the separation process including protein adsorption. In this study, the electrospun Nylon fibers was incorporated with positively charged zinc doped hydroxyapatite (HAp) nanoparticles to study the adsorption of negatively charged proteins, namely bovine serum albumin (BSA). Effects of zinc amount within the atomic structure of HAp (nZH; n=0, 4, 8 At.%) was evaluated on produced scaffolds and consequently protein adsorption. The results showed that the ability of Nylon membrane to adsorb BSA increased with incorporation of nZH nanoparticles within the nylon structure. This phenomenon is appeared to be relate to different electrostatic charge and not to physical characteristic of scaffolds. The incorporated membrane (N-4ZH) by nanoparticles with highest zeta (ξ) potential adsorbed the maximum amount of protein. The adsorption of BSA was best fitted with pseudo-second order kinetic model. The experimental isotherm data were further analyzed by using Langmuir and Freundlich equations. By comparing the correlation coefficients obtained for each linear transformation of isotherm analysis, it was found that the Langmuir equation was the best fit equilibrium model that described the adsorption of BSA on these membranes. Copyright © 2014 Elsevier Inc. All rights reserved.

Using an Engineered Protein Model to Constrain Protein-Mineral Interactions

NASA Astrophysics Data System (ADS)

Chacon, S. S.; Reardon, P. N.; Washton, N.; Kleber, M.

2015-12-01

Exoenzymes are proteins that can catalyze the depolymerization of soil organic matter (SOM). Proteins can also be an important source of organic N for microorganisms, but must be fragmented into small peptides in order to be transported through their membranes. An exoenzyme's affinity to mineral surfaces found in soil affects their capacity to degrade SOM or other proteins. Our goal was to determine the range of modifications on proteins when they interact with a mineral surface. We hypothesized that pedogenic oxides would fragment or promote greater chemical modifications to a protein than phyllosilicates. A well-characterized protein proxy (Gb1, IEP 4.0, 6.2 kDA) was adsorbed onto functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnesite) at pH 5 and pH 7. We then generated three engineered proxies of Gb1 by inserting either negatively charged, positively charged or aromatic amino acids into the second loop. We used liquid chromatography coupled with a mass spectrometer (LC-MS/MS) and solution-state Heteronuclear Single Quantum Coherence Spectroscopy Nuclear Magnetic Resonance (HSQC NMR) to observe modifications to Gb1 that was allowed to equilibrate during the adsorption process for kaolinite, goethite, birnessite, and montmorillonite. We also used Helium Ion Microscopy (HIM) to determine which surface archetypes Gb1 preferentially adsorbed to as a function of the mineral type. The three engineered proxies were used to determine how variation of the amino acid sequence affects a protein interaction with a mineral surface. Preliminary results in the LC-MS/MS indicate that birnessite hydrolytically fragments Gb1 into polypeptides. Our results suggest that not all mineral surfaces in soil may act as sorbents for EEs and that chemical modification of their structure should also be considered as an explanation for decrease in EE activity. Our results also indicate an abiotic pathway for the turnover of proteins, although its relative contribution to protein turnover in soil must be further investigated.

Structural Insights into the Unusually Strong ATPase Activity of the AAA Domain of the Caenorhabditis elegans Fidgetin-like 1 (FIGL-1) Protein*

PubMed Central

Peng, Wentao; Lin, Zhijie; Li, Weirong; Lu, Jing; Shen, Yuequan; Wang, Chunguang

2013-01-01

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function. PMID:23979136

Structural insights into the unusually strong ATPase activity of the AAA domain of the Caenorhabditis elegans fidgetin-like 1 (FIGL-1) protein.

PubMed

Peng, Wentao; Lin, Zhijie; Li, Weirong; Lu, Jing; Shen, Yuequan; Wang, Chunguang

2013-10-11

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function.

The chaperone-histone partnership: for the greater good of histone traffic and chromatin plasticity.

PubMed

Hondele, Maria; Ladurner, Andreas G

2011-12-01

Histones are highly positively charged proteins that wrap our genome. Their surface properties also make them prone to nonspecific interactions and aggregation. A class of proteins known as histone chaperones is dedicated to safeguard histones by aiding their proper incorporation into nucleosomes. Histone chaperones facilitate ordered nucleosome assembly and disassembly reactions through the formation of semi-stable histone-chaperone intermediates without requiring ATP, but merely providing a complementary protein surface for histones to dynamically interact with. Recurrent 'chaperoning' mechanisms involve the masking of the histone's positive charge and the direct blocking of crucial histone surface sites, including those required for H3-H4 tetramerization or the binding of nucleosomal DNA. This shielding prevents histones from engaging in premature or unwanted interactions with nucleic acids and other cellular components. In this review, we analyze recent structural studies on chaperone-histone interactions and discuss the implications of this vital partnership for nucleosome assembly and disassembly pathways. Copyright © 2011 Elsevier Ltd. All rights reserved.

Electrostatic interaction of positive charges on the surface of Psb31 with photosystem II in the diatom Chaetoceros gracilis.

PubMed

Nagao, Ryo; Suzuki, Takehiro; Okumura, Akinori; Kihira, Tomohiro; Toda, Ayaka; Dohmae, Naoshi; Nakazato, Katsuyoshi; Tomo, Tatsuya

2017-09-01

Psb31, a novel extrinsic protein found in diatom photosystem II (PSII), directly binds to PSII core subunits, independent of the other extrinsic proteins, and functions to maintain optimum oxygen evolution. However, how Psb31 electrostatically interacts with PSII intrinsic proteins remains to be clarified. In this study, we examined electrostatic interaction of Psb31 with PSII complexes isolated from the diatom Chaetoceros gracilis. Positive or negative charges of isolated Psb31 proteins were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME), respectively, resulting in formation of uncharged groups. NSP-modified Psb31 did not bind to PSII with a concomitant increase in NSP concentration, whereas GME-modified Psb31 clearly bound to PSII with retention of oxygen-evolving activity, indicating that positive charges of Lys residues and the N-terminus on the surface of Psb31 are involved in electrostatic interactions with PSII intrinsic proteins. Mass spectrometry analysis of NSP-modified Psb31 and sequence comparisons of Psb31 from C. gracilis with other chromophyte algae led to identification of three Lys residues as possible binding sites to PSII. Based on these findings, together with our previous cross-linking study in diatom PSII and a red algal PSII structure, we discuss binding properties of Psb31 with PSII core proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Mina; Moreland, K. Trent; Chen, Jiajun

Here, we investigated the roles of three proteins associated with the formation of otoconia including fetuin A, osteopontin (OPN), and otoconin 90 (OC90). In situ atomic force microscopy (AFM) studies of the effects of these proteins on the growth of atomic steps on calcite surfaces were performed to obtain insight into their effects on the growth kinetics. We also used scanning electron microscopy to examine the effects of these proteins on crystal morphology. All three proteins were found to be potent inhibitors of calcite growth, although fetuin A promoted growth at concentrations below about 40 nM and only became anmore » inhibitor at higher concentrations. We then used in situ optical microscopy to observe calcite nucleation on films of these proteins adsorbed onto mica surfaces. By measuring the calcite nucleation rate as a function of supersaturation, the value of the interfacial energy that controls the free energy barrier to heterogeneous nucleation was determined for each protein. OPN and OC90 films led to significantly reduced interfacial energies as compared to the value for homogeneous calcite nucleation in bulk solution. The value for fetuin A was equal to that for bulk solution within experimental error. Zeta potential measurements showed all of the proteins possessed negative surface charge and varied in magnitude according to sequence fetuin A > OC90 > OPN. In addition, the interfacial energies exhibited an inverse scaling with the zeta potential. In analogy to previous measurements on polysaccharide films, this scaling indicates the differences between the proteins arise from the effect of protein surface charge on the solution–substrate interfacial energy.« less

Study of the adhesion of neurodegenerative proteins on plasma-modified and coated polypropylene surfaces.

PubMed

Poncin-Epaillard, F; Mille, C; Debarnot, D; Zorzi, W; El Moualij, B; Coudreuse, A; Legeay, G; Quadrio, I; Perret-Liaudet, A

2012-01-01

The inner polymeric surface of an ELISA titration well is plasma-modified and coated with different surfactant molecules. The titration of neurodegenerative proteins markers (prion, Tau and β-synuclein), previously demonstrated as more efficient with such modified tubes, is related to the adhesion behaviour of these proteins and their corresponding capture antibodies. The adhesion process is studied in terms of anchoring and specific mechanisms. The proteins and antibodies binding onto such modified surfaces is related to the substrate hydrophilic character calculated from the angle contact measure, to the polymer surface charge measured through the streaming potential determination at different pH and the inner surface roughness determined from AFM images. Furthermore, the influence of the blocking agent used during the ELISA titration is also studied.

Secondary Structures of Ubiquitin Ions Soft-Landed onto Self-Assembled Monolayer Surfaces

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Qichi; Laskin, Julia

2016-06-09

The secondary structures of multiply charged ubiquitin ions soft-landed onto self-assembled monolayer (SAM) surfaces were studied using in situ infrared reflection-absorption spectroscopy (IRRAS). Two charge states of ubiquitin, 5+ and 13+, were mass selected separately from a mixture of different charge states produced by electrospray ionization (ESI). The low 5+ charge state represents a native-like folded state of ubiquitin, while the high 13+ charge state assumes an extended, almost linear conformation. Each of the two charge states was soft-landed onto a CH 3- and COOH-terminated SAM of alkylthiols on gold (HSAM and COOH-SAM). HSAM is a hydrophobic surface known tomore » stabilize helical conformations of soft-landed protonated peptides, whereas COOH-SAM is a hydrophilic surface that preferentially stabilizes β-sheet conformations. IRRAS spectra of the soft-landed ubiquitin ions were acquired as a function of time during and after ion soft-landing. Similar to smaller peptide ions, helical conformations of ubiquitin are found to be more abundant on HSAM, while the relative abundance of β-sheet conformations increases on COOH-SAM. The initial charge state of ubiquitin also has a pronounced effect on its conformation on the surface. Specifically, on both surfaces, a higher relative abundance of helical conformations and lower relative abundance of β-sheet conformations is observed for the 13+ charge state compared to the 5+ charge state. Time-resolved experiments indicate that the α-helical band in the spectrum of the 13+ charge state slowly increases with time on the HSAM surface and decreases in the spectrum of the 13+ charge state on COOH-SAM. These results further support the preference of the hydrophobic HSAM surface toward helical conformations and demonstrate that soft-landed protein ions may undergo slow conformational changes during and after deposition.« less

Molecular dynamics and Monte Carlo simulations resolve apparent diffusion rate differences for proteins confined in nanochannels

DOE PAGES

Tringe, J. W.; Ileri, N.; Levie, H. W.; ...

2015-08-01

We use Molecular Dynamics and Monte Carlo simulations to examine molecular transport phenomena in nanochannels, explaining four orders of magnitude difference in wheat germ agglutinin (WGA) protein diffusion rates observed by fluorescence correlation spectroscopy (FCS) and by direct imaging of fluorescently-labeled proteins. We first use the ESPResSo Molecular Dynamics code to estimate the surface transport distance for neutral and charged proteins. We then employ a Monte Carlo model to calculate the paths of protein molecules on surfaces and in the bulk liquid transport medium. Our results show that the transport characteristics depend strongly on the degree of molecular surface coverage.more » Atomic force microscope characterization of surfaces exposed to WGA proteins for 1000 s show large protein aggregates consistent with the predicted coverage. These calculations and experiments provide useful insight into the details of molecular motion in confined geometries.« less

Surface and anti-fouling properties of a polyampholyte hydrogel grafted onto a polyethersulfone membrane.

PubMed

Zhang, Wei; Yang, Zhe; Kaufman, Yair; Bernstein, Roy

2018-05-01

Zwitterion polymers have anti-fouling properties; therefore, grafting new zwitterions to surfaces, particularly as hydrogels, is one of the leading research directions for preventing fouling. Specifically, polyampholytes, polymers of random mixed charged subunits with a net-electric charge, offer a synthetically easy alternative for studying new zwitterions with a broad spectrum of charged moieties. Here, a novel polyampholyte hydrogel was grafted onto the surface of polyethersulfone membrane by copolymerizing a mixture of vinylsulfonic acid (VSA) and [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METMAC) as the negatively and positively charged monomers, respectively, using various monomer ratios in the polymerization solution, and with N,N'-methylenebisacrylamide as the crosslinker. The physicochemical, morphological and anti-fouling properties of the modified membranes were systematically investigated. Hydrophilic hydrogels were successfully grafted using monomers at different molar ratios. A thin-film zwitterion hydrogel (∼90 nm) was achieved at a 3:1 [VSA:METMAC] molar ratio in the polymerization solution. Among all examined membranes, the zwitterion polyampholyte-modified membrane demonstrated the lowest adsorption of proteins, humic acid, and sodium alginate. It also had low fouling and high flux recovery following filtration with a protein or with an extracellular polymeric substance solution. These findings suggest that this polyampholyte hydrogel is applicable as a low fouling surface coating. Copyright © 2018 Elsevier Inc. All rights reserved.

Influence of surface groups of proteins on water studied by freezing/thawing hysteresis and infrared spectroscopy.

PubMed

Zelent, Bogumil; Bryan, Michael A; Sharp, Kim A; Vanderkooi, Jane M

2009-05-01

The influence of proteins and solutes on hysteresis of freezing and melting of water was measured by infrared (IR) spectroscopy. Of the solutes examined, poly-L-arginine and flounder antifreeze protein produced the largest freezing point depression of water, with little effect on the melting temperature. Poly-L-lysine, poly-L-glutamate, cytochrome c and bovine serum albumin had less effect on the freezing of water. Small compounds used to mimic non-polar (trimethylamine N-oxide, methanol), positively charged (guanidinium chloride, NH(4)Cl, urea) and negatively charged (Na acetate) groups on protein surfaces were also examined. These molecules and ions depress water's freezing point and the melting profiles became broad. Since infrared absorption measures both bulk solvent and solvent bound to the solutes, this result is consistent with solutes interacting with liquid water. The amide I absorption bands of antifreeze protein and poly-L-arginine do not detectably change with the phase transition of water. An interpretation is that the antifreeze protein and poly-L-arginine order liquid water such that the water around the group is ice-like.

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties.

PubMed

Doktorova, Milka; Heberle, Frederick A; Kingston, Richard L; Khelashvili, George; Cuendet, Michel A; Wen, Yi; Katsaras, John; Feigenson, Gerald W; Vogt, Volker M; Dick, Robert A

2017-11-07

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein's matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here, using a broad set of in vitro and in silico techniques we addressed molecular mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein

PubMed Central

Pan, Timothy; Tzeng, Huey-Fen

2017-01-01

Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-associated gastric inflammation. HP-NAP is also a vaccine candidate, a possible drug target, and a potential diagnostic marker for H. pylori-associated diseases. Previously, we purified recombinant HP-NAP by one-step diethylaminoethyl (DEAE) negative mode chromatography by collecting the unbound fraction at pH 8.0 at 4°C. It remains unclear why HP-NAP does not bind to DEAE resins at the pH above its isoelectric point during the purification. To investigate how pH affects the surface net charge of HP-NAP and its binding to DEAE resins during the purification, recombinant HP-NAP expressed in Escherichia coli was subjected to DEAE negative mode chromatography at pH ranging from 7.0 to 9.0 at 25°C and the surface charge of purified HP-NAP was determined by capillary electrophoresis. A minimal amount of HP-NAP was detected in the elution fraction of DEAE Sepharose resin at pH 8.5, whereas recombinant HP-NAP was detected in the elution fraction of DEAE Sephadex resin only at pH 7.0 and 8.0. The purified recombinant HP-NAP obtained from the unbound fractions was not able to bind to DEAE resins at pH 7.0 to 9.0. In addition, the surface charge of the purified HP-NAP was neutral at pH 7.0 to 8.0 and was either neutral or slightly negative at pH 8.5 and 9.0. However, recombinant HP-NAP purified from gel-filtration chromatography was able to bind to DEAE Sepharose resin at pH 7.0 to 9.0 and DEAE Sephadex resin at pH 7.0. At pH 8.5 and 9.0, only the negatively charged species of HP-NAP were found. Thus, recombinant HP-NAP with different charge status can be differentially purified by DEAE negative mode chromatography and gel-filtration chromatography. Furthermore, the charge distribution on the surface of HP-NAP, the presence of impure proteins, and the overall net charge of the resins all affect the binding of HP-NAP to DEAE resins during the negative purification. PMID:28328957

Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein.

PubMed

Hong, Zhi-Wei; Yang, Yu-Chi; Pan, Timothy; Tzeng, Huey-Fen; Fu, Hua-Wen

2017-01-01

Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-associated gastric inflammation. HP-NAP is also a vaccine candidate, a possible drug target, and a potential diagnostic marker for H. pylori-associated diseases. Previously, we purified recombinant HP-NAP by one-step diethylaminoethyl (DEAE) negative mode chromatography by collecting the unbound fraction at pH 8.0 at 4°C. It remains unclear why HP-NAP does not bind to DEAE resins at the pH above its isoelectric point during the purification. To investigate how pH affects the surface net charge of HP-NAP and its binding to DEAE resins during the purification, recombinant HP-NAP expressed in Escherichia coli was subjected to DEAE negative mode chromatography at pH ranging from 7.0 to 9.0 at 25°C and the surface charge of purified HP-NAP was determined by capillary electrophoresis. A minimal amount of HP-NAP was detected in the elution fraction of DEAE Sepharose resin at pH 8.5, whereas recombinant HP-NAP was detected in the elution fraction of DEAE Sephadex resin only at pH 7.0 and 8.0. The purified recombinant HP-NAP obtained from the unbound fractions was not able to bind to DEAE resins at pH 7.0 to 9.0. In addition, the surface charge of the purified HP-NAP was neutral at pH 7.0 to 8.0 and was either neutral or slightly negative at pH 8.5 and 9.0. However, recombinant HP-NAP purified from gel-filtration chromatography was able to bind to DEAE Sepharose resin at pH 7.0 to 9.0 and DEAE Sephadex resin at pH 7.0. At pH 8.5 and 9.0, only the negatively charged species of HP-NAP were found. Thus, recombinant HP-NAP with different charge status can be differentially purified by DEAE negative mode chromatography and gel-filtration chromatography. Furthermore, the charge distribution on the surface of HP-NAP, the presence of impure proteins, and the overall net charge of the resins all affect the binding of HP-NAP to DEAE resins during the negative purification.

Solvent Reaction Field Potential inside an Uncharged Globular Protein: A Bridge between Implicit and Explicit Solvent Models?

PubMed Central

Baker, Nathan A.; McCammon, J. Andrew

2008-01-01

The solvent reaction field potential of an uncharged protein immersed in Simple Point Charge/Extended (SPC/E) explicit solvent was computed over a series of molecular dynamics trajectories, intotal 1560 ns of simulation time. A finite, positive potential of 13 to 24 kbTec−1 (where T = 300K), dependent on the geometry of the solvent-accessible surface, was observed inside the biomolecule. The primary contribution to this potential arose from a layer of positive charge density 1.0 Å from the solute surface, on average 0.008 ec/Å3, which we found to be the product of a highly ordered first solvation shell. Significant second solvation shell effects, including additional layers of charge density and a slight decrease in the short-range solvent-solvent interaction strength, were also observed. The impact of these findings on implicit solvent models was assessed by running similar explicit-solvent simulations on the fully charged protein system. When the energy due to the solvent reaction field in the uncharged system is accounted for, correlation between per-atom electrostatic energies for the explicit solvent model and a simple implicit (Poisson) calculation is 0.97, and correlation between per-atom energies for the explicit solvent model and a previously published, optimized Poisson model is 0.99. PMID:17949217

Solvent reaction field potential inside an uncharged globular protein: A bridge between implicit and explicit solvent models?

NASA Astrophysics Data System (ADS)

Cerutti, David S.; Baker, Nathan A.; McCammon, J. Andrew

2007-10-01

The solvent reaction field potential of an uncharged protein immersed in simple point charge/extended explicit solvent was computed over a series of molecular dynamics trajectories, in total 1560ns of simulation time. A finite, positive potential of 13-24 kbTec-1 (where T =300K), dependent on the geometry of the solvent-accessible surface, was observed inside the biomolecule. The primary contribution to this potential arose from a layer of positive charge density 1.0Å from the solute surface, on average 0.008ec/Å3, which we found to be the product of a highly ordered first solvation shell. Significant second solvation shell effects, including additional layers of charge density and a slight decrease in the short-range solvent-solvent interaction strength, were also observed. The impact of these findings on implicit solvent models was assessed by running similar explicit solvent simulations on the fully charged protein system. When the energy due to the solvent reaction field in the uncharged system is accounted for, correlation between per-atom electrostatic energies for the explicit solvent model and a simple implicit (Poisson) calculation is 0.97, and correlation between per-atom energies for the explicit solvent model and a previously published, optimized Poisson model is 0.99.

Modifications in nanoparticle-protein interactions by varying the protein conformation

NASA Astrophysics Data System (ADS)

Kumar, Sugam; Yadav, I.; Aswal, V. K.; Kohlbrecher, J.

2017-05-01

Small-angle neutron scattering has been used to study the interaction of silica nanoparticle with Bovine Serum Albumin (BSA) protein without and with a protein denaturing agent urea. The measurements have been carried out at pH 7 where both the components (nanoparticle and protein) are similarly charged. We show that the interactions in nanoparticle-protein system can be modified by changing the conformation of protein through the presence of urea. In the absence of urea, the strong electrostatic repulsion between the nanoparticle and protein prevents protein adsorption on nanoparticle surface. This non-adsorption, in turn gives rise to depletion attraction between nanoparticles. However, with addition of urea the depletion attraction is completely suppressed. Urea driven denaturation of protein is utilized to expose the positively charged patched of the BSA molecules which eventually leads to adsorption of BSA on nanoparticles eliminating the depletion interaction.

Effect of otoconial proteins fetuin A, osteopontin, and otoconin 90 on the nucleation and growth of calcite

DOE PAGES

Hong, Mina; Moreland, K. Trent; Chen, Jiajun; ...

2014-10-30

Here, we investigated the roles of three proteins associated with the formation of otoconia including fetuin A, osteopontin (OPN), and otoconin 90 (OC90). In situ atomic force microscopy (AFM) studies of the effects of these proteins on the growth of atomic steps on calcite surfaces were performed to obtain insight into their effects on the growth kinetics. We also used scanning electron microscopy to examine the effects of these proteins on crystal morphology. All three proteins were found to be potent inhibitors of calcite growth, although fetuin A promoted growth at concentrations below about 40 nM and only became anmore » inhibitor at higher concentrations. We then used in situ optical microscopy to observe calcite nucleation on films of these proteins adsorbed onto mica surfaces. By measuring the calcite nucleation rate as a function of supersaturation, the value of the interfacial energy that controls the free energy barrier to heterogeneous nucleation was determined for each protein. OPN and OC90 films led to significantly reduced interfacial energies as compared to the value for homogeneous calcite nucleation in bulk solution. The value for fetuin A was equal to that for bulk solution within experimental error. Zeta potential measurements showed all of the proteins possessed negative surface charge and varied in magnitude according to sequence fetuin A > OC90 > OPN. In addition, the interfacial energies exhibited an inverse scaling with the zeta potential. In analogy to previous measurements on polysaccharide films, this scaling indicates the differences between the proteins arise from the effect of protein surface charge on the solution–substrate interfacial energy.« less

pH-Switchable Interaction of a Carboxybetaine Ester-Based SAM with DNA and Gold Nanoparticles.

PubMed

Filip, Jaroslav; Popelka, Anton; Bertok, Tomas; Holazova, Alena; Osicka, Josef; Kollar, Jozef; Ilcikova, Marketa; Tkac, Jan; Kasak, Peter

2017-07-11

We describe a self-assembled monolayer (SAM) on a gold surface with a carboxybetaine ester functionality to control the interaction between DNA and gold nanoparticles via pH. The negatively charged phosphate backbone of DNA interacts with and adsorbs to the positively charged carboxybetaine esters on the SAM. DNA release can be achieved by the hydrolysis of carboxybetaine ester (CBE) to a zwitterionic carboxybetaine state. Furthermore, the adsorption of negatively charged citrate-capped gold nanoparticles to a SAM-modified plain gold surface can be controlled by the pH. The SAM based on carboxybetaine ester allows for the homogeneous adsorption of particles, whereas the SAM after hydrolysis at high pH repels AuNP adsorption. The antifouling surface properties of the surface modified with carboxybetaine were investigated with protein samples.

D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function

PubMed Central

Burtscher, Laura; Hajdu, Dorottya; Muñoz, Alberto; Gáspári, Zoltán; Read, Nick D.; Batta, Gyula; Marx, Florentine

2017-01-01

The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF. PMID:28072824

Electrostatic Ratchet in the Protective Antigen Channel Promotes Anthrax Toxin Translocation*

PubMed Central

Wynia-Smith, Sarah L.; Brown, Michael J.; Chirichella, Gina; Kemalyan, Gigi; Krantz, Bryan A.

2012-01-01

Central to the power-stroke and Brownian-ratchet mechanisms of protein translocation is the process through which nonequilibrium fluctuations are rectified or ratcheted by the molecular motor to transport substrate proteins along a specific axis. We investigated the ratchet mechanism using anthrax toxin as a model. Anthrax toxin is a tripartite toxin comprised of the protective antigen (PA) component, a homooligomeric transmembrane translocase, which translocates two other enzyme components, lethal factor (LF) and edema factor (EF), into the cytosol of the host cell under the proton motive force (PMF). The PA-binding domains of LF and EF (LFN and EFN) possess identical folds and similar solution stabilities; however, EFN translocates ∼10–200-fold slower than LFN, depending on the electrical potential (Δψ) and chemical potential (ΔpH) compositions of the PMF. From an analysis of LFN/EFN chimera proteins, we identified two 10-residue cassettes comprised of charged sequence that were responsible for the impaired translocation kinetics of EFN. These cassettes have nonspecific electrostatic requirements: one surprisingly prefers acidic residues when driven by either a Δψ or a ΔpH; the second requires basic residues only when driven by a Δψ. Through modeling and experiment, we identified a charged surface in the PA channel responsible for charge selectivity. The charged surface latches the substrate and promotes PMF-driven transport. We propose an electrostatic ratchet in the channel, comprised of opposing rings of charged residues, enforces directionality by interacting with charged cassettes in the substrate, thereby generating forces sufficient to drive unfolding. PMID:23115233

Contributions of depth filter components to protein adsorption in bioprocessing.

PubMed

Khanal, Ohnmar; Singh, Nripen; Traylor, Steven J; Xu, Xuankuo; Ghose, Sanchayita; Li, Zheng J; Lenhoff, Abraham M

2018-04-16

Depth filtration is widely used in downstream bioprocessing to remove particulate contaminants via depth straining and is therefore applied to harvest clarification and other processing steps. However, depth filtration also removes proteins via adsorption, which can contribute variously to impurity clearance and to reduction in product yield. The adsorption may occur on the different components of the depth filter, that is, filter aid, binder, and cellulose filter. We measured adsorption of several model proteins and therapeutic proteins onto filter aids, cellulose, and commercial depth filters at pH 5-8 and ionic strengths <50 mM and correlated the adsorption data to bulk measured properties such as surface area, morphology, surface charge density, and composition. We also explored the role of each depth filter component in the adsorption of proteins with different net charges, using confocal microscopy. Our findings show that a complete depth filter's maximum adsorptive capacity for proteins can be estimated by its protein monolayer coverage values, which are of order mg/m 2 , depending on the protein size. Furthermore, the extent of adsorption of different proteins appears to depend on the nature of the resin binder and its extent of coating over the depth filter surface, particularly in masking the cation-exchanger-like capacity of the siliceous filter aids. In addition to guiding improved depth filter selection, the findings can be leveraged in inspiring a more intentional selection of components and design of depth filter construction for particular impurity removal targets. © 2018 Wiley Periodicals, Inc.

Protein-protein interactions in paralogues: Electrostatics modulates specificity on a conserved steric scaffold

PubMed Central

Huber, Roland G.; Bond, Peter J.

2017-01-01

An improved knowledge of protein-protein interactions is essential for better understanding of metabolic and signaling networks, and cellular function. Progress tends to be based on structure determination and predictions using known structures, along with computational methods based on evolutionary information or detailed atomistic descriptions. We hypothesized that for the case of interactions across a common interface, between proteins from a pair of paralogue families or within a family of paralogues, a relatively simple interface description could distinguish between binding and non-binding pairs. Using binding data for several systems, and large-scale comparative modeling based on known template complex structures, it is found that charge-charge interactions (for groups bearing net charge) are generally a better discriminant than buried non-polar surface. This is particularly the case for paralogue families that are less divergent, with more reliable comparative modeling. We suggest that electrostatic interactions are major determinants of specificity in such systems, an observation that could be used to predict binding partners. PMID:29016650

Protein-protein interactions in paralogues: Electrostatics modulates specificity on a conserved steric scaffold.

PubMed

Ivanov, Stefan M; Cawley, Andrew; Huber, Roland G; Bond, Peter J; Warwicker, Jim

2017-01-01

An improved knowledge of protein-protein interactions is essential for better understanding of metabolic and signaling networks, and cellular function. Progress tends to be based on structure determination and predictions using known structures, along with computational methods based on evolutionary information or detailed atomistic descriptions. We hypothesized that for the case of interactions across a common interface, between proteins from a pair of paralogue families or within a family of paralogues, a relatively simple interface description could distinguish between binding and non-binding pairs. Using binding data for several systems, and large-scale comparative modeling based on known template complex structures, it is found that charge-charge interactions (for groups bearing net charge) are generally a better discriminant than buried non-polar surface. This is particularly the case for paralogue families that are less divergent, with more reliable comparative modeling. We suggest that electrostatic interactions are major determinants of specificity in such systems, an observation that could be used to predict binding partners.

A label-free approach to detect ligand binding to cell surface proteins in real time.

PubMed

Burtscher, Verena; Hotka, Matej; Li, Yang; Freissmuth, Michael; Sandtner, Walter

2018-04-26

Electrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a long-range conformational change. The Gouy-Chapman model predicts that adsorption of charged ligands to surface proteins must produce displacement currents and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance. © 2018, Burtscher et al.

Effect of protein properties on display efficiency using the M13 phage display system.

PubMed

Imai, S; Mukai, Y; Takeda, T; Abe, Y; Nagano, K; Kamada, H; Nakagawa, S; Tsunoda, S; Tsutsumi, Y

2008-10-01

The M13 phage display system is a powerful technology for engineering proteins such as functional mutant proteins and peptides. In this system, it is necessary that the protein is displayed on the phage surface. Therefore, its application is often limited when a protein is poorly displayed. In this study, we attempted to understand the relationship between a protein's properties and its display efficiency using the well-known pIII and pVIII type phage display system. The display of positively charged SV40 NLS and HIV-1 Tat peptides on pill was less efficient than that of the neutrally charged RGDS peptide. When different molecular weight proteins (1.5-58 kDa) were displayed on pIII and pVIII, their display efficiencies were directly influenced by their molecular weights. These results indicate the usefulness in predicting a desired protein's compatibility with protein and peptide engineering using the phage display system.

DNA mimic proteins: functions, structures, and bioinformatic analysis.

PubMed

Wang, Hao-Ching; Ho, Chun-Han; Hsu, Kai-Cheng; Yang, Jinn-Moon; Wang, Andrew H-J

2014-05-13

DNA mimic proteins have DNA-like negative surface charge distributions, and they function by occupying the DNA binding sites of DNA binding proteins to prevent these sites from being accessed by DNA. DNA mimic proteins control the activities of a variety of DNA binding proteins and are involved in a wide range of cellular mechanisms such as chromatin assembly, DNA repair, transcription regulation, and gene recombination. However, the sequences and structures of DNA mimic proteins are diverse, making them difficult to predict by bioinformatic search. To date, only a few DNA mimic proteins have been reported. These DNA mimics were not found by searching for functional motifs in their sequences but were revealed only by structural analysis of their charge distribution. This review highlights the biological roles and structures of 16 reported DNA mimic proteins. We also discuss approaches that might be used to discover new DNA mimic proteins.

Role of Transbilayer Distribution of Lipid Molecules on the Structure and Protein-Lipid Interaction of an Amyloidogenic Protein on the Membrane Surface

NASA Astrophysics Data System (ADS)

Cheng, Kwan; Cheng, Sara

We used molecular dynamics simulations to examine the effects of transbilayer distribution of lipid molecules, particularly anionic lipids with negatively charged headgroups, on the structure and binding kinetics of an amyloidogenic protein on the membrane surface and subsequent protein-induced structural disruption of the membrane. Our systems consisted of a model beta-sheet rich dimeric protein absorbed on asymmetric bilayers with neutral and anionic lipids and symmetric bilayers with neutral lipids. We observed larger folding, domain aggregation, and tilt angle of the absorbed protein on the asymmetric bilayer surfaces. We also detected more focused bilayer thinning in the asymmetric bilayer due to weak lipid-protein interactions. Our results support the mechanism that the higher lipid packing in the protein-contacting lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions of an amyloidogenic protein on the membrane surface. We speculate that the observed surface-induced structural and protein-lipid interaction of our model amyloidogenic protein may play a role in the early membrane-associated amyloid cascade pathway that leads to membrane structural damage of neurons in Alzheimer's disease. NSF ACI-1531594.

Free energy of solvated salt bridges: a simulation and experimental study.

PubMed

White, Andrew D; Keefe, Andrew J; Ella-Menye, Jean-Rene; Nowinski, Ann K; Shao, Qing; Pfaendtner, Jim; Jiang, Shaoyi

2013-06-20

Charged amino acids are the most common on surfaces of proteins and understanding the interactions between these charged amino acids, salt bridging, is crucial for understanding protein-protein interactions. Previous simulations have been limited to implicit solvent or fixed binding geometry due to the sampling required for converged free energies. Using well-tempered metadynamics, we have calculated salt bridge free energy surfaces in water and confirmed the results with NMR experiments. The simulations give binding free energies, quantitative ranking of salt bridging strength, and insights into the hydration of the salt bridges. The arginine-aspartate salt bridge was found to be the weakest and arginine-glutamate the strongest, showing that arginine can discriminate between aspartate and glutamate, whereas the salt bridges with lysine are indistinguishable in their free energy. The salt bridging hydration is found to be complementary to salt bridge orientation with arginine having specific orientations.

Probing the Selectivity and Protein•Protein Interactions of a Non-Reducing Fungal Polyketide Synthase Using Mechanism-Based Crosslinkers

PubMed Central

Bruegger, Joel; Haushalter, Bob; Vagstad, Anna; Shakya, Gaurav; Mih, Nathan; Townsend, Craig A.; Burkart, Michael D.; Tsai, Shiou-Chuan

2013-01-01

SUMMARY Protein•protein interactions, which often involve interactions between an acyl carrier protein (ACP) and its partner enzymes, are important for coordinating polyketide biosynthesis. However, the nature of such interactions is not well understood, especially in the fungal non-reducing polyketide synthases (NR-PKSs) that biosynthesize toxic and pharmaceutically important polyketides. Here, we employ a mechanism-based crosslinker to successfully probe ACP and ketosynthase (KS) domain interactions in NR-PKSs. We found that crosslinking efficiency is closely correlated with the strength of ACP•KS interactions, and that KS demonstrates strong starter unit selectivity. We further identified positively charged surface residues by KS mutagenesis, which mediate key interactions with the negatively-charged ACP surface. Such complementary/matching contact pairs can serve as “adapter surfaces” for future efforts to generate new polyketides using NR-PKSs. PMID:23993461

Physicochemical Study of Viral Nanoparticles at the Air/Water Interface.

PubMed

Torres-Salgado, Jose F; Comas-Garcia, Mauricio; Villagrana-Escareño, Maria V; Durán-Meza, Ana L; Ruiz-García, Jaime; Cadena-Nava, Ruben D

2016-07-07

The assembly of most single-stranded RNA (ssRNA) viruses into icosahedral nucleocapsids is a spontaneous process driven by protein-protein and RNA-protein interactions. The precise nature of these interactions results in the assembly of extremely monodisperse and structurally indistinguishable nucleocapsids. In this work, by using a ssRNA plant virus (cowpea chlorotic mottle virus [CCMV]) as a charged nanoparticle we show that the diffusion of these nanoparticles from the bulk solution to the air/water interface is an irreversible adsorption process. By using the Langmuir technique, we measured the diffusion and adsorption of viral nucleocapsids at the air/water interface at different pH conditions. The pH changes, and therefore in the net surface charge of the virions, have a great influence in the diffusion rate from the bulk solution to the air/water interface. Moreover, assembly of mesoscopic and microscopic viral aggregates at this interface depends on the net surface charge of the virions and the surface pressure. By using Brewster's angle microscopy we characterized these structures at the interface. Most common structures observed were clusters of virions and soap-frothlike micron-size structures. Furthermore, the CCMV films were compressed to form monolayers and multilayers from moderate to high surface pressures, respectively. After transferring the films from the air/water interface onto mica by using the Langmuir-Blodgett technique, their morphology was characterized by atomic force microscopy. These viral monolayers showed closed-packing nano- and microscopic arrangements.

Poisson-Boltzmann model for protein-surface electrostatic interactions and grid-convergence study using the PyGBe code

NASA Astrophysics Data System (ADS)

Cooper, Christopher D.; Barba, Lorena A.

2016-05-01

Interactions between surfaces and proteins occur in many vital processes and are crucial in biotechnology: the ability to control specific interactions is essential in fields like biomaterials, biomedical implants and biosensors. In the latter case, biosensor sensitivity hinges on ligand proteins adsorbing on bioactive surfaces with a favorable orientation, exposing reaction sites to target molecules. Protein adsorption, being a free-energy-driven process, is difficult to study experimentally. This paper develops and evaluates a computational model to study electrostatic interactions of proteins and charged nanosurfaces, via the Poisson-Boltzmann equation. We extended the implicit-solvent model used in the open-source code PyGBe to include surfaces of imposed charge or potential. This code solves the boundary integral formulation of the Poisson-Boltzmann equation, discretized with surface elements. PyGBe has at its core a treecode-accelerated Krylov iterative solver, resulting in O(N log N) scaling, with further acceleration on hardware via multi-threaded execution on GPUs. It computes solvation and surface free energies, providing a framework for studying the effect of electrostatics on adsorption. We derived an analytical solution for a spherical charged surface interacting with a spherical dielectric cavity, and used it in a grid-convergence study to build evidence on the correctness of our approach. The study showed the error decaying with the average area of the boundary elements, i.e., the method is O(1 / N) , which is consistent with our previous verification studies using PyGBe. We also studied grid-convergence using a real molecular geometry (protein G B1 D4‧), in this case using Richardson extrapolation (in the absence of an analytical solution) and confirmed the O(1 / N) scaling. With this work, we can now access a completely new family of problems, which no other major bioelectrostatics solver, e.g. APBS, is capable of dealing with. PyGBe is open-source under an MIT license and is hosted under version control at https://github.com/barbagroup/pygbe. To supplement this paper, we prepared ;reproducibility packages; consisting of running and post-processing scripts in Python for replicating the grid-convergence studies, all the way to generating the final plots, with a single command.

Electrostatic Interactions between OmpG Nanopore and Analyte Protein Surface Can Distinguish between Glycosylated Isoforms.

PubMed

Fahie, Monifa A; Chen, Min

2015-08-13

The flexible loops decorating the entrance of OmpG nanopore move dynamically during ionic current recording. The gating caused by these flexible loops changes when a target protein is bound. The gating is characterized by parameters including frequency, duration, and open-pore current, and these features combine to reveal the identity of a specific analyte protein. Here, we show that OmpG nanopore equipped with a biotin ligand can distinguish glycosylated and deglycosylated isoforms of avidin by their differences in surface charge. Our studies demonstrate that the direct interaction between the nanopore and analyte surface, induced by the electrostatic attraction between the two molecules, is essential for protein isoform detection. Our technique is remarkably sensitive to the analyte surface, which may provide a useful tool for glycoprotein profiling.

Patch Finder Plus (PFplus): a web server for extracting and displaying positive electrostatic patches on protein surfaces.

PubMed

Shazman, Shula; Celniker, Gershon; Haber, Omer; Glaser, Fabian; Mandel-Gutfreund, Yael

2007-07-01

Positively charged electrostatic patches on protein surfaces are usually indicative of nucleic acid binding interfaces. Interestingly, many proteins which are not involved in nucleic acid binding possess large positive patches on their surface as well. In some cases, the positive patches on the protein are related to other functional properties of the protein family. PatchFinderPlus (PFplus) http://pfp.technion.ac.il is a web-based tool for extracting and displaying continuous electrostatic positive patches on protein surfaces. The input required for PFplus is either a four letter PDB code or a protein coordinate file in PDB format, provided by the user. PFplus computes the continuum electrostatics potential and extracts the largest positive patch for each protein chain in the PDB file. The server provides an output file in PDB format including a list of the patch residues. In addition, the largest positive patch is displayed on the server by a graphical viewer (Jmol), using a simple color coding.

Patch Finder Plus (PFplus): A web server for extracting and displaying positive electrostatic patches on protein surfaces

PubMed Central

Shazman, Shula; Celniker, Gershon; Haber, Omer; Glaser, Fabian; Mandel-Gutfreund, Yael

2007-01-01

Positively charged electrostatic patches on protein surfaces are usually indicative of nucleic acid binding interfaces. Interestingly, many proteins which are not involved in nucleic acid binding possess large positive patches on their surface as well. In some cases, the positive patches on the protein are related to other functional properties of the protein family. PatchFinderPlus (PFplus) http://pfp.technion.ac.il is a web-based tool for extracting and displaying continuous electrostatic positive patches on protein surfaces. The input required for PFplus is either a four letter PDB code or a protein coordinate file in PDB format, provided by the user. PFplus computes the continuum electrostatics potential and extracts the largest positive patch for each protein chain in the PDB file. The server provides an output file in PDB format including a list of the patch residues. In addition, the largest positive patch is displayed on the server by a graphical viewer (Jmol), using a simple color coding. PMID:17537808

Surface charge engineering of a Bacillus gibsonii subtilisin protease.

PubMed

Jakob, Felix; Martinez, Ronny; Mandawe, John; Hellmuth, Hendrik; Siegert, Petra; Maurer, Karl-Heinz; Schwaneberg, Ulrich

2013-08-01

In proteins, a posttranslational deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartic (Asp) and glutamic acid (Glu), respectively. This process changes the protein net charge affecting enzyme activity, pH optimum, and stability. Understanding the principles which affect these enzyme properties would be valuable for protein engineering in general. In this work, three criteria for selecting amino acid substitutions of the deamidation type in the Bacillus gibsonii alkaline protease (BgAP) are proposed and systematically studied in their influence on pH-dependent activity and thermal resistance. Out of 113 possible surface amino acids, 18 (11 Asn and 7 Gln) residues of BgAP were selected and evaluated based on three proposed criteria: (1) The Asn or Gln residues should not be conserved, (2) should be surface exposed, and (3) neighbored by glycine. "Deamidation" in five (N97, N253, Q37, Q200, and Q256) out of eight (N97, N154, N250, N253, Q37, Q107, Q200, and Q256) amino acids meeting all criteria resulted in increased proteolytic activity. In addition, pH activity profiles of the variants N253D and Q256E and the combined variant N253DQ256E were dramatically shifted towards higher activity at lower pH (range of 8.5-10). Variant N253DQ256E showed twice the specific activity of wild-type BgAP and its thermal resistance increased by 2.4 °C at pH 8.5. These property changes suggest that mimicking surface deamidation by substituting Gln by Glu and/or Asn by Asp might be a simple and fast protein reengineering approach for modulating enzyme properties such as activity, pH optimum, and thermal resistance.

Treatment of charge singularities in implicit solvent models.

PubMed

Geng, Weihua; Yu, Sining; Wei, Guowei

2007-09-21

This paper presents a novel method for solving the Poisson-Boltzmann (PB) equation based on a rigorous treatment of geometric singularities of the dielectric interface and a Green's function formulation of charge singularities. Geometric singularities, such as cusps and self-intersecting surfaces, in the dielectric interfaces are bottleneck in developing highly accurate PB solvers. Based on an advanced mathematical technique, the matched interface and boundary (MIB) method, we have recently developed a PB solver by rigorously enforcing the flux continuity conditions at the solvent-molecule interface where geometric singularities may occur. The resulting PB solver, denoted as MIBPB-II, is able to deliver second order accuracy for the molecular surfaces of proteins. However, when the mesh size approaches half of the van der Waals radius, the MIBPB-II cannot maintain its accuracy because the grid points that carry the interface information overlap with those that carry distributed singular charges. In the present Green's function formalism, the charge singularities are transformed into interface flux jump conditions, which are treated on an equal footing as the geometric singularities in our MIB framework. The resulting method, denoted as MIBPB-III, is able to provide highly accurate electrostatic potentials at a mesh as coarse as 1.2 A for proteins. Consequently, at a given level of accuracy, the MIBPB-III is about three times faster than the APBS, a recent multigrid PB solver. The MIBPB-III has been extensively validated by using analytically solvable problems, molecular surfaces of polyatomic systems, and 24 proteins. It provides reliable benchmark numerical solutions for the PB equation.

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doktorova, Milka; Heberle, Frederick A.; Kingston, Richard L.

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein’s matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here in this paper, using a broad set of in vitro and in silico techniques we addressed molecularmore » mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins.« less

Treatment of charge singularities in implicit solvent models

NASA Astrophysics Data System (ADS)

Geng, Weihua; Yu, Sining; Wei, Guowei

2007-09-01

This paper presents a novel method for solving the Poisson-Boltzmann (PB) equation based on a rigorous treatment of geometric singularities of the dielectric interface and a Green's function formulation of charge singularities. Geometric singularities, such as cusps and self-intersecting surfaces, in the dielectric interfaces are bottleneck in developing highly accurate PB solvers. Based on an advanced mathematical technique, the matched interface and boundary (MIB) method, we have recently developed a PB solver by rigorously enforcing the flux continuity conditions at the solvent-molecule interface where geometric singularities may occur. The resulting PB solver, denoted as MIBPB-II, is able to deliver second order accuracy for the molecular surfaces of proteins. However, when the mesh size approaches half of the van der Waals radius, the MIBPB-II cannot maintain its accuracy because the grid points that carry the interface information overlap with those that carry distributed singular charges. In the present Green's function formalism, the charge singularities are transformed into interface flux jump conditions, which are treated on an equal footing as the geometric singularities in our MIB framework. The resulting method, denoted as MIBPB-III, is able to provide highly accurate electrostatic potentials at a mesh as coarse as 1.2Å for proteins. Consequently, at a given level of accuracy, the MIBPB-III is about three times faster than the APBS, a recent multigrid PB solver. The MIBPB-III has been extensively validated by using analytically solvable problems, molecular surfaces of polyatomic systems, and 24 proteins. It provides reliable benchmark numerical solutions for the PB equation.

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties

DOE PAGES

Doktorova, Milka; Heberle, Frederick A.; Kingston, Richard L.; ...

2017-11-07

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein’s matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here in this paper, using a broad set of in vitro and in silico techniques we addressed molecularmore » mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins.« less

Electrostatic Interactions and Self-Assembly in Polymeric Systems

NASA Astrophysics Data System (ADS)

Dobrynin, Andrey

Electrostatic interactions between macroions play an important role in different areas ranging from materials science to biophysics. They are main driving forces behind layer-by-layer assembly technique that allows self-assembly of multilayer films from synthetic polyelectrolytes, DNA, proteins and nanoparticles. They are responsible for complexation and reversible gelation between polyelectrolytes and proteins. In this talk, using results of the molecular dynamics simulations and analytical calculations, I will demonstrate what effect electrostatic interactions, counterion condensation and polymer solvent affinity have on a collapse of polyelectrolyte chain in a poor solvent conditions for the polymer backbone, on complexations and reversible gelation between polyelectrolytes and polyamholytes (unstructured proteins), on microphase separation transitions in spherical and planar charged brushes, and on a layer-by-layer assembly of charged nanoparticles and linear polyelectrolytes on charged surfaces. NSF DMR-1004576 DMR-1409710.

Protein adsorption onto CF(3)-terminated oligo(ethylene glycol) containing self-assembled monolayers (SAMs): the influence of ionic strength and electrostatic forces.

PubMed

Bonnet, Nelly; O'Hagan, David; Hähner, Georg

2010-05-07

Oligo(ethylene glycol) (OEG) containing self-assembled monolayers (SAMs) on gold are known for their protein resistant properties. The underlying molecular mechanisms and the contributions of the interactions involved, however, are still not completely understood. It is known that electrostatic, van der Waals, hydrophobic, and hydration forces all play a role in the interaction between proteins and surfaces, but it is difficult to study their influence separately and to quantify their contributions. In the present study we investigate five different OEG containing SAMs and the influence of the ionic strength and the electrostatic component on the amount of a negatively charged protein (fibrinogen) that adsorbs onto them. Atomic force microscopy (AFM) was employed to record force-distance curves with hydrophobic probes depending on the ion concentration, and the amount of the protein that adsorbs relative to a hydrophobic surface was quantified using ellipsometry. The findings suggest that electrostatic forces can create a very low energy barrier thus only slightly decreasing the number of negatively charged proteins in solution with sufficient energy to approach the surface closely, and have a rather small influence on the amount that adsorbs. The films we investigated were not protein resistant. This supports other studies, reporting that a strong short-range repulsion as for example caused by hydration forces is required to make these films resistant to the non-specific adsorption of proteins.

Surfactant protein B: lipid interactions of synthetic peptides representing the amino-terminal amphipathic domain.

PubMed Central

Bruni, R; Taeusch, H W; Waring, A J

1991-01-01

The mechanisms by which pulmonary surfactant protein B (SP-B) affects the surface activity of surfactant lipids are unclear. We have studied the peptide/lipid interactions of the amino-terminal amphipathic domain of SP-B by comparing the secondary conformations and surface activities of a family of synthetic peptides based on the native human SP-B sequence, modified by site-specific amino acid substitutions. Circular dichroism measurements show an alpha-helical structure correlating with the ability of the peptides to interact with lipids and with the surface activity of peptide/lipid dispersions. Amino acid substitutions altering either the charge or the hydrophobicity of the residues lowered the helical content and reduced the association of the aminoterminal segment with lipid dispersions. Surface activity of peptide/lipid mixtures was maximally altered by reversal of charge in synthetic peptides. These observations indicate that electrostatic interactions and hydrophobicity are important factors in determining optimal structure and function of surfactant peptides in lipid dispersions. Images PMID:1871144

Relating saturation capacity to charge density in strong cation exchangers.

PubMed

Steinebach, Fabian; Coquebert de Neuville, Bertrand; Morbidelli, Massimo

2017-07-21

In this work the relation between physical and chemical resin characteristics and the total amount of adsorbed protein (saturation capacity) for ion-exchange resins is discussed. Eleven different packing materials with a sulfo-functionalization and one multimodal resin were analyzed in terms of their porosity, pore size distribution, ligand density and binding capacity. By specifying the ligand density and binding capacity by the total and accessible surface area, two different groups of resins were identified: Below a ligand density of approx. 2.5μmol/m 2 area the ligand density controls the saturation capacity, while above this limit the accessible surface area becomes the limiting factor. This results in a maximum protein uptake of around 2.5mg/m 2 of accessible surface area. The obtained results allow estimating the saturation capacity from independent resin characteristics like the saturation capacity mainly depends on "library data" such as the accessible and total surface area and the charge density. Hence these results give an insight into the fundamentals of protein adsorption and help to find suitable resins, thus limiting the experimental effort in early process development stages. Copyright © 2017 Elsevier B.V. All rights reserved.

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions

PubMed Central

Gokarn, Yatin R; Fesinmeyer, R Matthew; Saluja, Atul; Razinkov, Vladimir; Chase, Susan F; Laue, Thomas M; Brems, David N

2011-01-01

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li+, Na+, K+, Rb+, Cs+, GdnH+, and Ca2+), identity. The Q* decreased in the order F− > Cl− > Br− > NO3− ∼ I− > SCN− > ClO4− ≫ SO42−, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO42− anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface. PMID:21432935

Protein adsorption at the electrified air-water interface: implications on foam stability.

PubMed

Engelhardt, Kathrin; Rumpel, Armin; Walter, Johannes; Dombrowski, Jannika; Kulozik, Ulrich; Braunschweig, Björn; Peukert, Wolfgang

2012-05-22

The surface chemistry of ions, water molecules, and proteins as well as their ability to form stable networks in foams can influence and control macroscopic properties such as taste and texture of dairy products considerably. Despite the significant relevance of protein adsorption at liquid interfaces, a molecular level understanding on the arrangement of proteins at interfaces and their interactions has been elusive. Therefore, we have addressed the adsorption of the model protein bovine serum albumin (BSA) at the air-water interface with vibrational sum-frequency generation (SFG) and ellipsometry. SFG provides specific information on the composition and average orientation of molecules at interfaces, while complementary information on the thickness of the adsorbed layer can be obtained with ellipsometry. Adsorption of charged BSA proteins at the water surface leads to an electrified interface, pH dependent charging, and electric field-induced polar ordering of interfacial H(2)O and BSA. Varying the bulk pH of protein solutions changes the intensities of the protein related vibrational bands substantially, while dramatic changes in vibrational bands of interfacial H(2)O are simultaneously observed. These observations have allowed us to determine the isoelectric point of BSA directly at the electrolyte-air interface for the first time. BSA covered air-water interfaces with a pH near the isoelectric point form an amorphous network of possibly agglomerated BSA proteins. Finally, we provide a direct correlation of the molecular structure of BSA interfaces with foam stability and new information on the link between microscopic properties of BSA at water surfaces and macroscopic properties such as the stability of protein foams.

Dissociation of hydrophobic and charged nano particles in aqueous guanidinium chloride and urea solutions: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Li, Weifeng; Mu, Yuguang

2012-02-01

It has been a long history that urea and guanidinium chloride (GdmCl) are used as agents for denaturing proteins. The underlying mechanism has been extensively studied in the past several decades. However, the question regarding why GdmCl is much stronger than urea has seldom been touched. Here, through molecular dynamics simulations, we show that a 4 M GdmCl solution is more able than 7 M urea solution to dissociate both hydrophobic and charged nano-particles (NP). Both urea and GdmCl affect the NPs' aggregation through direct binding to the NP surface. The advantages of GdmCl originate from the net charge of bound guanidinium ions which can generate a local positively charged environment around hydrophobic and negatively charged NPs. This effective coating can introduce Coulombic repulsion between all the NPs. Urea shows certain ability to dissociate hydrophobic NPs. However, in the case of charged NPs, urea molecules located between two opposite-charged NPs will form ordered hydrogen bonds, acting like ``glue'' which prevents separation of the NPs. Although urea can form hydrogen bonds with either hydrophilic amino acids or the protein backbone, which are believed to contribute to protein denaturation, our findings strongly suggest that this property does not always contribute positively to urea's denaturation power.

Free energy landscapes of encounter complexes in protein-protein association.

PubMed

Camacho, C J; Weng, Z; Vajda, S; DeLisi, C

1999-03-01

We report the computer generation of a high-density map of the thermodynamic properties of the diffusion-accessible encounter conformations of four receptor-ligand protein pairs, and use it to study the electrostatic and desolvation components of the free energy of association. Encounter complex conformations are generated by sampling the translational/rotational space of the ligand around the receptor, both at 5-A and zero surface-to-surface separations. We find that partial desolvation is always an important effect, and it becomes dominant for complexes in which one of the reactants is neutral or weakly charged. The interaction provides a slowly varying attractive force over a small but significant region of the molecular surface. In complexes with no strong charge complementarity this region surrounds the binding site, and the orientation of the ligand in the encounter conformation with the lowest desolvation free energy is similar to the one observed in the fully formed complex. Complexes with strong opposite charges exhibit two types of behavior. In the first group, represented by barnase/barstar, electrostatics exerts strong orientational steering toward the binding site, and desolvation provides some added adhesion within the local region of low electrostatic energy. In the second group, represented by the complex of kallikrein and pancreatic trypsin inhibitor, the overall stability results from the rather nonspecific electrostatic attraction, whereas the affinity toward the binding region is determined by desolvation interactions.

Spiers Memorial Lecture. Ions at aqueous interfaces.

PubMed

Jungwirth, Pavel

2009-01-01

Studies of aqueous interfaces and of the behavior of ions therein have been profiting from a recent remarkable progress in surface selective spectroscopies, as well as from developments in molecular simulations. Here, we summarize and place in context our investigations of ions at aqueous interfaces employing molecular dynamics simulations and electronic structure methods, performed in close contact with experiment. For the simplest of these interfaces, i.e. the open water surface, we demonstrate that the traditional picture of an ion-free surface is not valid for large, soft (polarizable) ions such as the heavier halides. Both simulations and spectroscopic measurements indicate that these ions can be present and even enhanced at surface of water. In addition we show that the ionic product of water exhibits a peculiar surface behavior with hydronium but not hydroxide accumulating at the air/water and alkane/water interfaces. This result is supported by surface-selective spectroscopic experiments and surface tension measurements. However, it contradicts the interpretation of electrophoretic and titration experiments in terms of strong surface adsorption of hydroxide; an issue which is further discussed here. The applicability of the observed behavior of ions at the water surface to investigations of their affinity for the interface between proteins and aqueous solutions is explored. Simulations show that for alkali cations the dominant mechanism of specific interactions with the surface of hydrated proteins is via ion pairing with negatively charged amino acid residues and with the backbone amide groups. As far as halide anions are concerned, the lighter ones tend to pair with positively charged amino acid residues, while heavier halides exhibit affinity to the amide group and to non-polar protein patches, the latter resembling their behavior at the air/water interface. These findings, together with results for more complex molecular ions, allow us to formulate a local model of interactions of ions with proteins with the aim to rationalize at the molecular level ion-specific Hofmeister effects, e.g. the salting out of proteins.

What interactions drive the salivary mucosal pellicle formation?

PubMed Central

Gibbins, Hannah L.; Yakubov, Gleb E.; Proctor, Gordon B.; Wilson, Stephen; Carpenter, Guy H.

2014-01-01

The bound salivary pellicle is essential for protection of both the enamel and mucosa in the oral cavity. The enamel pellicle formation is well characterised, however the mucosal pellicle proteins have only recently been clarified and what drives their formation is still unclear. The aim of this study was to examine the salivary pellicle on particles with different surface properties (hydrophobic or hydrophilic with a positive or negative charge), to determine a suitable model to mimic the mucosal pellicle. A secondary aim was to use the model to test how transglutaminase may alter pellicle formation. Particles were incubated with resting whole mouth saliva, parotid saliva and submandibular/sublingual saliva. Following incubation and two PBS and water washes bound salivary proteins were eluted with two concentrations of SDS, which were later analysed using SDS-PAGE and Western blotting. Experiments were repeated with purified transglutaminase to determine how this epithelial-derived enzyme may alter the bound pellicle. Protein pellicles varied according to the starting salivary composition and the particle chemistry. Amylase, the single most abundant protein in saliva, did not bind to any particle indicating specific protein binding. Most proteins bound through hydrophobic interactions and a few according to their charges. The hydrophobic surface most closely matched the known salivary mucosal pellicle by containing mucins, cystatin and statherin but an absence of amylase and proline-rich proteins. This surface was further used to examine the effect of added transglutaminase. At the concentrations used only statherin showed any evidence of crosslinking with itself or another saliva protein. In conclusion, the formation of the salivary mucosal pellicle is probably mediated, at least in part, by hydrophobic interactions to the epithelial cell surface. PMID:24921197

A New Approach for Detection Improvement of the Creutzfeldt-Jakob Disorder through a Specific Surface Chemistry Applied onto Titration Well

PubMed Central

Mille, Caroline; Debarnot, Dominique; Zorzi, Willy; Moualij, Benaissa El; Quadrio, Isabelle; Perret-Liaudet, Armand; Coudreuse, Arnaud; Legeay, Gilbert; Poncin-Epaillard, Fabienne

2012-01-01

This work illustrates the enhancement of the sensitivity of the ELISA titration for recombinant human and native prion proteins, while reducing other non-specific adsorptions that could increase the background signal and lead to a low sensitivity and false positives. It is achieved thanks to the association of plasma chemistry and coating with different amphiphilic molecules bearing either ionic charges and/or long hydrocarbon chains. The treated support by 3-butenylamine hydrochloride improves the signal detection of recombinant protein, while surface modification with the 3,7-dimethylocta-2,6-dien-1-diamine (geranylamine) enhances the sensitivity of the native protein. Beside the surface chemistry effect, these different results are associated with protein conformation. PMID:25586034

Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags.

PubMed

Mack, Laura; Brill, Boris; Delis, Natalia; Groner, Bernd

2014-12-01

The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Modulation of Protein Fouling and Interfacial Properties at Carbon Surfaces via Immobilization of Glycans Using Aryldiazonium Chemistry

PubMed Central

Zen, Federico; Angione, M. Daniela; Behan, James A.; Cullen, Ronan J.; Duff, Thomas; Vasconcelos, Joana M.; Scanlan, Eoin M.; Colavita, Paula E.

2016-01-01

Carbon materials and nanomaterials are of great interest for biological applications such as implantable devices and nanoparticle vectors, however, to realize their potential it is critical to control formation and composition of the protein corona in biological media. In this work, protein adsorption studies were carried out at carbon surfaces functionalized with aryldiazonium layers bearing mono- and di-saccharide glycosides. Surface IR reflectance absorption spectroscopy and quartz crystal microbalance were used to study adsorption of albumin, lysozyme and fibrinogen. Protein adsorption was found to decrease by 30–90% with respect to bare carbon surfaces; notably, enhanced rejection was observed in the case of the tested di-saccharide vs. simple mono-saccharides for near-physiological protein concentration values. ζ-potential measurements revealed that aryldiazonium chemistry results in the immobilization of phenylglycosides without a change in surface charge density, which is known to be important for protein adsorption. Multisolvent contact angle measurements were used to calculate surface free energy and acid-base polar components of bare and modified surfaces based on the van Oss-Chaudhury-Good model: results indicate that protein resistance in these phenylglycoside layers correlates positively with wetting behavior and Lewis basicity. PMID:27108562

Modulation of Protein Fouling and Interfacial Properties at Carbon Surfaces via Immobilization of Glycans Using Aryldiazonium Chemistry

NASA Astrophysics Data System (ADS)

Zen, Federico; Angione, M. Daniela; Behan, James A.; Cullen, Ronan J.; Duff, Thomas; Vasconcelos, Joana M.; Scanlan, Eoin M.; Colavita, Paula E.

2016-04-01

Carbon materials and nanomaterials are of great interest for biological applications such as implantable devices and nanoparticle vectors, however, to realize their potential it is critical to control formation and composition of the protein corona in biological media. In this work, protein adsorption studies were carried out at carbon surfaces functionalized with aryldiazonium layers bearing mono- and di-saccharide glycosides. Surface IR reflectance absorption spectroscopy and quartz crystal microbalance were used to study adsorption of albumin, lysozyme and fibrinogen. Protein adsorption was found to decrease by 30-90% with respect to bare carbon surfaces; notably, enhanced rejection was observed in the case of the tested di-saccharide vs. simple mono-saccharides for near-physiological protein concentration values. ζ-potential measurements revealed that aryldiazonium chemistry results in the immobilization of phenylglycosides without a change in surface charge density, which is known to be important for protein adsorption. Multisolvent contact angle measurements were used to calculate surface free energy and acid-base polar components of bare and modified surfaces based on the van Oss-Chaudhury-Good model: results indicate that protein resistance in these phenylglycoside layers correlates positively with wetting behavior and Lewis basicity.

Reduced Point Charge Models of Proteins: Effect of Protein-Water Interactions in Molecular Dynamics Simulations of Ubiquitin Systems.

PubMed

Leherte, Laurence; Vercauteren, Daniel P

2017-10-26

We investigate the influence of various solvent models on the structural stability and protein-water interface of three ubiquitin complexes (PDB access codes: 1Q0W , 2MBB , 2G3Q ) modeled using the Amber99sb force field (FF) and two different point charge distributions. A previously developed reduced point charge model (RPCM), wherein each amino acid residue is described by a limited number of point charges, is tested and compared to its all-atom (AA) version. The complexes are solvated in TIP4P-Ew or TIP3P type water molecules, involving either the scaling of the Lennard-Jones protein-O water interaction parameters, or the coarse-grain (CG) SIRAH water description. The best agreements between the RPCM and AA models were obtained for structural, protein-water, and ligand-ubiquitin properties when using the TIP4P-Ew water FF with a scaling factor γ of 0.7. At the RPCM level, a decrease in γ, or the inclusion of SIRAH particles, allows weakening of the protein-water interactions. It results in a slight collapse of the protein structure and a less compact hydration shell and, thus, in a decrease in the number of protein-water and water-water H-bonds. The dynamics of the surface protein atoms and of the water shell molecules are also slightly refrained, which allow the generation of stable RPCM trajectories.

Electro-induced protein deposition on low-fouling surfaces

NASA Astrophysics Data System (ADS)

Cole, M. A.; Voelcker, N. H.; Thissen, H.

2007-12-01

Control over protein adsorption is a key issue for numerous biomedical applications ranging from diagnostic microarrays to tissue-engineered medical devices. Here, we describe a method for creating surfaces that prevent non-specific protein adsorption, which upon application of an external trigger can be transformed into surfaces showing high protein adsorption on demand. Silicon wafers were used as substrate materials upon which thin functional coatings were constructed by the deposition of an allylamine plasma polymer followed by high-density grafting of poly(ethylene oxide) aldehyde, resulting in a low-fouling surface. When the underlying highly doped silicon substrate was used as an electrode, the resulting electrostatic attraction between the electrode and charged proteins in solution induced protein deposition at the low-fouling interface. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were used to characterize the surface modifications. Controlled protein adsorption experiments were carried out using horseradish peroxidase. The amount of protein deposited at the surface was then investigated by means of a colorimetric assay. It is expected that the concept described here will find use in a variety of biotechnological and biomedical applications, particularly in the area of biochips.

Quantum mechanical/molecular mechanical/continuum style solvation model: second order Møller-Plesset perturbation theory.

PubMed

Thellamurege, Nandun M; Si, Dejun; Cui, Fengchao; Li, Hui

2014-05-07

A combined quantum mechanical/molecular mechanical/continuum (QM/MM/C) style second order Møller-Plesset perturbation theory (MP2) method that incorporates induced dipole polarizable force field and induced surface charge continuum solvation model is established. The Z-vector method is modified to include induced dipoles and induced surface charges to determine the MP2 response density matrix, which can be used to evaluate MP2 properties. In particular, analytic nuclear gradient is derived and implemented for this method. Using the Assisted Model Building with Energy Refinement induced dipole polarizable protein force field, the QM/MM/C style MP2 method is used to study the hydrogen bonding distances and strengths of the photoactive yellow protein chromopore in the wild type and the Glu46Gln mutant.

Surface Oxide Net Charge of a Titanium Alloy; Comparison Between Effects of Treatment With Heat or Radiofrequency Plasma Glow Discharge

PubMed Central

MacDonald, Daniel E.; Rapuano, Bruce E.; Schniepp, Hannes C.

2010-01-01

In the current study, we have compared the effects of heat and radiofrequency plasma glow discharge (RFGD) treatment of a Ti6Al4V alloy on the physico-chemical properties of the alloy’s surface oxide. Titanium alloy (Ti6Al4V) disks were passivated alone, heated to 600 °C, or RFGD plasma treated in pure oxygen. RFGD treatment did not alter the roughness, topography, elemental composition or thickness of the alloy’s surface oxide layer. In contrast, heat treatment altered oxide topography by creating a pattern of oxide elevations approximately 50–100 nm in diameter. These nanostructures exhibited a three-fold increase in roughness compared to untreated surfaces when RMS roughness was calculated after applying a spatial high-pass filter with a 200 nm cutoff wavelength. Heat treatment also produced a surface enrichment in aluminum and vanadium oxides. Both RFGD and heat treatment produced similar increases in oxide wettability. Atomic force microscopy (AFM) measurements of metal surface oxide net charge signified by a long range force of attraction to or repulsion from a (negatively charged) silicon nitride AFM probe were also obtained for all three experimental groups. Force measurements showed that the RFGD-treated Ti6Al4V samples demonstrated a higher net positive surface charge at pH values below 6 and a higher net negative surface charge at physiological pH (pH values between 7 and 8) compared to control and heat-treated samples These findings suggest that RFGD treatment of metallic implant materials can be used to study the role of negatively charged surface oxide functional groups in protein bioactivity, osteogenic cell behavior and osseointegration independently of oxide topography. PMID:20880672

The structure and dipole moment of globular proteins in solution and crystalline states: use of NMR and X-ray databases for the numerical calculation of dipole moment.

PubMed

Takashima, S

2001-04-05

The large dipole moment of globular proteins has been well known because of the detailed studies using dielectric relaxation and electro-optical methods. The search for the origin of these dipolemoments, however, must be based on the detailed knowledge on protein structure with atomic resolutions. At present, we have two sources of information on the structure of protein molecules: (1) x-ray databases obtained in crystalline state; (2) NMR databases obtained in solution state. While x-ray databases consist of only one model, NMR databases, because of the fluctuation of the protein folding in solution, consist of a number of models, thus enabling the computation of dipole moment repeated for all these models. The aim of this work, using these databases, is the detailed investigation on the interdependence between the structure and dipole moment of protein molecules. The dipole moment of protein molecules has roughly two components: one dipole moment is due to surface charges and the other, core dipole moment, is due to polar groups such as N--H and C==O bonds. The computation of surface charge dipole moment consists of two steps: (A) calculation of the pK shifts of charged groups for electrostatic interactions and (B) calculation of the dipole moment using the pK corrected for electrostatic shifts. The dipole moments of several proteins were computed using both NMR and x-ray databases. The dipole moments of these two sets of calculations are, with a few exceptions, in good agreement with one another and also with measured dipole moments.

Anionic deep cavitands enable the adhesion of unmodified proteins at a membrane bilayer.

PubMed

Ghang, Yoo-Jin; Perez, Lizeth; Morgan, Melissa A; Si, Fang; Hamdy, Omar M; Beecher, Consuelo N; Larive, Cynthia K; Julian, Ryan R; Zhong, Wenwan; Cheng, Quan; Hooley, Richard J

2014-12-28

An anionic self-folding deep cavitand is capable of immobilizing unmodified proteins and enzymes at a supported lipid bilayer interface, providing a simple, soft bioreactive surface that allows enzymatic function under mild conditions. The adhesion is based on complementary charge interactions, and the hosts are capable of binding enzymes such as trypsin at the bilayer interface: the catalytic activity is retained upon adhesion, allowing selective reactions to be performed at the membrane surface.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osipiuk, J.; Gornicki, P.; Maj, L.

The structure of the YlxR protein of unknown function from Streptococcus pneumonia was determined to 1.35 Angstroms. YlxR is expressed from the nusA/infB operon in bacteria and belongs to a small protein family (COG2740) that shares a conserved sequence motif GRGA(Y/W). The family shows no significant amino-acid sequence similarity with other proteins. Three-wavelength diffraction MAD data were collected to 1.7 Angstroms from orthorhombic crystals using synchrotron radiation and the structure was determined using a semi-automated approach. The YlxR structure resembles a two-layer {alpha}/{beta} sandwich with the overall shape of a cylinder and shows no structural homology to proteins of knownmore » structure. Structural analysis revealed that the YlxR structure represents a new protein fold that belongs to the {alpha}-{beta} plait superfamily. The distribution of the electrostatic surface potential shows a large positively charged patch on one side of the protein, a feature often found in nucleic acid-binding proteins. Three sulfate ions bind to this positively charged surface. Analysis of potential binding sites uncovered several substantial clefts, with the largest spanning 3/4 of the protein. A similar distribution of binding sites and a large sharply bent cleft are observed in RNA-binding proteins that are unrelated in sequence and structure. It is proposed that YlxR is an RNA-binding protein.« less

Streptococcus pneumonia YlxR at 1.35 A shows a putative new fold.

PubMed

Osipiuk, J; Górnicki, P; Maj, L; Dementieva, I; Laskowski, R; Joachimiak, A

2001-11-01

The structure of the YlxR protein of unknown function from Streptococcus pneumonia was determined to 1.35 A. YlxR is expressed from the nusA/infB operon in bacteria and belongs to a small protein family (COG2740) that shares a conserved sequence motif GRGA(Y/W). The family shows no significant amino-acid sequence similarity with other proteins. Three-wavelength diffraction MAD data were collected to 1.7 A from orthorhombic crystals using synchrotron radiation and the structure was determined using a semi-automated approach. The YlxR structure resembles a two-layer alpha/beta sandwich with the overall shape of a cylinder and shows no structural homology to proteins of known structure. Structural analysis revealed that the YlxR structure represents a new protein fold that belongs to the alpha-beta plait superfamily. The distribution of the electrostatic surface potential shows a large positively charged patch on one side of the protein, a feature often found in nucleic acid-binding proteins. Three sulfate ions bind to this positively charged surface. Analysis of potential binding sites uncovered several substantial clefts, with the largest spanning 3/4 of the protein. A similar distribution of binding sites and a large sharply bent cleft are observed in RNA-binding proteins that are unrelated in sequence and structure. It is proposed that YlxR is an RNA-binding protein.

Positively charged and bipolar layered poly(ether imide) nanofiltration membranes for water softening applications

NASA Astrophysics Data System (ADS)

Gassara, S.; Abdelkafi, A.; Quémener, D.; Amar, R. Ben; Deratani, A.

2015-07-01

Poly(ether imide) (PEI) ultrafiltration membranes were chemically modified with branched poly(ethyleneimine) to obtain nanofiltration (NF) membrane Cat PEI with a positive charge in the pH range below 9. An oppositely charged polyelectrolyte layer was deposited on the resulting membrane surface by using sodium polystyrene sulfonate (PSSNa) and sodium polyvinyl sulfonate (PVSNa) to prepare a bipolar layered membrane NF Cat PEI_PSS and Cat PEI_PVS having a negatively charged surface and positively charged pores. Cat PEI exhibited good performance to remove multivalent cations (more than 90% of Ca2+) from single salt solutions except in presence of sulfate ions. Adding an anionic polyelectrolyte layer onto the positively charged surface resulted in a significant enhancement of rejection performance even in presence of sulfate anions. Application of the prepared membranes in water softening of natural complex mixtures was successful for the different studied membranes and a large decrease of hardness was obtained. Moreover, Cat PEI_PSS showed a good selectivity for nitrate removal. Fouling experiments were carried out with bovine serum albumin, as model protein foulant. Cat PEI_PSS showed much better fouling resistance than Cat PEI with a quantitative flux recovery ratio.

Semiconductor sensor embedded microfluidic chip for protein biomarker detection using a bead-based immunoassay combined with deoxyribonucleic acid strand labeling.

PubMed

Lin, Yen-Heng; Peng, Po-Yu

2015-04-15

Two major issues need to be addressed in applying semiconductor biosensors to detecting proteins in immunoassays. First, the length of the antibody on the sensor surface surpasses the Debye lengths (approximately 1 nm, in normal ionic strength solution), preventing certain specifically bound proteins from being tightly attached to the sensor surface. Therefore, these proteins do not contribute to the sensor's surface potential change. Second, these proteins carry a small charge and can be easily affected by the pH of the surrounding solution. This study proposes a magnetic bead-based immunoassay using a secondary antibody to label negatively charged DNA fragments for signal amplification. An externally imposed magnetic force attaches the analyte tightly to the sensor surface, thereby effectively solving the problem of the analyte protein's distance to the sensor surface surpassing the Debye lengths. In addition, a normal ion intensity buffer can be used without dilution for the proposed method. Experiments revealed that the sensitivity can be improved by using a longer DNA fragment for labeling and smaller magnetic beads as solid support for the antibody. By using a 90 base pair DNA label, the signal was 15 times greater than that without labeling. In addition, by using a 120 nm magnetic bead, a minimum detection limit of 12.5 ng mL(-1) apolipoprotein A1 can be measured. Furthermore, this study integrates a semiconductor sensor with a microfluidic chip. With the help of microvalves and micromixers in the chip, the length of the mixing step for each immunoassay has been reduced from 1h to 20 min, and the sample volume has been reduced from 80 μL to 10 μL. In practice, a protein biomarker in a urinary bladder cancer patient's urine was successfully measured using this technique. This study provides a convenient and effective method to measure protein using a semiconductor sensor. Copyright © 2015 Elsevier B.V. All rights reserved.

Evaluation of reduced point charge models of proteins through Molecular Dynamics simulations: application to the Vps27 UIM-1-Ubiquitin complex.

PubMed

Leherte, Laurence; Vercauteren, Daniel P

2014-02-01

Reduced point charge models of amino acids are designed, (i) from local extrema positions in charge density distribution functions built from the Poisson equation applied to smoothed molecular electrostatic potential (MEP) functions, and (ii) from local maxima positions in promolecular electron density distribution functions. Corresponding charge values are fitted versus all-atom Amber99 MEPs. To easily generate reduced point charge models for protein structures, libraries of amino acid templates are built. The program GROMACS is used to generate stable Molecular Dynamics trajectories of an Ubiquitin-ligand complex (PDB: 1Q0W), under various implementation schemes, solvation, and temperature conditions. Point charges that are not located on atoms are considered as virtual sites with a nul mass and radius. The results illustrate how the intra- and inter-molecular H-bond interactions are affected by the degree of reduction of the point charge models and give directions for their implementation; a special attention to the atoms selected to locate the virtual sites and to the Coulomb-14 interactions is needed. Results obtained at various temperatures suggest that the use of reduced point charge models allows to probe local potential hyper-surface minima that are similar to the all-atom ones, but are characterized by lower energy barriers. It enables to generate various conformations of the protein complex more rapidly than the all-atom point charge representation. Copyright © 2013 Elsevier Inc. All rights reserved.

An Electrostatic Charge Partitioning Model for the Dissociation of Protein Complexes in the Gas Phase

NASA Astrophysics Data System (ADS)

Sciuto, Stephen V.; Liu, Jiangjiang; Konermann, Lars

2011-10-01

Electrosprayed multi-protein complexes can be dissociated by collisional activation in the gas phase. Typically, these processes follow a mechanism whereby a single subunit gets ejected with a disproportionately high amount of charge relative to its mass. This asymmetric behavior suggests that the departing subunit undergoes some degree of unfolding prior to being separated from the residual complex. These structural changes occur concomitantly with charge (proton) transfer towards the subunit that is being unraveled. Charge accumulation takes place up to the point where the subunit loses physical contact with the residual complex. This work develops a simple electrostatic model for studying the relationship between conformational changes and charge enrichment during collisional activation. Folded subunits are described as spheres that carry continuum surface charge. The unfolded chain is envisioned as random coil bead string. Simulations are guided by the principle that the system will adopt the charge configuration with the lowest potential energy for any backbone conformation. A finite-difference gradient algorithm is used to determine the charge on each subunit throughout the dissociation process. Both dimeric and tetrameric protein complexes are investigated. The model reproduces the occurrence of asymmetric charge partitioning for dissociation events that are preceded by subunit unfolding. Quantitative comparisons of experimental MS/MS data with model predictions yield estimates of the structural changes that occur during collisional activation. Our findings suggest that subunit separation can occur over a wide range of scission point structures that correspond to different degrees of unfolding.

Electrostatic lipid-protein interactions sequester the curli amyloid fold on the lipopolysaccharide membrane surface.

PubMed

Swasthi, Hema M; Mukhopadhyay, Samrat

2017-12-01

Curli is a functional amyloid protein in the extracellular matrix of enteric Gram-negative bacteria. Curli is assembled at the cell surface and consists of CsgA, the major subunit of curli, and a membrane-associated nucleator protein, CsgB. Oligomeric intermediates that accumulate during the lag phase of amyloidogenesis are generally toxic, but the underlying mechanism by which bacterial cells overcome this toxicity during curli assembly at the surface remains elusive. Here, we elucidated the mechanism of curli amyloidogenesis and provide molecular insights into the strategy by which bacteria can potentially bypass the detrimental consequences of toxic amyloid intermediates. Using a diverse range of biochemical and biophysical tools involving circular dichroism, fluorescence, Raman spectroscopy, and atomic force microscopy imaging, we characterized the molecular basis of the interaction of CsgB with a membrane-mimetic anionic surfactant as well as with lipopolysaccharide (LPS) constituting the outer leaflet of Gram-negative bacteria. Aggregation studies revealed that the electrostatic interaction of the positively charged C-terminal region of the protein with a negatively charged head group of surfactant/LPS promotes a protein-protein interaction that results in facile amyloid formation without a detectable lag phase. We also show that CsgB, in the presence of surfactant/LPS, accelerates the fibrillation rate of CsgA by circumventing the lag phase during nucleation. Our findings suggest that the electrostatic interactions between lipid and protein molecules play a pivotal role in efficiently sequestering the amyloid fold of curli on the membrane surface without significant accumulation of toxic oligomeric intermediates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemical and genetic wrappers for improved phage and RNA display.

PubMed

Lamboy, Jorge A; Tam, Phillip Y; Lee, Lucie S; Jackson, Pilgrim J; Avrantinis, Sara K; Lee, Hye J; Corn, Robert M; Weiss, Gregory A

2008-11-24

An Achilles heel inherent to all molecular display formats, background binding between target and display system introduces false positives into screens and selections. For example, the negatively charged surfaces of phage, mRNA, and ribosome display systems bind with unacceptably high nonspecificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. Here we report the first systematic attempt to understand why a broad class of molecular display selections fail, and then solve the underlying problem for both phage and RNA display. Firstly, a genetic strategy was used to introduce a short, charge-neutralizing peptide into the solvent-exposed, negatively charged phage coat. The modified phage (KO7(+)) reduced or eliminated nonspecific binding to the problematic high-pI proteins. In the second, chemical approach, nonspecific interactions were blocked by oligolysine wrappers in the cases of phage and total RNA. For phage display applications, the peptides Lys(n) (where n=16 to 24) emerged as optimal for wrapping the phage. Lys(8), however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked nonspecific binding to allow successful selections, screens, and assays with five previously unworkable protein targets.

Enhanced protein adsorption and patterning on nanostructured latex-coated paper.

PubMed

Juvonen, Helka; Määttänen, Anni; Ihalainen, Petri; Viitala, Tapani; Sarfraz, Jawad; Peltonen, Jouko

2014-06-01

Specific interactions of extracellular matrix proteins with cells and their adhesion to the substrate are important for cell growth. A nanopatterned latex-coated paper substrate previously shown to be an excellent substrate for cell adhesion and 2D growth was studied for directed immobilization of proteins. The nanostructured latex surface was formed by short-wavelength IR irradiation of a two-component latex coating consisting of a hydrophilic film-forming styrene butadiene acrylonitrile copolymer and hydrophobic polystyrene particles. The hydrophobic regions of the IR-treated latex coating showed strong adhesion of bovine serum albumin (cell repelling protein), fibronectin (cell adhesive protein) and streptavidin. Opposite to the IR-treated surface, fibronectin and streptavidin had a poor affinity toward the untreated pristine latex coating. Detailed characterization of the physicochemical surface properties of the latex-coated substrates revealed that the observed differences in protein affinity were mainly due to the presence or absence of the protein repelling polar and charged surface groups. The protein adsorption was assisted by hydrophobic (dehydration) interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

Energetics and kinetics of primary charge separation in bacterial photosynthesis.

PubMed

LeBard, David N; Kapko, Vitaliy; Matyushov, Dmitry V

2008-08-21

We report the results of molecular dynamics (MD) simulations and formal modeling of the free-energy surfaces and reaction rates of primary charge separation in the reaction center of Rhodobacter sphaeroides. Two simulation protocols were used to produce MD trajectories. Standard force-field potentials were employed in the first protocol. In the second protocol, the special pair was made polarizable to reproduce a high polarizability of its photoexcited state observed by Stark spectroscopy. The charge distribution between covalent and charge-transfer states of the special pair was dynamically adjusted during the simulation run. We found from both protocols that the breadth of electrostatic fluctuations of the protein/water environment far exceeds previous estimates, resulting in about 1.6 eV reorganization energy of electron transfer in the first protocol and 2.5 eV in the second protocol. Most of these electrostatic fluctuations become dynamically frozen on the time scale of primary charge separation, resulting in much smaller solvation contributions to the activation barrier. While water dominates solvation thermodynamics on long observation times, protein emerges as the major thermal bath coupled to electron transfer on the picosecond time of the reaction. Marcus parabolas were obtained for the free-energy surfaces of electron transfer by using the first protocol, while a highly asymmetric surface was obtained in the second protocol. A nonergodic formulation of the diffusion-reaction electron-transfer kinetics has allowed us to reproduce the experimental results for both the temperature dependence of the rate and the nonexponential decay of the population of the photoexcited special pair.

Structural Characterization and Determinants of Specificity of Single-Chain Antibody Inhibitors of Membrane-Type Serine Protease 1

DTIC Science & Technology

2008-03-01

Colman, P. M. (1993). Shape complementarity at protein / protein interfaces . J Mol Biol 234, 946-50. 26. Huang, M., Syed, R., Stura, E. A., Stone, M. J...Å2 of surface area (Table 1). In the apo MT-SP1 structure20, Asp96 forms the bottom of the S4 pocket, allowing a positively charged substrate P4...of surface area that E2 buries on MT-SP1 is larger than the typical antibody/ protein antigen interaction, which averages about 875 Å2 26; 27. This

Defining Protein Electrostatic Recognition Processes

DTIC Science & Technology

1989-11-30

of the electrostatic potentiai on the molecular surface of negatively charged Asp-101 in the fifth residue of JH1. the hapten and the V regions of...making and aligning expanded molecular dot surfaces for each molecule and checking these surfaces for interpenetration. The program TURNIP used these...the molecular surfaces are separated by 6 and 12A. All orientations have the exposed heme edge of cytochrome c facing the acidic patch of plastocyanin

Phenylboronate chromatography selectively separates glycoproteins through the manipulation of electrostatic, charge transfer, and cis-diol interactions.

PubMed

Carvalho, Rimenys J; Woo, James; Aires-Barros, M Raquel; Cramer, Steven M; Azevedo, Ana M

2014-10-01

Phenylboronate chromatography (PBC) has been applied for several years, however details regarding the mechanisms of interactions between the ligand and biomolecules are still scarce. The goal of this work is to investigate the various chemical interactions between proteins and their ligands, using a protein library containing both glycosylated and nonglycosylated proteins. Differences in the adsorption of these proteins over a pH range from 4 to 9 were related to two main properties: charge and presence of glycans. Acidic or neutral proteins were strongly adsorbed below pH 8 although the uncharged trigonal form of phenylboronate (PB) is less susceptible to forming electrostatic and cis-diol interactions with proteins. The glycosylated proteins were only adsorbed above pH 8 when the electrostatic repulsion between the boronate anion and the protein surface was mitigated (at 200 mM NaCl). All basic proteins were highly adsorbed above pH 8 with PB also acting as a cation-exchanger with binding occurring through electrostatic interactions. Batch adsorption performed at acidic conditions in the presence of Lewis base showed that charge-transfer interactions are critical for protein retention. This study demonstrates the multimodal interaction of PBC, which can be a selective tool for separation of different classes of proteins. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The protein corona of circulating PEGylated liposomes.

PubMed

Palchetti, Sara; Colapicchioni, Valentina; Digiacomo, Luca; Caracciolo, Giulio; Pozzi, Daniela; Capriotti, Anna Laura; La Barbera, Giorgia; Laganà, Aldo

2016-02-01

Following systemic administration, liposomes are covered by a 'corona' of proteins, and preserving the surface functionality is challenging. Coating the liposome surface with polyethylene glycol (PEG) is the most widely used anti-opsonization strategy, but it cannot fully preclude protein adsorption. To date, protein binding has been studied following in vitro incubation to predict the fate of liposomes in vivo, while dynamic incubation mimicking in vivo conditions remains largely unexplored. The main aim of this investigation was to determine whether shear stress, produced by physiologically relevant dynamic flow, could influence the liposome-protein corona. The corona of circulating PEGylated liposome was thoroughly compared with that formed by incubation in vitro. Systematic comparison in terms of size, surface charge and quantitative composition was made by dynamic light scattering, microelectrophoresis and nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). Size of coronas formed under static vs. dynamic incubation did not appreciably differ from each other. On the other side, the corona of circulating liposomes was more negatively charged than its static counterpart. Of note, the variety of protein species in the corona formed in a dynamic flow was significantly wider. Collectively, these results demonstrated that the corona of circulating PEGylated liposomes can be considerably different from that formed in a static fluid. This seems to be a key factor to predict the biological activity of a liposomal formulation in a physiological environment. Copyright © 2015 Elsevier B.V. All rights reserved.

Adsorption Kinetics, Conformation, and Mobility of the Growth Hormone and Lysozyme on Solid Surfaces, Studied with TIRF

PubMed

Buijs; Hlady

1997-06-01

Interactions of recombinant human growth hormone and lysozyme with solid surfaces are studied using total internal reflection fluorescence (TIRF) and monitoring the protein's intrinsic tryptophan fluorescence. The intensity, spectra, quenching, and polarization of the fluorescence emitted by the adsorbed proteins are monitored and related to adsorption kinetics, protein conformation, and fluorophore rotational mobility. To study the influence of electrostatic and hydrophobic interactions on the adsorption process, three sorbent surfaces are used which differ in charge and hydrophobicity. The chemical surface groups are silanol, methyl, and quaternary amine. Results indicate that adsorption of hGH is dominated by hydrophobic interactions. Lysozyme adsoption is strongly affected by the ionic strength. This effect is probably caused by an ionic strength dependent conformational state in solution which, in turn, influences the affinity for adsorption. Both proteins are more strongly bound to hydrophobic surfaces and this strong interaction is accompanied by a less compact conformation. Furthermore, it was seen that regardless of the characteristics of the sorbent surface, the rotational mobility of both proteins' tryptophans is largely reduced upon adsorption.

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

PubMed Central

Kisley, Lydia; Chen, Jixin; Mansur, Andrea P.; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Chen, Wen-Hsiang; Dhamane, Sagar; Willson, Richard C.; Landes, Christy F.

2014-01-01

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally. PMID:24459184

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations.

PubMed

Kisley, Lydia; Chen, Jixin; Mansur, Andrea P; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Chen, Wen-Hsiang; Dhamane, Sagar; Willson, Richard C; Landes, Christy F

2014-02-11

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

Effect of phosphorylation of myelin basic protein by MAPK on its interactions with actin and actin binding to a lipid membrane in vitro.

PubMed

Boggs, Joan M; Rangaraj, Godha; Gao, Wen; Heng, Yew-Meng

2006-01-17

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is most likely responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also polymerize actin, bundle F-actin filaments, and bind actin filaments to lipid bilayers through electrostatic interactions. MBP consists of a number of posttranslationally modified isomers of varying charge, some resulting from phosphorylation at several sites by different kinases, including mitogen-activated protein kinase (MAPK). Phosphorylation of MBP in oligodendrocytes occurs in response to various extracellular stimuli. Phosphorylation/dephosphorylation of MBP also occurs in the myelin sheath in response to electrical activity in the brain. Here we investigate the effect of phosphorylation of MBP on its interaction with actin in vitro by phosphorylating the most highly charged unmodified isomer, C1, at two sites with MAPK. Phosphorylation decreased the ability of MBP to polymerize actin and to bundle actin filaments but had no effect on the dissociation constant of the MBP-actin complex or on the ability of Ca2+-calmodulin to dissociate the complex. The most significant effect of phosphorylation on the MBP-actin complex was a dramatic reduction in its ability to bind to negatively charged lipid bilayers. The effect was much greater than that reported earlier for another charge isomer of MBP, C8, in which six arginines were deiminated to citrulline, resulting in a reduction of net positive charge of 6. These results indicate that although average electrostatic forces are the primary determinant of the interaction of MBP with actin, phosphorylation may have an additional effect due to a site-specific electrostatic effect or to a conformational change. Thus, phosphorylation of MBP, which occurs in response to various extracellular signals in both myelin and oligodendrocytes, attenuates the ability of MBP to polymerize and bundle actin and to bind it to a negatively charged membrane.

Noninvasive noble metal nanoparticle arrays for surface-enhanced Raman spectroscopy of proteins

NASA Astrophysics Data System (ADS)

Inya-Agha, Obianuju; Forster, Robert J.; Keyes, Tia E.

2007-02-01

Noble metal nanoparticles arrays are well established substrates for surface enhanced Raman spectroscopy (SERS). Their ability to enhance optical fields is based on the interaction of their surface valence electrons with incident electromagnetic radiation. In the array configuration, noble metal nanoparticles have been used to produce SER spectral enhancements of up to 10 8 orders of magnitude, making them useful for the trace analysis of physiologically relevant analytes such as proteins and peptides. Electrostatic interactions between proteins and metal surfaces result in the preferential adsorption of positively charged protein domains onto metal surfaces. This preferential interaction has the effect of disrupting the native conformation of the protein fold, with a concomitant loss of protein function. A major historic advantage of Raman microspectroscopy has been is its non-invasive nature; protein denaturation on the metal surfaces required for SER spectroscopy renders it a much more invasive technique. Further, part of the analytical power of Raman spectroscopy lies in its use as a secondary conformation probe. The protein structural loss which occurs on the metal surface results in secondary conformation readings which are not true to the actual native state of the analyte. This work presents a method for chemical fabrication of noble metal SERS arrays with surface immobilized layers which can protect protein native conformation without excessively mitigating the electromagnetic enhancements of spectra. Peptide analytes are used as model systems for proteins. Raman spectra of alpha lactalbumin on surfaces and when immobilized on these novel arrays are compared. We discuss the ability of the surface layer to protect protein structure whilst improving signal intensity.

The Role of Ionic Interactions in the Adherence of the S. epidermidis Adhesin SdrF to Prosthetic Material

PubMed Central

Toba, Faustino A.; Visai, Livia; Trivedi, Sheetal; Lowy, Franklin D.

2012-01-01

Staphylococcus epidermidis infections are common complications of prosthetic device implantation. SdrF, a surface protein, appears to play a critical role in the initial colonization step by adhering to type I collagen and Dacron™. The role of ionic interactions in S. epidermidis adherence to prosthetic material was examined. SdrF was cloned and expressed in Lactococcus lactis. The effect of pH, cation concentration and detergents on adherence to different types of plastic surfaces was assessed by crystal violet staining and bacterial cell counting. SdrF, in contrast with controls and other S. epidermidis surface proteins, bound to hydrophobic materials such as polystyrene. Binding was an ionic interaction and was affected by surface charge of the plastic, pH and cation concentration. Adherence of the SdrF construct was increased to positively charged plastics and was reduced by increasing concentrations of Ca2+ and Na+. Binding was optimal at pH 7.4. Kinetic studies demonstrated that the SdrF B domain, as well as one of the B subdomains was sufficient to mediate binding. The SdrF construct also bound more avidly to Goretex™ than the lacotococcal control. SdrF is a multifunctional protein that contributes to prosthetic devices infections by ionic, as well as specific receptor-ligand interactions. PMID:23039791

Partial molar volume of proteins studied by the three-dimensional reference interaction site model theory.

PubMed

Imai, Takashi; Kovalenko, Andriy; Hirata, Fumio

2005-04-14

The three-dimensional reference interaction site model (3D-RISM) theory is applied to the analysis of hydration effects on the partial molar volume of proteins. For the native structure of some proteins, the partial molar volume is decomposed into geometric and hydration contributions using the 3D-RISM theory combined with the geometric volume calculation. The hydration contributions are correlated with the surface properties of the protein. The thermal volume, which is the volume of voids around the protein induced by the thermal fluctuation of water molecules, is directly proportional to the accessible surface area of the protein. The interaction volume, which is the contribution of electrostatic interactions between the protein and water molecules, is apparently governed by the charged atomic groups on the protein surface. The polar atomic groups do not make any contribution to the interaction volume. The volume differences between low- and high-pressure structures of lysozyme are also analyzed by the present method.

Observation of Iron Specific Interaction with a Charge Neutral Phospholipid

NASA Astrophysics Data System (ADS)

Wang, Wenjie; Zhang, Honghu; Feng, Shuren; San Emeterio, Josue; Kuzmenko, Ivan; Nilsen-Hamilton, Marit; Mallapragada, Surya; Vaknin, David

2015-03-01

Using surface sensitive X-ray scattering and spectroscopic techniques we show that phosphatidyl choline (PC) head groups attract positively charged iron ions and complexes even at pH values that are lower than 3. DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) is a zwitterionic lipid typically used as a model system for cell membranes. Within a large pH range (3 -11), it carries a negative charge on the phosphate group and a positive charge on the quaternary ammonium cation, thus appears charge neutral. Further lowering the pH, i.e. adding a proton to the phosphate group, results in a positively charged headgroup. Surprisingly, we detect significant enrichment of iron at the interface of the DPPC monolayer and the aqueous subphase with the pH maintained at 3 or even lower. With a supposedly charge neutral or even positive surface, the observation of surface bound, charge positive iron ions or iron hydroxides is counter-intuitive and suggests iron-specific interaction with the phospholipid headgroup, which is not governed by electrostatic interaction. The effect of the integration of Mms6, a membrane protein that promotes the formation of magnetic nanocrystals, into the DPPC monolayer will also be discussed. Research supported by the U.S. Department of Energy under Contract No. DE-AC02-07CH11358 and DE-AC02-06CH11357.

Quantum mechanical/molecular mechanical/continuum style solvation model: Second order Møller-Plesset perturbation theory

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thellamurege, Nandun M.; Si, Dejun; Cui, Fengchao

A combined quantum mechanical/molecular mechanical/continuum (QM/MM/C) style second order Møller-Plesset perturbation theory (MP2) method that incorporates induced dipole polarizable force field and induced surface charge continuum solvation model is established. The Z-vector method is modified to include induced dipoles and induced surface charges to determine the MP2 response density matrix, which can be used to evaluate MP2 properties. In particular, analytic nuclear gradient is derived and implemented for this method. Using the Assisted Model Building with Energy Refinement induced dipole polarizable protein force field, the QM/MM/C style MP2 method is used to study the hydrogen bonding distances and strengths ofmore » the photoactive yellow protein chromopore in the wild type and the Glu46Gln mutant.« less

Reversibly immobilized biological materials in monolayer films on electrodes

DOEpatents

Weaver, P.F.; Frank, A.J.

1993-05-04

Methods and techniques are described for reversibly binding charged biological particles in a fluid medium to an electrode surface. The methods are useful in a variety of applications. The biological materials may include microbes, proteins, and viruses. The electrode surface may consist of reversibly electroactive materials such as polyvinylferrocene, silicon-linked ferrocene or quinone.

Reversibly immobilized biological materials in monolayer films on electrodes

DOEpatents

Weaver, Paul F.; Frank, Arthur J.

1993-01-01

Methods and techniques are described for reversibly binding charged biological particles in a fluid medium to an electrode surface. The methods are useful in a variety of applications. The biological materials may include microbes, proteins, and viruses. The electrode surface may consist of reversibly electroactive materials such as polyvinylferrocene, silicon-linked ferrocene or quinone.

Electrostatic interaction based approach to thrombin detection by surface-enhanced Raman spectroscopy.

PubMed

Hu, Juan; Zheng, Peng-Cheng; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin; Liu, Guo-Kun

2009-01-01

We have developed an electrostatic interaction based biosensor for thrombin detection using surface-enhanced Raman spectroscopy (SERS). This method utilized the electrostatic interaction between capture (thrombin aptamer) and probe (crystal violet, CV) molecules. The specific interaction between thrombin and aptamer could weaken the electrostatic barrier effect from the negative charged aptamer SAMs to the diffusion process of the positively charged CV from the bulk solution to the Au nanoparticle surface. Therefore, the more the bound thrombin, the more the CV molecules near the Au nanoparticle surface and the stronger the observed Raman signal of CV, provided the Raman detections were set at the same time point for each case. This procedure presented a highly specific selectivity and a linear detection of thrombin in the range from 0.1 nM to 10 nM with a detection limit of about 20 pM and realized the thrombin detection in human blood serum solution directly. The electrostatic interaction based technique provides an easy and fast-responding optical platform for a "signal-on" detection of proteins, which might be applicable for the real time assay of proteins.

Inferring the microscopic surface energy of protein-protein interfaces from mutation data.

PubMed

Moal, Iain H; Dapkūnas, Justas; Fernández-Recio, Juan

2015-04-01

Mutations at protein-protein recognition sites alter binding strength by altering the chemical nature of the interacting surfaces. We present a simple surface energy model, parameterized with empirical ΔΔG values, yielding mean energies of -48 cal mol(-1) Å(-2) for interactions between hydrophobic surfaces, -51 to -80 cal mol(-1) Å(-2) for surfaces of complementary charge, and 66-83 cal mol(-1) Å(-2) for electrostatically repelling surfaces, relative to the aqueous phase. This places the mean energy of hydrophobic surface burial at -24 cal mol(-1) Å(-2) . Despite neglecting configurational entropy and intramolecular changes, the model correlates with empirical binding free energies of a functionally diverse set of rigid-body interactions (r = 0.66). When used to rerank docking poses, it can place near-native solutions in the top 10 for 37% of the complexes evaluated, and 82% in the top 100. The method shows that hydrophobic burial is the driving force for protein association, accounting for 50-95% of the cohesive energy. The model is available open-source from http://life.bsc.es/pid/web/surface_energy/ and via the CCharpPPI web server http://life.bsc.es/pid/ccharppi/. © 2015 Wiley Periodicals, Inc.

Crystal structure of class III chitinase from pomegranate provides the insight into its metal storage capacity.

PubMed

Masuda, Taro; Zhao, Guanghua; Mikami, Bunzo

2015-01-01

Chitinase hydrolyzes the β-1,4-glycosidic bond in chitin. In higher plants, this enzyme has been regarded as a pathogenesis-related protein. Recently, we identified a class III chitinase, which functions as a calcium storage protein in pomegranate (Punica granatum) seed (PSC, pomegranate seed chitinase). Here, we solved a crystal structure of PSC at 1.6 Å resolution. Although its overall structure, including the structure of catalytic site and non-proline cis-peptides, was closely similar to those of other class III chitinases, PSC had some unique structural characteristics. First, there were some metal-binding sites with coordinated water molecules on the surface of PSC. Second, many unconserved aspartate residues were present in the PSC sequence which rendered the surface of PSC negatively charged. This acidic electrostatic property is in contrast to that of hevamine, well-characterized plant class III chitinase, which has rather a positively charged surface. Thus, the crystal structure provides a clue for metal association property of PSC.

Brownian dynamics simulations of interactions between aldolase and G- or F-actin.

PubMed Central

Ouporov, I V; Knull, H R; Thomasson, K A

1999-01-01

Compartmentation of proteins in cells is important to proper cell function. Interactions of F-actin and glycolytic enzymes is one mechanism by which glycolytic enzymes can compartment. Brownian dynamics (BD) simulations of the binding of the muscle form of the glycolytic enzyme fructose-1,6-bisphosphate aldolase (aldolase) to F- or G-actin provide first-encounter snapshots of these interactions. Using x-ray structures of aldolase, G-actin, and three-dimensional models of F-actin, the electrostatic potential about each protein was predicted by solving the linearized Poisson-Boltzmann equation for use in BD simulations. The BD simulations provided solution complexes of aldolase with F- or G-actin. All complexes demonstrate the close contacts between oppositely charged regions of the protein surfaces. Positively charged surface regions of aldolase (residues Lys 13, 27, 288, 293, and 341 and Arg 257) are attracted to the negatively charged amino terminus (Asp 1 and Glu 2 and 4) and other patches (Asp 24, 25, and 363 and Glu 361, 364, 99, and 100) of actin subunits. According to BD results, the most important factor for aldolase binding to actin is the quaternary structure of aldolase and actin. Two pairs of adjacent aldolase subunits greatly add to the positive electrostatic potential of each other creating a region of attraction for the negatively charged subdomain 1 of the actin subunit that is exposed to solvent in the quaternary F-actin structure. PMID:9876119

Interaction of fibrinogen and albumin with titanium dioxide nanoparticles of different crystalline phases

NASA Astrophysics Data System (ADS)

Marucco, Arianna; Fenoglio, Ivana; Turci, Francesco; Fubini, Bice

2013-04-01

TiO2 nanoparticles (NPs) are contained in different kinds of industrial products including paints, self-cleaning glasses, sunscreens. TiO2 is also employed in photocatalysis and it has been proposed for waste water treatment. Micrometric TiO2 is generally considered a safe material, while there is concern on the possible health effects of nanometric titania. Due to their small size NPs may migrate within the human body possibly entering in the blood stream. Therefore studies on the interaction of NPs with plasma proteins are needed. In fact, the interaction with proteins is believed to ultimately influences the NPs biological fate. Fibrinogen and albumin are two of the most abundant plasma proteins. They are involved in several important physiological functions. Furthermore, fibrinogen is known to trigger platelet adhesion and inflammation. For these reasons the study of the interaction between these protein and nanoparticles is an important step toward the understanding of the behavior of NPs in the body. In this study we investigated the interaction of albumin and fibrinogen with TiO2 nanoparticles of different crystal phases (rutile and anatase) using an integrated set of techniques. The amount of adsorbed fibrinogen and albumin for each TiO2 surface was investigated by using the bicinchoninic acid assay (BCA). The variation of the surface charge of the NP-protein conjugates respect to the naked NPs was used to indirectly estimate both surface coverage and reversibility of the adsorption upon dilution. Surface charge was monitored by measuring the ζ potential with a conventional electrophoretic light scattering (ELS) system. The extent of protein deformation was evaluated by Raman Spectroscopy. We found that both proteins adsorb irreversibly against electrostatic repulsion, likely undergoing conformational changes or selective orientation upon adsorption. The size of primary particles and the particles aggregation rather than the crystal phase modulate the affinity of fibrinogen for the TiO2 surfaces.

Structural features and activity of Brazzein and its mutants upon substitution of a surfaced exposed alanine.

PubMed

Ghanavatian, Parisa; Khalifeh, Khosrow; Jafarian, Vahab

2016-12-01

Brazzein (Brz) is a member of sweet-tasting protein containing four disulfide bonds. It was reported as a compact and heat-resistant protein. Here, we have used site-directed mutagenesis and replaced a surface-exposed alanine with aspartic acid (A19D mutant), lysine (A19K mutant) and glycine (A19G mutant). Activity comparisons of wild-type (WT) and mutants using taste panel test procedure showed that A19G variant has the same activity as WT protein. However, introduction of a positive charge in A19K mutant led to significant increase in Brz's sweetness, while A19D has reduced sweetness compared to WT protein. Docking studies showed that mutation at position 19 results in slight chain mobility of protein at the binding surface and changing the patterns of interactions toward more effective binding of E9K variant in the concave surface of sweet taste receptor. Far-UV CD data spectra have a characteristic shape of beta structure for all variants, however different magnitudes of spectra suggest that beta-sheet structure in WT and A19G is more stable than that of A19D and A19K. Equilibrium unfolding studies with fluorescence spectroscopy and using urea and dithiothritol (DTT) as chemical denaturants indicates that A19G mutant gains more stability against urea denaturation; while conformational stability of A19D and A19K decreases when compared with WT and A19G variants. We concluded that the positive charge at the surface of protein is important factor responsible for the interaction of protein with the human sweet receptor and Ala 19 can be considered as a key region for investigating the mechanism of the interaction of Brz with corresponding receptor. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Synthetic peptides derived from salivary proteins and the control of surface charge densities of dental surfaces improve the inhibition of dental calculus formation.

PubMed

Grohe, Bernd

2017-08-01

Peptides descended from the salivary proteins statherin and histatin were recently identified in saliva and the acquired enamel pellicle (AEP), a proteomic layer coated on enamel. In particular, the statherin phosphopeptide DpSpSEEKFLR (DSS) was found to adsorb to enamel-like hydroxyapatite and inhibit plaque-related crystal formation. To determine the mechanism of these processes, we studied peptide-crystal interactions based on the sequences DSS and RKFHEKHHSHRGYR (RKF). The latter is a basic histatin sequence showing antimicrobial effects. To initiate crystallization we used calcium oxalate monohydrate (COM), a rather secondary phase in the oral environment, however highly amenable to experimental analyses of nucleation and growth processes. Using electron microscopy we found that the peptides DSS, DSS-RKF and DSS-DSS all inhibit crystal formation; with DSS-DSS showing the strongest effects while RKF showed no effect. In addition, using either enamel-like or mica substrates, we found that the ratio of the substrate's surface charge densities was directly correlated with the ratio of COM nucleation rates on theses surfaces. The findings suggest that mineralization processes on enamel/AEP-films are controllable by the degree of peptide phosphorylation/acidity and the level of the enamel surface charge density. Both parameters can, when well adjusted, help to overcome periodontal disease and dental calculus formation. In addition, the presence of antimicrobial RKF will reduce the buildup of bacterial plaque. Copyright © 2017 Elsevier B.V. All rights reserved.

Probing the interplay between amyloidogenic proteins and membranes using lipid monolayers and bilayers.

PubMed

Relini, Annalisa; Marano, Nadia; Gliozzi, Alessandra

2014-05-01

Many degenerative diseases such as Alzheimer's and Parkinson's involve proteins that have a tendency to misfold and aggregate eventually forming amyloid fibers. This review describes the use of monolayers, bilayers, supported membranes, and vesicles as model systems that have helped elucidate the mechanisms and consequences of the interactions between amyloidogenic proteins and membranes. These are twofold: membranes favor the formation of amyloid structures and these induce damage in those membranes. We describe studies that show how interfaces, especially charged ones, favor amyloidogenic protein aggregation by several means. First, surfaces increase the effective protein concentration reducing a three-dimensional system to a two-dimensional one. Second, charged surfaces allow electrostatic interactions with the protein. Anionic lipids as well as rafts, rich in cholesterol and gangliosides, prove to play an especially important role. Finally, these amphipathic systems also offer a hydrophobic environment favoring conformational changes, oligomerization, and eventual formation of mature fibers. In addition, we examine several models for membrane permeabilization: protein pores, leakage induced by extraction of lipids, chaotic pores, and membrane tension, presenting illustrative examples of experimental evidence in support of these models. The picture that emerges from recent work is one where more than one mechanism is in play. Which mechanism prevails depends on the protein, its aggregation state, and the lipid environment in which the interactions occur. © 2013.

Protein-lipid interactions at the air/water interface.

PubMed

Lad, Mitaben D; Birembaut, Fabrice; Frazier, Richard A; Green, Rebecca J

2005-10-07

Surface pressure measurements and external reflection FTIR spectroscopy have been used to probe protein-lipid interactions at the air/water interface. Spread monomolecular layers of stearic acid and phosphocholine were prepared and held at different compressed phase states prior to the introduction of protein to the buffered subphase. Contrasting interfacial behaviour of the proteins, albumin and lysozyme, was observed and revealed the role of both electrostatic and hydrophobic interactions in protein adsorption. The rate of adsorption of lysozyme to the air/water interface increased dramatically in the presence of stearic acid, due to strong electrostatic interactions between the negatively charged stearic acid head group and lysozyme, whose net charge at pH 7 is positive. Introduction of albumin to the subphase resulted in solubilisation of the stearic acid via the formation of an albumin-stearic acid complex and subsequent adsorption of albumin. This observation held for both human and bovine serum albumin. Protein adsorption to a PC layer held at low surface pressure revealed adsorption rates similar to adsorption to the bare air/water interface and suggested very little interaction between the protein and the lipid. For PC layers in their compressed phase state some adsorption of protein occurred after long adsorption times. Structural changes of both lysozyme and albumin were observed during adsorption, but these were dramatically reduced in the presence of a lipid layer compared to that of adsorption to the pure air/water interface.

Modulating protein adsorption onto hydroxyapatite particles using different amino acid treatments

PubMed Central

Lee, Wing-Hin; Loo, Ching-Yee; Van, Kim Linh; Zavgorodniy, Alexander V.; Rohanizadeh, Ramin

2012-01-01

Hydroxyapatite (HA) is a material of choice for bone grafts owing to its chemical and structural similarities to the mineral phase of hard tissues. The combination of osteogenic proteins with HA materials that carry and deliver the proteins to the bone-defective areas will accelerate bone regeneration. The study investigated the treatment of HA particles with different amino acids such as serine (Ser), asparagine (Asn), aspartic acid (Asp) and arginine (Arg) to enhance the adsorption ability of HA carrier for delivering therapeutic proteins to the body. The crystallinity of HA reduced when amino acids were added during HA preparation. Depending on the types of amino acid, the specific surface area of the amino acid-functionalized HA particles varied from 105 to 149 m2 g–1. Bovine serum albumin (BSA) and lysozyme were used as model proteins for adsorption study. The protein adsorption onto the surface of amino acid-functionalized HA depended on the polarities of HA particles, whereby, compared with lysozyme, BSA demonstrated higher affinity towards positively charged Arg-HA. Alternatively, the binding affinity of lysozyme onto the negatively charged Asp-HA was higher when compared with BSA. The BSA and lysozyme adsorptions onto the amino acid-functionalized HA fitted better into the Freundlich than Langmuir model. The amino acid-functionalized HA particles that had higher protein adsorption demonstrated a lower protein-release rate. PMID:21957116

Investigation of ultrahigh sensitivity in GaInAsP nanolaser biosensor

NASA Astrophysics Data System (ADS)

Saijo, Yoshito; Watanabe, Takumi; Hasegawa, Yu; Nishijima, Yoshiaki; Baba, Toshihiko

2018-02-01

We have developed GaInAsP semiconductor photonic crystal nanolaser biosensor and demonstrated the detection of ultralow-concentration (fM to aM) proteins and deoxyribonucleic acids (DNAs) adsorbed on the device surface. In general, this type of photonic sensors exploiting optical resonance has been considered to detect the refractive index of biomolecules via the wavelength shift. However, this principle cannot explain the detection of such ultralowconcentration. Therefore, we investigated another candidate principle, i.e., ion sensitivity. We consider such a process that 1) the electric charge of biomolecules changes the nanolaser's surface charge, 2) the Schottky barrier near the semiconductor surface is increased or decreased, 3) the distribution of photopumped carriers is modified by the barrier, 4) the refractive index of the semiconductor is changed by the carrier effects, and 5) the laser wavelength shifts. To confirm this process, we electrochemically measured the zeta and flatband potentials when charged electrolyte polymers were adsorbed in water. We clearly observed that these potentials temporally behaved consistently with that of the laser wavelength, which suggests that polymers significantly acted on the Schottky barrier. The same behaviors were also observed for the adsorption of 1 fM DNA. We consider that a limited number of charged DNA changed the surface functional group of the entire device surface. Such charge effects will be the key that achieves the ultrahigh sensitivity in the nanolaser biosensor.

Novel computational approach for studying ph effects, excluded volume and ion-ion correlations in electrical double layers around polyelectrolytes and nanoparticles

NASA Astrophysics Data System (ADS)

Ovanesyan, Zaven

Highly charged cylindrical and spherical objects (macroions) are probably the simplest structures for modeling nucleic acids, proteins and nanoparticles. Their ubiquitous presence within biophysical systems ensures that Coulomb forces are among the most important interactions that regulate the behavior of these systems. In these systems, ions position themselves in a strongly correlated manner near the surface of a macroion and form electrical double layers (EDLs). These EDLs play an important role in many biophysical and biochemical processes. For instance, the macroion's net charge can change due to the binding of many multivalent ions to its surface. Thus, proper description of EDLs near the surface of a macroion may reveal a counter-intuitive charge inversion behavior, which can generate attraction between like-charged objects. This is relevant for the variety of fields such as self-assembly of DNA and RNA folding, as well as for protein aggregation and neurodegenerative diseases. Certainly, the key factors that contribute to these phenomena cannot be properly understood without an accurate solvation model. With recent advancements in computer technologies, the possibility to use computational tools for fundamental understanding of the role of EDLs around biomolecules and nanoparticles on their physical and chemical properties is becoming more feasible. Establishing the impact of the excluded volume and ion-ion correlations, ionic strength and pH of the electrolyte on the EDL around biomolecules and nanoparticles, and how changes in these properties consequently affect the Zeta potential and surface charge density are still not well understood. Thus, modeling and understanding the role of these properties on EDLs will provide more insights on the stability, adsorption, binding and function of biomolecules and nanoparticles. Existing mean-field theories such as Poisson Boltzmann (PB) often neglect the ion-ion correlations, solvent and ion excluded volume effects, which are important details for proper description of EDL properties. In this thesis, we implement an efficient and accurate classical solvation density functional theory (CDSFT) for EDLs of spherical macroions and cylindrical polyelectrolytes embedded in aqueous electrolytes. This approach extends the capabilities of mean field approximations by taking into account electrostatic ion-ion correlations, size asymmetry and excluded volume effects without compromising the computational cost. We apply the computational tool to study the structural and thermodynamic properties of the ionic atmosphere around B-DNA and spherical nanoparticles. We demonstrate that the presence of solvent molecules at experimental concentration and size values has a significant impact on the layering of ions. This layering directly influences the integrated charge and mean electrostatic potential in the diffuse region of the spherical electrical double layer (SEDL) and have a noticeable impact on the behavior of zeta potential (ZP). Recently, we have extended the aforementioned CSDFT to account for the charge-regulated mechanisms of the macroion surface on the structural and thermodynamic properties of spherical EDLs. In the approach, the CSDFT is combined with a surface complexation model to account for ion correlation and excluded volume effects on the surface titration of spherical macroions. We apply the proposed computational approach to describe the role that the ion size and solvent excluded volume play on the surface titration properties of silica nanoparticles. We analyze the effects of the nanoparticle size, pH and salt concentration of the aqueous solution on the nanoparticle's surface charge and zeta potential. The results reveal that surface charge density and zeta potential significantly depend on excluded volume and ion-ion correlation effects as well as on pH for monovalent ion species at high salt concentrations. Overall, our results are in good agreement with Monte Carlo simulations and available experimental data. We discuss future directions of this work, which includes extension of the solvation model for studying the flexibility properties of rigid peptides and globular proteins, and describes benefits that this research can potentially bring to scientific and non scientific communities.

Nonlocal continuum electrostatic theory predicts surprisingly small energetic penalties for charge burial in proteins.

PubMed

Bardhan, Jaydeep P

2011-09-14

We study the energetics of burying charges, ion pairs, and ionizable groups in a simple protein model using nonlocal continuum electrostatics. Our primary finding is that the nonlocal response leads to markedly reduced solvent screening, comparable to the use of application-specific protein dielectric constants. Employing the same parameters as used in other nonlocal studies, we find that for a sphere of radius 13.4 Å containing a single +1e charge, the nonlocal solvation free energy varies less than 18 kcal/mol as the charge moves from the surface to the center, whereas the difference in the local Poisson model is ∼35 kcal/mol. Because an ion pair (salt bridge) generates a comparatively more rapidly varying Coulomb potential, energetics for salt bridges are even more significantly reduced in the nonlocal model. By varying the central parameter in nonlocal theory, which is an effective length scale associated with correlations between solvent molecules, nonlocal-model energetics can be varied from the standard local results to essentially zero; however, the existence of the reduction in charge-burial penalties is quite robust to variations in the protein dielectric constant and the correlation length. Finally, as a simple exploratory test of the implications of nonlocal response, we calculate glutamate pK(a) shifts and find that using standard protein parameters (ε(protein) = 2-4), nonlocal results match local-model predictions with much higher dielectric constants. Nonlocality may, therefore, be one factor in resolving discrepancies between measured protein dielectric constants and the model parameters often used to match titration experiments. Nonlocal models may hold significant promise to deepen our understanding of macromolecular electrostatics without substantially increasing computational complexity. © 2011 American Institute of Physics

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment.

PubMed

Austerberry, James I; Dajani, Rana; Panova, Stanislava; Roberts, Dorota; Golovanov, Alexander P; Pluen, Alain; van der Walle, Christopher F; Uddin, Shahid; Warwicker, Jim; Derrick, Jeremy P; Curtis, Robin

2017-06-01

The aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations. Copyright © 2017. Published by Elsevier B.V.

The importance of electrostatic potential in the interaction of sweet proteins with the sweet taste receptor.

PubMed

Esposito, Veronica; Gallucci, Roberta; Picone, Delia; Saviano, Gabriella; Tancredi, Teodorico; Temussi, Piero A

2006-07-07

In addition to many small molecular mass sweeteners there are in nature a few sweet proteins. The molecular volume of sweet proteins is so different from that of common sweeteners that it was difficult to understand how molecules as large as proteins can activate a receptor designed to host small molecules. We have recently shown that sweet proteins can activate the sweet receptor by a mechanism of interaction, called ''wedge model", in which proteins fit a large cavity of the receptor with wedge-shaped surfaces of their structures. In order to substantiate this model we have designed, expressed and characterized seven mutants of MNEI, a single chain monellin. Three uncharged residues of the interaction surface, Met42, Tyr63 and Tyr65, were changed either into acidic or basic residues whereas Asp68, a key acidic residue, was changed into a basic one. As a general trend, we observe that an increase of the negative charge is much more detrimental for sweetness than an increase of positive charge. In addition we show that by a careful choice of a residue at the center of the interface between MNEI and receptor, it is possible even to increase the sweetness of MNEI. These results are fully consistent with the wedge model.

Evolutionary conservativeness of electric field in the Cu,Zn superoxide dismutase active site. Evidence for co-ordinated mutation of charged amino acid residues.

PubMed

Desideri, A; Falconi, M; Polticelli, F; Bolognesi, M; Djinovic, K; Rotilio, G

1992-01-05

Equipotential lines were calculated, using the Poisson-Boltzmann equation, for six Cu,Zn superoxide dismutases with different protein electric charge and various degrees of sequence homology, namely those from ox, pig, sheep, yeast, and the isoenzymes A and B from the amphibian Xenopus laevis. The three-dimensional structures of the porcine and ovine superoxide dismutases were obtained by molecular modelling reconstruction using the structure of the highly homologous bovine enzyme as a template. The three-dimensional structure of the evolutionary distant yeast Cu,Zn superoxide dismutase was recently resolved by us, while computer-modelled structures are available for X. laevis isoenzymes. The six proteins display large differences in the net protein charge and distribution of electrically charged surface residues but the trend of the equipotential lines in the proximity of the active sites was found to be constant in all cases. These results are in line with the very similar catlytic rate constants experimentally measured for the corresponding enzyme activities. This analysis shows that electrostatic guidance for the enzyme-substrate interaction in Cu,Zn superoxide dismutases is related to a spatial distribution of charges, arranged so as to maintain, in the area surrounding the active sites, an identical electrostatic potential distribution, which is conserved in the evolution of this protein family.

Microbead-based immunoassay using the outer membrane layer of Escherichia coli combined with autodisplayed Z-domains

NASA Astrophysics Data System (ADS)

Kim, Do-Hoon; Bong, Ji-Hong; Yoo, Gu; Chang, Seo-Yoon; Park, Min; Chang, Young Wook; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2016-01-01

The Z-domain has the potential to control the orientation of immobilized antibodies because of its binding affinity to the Fc regions of antibodies (IgGs). In this work, Z-domains were autodisplayed on the outer membrane (OM) of Escherichia coli. OM particles were isolated and coated onto microbeads with positive, neutral, or negative surface charges. Other conditions such as incubation time and initial OM concentration were also optimized for the OM coating to obtain maximum antibody-binding. Using three kinds of model proteins with different isoelectric points (pI), streptavidin (pI = 5, negative charge at pH 7), horseradish peroxidase (pI = 7, neutral charge at pH 7), and avidin (pI = 10, positive charge at pH 7), protein immobilization onto the microbeads was carried out through physical adsorption and electrostatic interactions. Using fluorescently labeled antibodies and fluorescence-activated cell sorting, it was determined that the neutral and the positively charged microbeads effectively bound antibodies while minimizing non-specific protein binding. The OM-coated microbeads with autodisplayed Z-domains were applied to C-reactive protein immunoassay. This immunoassay achieved 5-fold improved sensitivity compared to conventional immunoassay based on physical adsorption of antibodies at the cutoff concentration of medical diagnosis of inflammatory diseases (1000 ng/ml) and cardiovascular diseases (200 ng/ml).

Lysine-based polycation:heparin coacervate for controlled protein delivery.

PubMed

Johnson, Noah Ray; Ambe, Trisha; Wang, Yadong

2014-01-01

Polycations have good potential as carriers of proteins and genetic material. However, poor control over the release rate and safety issues currently limit their use as delivery vehicles. Here we introduce a new lysine-based polycation, poly(ethylene lysinylaspartate diglyceride) (PELD), which exhibits high cytocompatibility. PELD self-assembles with the biological polyanion heparin into a coacervate that incorporates proteins with high loading efficiency. Coacervates of varying surface charge were obtained by simple alteration of the PELD:heparin ratio and resulted in diverse release profiles of the model protein bovine serum albumin. Therefore, coacervate charge represents a direct means of control over release rate and duration. The PELD coacervate also rapidly adsorbed onto a porous polymeric scaffold, demonstrating potential use in tissue engineering applications. This coacervate represents a safe and tunable protein delivery system for biomedical applications. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Structure and assembly of scalable porous protein cages

NASA Astrophysics Data System (ADS)

Sasaki, Eita; Böhringer, Daniel; van de Waterbeemd, Michiel; Leibundgut, Marc; Zschoche, Reinhard; Heck, Albert J. R.; Ban, Nenad; Hilvert, Donald

2017-03-01

Proteins that self-assemble into regular shell-like polyhedra are useful, both in nature and in the laboratory, as molecular containers. Here we describe cryo-electron microscopy (EM) structures of two versatile encapsulation systems that exploit engineered electrostatic interactions for cargo loading. We show that increasing the number of negative charges on the lumenal surface of lumazine synthase, a protein that naturally assembles into a ~1-MDa dodecahedron composed of 12 pentamers, induces stepwise expansion of the native protein shell, giving rise to thermostable ~3-MDa and ~6-MDa assemblies containing 180 and 360 subunits, respectively. Remarkably, these expanded particles assume unprecedented tetrahedrally and icosahedrally symmetric structures constructed entirely from pentameric units. Large keyhole-shaped pores in the shell, not present in the wild-type capsid, enable diffusion-limited encapsulation of complementarily charged guests. The structures of these supercharged assemblies demonstrate how programmed electrostatic effects can be effectively harnessed to tailor the architecture and properties of protein cages.

Structural Study of the C-Terminal Domain of Nonstructural Protein 1 from Japanese Encephalitis Virus.

PubMed

Poonsiri, Thanalai; Wright, Gareth S A; Diamond, Michael S; Turtle, Lance; Solomon, Tom; Antonyuk, Svetlana V

2018-04-01

Japanese encephalitis virus (JEV) is a mosquito-transmitted flavivirus that is closely related to other emerging viral pathogens, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout Southeast Asia, the Pacific Islands, and the Far East, causing around 68,000 cases of infection worldwide each year. In this report, we present a 2.1-Å-resolution crystal structure of the C-terminal β-ladder domain of JEV nonstructural protein 1 (NS1-C). The surface charge distribution of JEV NS1-C is similar to those of WNV and ZIKV but differs from that of DENV. Analysis of the JEV NS1-C structure, with in silico molecular dynamics simulation and experimental solution small-angle X-ray scattering, indicates extensive loop flexibility on the exterior of the protein. This, together with the surface charge distribution, indicates that flexibility influences the protein-protein interactions that govern pathogenicity. These factors also affect the interaction of NS1 with the 22NS1 monoclonal antibody, which is protective against West Nile virus infection. Liposome and heparin binding assays indicate that only the N-terminal region of NS1 mediates interaction with membranes and that sulfate binding sites common to NS1 structures are not glycosaminoglycan binding interfaces. This report highlights several differences between flavivirus NS1 proteins and contributes to our understanding of their structure-pathogenic function relationships. IMPORTANCE JEV is a major cause of viral encephalitis in Asia. Despite extensive vaccination, epidemics still occur. Nonstructural protein 1 (NS1) plays a role in viral replication, and, because it is secreted, it can exhibit a wide range of interactions with host proteins. NS1 sequence and protein folds are conserved within the Flavivirus genus, but variations in NS1 protein-protein interactions among viruses likely contribute to differences in pathogenesis. Here, we compared characteristics of the C-terminal β-ladder domain of NS1 between flaviviruses, including surface charge, loop flexibility, epitope cross-reactivity, membrane adherence, and glycosaminoglycan binding. These structural features are central to NS1 functionality and may provide insight into the development of diagnostic tests and therapeutics. Copyright © 2018 American Society for Microbiology.

Library of Antifouling Surfaces Derived From Natural Amino Acids by Click Reaction.

PubMed

Xu, Chen; Hu, Xin; Wang, Jie; Zhang, Ye-Min; Liu, Xiao-Jiu; Xie, Bin-Bin; Yao, Chen; Li, Yi; Li, Xin-Song

2015-08-12

Biofouling is of great concern in numerous applications ranging from ophthalmological implants to catheters, and from bioseparation to biosensors. In this report, a general and facile strategy to combat surface fouling is developed by grafting of amino acids onto polymer substrates to form zwitterionic structure through amino groups induced epoxy ring opening click reaction. First of all, a library of poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) hydrogels with zwitterionic surfaces were prepared, resulting in the formation of pairs of carboxyl anions and protonated secondary amino cations. The analysis of attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy confirmed the successful immobilization of amino acids on the hydrogel surfaces. After that, the contact angle and equilibrium water content of the modified hydrogels showed that the hydrogels exhibited improved hydrophilicity compared with the parent hydrogel. Furthermore, the protein deposition was evaluated by bicinchoninic acid assay using bovine serum albumin (BSA) and lysozyme as models. The results indicated that the performance of the hydrogels was determined by the nature of incorporated amino acid: the hydrogels incorporated with neutral amino acids had nonspecific antiadsorption capability to both BSA and lysozyme; the hydrogels incorporated with charged amino acids showed antiadsorption behaviors against protein with same charge and enhanced adsorption to the protein with opposite charge; the optimal antiadsorption performance was observed on the hydrogels incorporated with polar amino acids with a hydroxyl residual. The improvement of antiprotein fouling of the neutral amino acids grafted hydrogels can be ascribed to the formation of zwitterionic surfaces. Finally, a couple of soft contact lenses grafted with amino acids were fabricated having improved antifouling property and hydrophilicity. The result demonstrated the success of amino acids based zwitterionic antifouling strategy in ophthalmology. This strategy is also applicable to substrates including filtration membranes, microspheres and nanofibers as well. It is a versatile method for amino acids grafting onto polymer substrates to construct zwitterionic surfaces and achieve antifouling properties.

Modified interactions among globular proteins below isoelectric point in the presence of mono-, di- and tri-valent ions: A small angle neutron scattering study

NASA Astrophysics Data System (ADS)

Das, Kaushik; Kundu, Sarathi; Mehan, Sumit; Aswal, V. K.

2016-02-01

Both short range attraction and long range electrostatic repulsion exist among globular protein Bovine Serum Albumin in solution below its isoelectric point (pI ≈ 4.8). At pD ≈ 4.0, below pI, protein has a net positive surface charge although local charge inhomogeneity presents. Small angle neutron scattering study reveals that in the presence of both mono-(Na+) and di-(Ni2+) valent ions attractive interaction increases and repulsive interaction decreases with the increase of salt concentration. However, for tri-valent (Fe3+) ions, both attractive and repulsive interaction increases with increasing salt concentration but the relative strength of repulsion is more than the attraction.

Enthalpic Breakdown of Water Structure on Protein Active-Site Surfaces

PubMed Central

Haider, Kamran; Wickstrom, Lauren; Ramsey, Steven; Gilson, Michael K.; Kurtzman, Tom

2016-01-01

The principles underlying water reorganization around simple non-polar solutes are well understood and provide the framework for classical hydrophobic effect, whereby water molecules structure themselves around solutes so that they maintain favorable energetic contacts with both the solute and with other water molecules. However, for certain solute surface topographies, water molecules, due to their geometry and size, are unable to simultaneously maintain favorable energetic contacts with both the surface and neighboring water molecules. In this study, we analyze the solvation of ligand-binding sites for six structurally diverse proteins using hydration site analysis and measures of local water structure, in order to identify surfaces at which water molecules are unable to structure themselves in a way that maintains favorable enthalpy relative to bulk water. These surfaces are characterized by a high degree of enclosure, weak solute-water interactions, and surface constraints that induce unfavorable pair interactions between neighboring water molecules. Additionally, we find that the solvation of charged side-chains in an active site generally results in favorable enthalpy but can also lead to pair interactions between neighboring water molecules that are significantly unfavorable relative to bulk water. We find that frustrated local structure can occur not only in apolar and weakly polar pockets, where overall enthalpy tends to be unfavorable, but also in charged pockets, where overall water enthalpy tends to be favorable. The characterization of local water structure in these terms may prove useful for evaluating the displacement of water from diverse protein active-site environments. PMID:27169482

Ribosome surface properties may impose limits on the nature of the cytoplasmic proteome

PubMed Central

2017-01-01

Much of the molecular motion in the cytoplasm is diffusive, which possibly limits the tempo of processes. We studied the dependence of protein mobility on protein surface properties and ionic strength. We used surface-modified fluorescent proteins (FPs) and determined their translational diffusion coefficients (D) in the cytoplasm of Escherichia coli, Lactococcus lactis and Haloferax volcanii. We find that in E. coli D depends on the net charge and its distribution over the protein, with positive proteins diffusing up to 100-fold slower than negative ones. This effect is weaker in L. lactis and Hfx. volcanii due to electrostatic screening. The decrease in mobility is probably caused by interaction of positive FPs with ribosomes as shown in in vivo diffusion measurements and confirmed in vitro with purified ribosomes. Ribosome surface properties may thus limit the composition of the cytoplasmic proteome. This finding lays bare a paradox in the functioning of prokaryotic (endo)symbionts. PMID:29154755

Hemocompatible control of sulfobetaine-grafted polypropylene fibrous membranes in human whole blood via plasma-induced surface zwitterionization.

PubMed

Chen, Sheng-Han; Chang, Yung; Lee, Kueir-Rarn; Wei, Ta-Chin; Higuchi, Akon; Ho, Feng-Ming; Tsou, Chia-Chun; Ho, Hsin-Tsung; Lai, Juin-Yih

2012-12-21

In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood.

Proteins at the Biomaterial Electrolyte Interface

NASA Astrophysics Data System (ADS)

Tengvall, Pentti

2005-03-01

Proteins adsorb rapidly onto solid and polymeric surfaces because the association process is in the vast majority of cases energetically favourable, i.e. exothermic. The most common exceptions to this rule are hydrophilic interfaces with low net charge and high mobility, e.g. immobilized PEGs. Current research in the research area tries to understand and control unwanted and wanted adsorption by studying the adsorption kinetics, protein surface binding specificity, protein exchange at interfaces, and surface protein repulsion mechanisms. In blood plasma model systems humoral cascade reactions such as surface mediated coagulation and immune complement raise considerable interest due to the immediate association to blood compatibility, and in tissue applications the binding between surfaces and membrane receptors in cells and tissues. Thus, the understanding of interfacial events at the protein level is of large importance in applications such as blood and tissue contacting biomaterials, in vitro medical and biological diagnostics, food industry and in marine anti-fouling technology. Well described consequences of adsorption are a lowered system energy, increased system entropy, irreversible binding, conformational changes, specific surface/protein interactions, and in biomedical materials applications surface opsonization followed by cell-surface interactions and a host tissue response. This lecture will deal with some mechanisms known to be of importance for the adsorption processes, such as the influence of surface chemistry and surface energy, the composition of the protein solution, the Vroman effect, and residence time. Examples will be shown from ellipsometric experiments using different model surfaces in single/few protein solutions, and specific attention be given to blood serum and plasma experiments on coagulation and immune complement at interfaces.

An ultrasensitive universal detector based on neutralizer displacement

NASA Astrophysics Data System (ADS)

Das, Jagotamoy; Cederquist, Kristin B.; Zaragoza, Alexandre A.; Lee, Paul E.; Sargent, Edward H.; Kelley, Shana O.

2012-08-01

Diagnostic technologies that can provide the simultaneous detection of nucleic acids for gene expression, proteins for host response and small molecules for profiling the human metabolome will have a significant advantage in providing comprehensive patient monitoring. Molecular sensors that report changes in the electrostatics of a sensor's surface on analyte binding have shown unprecedented sensitivity in the detection of charged biomolecules, but do not lend themselves to the detection of small molecules, which do not carry significant charge. Here, we introduce the neutralizer displacement assay that allows charge-based sensing to be applied to any class of molecule irrespective of the analyte charge. The neutralizer displacement assay starts with an aptamer probe bound to a neutralizer. When analyte binding occurs the neutralizer is displaced, which results in a dramatic change in the surface charge for all types of analytes. We have tested the sensitivity, speed and specificity of this system in the detection of a panel of molecules: (deoxy)ribonucleic acid, ribonucleic acid, cocaine, adenosine triphosphate and thrombin.

Decreased bacteria activity on Si3N4 surfaces compared with PEEK or titanium

PubMed Central

Gorth, Deborah J; Puckett, Sabrina; Ercan, Batur; Webster, Thomas J; Rahaman, Mohamed; Bal, B Sonny

2012-01-01

A significant need exists for orthopedic implants that can intrinsically resist bacterial colonization. In this study, three biomaterials that are used in spinal implants – titanium (Ti), polyether-ether-ketone (PEEK), and silicon nitride (Si3N4) – were tested to understand their respective susceptibility to bacterial infection with Staphylococcus epidermidis, Staphlococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus. Specifically, the surface chemistry, wettability, and nanostructured topography of respective biomaterials, and the effects on bacterial biofilm formation, colonization, and growth were investigated. Ti and PEEK were received with as-machined surfaces; both materials are hydrophobic, with net negative surface charges. Two surface finishes of Si3N4 were examined: as-fired and polished. In contrast to Ti and PEEK, the surface of Si3N4 is hydrophilic, with a net positive charge. A decreased biofilm formation was found, as well as fewer live bacteria on both the as-fired and polished Si3N4. These differences may reflect differential surface chemistry and surface nanostructure properties between the biomaterials tested. Because protein adsorption on material surfaces affects bacterial adhesion, the adsorption of fibronectin, vitronectin, and laminin on Ti, PEEK, and Si3N4 were also examined. Significantly greater amounts of these proteins adhered to Si3N4 than to Ti or PEEK. The findings of this study suggest that surface properties of biomaterials lead to differential adsorption of physiologic proteins, and that this phenomenon could explain the observed in-vitro differences in bacterial affinity for the respective biomaterials. Intrinsic biomaterial properties as they relate to resistance to bacterial colonization may reflect a novel strategy toward designing future orthopedic implants. PMID:22973102

Denaturation of proteins near polar surfaces

NASA Astrophysics Data System (ADS)

Starzyk, Anna; Cieplak, Marek

2011-12-01

All-atom molecular dynamics simulations for proteins placed near a model mica surface indicate existence of two types of evolution. One type leads to the surface-induced unfolding and the other just to a deformation. The two behaviors are characterized by distinct properties of the radius of gyration and of a novel distortion parameter that distinguishes between elongated, globular, and planar shapes. They also differ in the nature of their single site diffusion and two-site distance fluctuations. The four proteins chosen for the studies, the tryptophan cage, protein G, hydrophobin and lyzozyme, are small to allow for a fair determination of the forces generated by the surface as the effects of finite cutoffs in the Coulombic interactions are thus minimized. When the net charge on the surface is set to zero artificially, infliction of deformation is seen to persists but no unfolding takes place. Unfolding may also be prevented by a cluster of disulfide bonds, as we observe in simulations of hydrophobin.

Nanowell-Trapped Charged Ligand-Bearing Nanoparticle Surfaces – A Novel Method of Enhancing Flow-Resistant Cell Adhesion

PubMed Central

Tran, Phat L.; Gamboa, Jessica R.; McCracken, Katherine E.; Riley, Mark R.

2014-01-01

Assuring cell adhesion to an underlying biomaterial surface is vital in implant device design and tissue engineering, particularly under circumstances where cells are subjected to potential detachment from overriding fluid flow. Cell-substrate adhesion is a highly regulated process involving the interplay of mechanical properties, surface topographic features, electrostatic charge, and biochemical mechanisms. At the nanoscale level the physical properties of the underlying substrate are of particular importance in cell adhesion. Conventionally, natural, pro-adhesive, and often thrombogenic, protein biomaterials are frequently utilized to facilitate adhesion. In the present study nanofabrication techniques are utilized to enhance the biological functionality of a synthetic polymer surface, polymethymethacrylate, with respect to cell adhesion. Specifically we examine the effect on cell adhesion of combining: 1. optimized surface texturing, 2. electrostatic charge and 3. cell adhesive ligands, uniquely assembled on the substrata surface, as an ensemble of nanoparticles trapped in nanowells. Our results reveal that the ensemble strategy leads to enhanced, more than simply additive, endothelial cell adhesion under both static and flow conditions. This strategy may be of particular utility for enhancing flow-resistant endothelialization of blood-contacting surfaces of cardiovascular devices subjected to flow-mediated shear. PMID:23225491

Tuning structure of oppositely charged nanoparticle and protein complexes

NASA Astrophysics Data System (ADS)

Kumar, Sugam; Aswal, V. K.; Callow, P.

2014-04-01

Small-angle neutron scattering (SANS) has been used to probe the structures of anionic silica nanoparticles (LS30) and cationic lyszyme protein (M.W. 14.7kD, I.P. ˜ 11.4) by tuning their interaction through the pH variation. The protein adsorption on nanoparticles is found to be increasing with pH and determined by the electrostatic attraction between two components as well as repulsion between protein molecules. We show the strong electrostatic attraction between nanoparticles and protein molecules leads to protein-mediated aggregation of nanoparticles which are characterized by fractal structures. At pH 5, the protein adsorption gives rise to nanoparticle aggregation having surface fractal morphology with close packing of nanoparticles. The surface fractals transform to open structures of mass fractal morphology at higher pH (7 and 9) on approaching isoelectric point (I.P.).

Proteins feel more than they see: fine-tuning of binding affinity by properties of the non-interacting surface.

PubMed

Kastritis, Panagiotis L; Rodrigues, João P G L M; Folkers, Gert E; Boelens, Rolf; Bonvin, Alexandre M J J

2014-07-15

Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct contributions from the interface, the non-interacting surface (NIS) also plays an important role in binding affinity, especially polar and charged residues. Their percentage on the NIS is conserved over orthologous complexes indicating an evolutionary selection pressure. Their effect on binding affinity can be explained by long-range electrostatic contributions and surface-solvent interactions that are known to determine the local frustration of the protein complex surface. Including these in a simple model significantly improves the affinity prediction of protein complexes from structural models. The impact of mutations outside the interacting surface on binding affinity is supported by experimental alanine scanning mutagenesis data. These results enable the development of more sophisticated and integrated biophysical models of binding affinity and open new directions in experimental control and modulation of biomolecular interactions. Copyright © 2014. Published by Elsevier Ltd.

Photosystem I assembly on chemically tailored SAM/ Au substrates for bio-hybrid device fabrication

NASA Astrophysics Data System (ADS)

Mukherjee, Dibyendu; Khomami, Bamin

2011-03-01

Photosystem I (PS I), a supra-molecular protein complex and a biological photodiode responsible for driving natural photosynthesis mechanism, charge separates upon exposure to light. Effective use of the photo-electrochemical activities of PS I for future bio-hybrid electronic devices requires controlled attachment of these proteins onto organic/ inorganic substrates. Our results indicate that various experimental parameters alter the surface topography of PS I deposited from colloidal aqueous buffer suspensions onto OH-terminated alkanethiolate SAM /Au substrates, thereby resulting in complex columnar structures that affect the electron capture pathway of PS I. Specifically, solution phase characterizations indicate that specific detergents used for PS I stabilization in buffer solutions drive the unique colloidal chemistry to tune protein-protein interactions and prevent aggregation, thereby allowing us to tailor the morphology of surface immobilized PS I. We present surface topographical, adsorption, and electrochemical characterizations of PSI /SAM/Au substrates to elucidate protein-surface attachment dynamics and its effect on the photo-activated electronic activities of surface immobilized PS I. Sustainable Energy Education and Research Center (SEERC).

Flexible Charged Macromolecules on Mixed Fluid Lipid Membranes: Theory and Monte Carlo Simulations

PubMed Central

Tzlil, Shelly; Ben-Shaul, Avinoam

2005-01-01

Fluid membranes containing charged lipids enhance binding of oppositely charged proteins by mobilizing these lipids into the interaction zone, overcoming the concomitant entropic losses due to lipid segregation and lower conformational freedom upon macromolecule adsorption. We study this energetic-entropic interplay using Monte Carlo simulations and theory. Our model system consists of a flexible cationic polyelectrolyte, interacting, via Debye-Hückel and short-ranged repulsive potentials, with membranes containing neutral lipids, 1% tetravalent, and 10% (or 1%) monovalent anionic lipids. Adsorption onto a fluid membrane is invariably stronger than to an equally charged frozen or uniform membrane. Although monovalent lipids may suffice for binding rigid macromolecules, polyvalent counter-lipids (e.g., phosphatidylinositol 4,5 bisphosphate), whose entropy loss upon localization is negligible, are crucial for binding flexible macromolecules, which lose conformational entropy upon adsorption. Extending Rosenbluth's Monte Carlo scheme we directly simulate polymer adsorption on fluid membranes. Yet, we argue that similar information could be derived from a biased superposition of quenched membrane simulations. Using a simple cell model we account for surface concentration effects, and show that the average adsorption probabilities on annealed and quenched membranes coincide at vanishing surface concentrations. We discuss the relevance of our model to the electrostatic-switch mechanism of, e.g., the myristoylated alanine-rich C kinase substrate protein. PMID:16126828

Electrostatic and dispersion interactions during protein adsorption on topographic nanostructures.

PubMed

Elter, Patrick; Lange, Regina; Beck, Ulrich

2011-07-19

Recently, biomaterials research has focused on developing functional implant surfaces with well-defined topographic nanostructures in order to influence protein adsorption and cellular behavior. To enhance our understanding of how proteins interact with such surfaces, we analyze the adsorption of lysozyme on an oppositely charged nanostructure using a computer simulation. We present an algorithm that combines simulated Brownian dynamics with numerical field calculation methods to predict the preferred adsorption sites for arbitrarily shaped substrates. Either proteins can be immobilized at their initial adsorption sites or surface diffusion can be considered. Interactions are analyzed on the basis of Derjaguin-Landau-Verway-Overbeek (DLVO) theory, including electrostatic and London dispersion forces, and numerical solutions are derived using the Poisson-Boltzmann and Hamaker equations. Our calculations show that for a grooved nanostructure (i.e., groove and plateau width 8 nm, height 4 nm), proteins first contact the substrate primarily near convex edges because of better geometric accessibility and increased electric field strengths. Subsequently, molecules migrate by surface diffusion into grooves and concave corners, where short-range dispersion interactions are maximized. In equilibrium, this mechanism leads to an increased surface protein concentration in the grooves, demonstrating that the total amount of protein per surface area can be increased if substrates have concave nanostructures.

Attachment dynamics of Photosystem I on nano-tailored surfaces for photovoltaic applications

NASA Astrophysics Data System (ADS)

Mukherjee, Dibyendu; Bruce, Barry D.; Khomami, Bamin

2010-03-01

Photosystem I (PSI), a biological photodiode, is a supra-molecular protein complex that charge separates upon exposure to light. Effective use of photo-electrochemical activities of PSI for hybrid photovoltaic (PV) device fabrications requires optimal encapsulation of these proteins onto organic/ inorganic substrates. Our results indicate that various experimental parameters alter the surface attachment dynamics of PSI deposited from colloidal aqueous buffer suspensions onto OH-terminated alkanethiolate/Au SAM substrates, thereby resulting in complex structural arrangements which affect the electron transfer and capture pathway of PSI. We present surface topographical, specific adsorption and polarization fluorescence characterizations of PSI/Au SAM substrates to elucidate the protein-surface interaction kinetics as well as the directional attachment dynamics of PSI. Our final goal is to enable site-specific homogeneous attachment of directionally aligned PSI onto chemically tailored nano-patterned substrates.

Perlinhibin, a Cysteine-, Histidine-, and Arginine-Rich Miniprotein from Abalone (Haliotis laevigata) Nacre, Inhibits In Vitro Calcium Carbonate Crystallization

PubMed Central

Mann, Karlheinz; Siedler, Frank; Treccani, Laura; Heinemann, Fabian; Fritz, Monika

2007-01-01

We have isolated a 4.785 Da protein from the nacreous layer of the sea snail Haliotis laevigata (greenlip abalone) shell after demineralization with acetic acid. The sequence of 41 amino acids was determined by Edman degradation supported by mass spectrometry. The most abundant amino acids were cysteine (19.5%), histidine (17%), and arginine (14.6%). The positively charged amino acids were almost counterbalanced by negatively charged ones resulting in a calculated isoelectric point of 7.86. Atomic-force microscopy studies of the interaction of the protein with calcite surfaces in supersaturated calcium carbonate solution or calcium chloride solution showed that the protein bound specifically to calcite steps, inhibiting further crystal growth at these sites in carbonate solution and preventing crystal dissolution when carbonate was substituted with chloride. Therefore this protein was named perlinhibin. X-ray diffraction investigation of the crystal after atomic-force microscopy growth experiments showed that the formation of aragonite was induced on the calcite substrate around holes caused by perlinhibin crystal-growth inhibition. The strong interaction of the protein with calcium carbonate was also shown by vapor diffusion crystallization. In the presence of the protein, the crystal surfaces were covered with holes due to protein binding and local inhibition of crystal growth. In addition to perlinhibin, we isolated and sequenced a perlinhibin-related protein, indicating that perlinhibin may be a member of a family of closely related proteins. PMID:17496038

Field effect sensors for PCR applications

NASA Astrophysics Data System (ADS)

Taing, Meng-Houit; Sweatman, Denis R.

2004-03-01

The use of field effect sensors for biological and chemical sensing is widely employed due to its ability to make detections based on charge and surface potential. Because proteins and DNA almost always carry a charge [1], silicon can be used to micro fabricate such a sensor. The EIS structure (Electrolyte on Insulator on Silicon) provides a novel, label-free and simple to fabricate way to make a field effect DNA detection sensor. The sensor responds to fluctuating capacitance caused by a depletion layer thickness change at the surface of the silicon substrate through DNA adsorption onto the dielectric oxide/PLL (Poly-L-Lysine) surface. As DNA molecules diffuse to the sensor surface, they are bound to their complimentary capture probes deposited on the surface. The negative charge exhibited by the DNA forces negative charge carriers in the substrate to move away from the surface. This causes an n-type depletion layer substrate to thicken and a p-type to thin. The depletion layer thickness can be measured by its capacitance using an LCR meter. This experiment is conducted using the ConVolt (constant voltage) approach. Nucleic acids are amplified by an on chip PCR (Polymerase Chain Reaction) system and then fed into the sensor. The low ionic solution strength will ensure that counter-ions do not affect the sensor measurements. The sensor surface contains capture probes that bind to the pathogen. The types of pathogens we"ll be detecting include salmonella, campylobacter and E.Coli DNA. They are held onto the sensor surface by the positively charged Poly-L-Lysine layer. The electrolyte is biased through a pseudo-reference electrode. Pseudo reference electrodes are usually made from metals such as Platinum or Silver. The problem associated with "floating" biasing electrodes is they cannot provide stable biasing potentials [2]. They drift due to surface charging effects and trapped charges on the surface. To eliminate this, a differential system consisting of 2 sensors that share a common pseudo-reference electrode is used to cancel out this effect. This paper will look at a differential system for multi-arrayed biosensors fabricated on silicon.

Plasmonic nanostructures for bioanalytical applications of SERS

NASA Astrophysics Data System (ADS)

Kahraman, Mehmet; Wachsmann-Hogiu, Sebastian

2016-03-01

Surface-enhanced Raman scattering (SERS) is a potential analytical technique for the detection and identification of chemicals and biological molecules and structures in the close vicinity of metallic nanostructures. We present a novel method to fabricate tunable plasmonic nanostructures and perform a comprehensive structural and optical characterization of the structures. Spherical latex particles are uniformly deposited on glass slides and used as templates to obtain nanovoid structures on polydimethylsiloxane surfaces. The diameter and depth of the nanovoids are controlled by the size of the latex particles. The nanovoids are coated with a thin Ag layer for fabrication of uniform plasmonic nanostructures. Structural characterization of the surfaces is performed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Optical properties of these plasmonic nanostructures are evaluated via UV/Vis spectroscopy, and SERS. The sample preparation step is the key point to obtain strong and reproducible SERS spectra from the biological structures. When the colloidal suspension is used as a SERS substrate for the protein detection, the electrostatic interaction of the proteins with the nanoparticles is described by the nature of their charge status, which influences the aggregation properties such as the size and shape of the aggregates, which is critical for the SERS experiment. However, when the solid SERS substrates are fabricated, SERS signal of the proteins that are background free and independent of the protein charge. Pros and cons of using plasmonic nano colloids and nanostructures as SERS substrate will be discussed for label-free detection of proteins using SERS.

Molecular-Scale Investigation of Heavy Metal Ions at a Charged Langmuir Monolayer

NASA Astrophysics Data System (ADS)

Rock, William; Qiao, Baofu; Uysal, Ahmet; Bu, Wei; Lin, Binhua

Solvent extraction - the surfactant-aided preferential transfer of a species from an aqueous to an organic phase - is an important technique used in heavy and precious metal refining and reprocessing. Solvent extraction requires transfer through an oil/water interface, and interfacial interactions are expected to control transfer kinetics and phase stability, yet these key interactions are poorly understood. Langmuir monolayers with charged headgroups atop concentrated salt solutions containing heavy metal ions act as a model of solvent extraction interfaces; studies of ions at a charged surface are also fundamentally important to many other phenomena including protein solvation, mineral surface chemistry, and electrochemistry. We probe these charged interfaces using a variety of surface-sensitive techniques - vibrational sum frequency generation (VSFG) spectroscopy, x-ray reflectivity (XRR), x-ray fluorescence near total reflection (XFNTR), and grazing incidence diffraction (GID). We integrate experiments with Molecular Dynamics (MD) simulations to uncover the molecular-level interfacial structure. This work is supported by the U.S. DOE, BES, Contract DE-AC02-06CH11357. ChemMatCARS is supported by NSF/CHE-1346572.

Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

PubMed Central

Kim, Young Eun; Kim, Yu-na; Kim, Jung A.; Kim, Ho Min; Jung, Yongwon

2015-01-01

Supramolecular protein assemblies offer novel nanoscale architectures with molecular precision and unparalleled functional diversity. A key challenge, however, is to create precise nano-assemblies of functional proteins with both defined structures and a controlled number of protein-building blocks. Here we report a series of supramolecular green fluorescent protein oligomers that are assembled in precise polygonal geometries and prepared in a monodisperse population. Green fluorescent protein is engineered to be self-assembled in cells into oligomeric assemblies that are natively separated in a single-protein resolution by surface charge manipulation, affording monodisperse protein (nano)polygons from dimer to decamer. Several functional proteins are multivalently displayed on the oligomers with controlled orientations. Spatial arrangements of protein oligomers and displayed functional proteins are directly visualized by a transmission electron microscope. By employing our functional protein assemblies, we provide experimental insight into multivalent protein–protein interactions and tools to manipulate receptor clustering on live cell surfaces. PMID:25972078

How to harvest solar energy with the photosynthetic reaction center

NASA Astrophysics Data System (ADS)

Balaeff, Alexander; Reyes, Justin

Photosynthetic reaction center (PRC) is a protein complex that performs a key step in photosynthesis: the electron-hole separation driven by photon absorbtion. The PRC has a great promise for applications in solar energy harvesting and photosensing. Such applications, however, are hampered by the difficulty in extracting the photogenerated electric charge from the PRC. To that end, it was proposed to attach the PRC to a molecular wire through which the charge could be collected. In order to find the attachment point for the wire that would maximize the rate of charge outflow from the PRC, we performed a computational study of the PRC from the R. virdis bacterium. An ensemble of PRC structures generated by a molecular dynamics simulation was used to calculate the rate of charge transport from the site of initial charge separation to several trial sites on the protein surface. The Pathways model was used to calculate the charge transfer rate in each step of the network of heme co-factors through which the charge transport was presumed to proceed. A simple kinetic model was then used to determine the overall rate of the multistep charge transport. The calculations revealed several candidate sites for the molecular wire attachment, recommended for experimental verification.

In vitro biocompatibility and electrical stability of thick-film platinum/gold alloy electrodes printed on alumina

NASA Astrophysics Data System (ADS)

Carnicer-Lombarte, Alejandro; Lancashire, Henry T.; Vanhoestenberghe, Anne

2017-06-01

Objective. High-density electrode arrays are a powerful tool in both clinical neuroscience and basic research. However, current manufacturing techniques require the use of specialised techniques and equipment, which are available to few labs. We have developed a high-density electrode array with customisable design, manufactured using simple printing techniques and with commercially available materials. Approach. Electrode arrays were manufactured by thick-film printing a platinum-gold alloy (Pt/Au) and an insulating dielectric on 96% alumina ceramic plates. Arrays were conditioned in serum and serum-free conditions, with and without 1 kHz, 200 µA, charge balanced stimulation for up to 21 d. Array biocompatibility was assessed using an extract assay and a PC-12 cell contact assay. Electrode impedance, charge storage capacity and charge injection capacity were before and after array conditioning. Main results. The manufactured Pt/Au electrodes have a highly porous surface and exhibit electrical properties comparable to arrays manufactured using alternative techniques. Materials used in array manufacture were found to be non-toxic to L929 fibroblasts by extract assay, and neuronal-like PC-12 cells adhered and extended neurites on the array surfaces. Arrays remained functional after long-term delivery of electrical pulses while exposed to protein-rich environments. Charge storage capacities and charge injection capacities increased following stimulation accounted for by an increase in surface index (real surface area) observed by vertical scanning interferometry. Further, we observed accumulation of proteins at the electrode sites following conditioning in the presence of serum. Significance. This study demonstrates the in vitro biocompatibility of commercially available thick-film printing materials. The printing technique is both simple and versatile, with layouts readily modified to produce customized electrode arrays. Thick-film electrode arrays are an attractive tool that may be implemented for general tissue engineering and neuroscience research.

Impact of silica nanoparticle surface chemistry on protein corona formation and consequential interactions with biological cells.

PubMed

Kurtz-Chalot, Andréa; Villiers, Christian; Pourchez, Jérémie; Boudard, Delphine; Martini, Matteo; Marche, Patrice N; Cottier, Michèle; Forest, Valérie

2017-06-01

Nanoparticles (NP) physico-chemical features greatly influence NP/cell interactions. NP surface functionalization is often used to improve NP biocompatibility or to enhance cellular uptake. But in biological media, the formation of a protein corona adds a level of complexity. The aim of this study was to investigate in vitro the influence of NP surface functionalization on their cellular uptake and the biological response induced. 50nm fluorescent silica NP were functionalized either with amine or carboxylic groups, in presence or in absence of polyethylene glycol (PEG). NP were incubated with macrophages, cellular uptake and cellular response were assessed in terms of cytotoxicity, pro-inflammatory response and oxidative stress. The NP protein corona was also characterized by protein mass spectroscopy. Results showed that NP uptake was enhanced in absence of PEG, while NP adsorption at the cell membrane was fostered by an initial positively charged NP surface. NP toxicity was not correlated with NP uptake. NP surface functionalization also influenced the formation of the protein corona as the profile of protein binding differed among the NP types. Copyright © 2017 Elsevier B.V. All rights reserved.

Interpreting the adsorption of serum albumin and lactoglobulin onto ZnS nanopaticles: effect of conformational rigidity of the proteins.

PubMed

Saikia, Jiban; Saha, Bedabrata; Das, Gopal

2014-02-15

The work we have undertaken is to investigate the adsorption of two different proteins (BSA and BLG) having near same IEP and differing in their conformational flexibility, onto the surface of ZnS nanoparticles (ZnS NPs). BSA and BLG both have an IEP value around pH~5. BSA is more prone to conformational deformation and considered "soft" while BLG holds the conformational rigidity and considered as "hard" protein. To ascertain the differences in surface coverage and conformation of the protein onto ZnS surface (PZC ~ 3.7), we have evaluated the adsorption profile at pH 7, where the entire surface behaves negatively. An integrated approach was taken by incorporating zeta (ζ) potential, fluorescence and CD for analyzing the adsorption process. In both systems, an increase in protein surface coverage was observed with the increase in free protein concentration in the solution and ζ values approaching that of native protein at high surface coverage. An alteration in the tertiary structure was observed for both BSA and BLG. The CD spectra analysis reveals that the secondary structure of the BSA was more deviated from the native protein structure, accommodating the increased adsorption value. For BLG no such prominent structural alteration was observed. These findings help us to understand better, how adjustment of the protein adsorption amount can be achieved onto the surface of nanoparticles having like charges. Copyright © 2013 Elsevier Inc. All rights reserved.

Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan.

PubMed

Wright, W W; Owen, C S; Vanderkooi, J M

1992-07-21

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)

Biocompatibilite des complexes proteines-nanoparticules: Perspectives sur la reponse cellulaire aux nanoparticules d'oxyde de fer fonctionnalisees, revetues d'un corona

NASA Astrophysics Data System (ADS)

Mbeh, Doris Antoinette

This thesis presents the study of the biocompatibility of nanoparticles (NPs) of iron oxide (Fe3O4) candidates for targeted delivery of therapeutic molecules. We especially devoted to study the impact of the surface composition of the NPs and protein adsorption at the surface thereof on the cellular responses. To do this, we first examined the toxic potential of magnetite with various functionalizations: one that is prepared with (1) a monolayer of oleic acid (Fe3O4@OA), which is then converted to (2) an envelope silane containing an amine (Fe3O4@NH 2), (3) a coating of silica (Fe3O4@SiO 2), and (4) an envelope containing a silane coating on amine silica (Fe3O4@SiO2@NH 2). The presence of these groups at the surface of the NPs was confirmed by XPS and transmission electron microscopy (TEM) analysis. We were able to prove that the toxic potential of NPs is dose-dependent and we determine the biocompatible doses for each surface functionalization. Microscopic observation of the morphology of the cells exposed to NPs, and their proinflammatory and mitochondrial activity showed that, in addition to surface features, the cell culture medium also affect the cytotoxicity of the NPs. These results clearly show that in order to use our NPs as pharmaceutical nanocarrier safely, we need to control the surface functionalization and the dynamic interaction between the NP and the physiological environment in which it is suspended. To understand the interaction between the NP and the culture medium, as a first step, we used three different culture media namely: DMEM, F-12K and DMEM / F12 (see Appendix A) and uncoated magnetite (Fe3O 4). These media were enriched with either fetal bovine serum (see Appendix B) or with a synthetic serum (SFMS). We have proved the presence of a protein corona on NPs suspended in culture media enriched with bovine serum. We also demonstrated that the formation of the corona depends on the composition of the culture medium and that the cytotoxic potential of the NPs is influenced by NP-protein interaction. In a second step, we used one culture medium (DMEM / F12) and the magnetite with three different surface compositions: uncoated SPIONs with hydroxyl groups (OH) on the surface; coated SPIONs with an amine group (NH2) on the surface and the last one with a carboxylic group (COOH) on the surface. The results show that the composition of the corona depends on the surface composition of the NP and cellular responses are also different from one surface to another. In fact, some proteins (e.g. albumin) are adsorbed on the coatings only positively charged (NH2), while others (e.g., fibrinogen) are adsorbed on the negatively charged coatings (OH and COOH). Cell proliferation is influenced by the surface chemistry and is dose-dependent. SPIONs coated with carboxylic groups are more biocompatible while those uncoated, having hydroxyl groups on the surface are the most cytotoxic. Exploring three possible mechanisms of cytotoxicity, i.e., the production of ions by the SPIONs in the culture media, reactive oxygen species and protein adsorption, we found that in our case, protein adsorption was behind the cytotoxicity of our NPs since oxidative stress have been proved non-existent and there are not enough ions in the culture media to be detected . From these results, we can make a first correlation between the chemical composition of the surface, the identity of the adsorbed proteins and cellular responses. But we must take into account many other parameters related either to the NPs such as the charge, the agglomeration status, or related to the culture medium as the density of each protein, or finally to the experimental conditions.

Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution.

PubMed

Shivange, Amol V; Hoeffken, Hans Wolfgang; Haefner, Stefan; Schwaneberg, Ulrich

2016-12-01

Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: ( i ) identification of conserved and highly variable regions; ( ii ) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; ( iii ) in vitro DNA recombination of synthetic genes; and ( iv ) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

Electrical passivation of nonselective bio molecules in carbon nanotubes: Effect of pulse train in serum

NASA Astrophysics Data System (ADS)

Kim, Seok Hyang; Woo, Jun-Myung; Choi, Seongwook; Park, Young June

2015-06-01

We present an experimental and simulation study about a desorption of albumin, a representative nonselective molecules in serum, on carbon nanotube (CNT) surface as an electrical bio sensing channel under the pulse train condition. The motivation of the study on binding kinetics between CNT surface and albumin is to suppress the adsorption of nonselective proteins in blood such as albumin, thereby enhancing the selectivity of the electrical biosensor. To theoretically model the behavior of molecules and ions under the step pulse bias, the physics on the reaction rate, mass transport, and the resulting surface pH-value are considered using the Poisson and drift-diffusion equations. For the simulation model, the phosphate buffered saline is considered as the electrolyte solution and albumin is considered as a representative charged molecule for nonspecific binding in serum. Both the transient simulation and experimental result indicate that the suppression of the nonspecific binding under the pulse train is due to the unsymmetrical field force experienced by the protein during the pulse transitions (high to low and low to high) and the non-symmetry is caused by the different transient times between the electric field and the charge/discharge of the protein according to the surface pH modulation in serum. The experimental and simulation results clearly indicate that the pulse bias suppresses the nonselective bio molecules adsorption at the CNT surface so that the selectivity of the electrical biosensor for detecting the target molecules can be enhanced.

Investigating the Role of Adducts in Protein Supercharging with Sulfolane

NASA Astrophysics Data System (ADS)

Douglass, Kevin Aart; Venter, Andre R.

2012-03-01

The supercharging effect of sulfolane on cytochrome c (cyt c) during electrospray ionization mass spectrometry (ESI-MS) in the absence of conformational effects was investigated. The addition of sulfolane on the order of 1 mM or greater to denaturing solutions of cyt c results in supercharging independent of protein concentration over the range of 0.1 to 10 μM. While supercharging was observed in the positive mode, no change in the charge state distribution was observed in the negative mode, ruling out polarity-independent factors such as conformational changes or surface tension effects. A series of sulfolane adducts observed with increasing intensity concurrent with increasing charge state suggests that a direct interaction between sulfolane and the charged sites of cyt c plays an important role in supercharging. We propose that charge delocalization occurring through large-scale dipole reordering of the highly polar supercharging reagent reduces the electrostatic barrier for proximal charging along the cyt c amino acid chain. Supporting this claim, supercharging was shown to increase with increasing dipole moment for several supercharging reagents structurally related to sulfolane.

Polymer-Based Surfaces Designed to Reduce Biofilm Formation: From Antimicrobial Polymers to Strategies for Long-Term Applications.

PubMed

Riga, Esther K; Vöhringer, Maria; Widyaya, Vania Tanda; Lienkamp, Karen

2017-10-01

Contact-active antimicrobial polymer surfaces bear cationic charges and kill or deactivate bacteria by interaction with the negatively charged parts of their cell envelope (lipopolysaccharides, peptidoglycan, and membrane lipids). The exact mechanism of this interaction is still under debate. While cationic antimicrobial polymer surfaces can be very useful for short-term applications, they lose their activity once they are contaminated by a sufficiently thick layer of adhering biomolecules or bacterial cell debris. This layer shields incoming bacteria from the antimicrobially active cationic surface moieties. Besides discussing antimicrobial surfaces, this feature article focuses on recent strategies that were developed to overcome the contamination problem. This includes bifunctional materials with simultaneously presented antimicrobial and protein-repellent moieties; polymer surfaces that can be switched from an antimicrobial, cell-attractive to a cell-repellent state; polymer surfaces that can be regenerated by enzyme action; degradable antimicrobial polymers; and antimicrobial polymer surfaces with removable top layers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Adsorption behavior of proteins on temperature-responsive resins.

PubMed

Poplewska, Izabela; Muca, Renata; Strachota, Adam; Piątkowski, Wojciech; Antos, Dorota

2014-01-10

The adsorption behavior of proteins on thermo-responsible resins based on poly(N-isopropylacrylamide) and its copolymer containing an anionic co-monomer has been investigated. The influence of the polymer composition, i.e., the content of the co-monomer and crosslinker on the thermo-sensitivity of the protein adsorption has been quantified. The properties of ungrafted polymer as well grafted onto the agarose matrix have been analyzed and compared. Batch and dynamic (column) experiments have been performed to measure the adsorption equilibrium of proteins and to quantify the phase transition process. As model proteins lysozyme, lactoferrin, α-chymotrypsinogen A and ovalbumin have been used. The adsorption process was found to be governed by ionic interactions between the negatively charged surface of resin and the protein, which enabled separation of proteins differing in electrostatic charge. The interactions enhanced with increase of temperature. Decrease of temperature facilitated desorption of proteins and reduced the salt usage in the desorption buffer. Grafted polymers exhibited markedly higher mechanical stability and, however, weaker temperature response compared to the ungrafted ones. Copyright © 2013 Elsevier B.V. All rights reserved.

Evidence that electrostatic interactions between vesicle-associated membrane protein 2 and acidic phospholipids may modulate the fusion of transport vesicles with the plasma membrane.

PubMed

Williams, Dumaine; Vicôgne, Jérome; Zaitseva, Irina; McLaughlin, Stuart; Pessin, Jeffrey E

2009-12-01

The juxtamembrane domain of vesicle-associated membrane protein (VAMP) 2 (also known as synaptobrevin2) contains a conserved cluster of basic/hydrophobic residues that may play an important role in membrane fusion. Our measurements on peptides corresponding to this domain determine the electrostatic and hydrophobic energies by which this domain of VAMP2 could bind to the adjacent lipid bilayer in an insulin granule or other transport vesicle. Mutation of residues within the juxtamembrane domain that reduce the VAMP2 net positive charge, and thus its interaction with membranes, inhibits secretion of insulin granules in beta cells. Increasing salt concentration in permeabilized cells, which reduces electrostatic interactions, also results in an inhibition of insulin secretion. Similarly, amphipathic weak bases (e.g., sphingosine) that reverse the negative electrostatic surface potential of a bilayer reverse membrane binding of the positively charged juxtamembrane domain of a reconstituted VAMP2 protein and inhibit membrane fusion. We propose a model in which the positively charged VAMP and syntaxin juxtamembrane regions facilitate fusion by bridging the negatively charged vesicle and plasma membrane leaflets.

Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge

PubMed Central

Maglia, Giovanni; Restrepo, Marcela Rincon; Mikhailova, Ellina; Bayley, Hagan

2008-01-01

Both protein and solid-state nanopores are under intense investigation for the analysis of nucleic acids. A crucial advantage of protein nanopores is that site-directed mutagenesis permits precise tuning of their properties. Here, by augmenting the internal positive charge within the α-hemolysin pore and varying its distribution, we increase the frequency of translocation of a 92-nt single-stranded DNA through the pore at +120 mV by ≈10-fold over the wild-type protein and dramatically lower the voltage threshold at which translocation occurs, e.g., by 50 mV for 1 event·s−1·μM−1. Further, events in which DNA enters the pore, but is not immediately translocated, are almost eliminated. These experiments provide a basis for improved nucleic acid analysis with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification. PMID:19060213

The effect of surface charge on the thermal stability and ice recrystallization inhibition activity of antifreeze protein III (AFP III).

PubMed

Deller, R C; Carter, B M; Zampetakis, I; Scarpa, F; Perriman, A W

2018-01-01

The aim of this study was to examine the effect of chemical cationization on the structure and function of antifreeze protein III (AFP III) over an extreme temperature range (-40°C to +90°C) using far-UV synchrotron radiation circular dichroism (SRCD) and ice recrystallization inhibition (IRI) assays. Chemical cationization was able to produce a modified AFP III with a net cationic charge at physiological pH that had enhanced resistance to denaturation at elevated temperatures, with no immediate negative impact on protein structure at subzero temperatures. Furthermore, cationized AFP III retained an IRI activity similar to that of native AFP III. Consequently, chemical cationization may provide a pathway to the development of more robust antifreeze proteins as supplementary cryoprotectants in the cryopreservation of clinically relevant cells. Copyright © 2017. Published by Elsevier Inc.

Use of hydrophilic polymer coatings for control of electroosmosis and protein adsorption

NASA Technical Reports Server (NTRS)

Harris, J. Milton

1987-01-01

The purpose of this project was to examine the utility of polyethylene glycol (PEG) and dextran coatings for control of electroosmosis and protein adsorption; electroosmosis is an important, deleterious process affecting electrophoretic separations, and protein adsorption is a factor which needs to be controlled during protein crystal growth to avoid multiple nucleation sites. Performance of the project required use of X-ray photoelectron spectroscopy to refine previously developed synthetic methods. The results of this spectroscopic examination are reported. Measurements of electroosmotic mobility of charged particles in appropriately coated capillaries reveals that a new, one-step route to coating capillaries gives a surface in which electroosmosis is dramatically reduced. Similarly, both PEG and dextran coatings were shown by protein adsorption measurements to be highly effective at reducing protein adsorption on solid surfaces. These results should have impact on future low-g electrophoretic and protein crystal growth experiments.

Fibrillar Structure and Charge Determine the Interaction of Polyglutamine Protein Aggregates with the Cell Surface*

PubMed Central

Trevino, R. Sean; Lauckner, Jane E.; Sourigues, Yannick; Pearce, Margaret M.; Bousset, Luc; Melki, Ronald; Kopito, Ron R.

2012-01-01

The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces. PMID:22753412

Cation specific binding with protein surface charges

PubMed Central

Hess, Berk; van der Vegt, Nico F. A.

2009-01-01

Biological organization depends on a sensitive balance of noncovalent interactions, in particular also those involving interactions between ions. Ion-pairing is qualitatively described by the law of “matching water affinities.” This law predicts that cations and anions (with equal valence) form stable contact ion pairs if their sizes match. We show that this simple physical model fails to describe the interaction of cations with (molecular) anions of weak carboxylic acids, which are present on the surfaces of many intra- and extracellular proteins. We performed molecular simulations with quantitatively accurate models and observed that the order K+ < Na+ < Li+ of increasing binding affinity with carboxylate ions is caused by a stronger preference for forming weak solvent-shared ion pairs. The relative insignificance of contact pair interactions with protein surfaces indicates that thermodynamic stability and interactions between proteins in alkali salt solutions is governed by interactions mediated through hydration water molecules. PMID:19666545

Fabrication of Biocompatible Potassium Sodium Niobate Piezoelectric Ceramic as an Electroactive Implant

PubMed Central

Chen, Wei; Yu, Zunxiong; Pang, Jinshan; Yu, Peng; Tan, Guoxin; Ning, Chengyun

2017-01-01

The discovery of piezoelectricity in natural bone has attracted extensive research in emulating biological electricity for various tissue regeneration. Here, we carried out experiments to build biocompatible potassium sodium niobate (KNN) ceramics. Then, influence substrate surface charges on bovine serum albumin (BSA) protein adsorption and cell proliferation on KNN ceramics surfaces was investigated. KNN ceramics with piezoelectric constant of ~93 pC/N and relative density of ~93% were fabricated. The adsorption of protein on the positive surfaces (Ps) and negative surfaces (Ns) of KNN ceramics with piezoelectric constant of ~93 pC/N showed greater protein adsorption capacity than that on non-polarized surfaces (NPs). Biocompatibility of KNN ceramics was verified through cell culturing and live/dead cell staining of MC3T3. The cells experiment showed enhanced cell growth on the positive surfaces (Ps) and negative surfaces (Ns) compared to non-polarized surfaces (NPs). These results revealed that KNN ceramics had great potential to be used to understand the effect of surface potential on cells processes and would benefit future research in designing piezoelectric materials for tissue regeneration. PMID:28772704

Fabrication of Biocompatible Potassium Sodium Niobate Piezoelectric Ceramic as an Electroactive Implant.

PubMed

Chen, Wei; Yu, Zunxiong; Pang, Jinshan; Yu, Peng; Tan, Guoxin; Ning, Chengyun

2017-03-26

The discovery of piezoelectricity in natural bone has attracted extensive research in emulating biological electricity for various tissue regeneration. Here, we carried out experiments to build biocompatible potassium sodium niobate (KNN) ceramics. Then, influence substrate surface charges on bovine serum albumin (BSA) protein adsorption and cell proliferation on KNN ceramics surfaces was investigated. KNN ceramics with piezoelectric constant of ~93 pC/N and relative density of ~93% were fabricated. The adsorption of protein on the positive surfaces (Ps) and negative surfaces (Ns) of KNN ceramics with piezoelectric constant of ~93 pC/N showed greater protein adsorption capacity than that on non-polarized surfaces (NPs). Biocompatibility of KNN ceramics was verified through cell culturing and live/dead cell staining of MC3T3. The cells experiment showed enhanced cell growth on the positive surfaces (Ps) and negative surfaces (Ns) compared to non-polarized surfaces (NPs). These results revealed that KNN ceramics had great potential to be used to understand the effect of surface potential on cells processes and would benefit future research in designing piezoelectric materials for tissue regeneration.

Dielectric boundary force and its crucial role in gramicidin

NASA Astrophysics Data System (ADS)

Nadler, Boaz; Hollerbach, Uwe; Eisenberg, R. S.

2003-08-01

In an electrostatic problem with nonuniform geometry, a charge Q in one region induces surface charges [called dielectric boundary charges (DBC)] at boundaries between different dielectrics. These induced surface charges, in return, exert a force [called dielectric boundary force (DBF)] on the charge Q that induced them. The DBF is often overlooked. It is not present in standard continuum theories of (point) ions in or near membranes and proteins, such as Gouy-Chapman, Debye-Huckel, Poisson-Boltzmann or Poisson-Nernst- Planck. The DBF is important when a charge Q is near dielectric interfaces, for example, when ions permeate through protein channels embedded in biological membranes. In this paper, we define the DBF and calculate it explicitly for a planar dielectric wall and for a tunnel geometry resembling the ionic channel gramicidin. In general, we formulate the DBF in a form useful for continuum theories, namely, as a solution of a partial differential equation with boundary conditions. The DBF plays a crucial role in the permeation of ions through the gramicidin channel. A positive ion in the channel produces a DBF of opposite sign to that of the fixed charge force (FCF) produced by the permanent charge of the gramicidin polypeptide, and so the net force on the positive ion is reduced. A negative ion creates a DBF of the same sign as the FCF and so the net (repulsive) force on the negative ion is increased. Thus, a positive ion can permeate the channel, while a negative ion is excluded from it. In gramicidin, it is this balance between the FCF and DBF that allows only singly charged positive ions to move into and through the channel. The DBF is not directly responsible, however, for selectivity between the alkali metal ions (e.g., Li+, Na+, K+): we prove that the DBF on a mobile spherical ion is independent of the ion’s radius.

An investigation of vibration-induced protein desorption mechanism using a micromachined membrane and PZT plate.

PubMed

Yeh, Po Ying; Le, Yevgeniya; Kizhakkedathu, Jayachandran N; Chiao, Mu

2008-10-01

A micromachined vibrating membrane is used to remove adsorbed proteins on a surface. A lead zirconate titanate (PZT) composite (3 x 1 x 0.5 mm) is attached to a silicon membrane (2,000 x 500 x 3 microm) and vibrates in a flexural plate wave (FPW) mode with wavelength of 4,000/3 microm at a resonant frequency of 308 kHz. The surface charge on the membrane and fluid shear stress contribute in minimizing the protein adsorption on the SiO(2) surface. In vitro characterization shows that 57 +/- 10% of the adsorbed bovine serum albumin (BSA), 47 +/- 13% of the immunoglobulin G (IgG), and 55.3~59.2 +/- 8% of the proteins from blood plasma are effectively removed from the vibrating surface. A simulation study of the vibration-frequency spectrum and vibrating amplitude distribution matches well with the experimental data. Potentially, a microelectromechanical system (MEMS)-based vibrating membrane could be the tool to minimize biofouling of in vivo MEMS devices.

Controlling Protein Surface Orientation by Strategic Placement of Oligo-Histidine Tags

PubMed Central

2017-01-01

We report oriented immobilization of proteins using the standard hexahistidine (His6)-Ni2+:NTA (nitrilotriacetic acid) methodology, which we systematically tuned to give control of surface coverage. Fluorescence microscopy and surface plasmon resonance measurements of self-assembled monolayers (SAMs) of red fluorescent proteins (TagRFP) showed that binding strength increased by 1 order of magnitude for each additional His6-tag on the TagRFP proteins. All TagRFP variants with His6-tags located on only one side of the barrel-shaped protein yielded a 1.5 times higher surface coverage compared to variants with His6-tags on opposite sides of the so-called β-barrel. Time-resolved fluorescence anisotropy measurements supported by polarized infrared spectroscopy verified that the orientation (and thus coverage and functionality) of proteins on surfaces can be controlled by strategic placement of a His6-tag on the protein. Molecular dynamics simulations show how the differently tagged proteins reside at the surface in “end-on” and “side-on” orientations with each His6-tag contributing to binding. Also, not every dihistidine subunit in a given His6-tag forms a full coordination bond with the Ni2+:NTA SAMs, which varied with the position of the His6-tag on the protein. At equal valency but different tag positions on the protein, differences in binding were caused by probing for Ni2+:NTA moieties and by additional electrostatic interactions between different fractions of the β-barrel structure and charged NTA moieties. Potential of mean force calculations indicate there is no specific single-protein interaction mode that provides a clear preferential surface orientation, suggesting that the experimentally measured preference for the end-on orientation is a supra-protein, not a single-protein, effect. PMID:28850777

Immobilization of E. coli with autodisplayed Z-domains to a surface-modified microplate for immunoassay.

PubMed

Yoo, Gu; Park, Min; Lee, Eun-Hang; Jose, Joachim; Pyun, Jae-Chul

2011-11-30

Escherichia coli with autodisplayed Z-domains was reported to improve the sensitivity of immunoassays by the orientation control of antibodies. In this work, a sensitive microplate-based immunoassay is presented by immobilizing E. coli cells to a surface-modified microplate. The microplate was prepared by coating parylene-H film with formyl groups, and then covalently coupling poly-L-lysine to the parylene-H film. The E. coli cells were bound to the microplate by charge interactions between the negatively charged E. coli outer membrane and the positively charged microplate surface. In this work, the preparation of the microplate coated with poly-L-lysine is presented. The immobilization efficiency of E. coli to the modified surface was estimated to be far higher than non-specific interaction by fluorescence microscope and the optical transmittance of the modified microplate was measured to be feasible for immunoassay. The microplate-based immunoassay is demonstrated to be feasible for medical diagnosis of inflammatory diseases by using C-reactive protein as a target analyte for the medical diagnosis of inflammatory diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

Attachment of Escherichia coli O157:H7 grown in tryptic soy broth and nutrient broth to apple and lettuce surfaces as related to cell hydrophobicity, surface charge, and capsule production.

PubMed

Hassan, A N; Frank, J F

2004-10-01

This study investigated the effect of growth in tryptic soy broth (TSB) and nutrient broth (NB) on the ability Escherichia coli O157:H7 to attach to lettuce and apple surfaces. In addition, cell surface hydrophobicity, charge and capsule production were determined on cells grown in these media. Cells grown in NB attached less to lettuce and apple surfaces than did those grown in TSB. TSB, but not NB, supported capsule production by E. coli O157:H7. Cells grown in TSB were more hydrophilic than those grown in NB. No difference was found in the electrokinetic properties of cells grown in these media. Electrostatic and hydrophobic interactions and surface proteins did not appear to play an important role in the attachment of E. coli O157:H7 to these surfaces. Of the factors studied, only capsule production was associated with attachment ability. Copyright 2003 Elsevier B.V.

Effects of protein conformational motions in the native form and non-uniform distribution of electrostatic interaction sites on interfacial water

NASA Astrophysics Data System (ADS)

Pal, Somedatta; Bandyopadhyay, Sanjoy

2013-07-01

Protein-water interactions and their influence on surrounding water is a long-standing problem. Despite its importance, the origin of differential water behavior at the protein surface is still elusive. We have performed molecular simulations of the protein barstar in aqueous medium. Efforts have been made to explore how the conformational motions of the protein segments in the native form and the heterogeneous electrostatic interactions with the polar and charged groups of the protein affect the interfacial water properties. The calculations reveal that reduced dimension of the hydration layer on freezing the protein's degrees of freedom does not modify the heterogeneous water distributions around the protein. However, turning off the protein-water electrostatic contribution leads to non-preferential near-uniform water arrangements at the surface. It is further shown that with protein-water electrostatic interactions turned on, the local structuring of water molecules around the segments are correlated with their degree of exposure to the solvent.

Chitosan-based ultrathin films as antifouling, anticoagulant and antibacterial protective coatings.

PubMed

Bulwan, Maria; Wójcik, Kinga; Zapotoczny, Szczepan; Nowakowska, Maria

2012-01-01

Ultrathin antifouling and antibacterial protective nanocoatings were prepared from ionic derivatives of chitosan using layer-by-layer deposition methodology. The surfaces of silicon, and glass protected by these nanocoatings were resistant to non-specific adsorption of proteins disregarding their net charges at physiological conditions (positively charged TGF-β1 growth factor and negatively charged bovine serum albumin) as well as human plasma components. The coatings also preserved surfaces from the formation of bacterial (Staphylococcus aureus) biofilm as shown using microscopic studies (SEM, AFM) and the MTT viability test. Moreover, the chitosan-based films adsorbed onto glass surface demonstrated the anticoagulant activity towards the human blood. The antifouling and antibacterial actions of the coatings were correlated with their physicochemical properties. The studied biologically relevant properties were also found to be dependent on the thickness of those nanocoatings. These materials are promising for biomedical applications, e.g., as protective coatings for medical devices, anticoagulant coatings and protective layers in membranes.

Discrimination between native and intentionally misfolded conformations of proteins: ES/IS, a new method for calculating conformational free energy that uses both dynamics simulations with an explicit solvent and an implicit solvent continuum model.

PubMed

Vorobjev, Y N; Almagro, J C; Hermans, J

1998-09-01

A new method for calculating the total conformational free energy of proteins in water solvent is presented. The method consists of a relatively brief simulation by molecular dynamics with explicit solvent (ES) molecules to produce a set of microstates of the macroscopic conformation. Conformational energy and entropy are obtained from the simulation, the latter in the quasi-harmonic approximation by analysis of the covariance matrix. The implicit solvent (IS) dielectric continuum model is used to calculate the average solvation free energy as the sum of the free energies of creating the solute-size hydrophobic cavity, of the van der Waals solute-solvent interactions, and of the polarization of water solvent by the solute's charges. The reliability of the solvation free energy depends on a number of factors: the details of arrangement of the protein's charges, especially those near the surface; the definition of the molecular surface; and the method chosen for solving the Poisson equation. Molecular dynamics simulation in explicit solvent relaxes the protein's conformation and allows polar surface groups to assume conformations compatible with interaction with solvent, while averaging of internal energy and solvation free energy tend to enhance the precision. Two recently developed methods--SIMS, for calculation of a smooth invariant molecular surface, and FAMBE, for solution of the Poisson equation via a fast adaptive multigrid boundary element--have been employed. The SIMS and FAMBE programs scale linearly with the number of atoms. SIMS is superior to Connolly's MS (molecular surface) program: it is faster, more accurate, and more stable, and it smooths singularities of the molecular surface. Solvation free energies calculated with these two programs do not depend on molecular position or orientation and are stable along a molecular dynamics trajectory. We have applied this method to calculate the conformational free energy of native and intentionally misfolded globular conformations of proteins (the EMBL set of deliberately misfolded proteins) and have obtained good discrimination in favor of the native conformations in all instances.

Protein Charge and Mass Contribute to the Spatio-temporal Dynamics of Protein-Protein Interactions in a Minimal Proteome

PubMed Central

Xu, Yu; Wang, Hong; Nussinov, Ruth; Ma, Buyong

2013-01-01

We constructed and simulated a ‘minimal proteome’ model using Langevin dynamics. It contains 206 essential protein types which were compiled from the literature. For comparison, we generated six proteomes with randomized concentrations. We found that the net charges and molecular weights of the proteins in the minimal genome are not random. The net charge of a protein decreases linearly with molecular weight, with small proteins being mostly positively charged and large proteins negatively charged. The protein copy numbers in the minimal genome have the tendency to maximize the number of protein-protein interactions in the network. Negatively charged proteins which tend to have larger sizes can provide large collision cross-section allowing them to interact with other proteins; on the other hand, the smaller positively charged proteins could have higher diffusion speed and are more likely to collide with other proteins. Proteomes with random charge/mass populations form less stable clusters than those with experimental protein copy numbers. Our study suggests that ‘proper’ populations of negatively and positively charged proteins are important for maintaining a protein-protein interaction network in a proteome. It is interesting to note that the minimal genome model based on the charge and mass of E. Coli may have a larger protein-protein interaction network than that based on the lower organism M. pneumoniae. PMID:23420643

Serum protein adsorption and platelet adhesion on aspartic-acid-immobilized polysulfone membranes.

PubMed

Higuchi, Akon; Hashiba, Hirokazu; Hayashi, Rika; Yoon, Boo Ok; Sakurai, Masaru; Hara, Mariko

2004-01-01

Polysulfone (PSf) membranes that covalently conjugated with aspartic acid (ASP-PSf) were prepared and analyzed for hemocompatability. Compared to PSf or other types of surface-modified PSf membranes, the ASP-PSf membranes had a reduced ability to adsorb protein from either a plasma solution or a mixed solution of albumin, globulin and fibrinogen. This appears to be due to the creation of a hydrophilic surface by the aspartic acid zwitterion immobilized on the ASP-PSf membranes. Furthermore, the analyses of membrane protein adsorption showed that a mixed protein solution recapitulates the cooperative adsorption of proteins that occurs in plasma. We also found that the number of adhering platelets was the lowest on the ASP-PSf membranes and, in general, that platelet adhesion decreased in parallel with fibrinogen adsorption. In summary, aspartic acid immobilized on the ASP-PSf membranes, which have zwitterions with a net zero charge, effectively contributes to the hydrophilic and hemocompatible sites on the surface of the hydrophobic PSf membranes.

Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

NASA Astrophysics Data System (ADS)

Bhattacharjee, Sourav; van Opstal, Edward J.; Alink, Gerrit M.; Marcelis, Antonius T. M.; Zuilhof, Han; Rietjens, Ivonne M. C. M.

2013-06-01

The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size 45 nm) and polystyrene nanoparticles (PSNPs/size 50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

Effect of electrolytes on proteins physisorption on ordered mesoporous silica materials.

PubMed

Salis, Andrea; Medda, Luca; Cugia, Francesca; Monduzzi, Maura

2016-01-01

This short review highlights the effect of electrolytes on the performance of proteins-mesoporous silica conjugates which can open interesting perspectives in biotechnological fields, particularly nanomedicine and biocatalysis. Indeed therapeutic proteins and peptides represent a challenging innovation for several kinds of diseases, but since their self-life in biological fluids is very short, they need a stealth protective carrier. Similarly, enzymes need a solid support to improve thermal stability and to allow for recycling. Ordered mesoporous silica materials represent a valid choice as widely demonstrated. Both proteins and silica mesoporous materials possess charged surfaces, and here, the crucial role of pH, buffer, ionic strength and electrolyte type is posed in relation with loading/release of proteins onto/from the silica support through the analysis of adsorption and release processes. A delicate interplay of electrostatic and van der Waals interactions arises from considering electrolytes' effects on the two different charged surfaces. Clear outcomes concern the effect of pH and ionic strength. Protein loading onto the silica matrix is favored by an adsorbing solution having a pH close to the protein pI, and by a high ionic strength that reduces the Debye length. Release is instead favored by an adsorbing solution characterized by an intermediate ionic strength, close to the physiological values. Significant specific ions effects are shown to affect both proteins and silica matrices, as well as protein adsorption onto silica matrices. Further work is needed to quantify specific ion effects on the preservation of the biological activity, and on the release performance. Copyright © 2015 Elsevier B.V. All rights reserved.

Probing the Interaction of Dielectric Nanoparticles with Supported Lipid Membrane Coatings on Nanoplasmonic Arrays

PubMed Central

Ferhan, Abdul Rahim; Ma, Gamaliel Junren; Jackman, Joshua A.; Sut, Tun Naw; Park, Jae Hyeon; Cho, Nam-Joon

2017-01-01

The integration of supported lipid membranes with surface-based nanoplasmonic arrays provides a powerful sensing approach to investigate biointerfacial phenomena at membrane interfaces. While a growing number of lipid vesicles, protein, and nucleic acid systems have been explored with nanoplasmonic sensors, there has been only very limited investigation of the interactions between solution-phase nanomaterials and supported lipid membranes. Herein, we established a surface-based localized surface plasmon resonance (LSPR) sensing platform for probing the interaction of dielectric nanoparticles with supported lipid bilayer (SLB)-coated, plasmonic nanodisk arrays. A key emphasis was placed on controlling membrane functionality by tuning the membrane surface charge vis-à-vis lipid composition. The optical sensing properties of the bare and SLB-coated sensor surfaces were quantitatively compared, and provided an experimental approach to evaluate nanoparticle–membrane interactions across different SLB platforms. While the interaction of negatively-charged silica nanoparticles (SiNPs) with a zwitterionic SLB resulted in monotonic adsorption, a stronger interaction with a positively-charged SLB resulted in adsorption and lipid transfer from the SLB to the SiNP surface, in turn influencing the LSPR measurement responses based on the changing spatial proximity of transferred lipids relative to the sensor surface. Precoating SiNPs with bovine serum albumin (BSA) suppressed lipid transfer, resulting in monotonic adsorption onto both zwitterionic and positively-charged SLBs. Collectively, our findings contribute a quantitative understanding of how supported lipid membrane coatings influence the sensing performance of nanoplasmonic arrays, and demonstrate how the high surface sensitivity of nanoplasmonic sensors is well-suited for detecting the complex interactions between nanoparticles and lipid membranes. PMID:28644423

Proteins detection by polymer optical fibers sensitised with overlayers of block and random copolymers

NASA Astrophysics Data System (ADS)

El Sachat, Alexandros; Meristoudi, Anastasia; Markos, Christos; Pispas, Stergios; Riziotis, Christos

2014-03-01

A low cost and low complexity optical detection method of proteins is presented by employing a detection scheme based on electrostatic interactions, and implemented by sensitization of a polymer optical fibers' (POF) surface by thin overlayers of properly designed sensitive copolymer materials with predesigned charges. This method enables the fast detection of proteins having opposite charge to the overlayer, and also the effective discrimination of differently charged proteins like lysozyme (LYS) and bovine serum albumin (BSA). As sensitive materials the block and the random copolymers of the same monomers were employed, namely the block copolymer poly(styrene-b-2vinylpyridine) (PS-b- P2VP) and the corresponding random copolymer poly(styrene-r-2vinylpyridine) (PS-r-P2VP), of similar composition and molecular weights. Results show systematically different response between the block and the random copolymers, although of the same order of magnitude, drawing thus important conclusions on their applications' techno-economic aspects given that they have significantly different associated manufacturing method and costs. The use of the POF platform, in combination with those adaptable copolymer sensing materials could lead to efficient low cost bio-detection schemes.

Surfactant-Modified Ultrafine Gold Nanoparticles with Magnetic Responsiveness for Reversible Convergence and Release of Biomacromolecules.

PubMed

Xu, Lu; Dong, Shuli; Hao, Jingcheng; Cui, Jiwei; Hoffmann, Heinz

2017-03-28

It is difficult to synthesize magnetic gold nanoparticles (AuNPs) with ultrafine sizes (<2 nm) based on a conventional method via coating AuNPs using magnetic particles, compounds, or ions. Here, magnetic cationic surfactants C 16 H 33 N + (CH 3 ) 3 [CeCl 3 Br] - (CTACe) and C 16 H 33 N + (CH 3 ) 3 [GdCl 3 Br] - (CTAGd) are prepared by a one-step coordination reaction, i.e., C 16 H 33 N + (CH 3 ) 3 Br - (CTABr) + CeCl 3 or GdCl 3 → CTACe or CTAGd. A simple strategy for fabricate ultrafine (<2 nm) magnetic gold nanoparticles (AuNPs) via surface modification with weak oxidizing paramagnetic cationic surfactants, CTACe or CTAGd, is developed. The resulting AuNPs can highly concentrate the charges of cationic surfactants on their surfaces, thereby presenting strong electrostatic interaction with negatively charged biomacromolecules, DNA, and proteins. As a consequence, they can converge DNA and proteins over 90% at a lower dosage than magnetic surfactants or existing magnetic AuNPs. The surface modification with these cationic surfactants endows AuNPs with strong magnetism, which allows them to magnetize and migrate the attached biomacromolecules with a much higher efficiency. The native conformation of DNA and proteins can be protected during the migration. Besides, the captured DNA and proteins could be released after adding sufficient inorganic salts such as at c NaBr = 50 mmol·L -1 . Our results could offer new guidance for a diverse range of systems including gene delivery, DNA transfection, and protein delivery and separation.

A Multi-technique Characterization of Adsorbed Protein Films: Orientation and Structure by ToF-SIMS, NEXAFS, SFG, and XPS

NASA Astrophysics Data System (ADS)

Baio, Joseph E.

There are many techniques that allow surface scientists to study interfaces. However, few are routinely applied to probe biological surfaces. The work presented here demonstrates how detailed information about the conformation, orientation, chemical state, and molecular structure of biological molecules immobilized onto a surface can be assessed by electron spectroscopy, mass spectrometry, and nonlinear vibrational spectroscopy techniques. This investigation began with the development of simple model systems (small proteins, and peptides) and evolved into a study of more complex --- real world systems. Initially, two model systems based on the chemical and electrostatic immobilization of a small rigid protein (Protein G B1 domain, 6kDa) were built to develop the capabilities of time-of-flight secondary ion mass spectrometry (ToFSIMS), near edge X-ray absorption fine structure spectroscopy (NEXAFS) and sum frequency generation (SFG) spectroscopy as tools to probe the structure of surface immobilized proteins. X-ray photoelectron spectroscopy (XPS) was used to measure the amount of immobilized protein and ToF-SIMS sampled the amino acid composition of the exposed surface of the protein film. Within the ToF-SIMS spectra, an enrichment of secondary ions from amino acids located at opposite ends of the proteins were used to describe protein orientation. SFG spectral peaks characteristic of ordered alpha-helix and beta-sheet elements were observed for both systems and the phase of the peaks indicated a predominantly upright orientation for both the covalent and electrostatic configurations. Polarization dependence of the NEXAFS signal from the N 1s to pi* transition of the peptide bonds that make up the beta-sheets also indicated protein ordering at the surface. Building upon the Protein G B1 studies, the orientation and structure of a surface immobilized antibody (HuLys Fv: variant of humanized anti-lysozyme variable fragment, 26kDa) was characterized across two immobilization schemes. This protein contained both a hexahistidine tag and a cysteine residue, introduced at opposite ends of the HuLys Fv, for immobilization onto nitrilotriacetic acid (NTA) and maleimide oligo- (ethylene glycol) (MEG)-terminated substrates. The thiol group on the cysteine residue selectively binds to the MEG groups, while the his-tag selectively binds to the Ni-loaded NTA groups. XPS was used to monitor protein coverage on both surfaces by following the change in the nitrogen atomic %. The ToF-SIMS data provided a clear differentiation between the two samples due to the intensity differences of secondary ions originating from asymmetrically located amino acids in HuLys Fv. Indicating that the HuLys Fv fragment when adsorbed into the NTA and MEG substrates will be induced into two different orientations. On the NTA substrate the protein's binding site is accessible, while on the MEG substrate the binding site is oriented towards the surface. By taking advantage of the electron pathway through the heme group in cytochrome c (CytoC) electrochemists have built sensors based upon CytoC immobilized onto functionalized metal electrodes. When immobilized onto a charged surface, CytoC, with its distribution of lysine and glutamate residues around its surface, should orient and form a well-ordered protein film. Here a detailed examination of CytoC orientation when electrostatically immobilized onto both amine (NH 3+) and carboxyl (COO-) functionalized gold is presented. Again, protein coverage, on both surfaces, was monitored by the change in the atomic % N, as determined by XPS. ToF-SIMS data demonstrated a clear separation between the two samples based on the intensity differences of secondary ions stemming from amino acids located asymmetrically within CytoC, indicating opposite orientations of the protein on the two different surfaces. Spectral features within the in situ sum frequency generation vibrational spectra, acquired for the protein interacting with positively and negatively charged surfaces, indicates that these electrostatic interactions do induce the protein into a well ordered film.

Adsorption effectiveness of β-lactoglobulin onto gold surface determined by quartz crystal microbalance.

PubMed

Jachimska, B; Świątek, S; Loch, J I; Lewiński, K; Luxbacher, T

2018-06-01

Bovine β-lactoglobulin (LGB) is a transport protein that can bind to its structure hydrophobic bioactive molecules. Due to the lack of toxicity, high stability and pH-dependent molecular binding mechanism, lactoglobulin can be used as a carrier of sparingly soluble drugs. Dynamic light scattering has confirmed LGB's tendency to create oligomeric forms. The hydrodynamic diameter of LGB molecules varies from 4 nm to 6 nm in the pH range of 2-10 and ionic strength I = 0.001-0.15 M, which corresponds to the presence of mono or dimeric LGB forms. The LGB zeta potential varies from 26.5 mV to -33.3 mV for I = 0.01 M and from 13.3 mV to -16 mV for I = 0.15 M in the pH range of 2-10. The isoelectric point is at pH 4.8. As a result of strong surface charge compensation, the maximum effective ionization degree of the LGB molecule is 35% for ionic strength I = 0.01 M and 22% for I = 0.15 M. The effectiveness of adsorption is linked with the properties of the protein, as well as those of the adsorption surface. The functionalization of gold surfaces with β-lactoglobulin (LGB) was studied using a quartz crystal microbalance with energy dissipation monitoring (QCM-D). The effectiveness of LGB adsorption correlates strongly with a charge of gold surface and the zeta potential of the molecule. The greatest value of the adsorbed mass was observed in the pH range in which LGB has a positive zeta potential values, below pH 4.8. This observation shows that electrostatic interactions play a dominant role in LGB adsorption on gold surfaces. Based on the adsorbed mass, protein orientation on gold surfaces was determined. The preferential side-on orientation of LGB molecules observed in the adsorption layer is consistent with the direction of the molecule dipole momentum determined by molecular dynamics simulations of the protein (MD). The use of the QCM-D method also allowed us to determine the effectiveness of adsorption of LGB on gold surface. Knowing the mechanism of LGB adsorption is significant importance for determining the optimum conditions for immobilizing this protein on solid surfaces. As β-lactoglobulin is a protein that binds various ligands, the binding properties of immobilized β-lactoglobulin can be used to design controlled protein structures for biomedical applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitative protein expression and cell surface characteristics of Escherichia coli MG1655 biofilms.

PubMed

Mukherjee, Joy; Ow, Saw Yen; Noirel, Josselin; Biggs, Catherine A

2011-02-01

Cell surface physicochemical characterization techniques were combined with quantitative changes in protein expression, to investigate the biological and biophysical changes of Escherichia coli MG1655 cells when grown as a biofilm (BIO). The overall surface charge of BIO cells was found to be less negative, highlighting the need for a lower electrophoretic mobility for attachment to occur. Comparison of the chemical functional groups on the cell surface showed similar profiles, with the absorbance intensity higher for proteins and carbohydrates in the BIO cells. Quantitative proteomic analysis demonstrated that 3 proteins were significantly increased, and 9 proteins significantly decreased in abundance, in cells grown as a BIO compared to their planktonic counterparts, with 7 of these total 12 proteins unique to this study. Proteins showing significant increased or decreased abundance include proteins involved in acid resistance, DNA protection and binding and ABC transporters. Further predictive analysis of the metabolic pathways showed an increased abundance of the amino acid metabolism and tricarboxylic acid (TCA) cycle, with a decrease in expression within the pentose phosphate and glycolysis pathways. It is therefore hypothesized that cells grown as a BIO are still energetically viable potentially using amino acids as an indirect carbon backbone source into the TCA cycle. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interaction of β-sheet folds with a gold surface.

PubMed

Hoefling, Martin; Monti, Susanna; Corni, Stefano; Gottschalk, Kay Eberhard

2011-01-01

The adsorption of proteins on inorganic surfaces is of fundamental biological importance. Further, biomedical and nanotechnological applications increasingly use interfaces between inorganic material and polypeptides. Yet, the underlying adsorption mechanism of polypeptides on surfaces is not well understood and experimentally difficult to analyze. Therefore, we investigate here the interactions of polypeptides with a gold(111) surface using computational molecular dynamics (MD) simulations with a polarizable gold model in explicit water. Our focus in this paper is the investigation of the interaction of polypeptides with β-sheet folds. First, we concentrate on a β-sheet forming model peptide. Second, we investigate the interactions of two domains with high β-sheet content of the biologically important extracellular matrix protein fibronectin (FN). We find that adsorption occurs in a stepwise mechanism both for the model peptide and the protein. The positively charged amino acid Arg facilitates the initial contact formation between protein and gold surface. Our results suggest that an effective gold-binding surface patch is overall uncharged, but contains Arg for contact initiation. The polypeptides do not unfold on the gold surface within the simulation time. However, for the two FN domains, the relative domain-domain orientation changes. The observation of a very fast and strong adsorption indicates that in a biological matrix, no bare gold surfaces will be present. Hence, the bioactivity of gold surfaces (like bare gold nanoparticles) will critically depend on the history of particle administration and the proteins present during initial contact between gold and biological material. Further, gold particles may act as seeds for protein aggregation. Structural re-organization and protein aggregation are potentially of immunological importance.

Separation techniques: Chromatography

PubMed Central

Coskun, Ozlem

2016-01-01

Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification. PMID:28058406

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles.

PubMed

Dijkstra, J; van Galen, M; Scherphof, G

1985-03-14

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library*

PubMed Central

Kiefer, Jonathan D.; Srinivas, Raja R.; Lobner, Elisabeth; Tisdale, Alison W.; Mehta, Naveen K.; Yang, Nicole J.; Tidor, Bruce; Wittrup, K. Dane

2016-01-01

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation. PMID:27582495

Protein-Ligand Interaction Detection with a Novel Method of Transient Induced Molecular Electronic Spectroscopy (TIMES): Experimental and Theoretical Studies.

PubMed

Zhang, Tiantian; Wei, Tao; Han, Yuanyuan; Ma, Heng; Samieegohar, Mohammadreza; Chen, Ping-Wei; Lian, Ian; Lo, Yu-Hwa

2016-11-23

Protein-ligand interaction detection without disturbances (e.g., surface immobilization, fluorescent labeling, and crystallization) presents a key question in protein chemistry and drug discovery. The emergent technology of transient induced molecular electronic spectroscopy (TIMES), which incorporates a unique design of microfluidic platform and integrated sensing electrodes, is designed to operate in a label-free and immobilization-free manner to provide crucial information for protein-ligand interactions in relevant physiological conditions. Through experiments and theoretical simulations, we demonstrate that the TIMES technique actually detects protein-ligand binding through signals generated by surface electric polarization. The accuracy and sensitivity of experiments were demonstrated by precise measurements of dissociation constant of lysozyme and N -acetyl-d-glucosamine (NAG) ligand and its trimer, NAG 3 . Computational fluid dynamics (CFD) computation is performed to demonstrate that the surface's electric polarization signal originates from the induced image charges during the transition state of surface mass transport, which is governed by the overall effects of protein concentration, hydraulic forces, and surface fouling due to protein adsorption. Hybrid atomistic molecular dynamics (MD) simulations and free energy computation show that ligand binding affects lysozyme structure and stability, producing different adsorption orientation and surface polarization to give the characteristic TIMES signals. Although the current work is focused on protein-ligand interactions, the TIMES method is a general technique that can be applied to study signals from reactions between many kinds of molecules.

Floating gate memory with charge storage dots array formed by Dps protein modified with site-specific binding peptides

NASA Astrophysics Data System (ADS)

Kamitake, Hiroki; Uenuma, Mutsunori; Okamoto, Naofumi; Horita, Masahiro; Ishikawa, Yasuaki; Yamashita, Ichro; Uraoka, Yukiharu

2015-05-01

We report a nanodot (ND) floating gate memory (NFGM) with a high-density ND array formed by a biological nano process. We utilized two kinds of cage-shaped proteins displaying SiO2 binding peptide (minTBP-1) on their outer surfaces: ferritin and Dps, which accommodate cobalt oxide NDs in their cavities. The diameters of the cobalt NDs were regulated by the cavity sizes of the proteins. Because minTBP-1 is strongly adsorbed on the SiO2 surface, high-density cobalt oxide ND arrays were obtained by a simple spin coating process. The densities of cobalt oxide ND arrays based on ferritin and Dps were 6.8 × 1011 dots cm-2 and 1.2 × 1012 dots cm-2, respectively. After selective protein elimination and embedding in a metal-oxide-semiconductor (MOS) capacitor, the charge capacities of both ND arrays were evaluated by measuring their C-V characteristics. The MOS capacitor embedded with the Dps ND array showed a wider memory window than the device embedded with the ferritin ND array. Finally, we fabricated an NFGM with a high-density ND array based on Dps, and confirmed its competent writing/erasing characteristics and long retention time.

Light-scattering studies of protein solutions: role of hydration in weak protein-protein interactions.

PubMed

Paliwal, A; Asthagiri, D; Abras, D; Lenhoff, A M; Paulaitis, M E

2005-09-01

We model the hydration contribution to short-range electrostatic/dispersion protein interactions embodied in the osmotic second virial coefficient, B(2), by adopting a quasi-chemical description in which water molecules associated with the protein are identified through explicit molecular dynamics simulations. These water molecules reduce the surface complementarity of highly favorable short-range interactions, and therefore can play an important role in mediating protein-protein interactions. Here we examine this quasi-chemical view of hydration by predicting the interaction part of B(2) and comparing our results with those derived from light-scattering measurements of B(2) for staphylococcal nuclease, lysozyme, and chymotrypsinogen at 25 degrees C as a function of solution pH and ionic strength. We find that short-range protein interactions are influenced by water molecules strongly associated with a relatively small fraction of the protein surface. However, the effect of these strongly associated water molecules on the surface complementarity of short-range protein interactions is significant, and must be taken into account for an accurate description of B(2). We also observe remarkably similar hydration behavior for these proteins despite substantial differences in their three-dimensional structures and spatial charge distributions, suggesting a general characterization of protein hydration.

Expression, purification, crystallization and structure determination of the N terminal domain of Fhb, a factor H binding protein from Streptococcus suis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Chunmao; Yu, You; Yang, Maojun, E-mail: maojunyang@tsinghua.edu.cn

2015-10-23

Fhb is a surface virulence protein from Streptococcus suis, which could aid bacterial evasion of host innate immune defense by recruiting complement regulator factor H to inactivate C3b deposited on bacterial surface in blood. Here we successfully expressed and purified the N terminal domain of Fhb (N-Fhb) and obtained crystals of the N-Fhb by sitting-drop vapor diffusion method with a resolution of 1.50 Å. The crystals belong to space group C2 with unit cell parameters a = 127.1 Å, b = 77.3 Å, c = 131.6 Å, α = 90°, β = 115.9°, γ = 90°. The structure of N-Fhb was determined by SAD method and the core structure of N-Fhb is a β sandwich. Wemore » speculated that binding of Fhb to human factor H may be mainly mediated by surface amino acids with negative charges. - Highlights: • We expressed N-Fhb as the soluble protein in Escherichia coli. • Crystals of N-Fhb were grown by sitting drop vapor diffusion method. • Crystals of N-Fhb could diffracted to 1.5 Å. • The core structure of N-Fhb was a β sandwich. • A part of the surface of N-Fhb was rich with negative charges.« less

Derivation of Poisson and Nernst-Planck equations in a bath and channel from a molecular model.

PubMed

Schuss, Z; Nadler, B; Eisenberg, R S

2001-09-01

Permeation of ions from one electrolytic solution to another, through a protein channel, is a biological process of considerable importance. Permeation occurs on a time scale of micro- to milliseconds, far longer than the femtosecond time scales of atomic motion. Direct simulations of atomic dynamics are not yet possible for such long-time scales; thus, averaging is unavoidable. The question is what and how to average. In this paper, we average a Langevin model of ionic motion in a bulk solution and protein channel. The main result is a coupled system of averaged Poisson and Nernst-Planck equations (CPNP) involving conditional and unconditional charge densities and conditional potentials. The resulting NP equations contain the averaged force on a single ion, which is the sum of two components. The first component is the gradient of a conditional electric potential that is the solution of Poisson's equation with conditional and permanent charge densities and boundary conditions of the applied voltage. The second component is the self-induced force on an ion due to surface charges induced only by that ion at dielectric interfaces. The ion induces surface polarization charge that exerts a significant force on the ion itself, not present in earlier PNP equations. The proposed CPNP system is not complete, however, because the electric potential satisfies Poisson's equation with conditional charge densities, conditioned on the location of an ion, while the NP equations contain unconditional densities. The conditional densities are closely related to the well-studied pair-correlation functions of equilibrium statistical mechanics. We examine a specific closure relation, which on the one hand replaces the conditional charge densities by the unconditional ones in the Poisson equation, and on the other hand replaces the self-induced force in the NP equation by an effective self-induced force. This effective self-induced force is nearly zero in the baths but is approximately equal to the self-induced force in and near the channel. The charge densities in the NP equations are interpreted as time averages over long times of the motion of a quasiparticle that diffuses with the same diffusion coefficient as that of a real ion, but is driven by the averaged force. In this way, continuum equations with averaged charge densities and mean-fields can be used to describe permeation through a protein channel.

Personalized protein corona on nanoparticles and its clinical implications.

PubMed

Corbo, Claudia; Molinaro, Roberto; Tabatabaei, Mateen; Farokhzad, Omid C; Mahmoudi, Morteza

2017-02-28

It is now well understood that once in contact with biological fluids, nanoscale objects lose their original identity and acquire a new biological character, referred to as a protein corona. The protein corona changes many of the physicochemical properties of nanoparticles, including size, surface charge, and aggregation state. These changes, in turn, affect the biological fate of nanoparticles, including their pharmacokinetics, biodistribution, and therapeutic efficacy. It is progressively being accepted that even slight variations in the composition of a protein source (e.g., plasma and serum) can substantially change the composition of the corona formed on the surface of the exact same nanoparticles. Recently it has been shown that the protein corona is strongly affected by the patient's specific disease. Therefore, the same nanomaterial incubated with plasma proteins of patients with different pathologies adsorb protein coronas with different compositions, giving rise to the concept of personalized protein corona. Herein, we review this concept along with recent advances on the topic, with a particular focus on clinical relevance.

Links between the charge model and bonded parameter force constants in biomolecular force fields

NASA Astrophysics Data System (ADS)

Cerutti, David S.; Debiec, Karl T.; Case, David A.; Chong, Lillian T.

2017-10-01

The ff15ipq protein force field is a fixed charge model built by automated tools based on the two charge sets of the implicitly polarized charge method: one set (appropriate for vacuum) for deriving bonded parameters and the other (appropriate for aqueous solution) for running simulations. The duality is intended to treat water-induced electronic polarization with an understanding that fitting data for bonded parameters will come from quantum mechanical calculations in the gas phase. In this study, we compare ff15ipq to two alternatives produced with the same fitting software and a further expanded data set but following more conventional methods for tailoring bonded parameters (harmonic angle terms and torsion potentials) to the charge model. First, ff15ipq-Qsolv derives bonded parameters in the context of the ff15ipq solution phase charge set. Second, ff15ipq-Vac takes ff15ipq's bonded parameters and runs simulations with the vacuum phase charge set used to derive those parameters. The IPolQ charge model and associated protocol for deriving bonded parameters are shown to be an incremental improvement over protocols that do not account for the material phases of each source of their fitting data. Both force fields incorporating the polarized charge set depict stable globular proteins and have varying degrees of success modeling the metastability of short (5-19 residues) peptides. In this particular case, ff15ipq-Qsolv increases stability in a number of α -helices, correctly obtaining 70% helical character in the K19 system at 275 K and showing appropriately diminishing content up to 325 K, but overestimating the helical fraction of AAQAA3 by 50% or more, forming long-lived α -helices in simulations of a β -hairpin, and increasing the likelihood that the disordered p53 N-terminal peptide will also form a helix. This may indicate a systematic bias imparted by the ff15ipq-Qsolv parameter development strategy, which has the hallmarks of strategies used to develop other popular force fields, and may explain some of the need for manual corrections in this force fields' evolution. In contrast, ff15ipq-Vac incorrectly depicts globular protein unfolding in numerous systems tested, including Trp cage, villin, lysozyme, and GB3, and does not perform any better than ff15ipq or ff15ipq-Qsolv in tests on short peptides. We analyze the free energy surfaces of individual amino acid dipeptides and the electrostatic potential energy surfaces of each charge model to explain the differences.

Reduced Capillary Length Scale in the Application of Ostwald Ripening Theory to the Coarsening of Charged Colloidal Crystals in Electrolyte Solutions

NASA Astrophysics Data System (ADS)

Rowe, Jeffrey D.; Baird, James K.

2007-06-01

A colloidal crystal suspended in an electrolyte solution will ordinarily exchange ions with the surrounding solution and develop a net surface charge density and a corresponding double layer. The interfacial tension of the charged surface has contributions arising from: (a) background interfacial tension of the uncharged surface, (b) the entropy associated with the adsorption of ions on the surface, and (c) the polarizing effect of the electrostatic field within the double layer. The adsorption and polarization effects make negative contributions to the surface free energy and serve to reduce the interfacial tension below the value to be expected for the uncharged surface. The diminished interfacial tension leads to a reduced capillary length scale. According to the Ostwald ripening theory of particle coarsening, the reduced capillary length will cause the solute supersaturation to decay more rapidly and the colloidal particles to be smaller in size and greater in number than in the absence of the double layer. Although the length scale for coarsening should be little affected in the case of inorganic colloids, such as AgI, it should be greatly reduced in the case of suspensions of protein crystals, such as apoferritin, catalase, and thaumatin.

Electrostatic model for protein adsorption in ion-exchange chromatography and application to monoclonal antibodies, lysozyme and chymotrypsinogen A.

PubMed

Guélat, Bertrand; Ströhlein, Guido; Lattuada, Marco; Morbidelli, Massimo

2010-08-27

A model for the adsorption equilibrium of proteins in ion-exchange chromatography explicitly accounting for the effect of pH and salt concentration in the limit of highly diluted systems was developed. It is based on the use of DLVO theory to estimate the electrostatic interactions between the charged surface of the ion-exchanger and the proteins. The corresponding charge distributions were evaluated as a function of pH and salt concentration using a molecular approach. The model was verified for the adsorption equilibrium of lysozyme, chymotrypsinogen A and four industrial monoclonal antibodies on two strong cation-exchangers. The adsorption equilibrium constants of these proteins were determined experimentally at various pH values and salt concentrations and the model was fitted with a good agreement using three adjustable parameters for each protein in the whole range of experimental conditions. Despite the simplifications of the model regarding the geometry of the protein-ion-exchanger system, the physical meaning of the parameters was retained. 2010 Elsevier B.V. All rights reserved.

Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours

NASA Astrophysics Data System (ADS)

Batoulis, Helena; Schmidt, Thomas H.; Weber, Pascal; Schloetel, Jan-Gero; Kandt, Christian; Lang, Thorsten

2016-04-01

Salts and proteins comprise two of the basic molecular components of biological materials. Kosmotropic/chaotropic co-solvation and matching ion water affinities explain basic ionic effects on protein aggregation observed in simple solutions. However, it is unclear how these theories apply to proteins in complex biological environments and what the underlying ionic binding patterns are. Using the positive ion Ca2+ and the negatively charged membrane protein SNAP25, we studied ion effects on protein oligomerization in solution, in native membranes and in molecular dynamics (MD) simulations. We find that concentration-dependent ion-induced protein oligomerization is a fundamental chemico-physical principle applying not only to soluble but also to membrane-anchored proteins in their native environment. Oligomerization is driven by the interaction of Ca2+ ions with the carboxylate groups of aspartate and glutamate. From low up to middle concentrations, salt bridges between Ca2+ ions and two or more protein residues lead to increasingly larger oligomers, while at high concentrations oligomers disperse due to overcharging effects. The insights provide a conceptual framework at the interface of physics, chemistry and biology to explain binding of ions to charged protein surfaces on an atomistic scale, as occurring during protein solubilisation, aggregation and oligomerization both in simple solutions and membrane systems.

DNA-Mediated Electrochemistry

PubMed Central

Gorodetsky, Alon A.; Buzzeo, Marisa C.

2009-01-01

The base pair stack of DNA has been demonstrated as a medium for long range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry. PMID:18980370

Electrostatic Control of Protein-Surface Interactions

DTIC Science & Technology

2013-10-21

occurs in numerous human diseases including Alzheimer’s, Parkinson’s, and type II diabetes (which are associated with the aggregation of the...demonstrates how examples of how a model α- helical peptide that contains positively charged lysine residues along one face and hydrophobic leucines and our

Crystal structure of human IPS-1/MAVS/VISA/Cardif caspase activation recruitment domain.

PubMed

Potter, Jane A; Randall, Richard E; Taylor, Garry L

2008-02-28

IPS-1/MAVS/VISA/Cardif is an adaptor protein that plays a crucial role in the induction of interferons in response to viral infection. In the initial stage of the intracellular antiviral response two RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-association gene 5 (MDA5), are independently able to bind viral RNA in the cytoplasm. The 62 kDa protein IPS-1/MAVS/VISA/Cardif contains an N-terminal caspase activation and recruitment (CARD) domain that associates with the CARD regions of RIG-I and MDA5, ultimately leading to the induction of type I interferons. As a first step towards understanding the molecular basis of this important adaptor protein we have undertaken structural studies of the IPS-1 MAVS/VISA/Cardif CARD region. The crystal structure of human IPS-1/MAVS/VISA/Cardif CARD has been determined to 2.1A resolution. The protein was expressed and crystallized as a maltose-binding protein (MBP) fusion protein. The MBP and IPS-1 components each form a distinct domain within the structure. IPS-1/MAVS/VISA/Cardif CARD adopts a characteristic six-helix bundle with a Greek-key topology and, in common with a number of other known CARD structures, contains two major polar surfaces on opposite sides of the molecule. One face has a surface-exposed, disordered tryptophan residue that may explain the poor solubility of untagged expression constructs. The IPS-1/MAVS/VISA/Cardif CARD domain adopts the classic CARD fold with an asymmetric surface charge distribution that is typical of CARD domains involved in homotypic protein-protein interactions. The location of the two polar areas on IPS-1/MAVS/VISA/Cardif CARD suggest possible types of associations that this domain makes with the two CARD domains of MDA5 or RIG-I. The N-terminal CARD domains of RIG-I and MDA5 share greatest sequence similarity with IPS-1/MAVS/VISA/Cardif CARD and this has allowed modelling of their structures. These models show a very different charge profile for the equivalent surfaces compared to IPS-1/MAVS/VISA/Cardif CARD.

Incorporating water-release and lateral protein interactions in modeling equilibrium adsorption for ion-exchange chromatography.

PubMed

Thrash, Marvin E; Pinto, Neville G

2006-09-08

The equilibrium adsorption of two albumin proteins on a commercial ion exchanger has been studied using a colloidal model. The model accounts for electrostatic and van der Waals forces between proteins and the ion exchanger surface, the energy of interaction between adsorbed proteins, and the contribution of entropy from water-release accompanying protein adsorption. Protein-surface interactions were calculated using methods previously reported in the literature. Lateral interactions between adsorbed proteins were experimentally measured with microcalorimetry. Water-release was estimated by applying the preferential interaction approach to chromatographic retention data. The adsorption of ovalbumin and bovine serum albumin on an anion exchanger at solution pH>pI of protein was measured. The experimental isotherms have been modeled from the linear region to saturation, and the influence of three modulating alkali chlorides on capacity has been evaluated. The heat of adsorption is endothermic for all cases studied, despite the fact that the net charge on the protein is opposite that of the adsorbing surface. Strong repulsive forces between adsorbed proteins underlie the endothermic heat of adsorption, and these forces intensify with protein loading. It was found that the driving force for adsorption is the entropy increase due to the release of water from the protein and adsorbent surfaces. It is shown that the colloidal model predicts protein adsorption capacity in both the linear and non-linear isotherm regions, and can account for the effects of modulating salt.

Conformational flexibility of domain III of annexin V: the effect of pH and binding to membrane-water interfaces

NASA Astrophysics Data System (ADS)

Sopkova, Jana; Vincent, Michel; Takahashi, Maza; Lewit-Bentley, Anita; Gallay, Jacques

1999-05-01

Steady-state and time-resolved fluorescence of the single tryptophan residue (W187) of annexin V show that the conformation and the dynamics of domain III are strongly modified upon binding of the protein to negatively charged phospholipid vesicles in the presence of calcium, or upon incorporation into reverse micelles of water/sodium bis(2- ethylhexyl) sulfosuccinate (AOT) in iso-octane. In the protein at neutral pH, W187 is slightly mobile and buried in a hydrophobic pocket. It becomes more mobile and is moved in a more polar environment when the protein interacts with the model membranes. In each condition, the heterogeneity of the fluorescence intensity decay of W187 is likely due to the co- existence of local conformers with different dynamics. A similar change of conformation and dynamics can be provoked by mild acidic pH. This suggests that electrostatic interactions are important for the folding of domain III. An interplay of salt bridges implying charged amino-acid side-chains at the protein surface in domain III can be observed in the crystal structure. Local pH modifications at the membrane surface can therefore be responsible for the observed conformational change. The high flexibility of domain III in the membrane- bound protein suggests moreover that this domain may not be crucial for the interaction of the protein with the membrane, in agreement with recent atomic force microscope results (Reviakine et al., 1998, J. Struct. Biol. 121, 356-362).

Hydroxyapatite formation on graphene oxide modified with amino acids: arginine versus glutamic acid

PubMed Central

Tavafoghi, M.; Brodusch, N.; Gauvin, R.; Cerruti, M.

2016-01-01

Hydroxyapatite (HA, Ca5(PO4)3OH) is the main inorganic component of hard tissues, such as bone and dentine. HA nucleation involves a set of negatively charged phosphorylated proteins known as non-collagenous proteins (NCPs). These proteins attract Ca2+ and PO43− ions and increase the local supersaturation to a level required for HA precipitation. Polar and charged amino acids (AAs) are highly expressed in NCPs, and seem to be responsible for the mineralizing effect of NCPs; however, the individual effect of these AAs on HA mineralization is still unclear. In this work, we investigate the effect of a negatively charged (Glu) and positively charged (Arg) AA bound to carboxylated graphene oxide (CGO) on HA mineralization in simulated body fluids (SBF). Our results show that Arg induces HA precipitation faster and in larger amounts than Glu. We attribute this to the higher stability of the complexes formed between Arg and Ca2+ and PO43− ions, and also to the fact that Arg exposes both carboxyl and amino groups on the surface. These can electrostatically attract both Ca2+ and PO43− ions, thus increasing local supersaturation more than Glu, which exposes carboxyl groups only. PMID:26791001

Hydroxyapatite formation on graphene oxide modified with amino acids: arginine versus glutamic acid.

PubMed

Tavafoghi, M; Brodusch, N; Gauvin, R; Cerruti, M

2016-01-01

Hydroxyapatite (HA, Ca5(PO4)3OH) is the main inorganic component of hard tissues, such as bone and dentine. HA nucleation involves a set of negatively charged phosphorylated proteins known as non-collagenous proteins (NCPs). These proteins attract Ca(2+) and PO4(3-) ions and increase the local supersaturation to a level required for HA precipitation. Polar and charged amino acids (AAs) are highly expressed in NCPs, and seem to be responsible for the mineralizing effect of NCPs; however, the individual effect of these AAs on HA mineralization is still unclear. In this work, we investigate the effect of a negatively charged (Glu) and positively charged (Arg) AA bound to carboxylated graphene oxide (CGO) on HA mineralization in simulated body fluids (SBF). Our results show that Arg induces HA precipitation faster and in larger amounts than Glu. We attribute this to the higher stability of the complexes formed between Arg and Ca(2+) and PO4(3-) ions, and also to the fact that Arg exposes both carboxyl and amino groups on the surface. These can electrostatically attract both Ca(2+) and PO4(3-) ions, thus increasing local supersaturation more than Glu, which exposes carboxyl groups only. © 2016 The Author(s).

Maintenance of coat protein N-terminal net charge and not primary sequence is essential for zucchini yellow mosaic virus systemic infectivity.

PubMed

Kimalov, Boaz; Gal-On, Amit; Stav, Ran; Belausov, Eduard; Arazi, Tzahi

2004-11-01

Zucchini yellow mosaic virus (ZYMV) surface exposed coat protein (CP) N-terminal domain (Nt) is 43 aa long and contains an equal number of positively and negatively charged amino acid residues (CP-Nt net charge = 0). A ZYMV-AGII truncation mutant lacking the first 20 aa of its CP-Nt (AGII-CP Delta 20; CP-Nt net charge = +2) was found to be systemically non-infectious even though AGII mutants harbouring larger CP-Nt deletions were previously demonstrated to be fully infectious. Nevertheless, AGII-CP Delta 20 infectivity was restored by fusion to its CP-Nt two Asp residues or a negatively charged Myc peptide, both predicted to neutralize CP-Nt net positive charge. To evaluate further the significance of CP-Nt net charge for AGII infectivity, a series of CP-Nt net charge mutants was generated and analysed for systemic infectivity of squash plants. AGII-CP(KKK) harbouring a CP-Nt amino fusion of three Lys residues (CP-Nt net charge = +3) was not systemically infectious. Addition of up to four Asp residues to CP-Nt did not abolish virus infectivity, although certain mutants were genetically unstable and had delayed infectivity. Addition of five negatively charged residues abolished infectivity (AGII-CP(DDDDD); CP-Nt net charge = -5) even though a recombinant CP(DDDDD) could assemble into potyviral-like particle in bacteria. Neutralization of CP-Nt net charge by fusing Asp or Lys residues recovered infectivity of AGII-CP(KKK) and AGII-CP(DDDDD). GFP-tagging of these mutants has demonstrated that both viruses have defective cell-to-cell movement. Together, these findings suggest that maintenance of CP-Nt net charge and not primary sequence is essential for ZYMV infectivity.

The interplay between pH sensitivity and label-free protein detection in immunologically modified nano-scaled field-effect transistor.

PubMed

Shalev, Gil; Rosenwaks, Yossi; Levy, Ilan

2012-01-15

We present experimental results in order to establish a correlation between pH sensitivity of immunologically modified nano-scaled field-effect transistor (NS-ImmunoFET) with their sensing capacity for label-free detection. The NS-ImmunoFETs are fabricated from silicon-on-insulator (SOI) wafers and are fully-depleted with thickness of ~20 nm. The data shows that higher sensitivity to pH entails enhanced sensitivity to analyte detection. This suggests that the mechanism of analyte detection as pure electrostatic perturbation induced by antibody-analyte interaction is over simplified. The fundamental assumption, in existing models for field-effect sensing mechanism assumes that the analyte molecules do not directly interact with the surface but rather stand 'deep' in the solution and away from the dielectric surface. Recent studies clearly provide contradicting evidence demonstrating that antibodies lie down flat on the surface. These observations led us to propose that the proteins that cover the gate area intimately interact with active sites on the surface thus forming a network of interacting sites. Since sensitivity to pH is directly correlated with the amount of amphoteric sites, we witness a direct correlation between sensitivity to pH and analyte detection. The highest and lowest threshold voltage shift for a label-free and specific detection of 6.5 nM IgG were 40 mV and 2.3 mV for NS-ImmunoFETs with pH sensitivity of 35 mV/decade and 15 mV/decade, respectively. Finally, physical modeling of the NS-ImmunoFET is presented and charge of a single IgG protein at pH 6 is calculated. The obtained value is consistent with charge of IgG protein cited in literature. Copyright © 2011 Elsevier B.V. All rights reserved.

Assembly of purple membranes on polyelectrolyte films.

PubMed

Saab, Marie-belle; Estephan, Elias; Cloitre, Thierry; Legros, René; Cuisinier, Frédéric J G; Zimányi, László; Gergely, Csilla

2009-05-05

The membrane protein bacteriorhodopsin in its native membrane bound form (purple membrane) was adsorbed and incorporated into polyelectrolyte multilayered films, and adsorption was in situ monitored by optical waveguide light-mode spectroscopy. The formation of a single layer or a double layer of purple membranes was observed when adsorbed on negatively or positively charged surfaces, respectively. The purple membrane patches adsorbed on the polyelectrolyte multilayers were also evidenced by atomic force microscopy images. The driving forces of the adsorption process were evaluated by varying the ionic strength of the solution as well as the purple membrane concentration. At high purple membrane concentration, interpenetrating polyelectrolyte loops might provide new binding sites for the adsorption of a second layer of purple membranes, whereas at lower concentrations only a single layer is formed. Negative surfaces do not promote a second protein layer adsorption. Driving forces other than just electrostatic ones, such as hydrophobic forces, should play a role in the polyelectrolyte/purple membrane layering. The subtle interplay of all these factors determines the formation of the polyelectrolyte/purple membrane matrix with a presumably high degree of orientation for the incorporated purple membranes, with their cytoplasmic, or extracellular side toward the bulk on negatively or positively charged polyelectrolyte, respectively. The structural stability of bacteriorhodopsin during adsorption onto the surface and incorporation into the polyelectrolyte multilayers was investigated by Fourier transform infrared spectroscopy in attenuated total reflection mode. Adsorption and incorporation of purple membranes within polyelectrolyte multilayers does not disturb the conformational majority of membrane-embedded alpha-helix structures of the protein, but may slightly alter the structure of the extramembraneous segments or their interaction with the environment. This high stability is different from the lower stability of the predominantly beta-sheet structures of numerous globular proteins when adsorbed onto surfaces.

The ASPP interaction network: electrostatic differentiation between pro- and anti-apoptotic proteins.

PubMed

Benyamini, Hadar; Friedler, Assaf

2011-01-01

The ASPP proteins are apoptosis regulators: ASPP1 and ASPP2 promote, while iASPP inhibits, apoptosis. The mechanism by which these different outcomes are achieved is still unknown. The C-terminal ankyrin repeats and SH3 domain (ANK-SH3) mediate the interactions of the ASPP proteins with major apoptosis regulators such as p53, Bcl-2, and NFκB. The structure of the complex between ASPP2(ANK-SH3) and the core domain of p53 (p53CD) was previously determined. We have recently characterized the individual interactions of ASPP2(ANK-SH3) with Bcl-2 and NFκB, as well as a regulatory intramolecular interaction with the proline rich domain of ASPP2. Here we compared the ASPP interactions at two levels: ASPP2(ANK-SH3) with different proteins, and different ASPP family members with each protein partner. We found that the binding sites of ASPP2 to p53CD, Bcl-2, and NFκB are different, yet lie on the same face of ASPP2(ANK-SH3) . The intramolecular binding site to the proline rich domain overlaps the three intermolecular binding sites. To reveal the basis of functional diversity in the ASPP family, we compared their protein-binding domains. A subset of surface-exposed residues differentiates ASPP1 and ASPP2 from iASPP: ASPP1/2 are more negatively charged in specific residues that contact positively charged residues of p53CD, Bcl-2, and NFκB. We also found a gain of positive charge at the non-protein binding face of ASPP1/2, suggesting a role in electrostatic direction towards the negatively charged protein binding face. The electrostatic differences in binding interfaces between the ASPP proteins may be one of the causes for their different function. Copyright © 2010 John Wiley & Sons, Ltd.

Subtle Change in the Charge Distribution of Surface Residues May Affect the Secondary Functions of Cytochrome c*

PubMed Central

Paul, Simanta Sarani; Sil, Pallabi; Haldar, Shubhasis; Mitra, Samaresh; Chattopadhyay, Krishnananda

2015-01-01

Although the primary function of cytochrome c (cyt c) is electron transfer, the protein caries out an additional secondary function involving its interaction with membrane cardiolipin (CDL), its peroxidase activity, and the initiation of apoptosis. Whereas the primary function of cyt c is essentially conserved, its secondary function varies depending on the source of the protein. We report here a detailed experimental and computational study, which aims to understand, at the molecular level, the difference in the secondary functions of cyt c obtained from horse heart (mammalian) and Saccharomyces cerevisiae (yeast). The conformational landscape of cyt c has been found to be heterogeneous, consisting of an equilibrium between the compact and extended conformers as well as the oligomeric species. Because the determination of relative populations of these conformers is difficult to obtain by ensemble measurements, we used fluorescence correlation spectroscopy (FCS), a method that offers single-molecule resolution. The population of different species is found to depend on multiple factors, including the protein source, the presence of CDL and urea, and their concentrations. The complex interplay between the conformational distribution and oligomerization plays a crucial role in the variation of the pre-apoptotic regulation of cyt c observed from different sources. Finally, computational studies reveal that the variation in the charge distribution at the surface and the charge reversal sites may be the key determinant of the conformational stability of cyt c. PMID:25873393

Using extremely halophilic bacteria to understand the role of surface charge and surface hydration in protein evolution, folding, and function

NASA Astrophysics Data System (ADS)

Hoff, Wouter; Deole, Ratnakar; Osu Collaboration

2013-03-01

Halophilic Archaea accumulate molar concentrations of KCl in their cytoplasm as an osmoprotectant, and have evolved highly acidic proteomes that only function at high salinity. We examine osmoprotection in the photosynthetic Proteobacteria Halorhodospira halophila. We find that H. halophila has an acidic proteome and accumulates molar concentrations of KCl when grown in high salt media. Upon growth of H. halophila in low salt media, its cytoplasmic K + content matches that of Escherichia coli, revealing an acidic proteome that can function in the absence of high cytoplasmic salt concentrations. These findings necessitate a reassessment of two central aspects of theories for understanding extreme halophiles. We conclude that proteome acidity is not driven by stabilizing interactions between K + ions and acidic side chains, but by the need for maintaining sufficient solvation and hydration of the protein surface at high salinity through strongly hydrated carboxylates. We propose that obligate protein halophilicity is a non-adaptive property resulting from genetic drift in which constructive neutral evolution progressively incorporates weakly stabilizing K + binding sites on an increasingly acidic protein surface.

Efficient in vitro encapsulation of protein cargo by an engineered protein container.

PubMed

Wörsdörfer, Bigna; Pianowski, Zbigniew; Hilvert, Donald

2012-01-18

An engineered variant of lumazine synthase, a nonviral capsid protein with a negatively charged luminal surface, is shown to encapsulate up to 100 positively supercharged green fluorescent protein (GFP) molecules in vitro. Packaging can be achieved starting either from intact, empty capsids or from capsid fragments by incubation with cargo in aqueous buffer. The yield of encapsulated GFP correlates directly with the host/guest mixing ratio, providing excellent control over packing density. Facile in vitro loading highlights the unusual structural dynamics of this novel nanocontainer and should facilitate diverse biotechnological and materials science applications. © 2011 American Chemical Society

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex.

PubMed

Perederina, Anna; Nevskaya, Natalia; Nikonov, Oleg; Nikulin, Alexei; Dumas, Philippe; Yao, Min; Tanaka, Isao; Garber, Maria; Gongadze, George; Nikonov, Stanislav

2002-12-01

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex.

PubMed Central

Perederina, Anna; Nevskaya, Natalia; Nikonov, Oleg; Nikulin, Alexei; Dumas, Philippe; Yao, Min; Tanaka, Isao; Garber, Maria; Gongadze, George; Nikonov, Stanislav

2002-01-01

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit. PMID:12515387

Physicochemical code for quinary protein interactions in Escherichia coli

PubMed Central

Mu, Xin; Choi, Seongil; Lang, Lisa; Mowray, David; Danielsson, Jens; Oliveberg, Mikael

2017-01-01

How proteins sense and navigate the cellular interior to find their functional partners remains poorly understood. An intriguing aspect of this search is that it relies on diffusive encounters with the crowded cellular background, made up of protein surfaces that are largely nonconserved. The question is then if/how this protein search is amenable to selection and biological control. To shed light on this issue, we examined the motions of three evolutionary divergent proteins in the Escherichia coli cytoplasm by in-cell NMR. The results show that the diffusive in-cell motions, after all, follow simplistic physical−chemical rules: The proteins reveal a common dependence on (i) net charge density, (ii) surface hydrophobicity, and (iii) the electric dipole moment. The bacterial protein is here biased to move relatively freely in the bacterial interior, whereas the human counterparts more easily stick. Even so, the in-cell motions respond predictably to surface mutation, allowing us to tune and intermix the protein’s behavior at will. The findings show how evolution can swiftly optimize the diffuse background of protein encounter complexes by just single-point mutations, and provide a rational framework for adjusting the cytoplasmic motions of individual proteins, e.g., for rescuing poor in-cell NMR signals and for optimizing protein therapeutics. PMID:28536196

Effect of cholesterol on electrostatics in lipid-protein films of a pulmonary surfactant.

PubMed

Finot, Eric; Leonenko, Yuri; Moores, Brad; Eng, Lukas; Amrein, Matthias; Leonenko, Zoya

2010-02-02

We report the changes in the electrical properties of the lipid-protein film of pulmonary surfactant produced by excess cholesterol. Pulmonary surfactant (PS) is a complex lipid-protein mixture that forms a molecular film at the interface of the lung's epithelia. The defined molecular arrangement of the lipids and proteins of the surfactant film gives rise to the locally highly variable electrical surface potential of the interface, which becomes considerably altered in the presence of cholesterol. With frequency modulation Kelvin probe force microscopy (FM-KPFM) and force measurements, complemented by theoretical analysis, we showed that excess cholesterol significantly changes the electric field around a PS film because of the presence of nanometer-sized electrostatic domains and affects the electrostatic interaction of an AFM probe with a PS film. These changes in the local electrical field would greatly alter the interaction of the surfactant film with charged species and would immediately impact the manner in which inhaled (often charged) airborne nanoparticles and fibers might interact with the lung interface.

Switching cell penetrating and CXCR4-binding activities of nanoscale-organized arginine-rich peptides.

PubMed

Favaro, Marianna Teixeira de Pinho; Serna, Naroa; Sánchez-García, Laura; Cubarsi, Rafael; Roldán, Mónica; Sánchez-Chardi, Alejandro; Unzueta, Ugutz; Mangues, Ramón; Ferrer-Miralles, Neus; Azzoni, Adriano Rodrigues; Vázquez, Esther; Villaverde, Antonio

2018-05-16

Arginine-rich protein motifs have been described as potent cell-penetrating peptides (CPPs) but also as rather specific ligands of the cell surface chemokine receptor CXCR4, involved in the infection by the human immunodeficiency virus (HIV). Polyarginines are commonly used to functionalize nanoscale vehicles for gene therapy and drug delivery, aimed to enhance cell penetrability of the therapeutic cargo. However, under which conditions these peptides do act as either unspecific or specific ligands is unknown. We have here explored the cell penetrability of differently charged polyarginines in two alternative presentations, namely as unassembled fusion proteins or assembled in multimeric protein nanoparticles. By this, we have observed that arginine-rich peptides switch between receptor-mediated and receptor-independent mechanisms of cell penetration. The relative weight of these activities is determined by the electrostatic charge of the construct and the oligomerization status of the nanoscale material, both regulatable by conventional protein engineering approaches. Copyright © 2018 Elsevier Inc. All rights reserved.

The influence of ligand charge and length on the assembly of Brome mosaic virus derived virus-like particles with magnetic core

NASA Astrophysics Data System (ADS)

Mieloch, Adam A.; Krecisz, Monika; Rybka, Jakub D.; Strugała, Aleksander; Krupiński, Michał; Urbanowicz, Anna; Kozak, Maciej; Skalski, Bohdan; Figlerowicz, Marek; Giersig, Michael

2018-03-01

Virus-like particles (VLPs) have sparked a great interest in the field of nanobiotechnology and nanomedicine. The introduction of superparamagnetic nanoparticles (SPIONs) as a core, provides potential use of VLPs in the hyperthermia therapy, MRI contrast agents and magnetically-powered delivery agents. Magnetite NPs also provide a significant improvement in terms of VLPs stability. Moreover employing viral structural proteins as self-assembling units has opened a new paths for targeted therapy, drug delivery systems, vaccines design, and many more. In many cases, the self-assembly of a virus strongly depends on electrostatic interactions between positively charged groups of the capsid proteins and negatively charged nucleic acid. This phenomenon imposes the negative net charge as a key requirement for the core nanoparticle. In our experiments, Brome mosaic virus (BMV) capsid proteins isolated from infected plants Hordeum vulgare were used. Superparamagnetic iron oxide nanoparticles (Fe3O4) with 15 nm in diameter were synthesized by thermal decomposition and functionalized with COOH-PEG-PL polymer or dihexadecylphosphate (DHP) in order to provide water solubility and negative charge required for the assembly. Nanoparticles were characterized by Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), Zeta Potential, Fourier Transformed Infrared Spectroscopy (FTIR) and Superconducting Quantum Interference Device (SQUID) magnetometry. TEM and DLS study were conducted to verify VLPs creation. This study demonstrates that the increase of negative surface charge is not a sufficient factor determining successful assembly. Additional steric interactions provided by longer ligands are crucial for the assembly of BMV SPION VLPs and may enhance the colloidal stability.

Drastic differences in glycosylation of related S-layer glycoproteins from moderate and extreme halophiles.

PubMed

Mengele, R; Sumper, M

1992-04-25

The outer surface of the moderate halophilic archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer glycoprotein. The polypeptide (794 amino acid residues) contains 7 N-glycosylation sites. Four of these sites were isolated as glycopeptides and the structure of one of the corresponding saccharides was determined. Oligosaccharides consisting of beta-1,4-linked glucose residues are attached to the protein via the linkage unit asparaginyl-glucose. In the related glycoprotein from the extreme halophile Halobacterium halobium, the glucose residues are replaced by sulfated glucuronic acid residues, causing a drastic increase in surface charge density. This is discussed in terms of a recent model explaining the stability of halophilic proteins.

Interaction of β-Sheet Folds with a Gold Surface

PubMed Central

Hoefling, Martin; Monti, Susanna; Corni, Stefano; Gottschalk, Kay Eberhard

2011-01-01

The adsorption of proteins on inorganic surfaces is of fundamental biological importance. Further, biomedical and nanotechnological applications increasingly use interfaces between inorganic material and polypeptides. Yet, the underlying adsorption mechanism of polypeptides on surfaces is not well understood and experimentally difficult to analyze. Therefore, we investigate here the interactions of polypeptides with a gold(111) surface using computational molecular dynamics (MD) simulations with a polarizable gold model in explicit water. Our focus in this paper is the investigation of the interaction of polypeptides with β-sheet folds. First, we concentrate on a β-sheet forming model peptide. Second, we investigate the interactions of two domains with high β-sheet content of the biologically important extracellular matrix protein fibronectin (FN). We find that adsorption occurs in a stepwise mechanism both for the model peptide and the protein. The positively charged amino acid Arg facilitates the initial contact formation between protein and gold surface. Our results suggest that an effective gold-binding surface patch is overall uncharged, but contains Arg for contact initiation. The polypeptides do not unfold on the gold surface within the simulation time. However, for the two FN domains, the relative domain-domain orientation changes. The observation of a very fast and strong adsorption indicates that in a biological matrix, no bare gold surfaces will be present. Hence, the bioactivity of gold surfaces (like bare gold nanoparticles) will critically depend on the history of particle administration and the proteins present during initial contact between gold and biological material. Further, gold particles may act as seeds for protein aggregation. Structural re-organization and protein aggregation are potentially of immunological importance. PMID:21687744

Cytochrome c at charged interfaces studied by resonance Raman and surface-enhanced resonance Raman spectroscopy

NASA Astrophysics Data System (ADS)

Hildebrandt, Peter

1991-05-01

The effect of electrostatic fields on the structure of cytochrome c bound to charged interfaces was studied by resonance Raman and surface enhanced resonance Raman spectroscopy. Binding of this heme protein to the Ag electrode or heteropolytungstates which may be regarded as simple model systems for biological interfaces establishes an equilibrium between two conformational states (I II). In state I the structure and the redox potential are the same as for the uncomplexed cytochrome c. In state II however the heme pocket assumes an open structure and the axial iron Met80 bond is weakened leading to thennal coordination equilibrium between the fivecoordinated high spin and the sixcoordinated low spin configuration. These structural changes are accompanied by a decrease of the redox potential by 420 mV. The structural rearrangement of the heme pocket in state II is presumably initiated by the dissociation of the internal salt bridge of Lys13 due to electrostatic interactions with the negatively charged surfaces of the model systems. From detailed Raman spectroscopic studies characteristic spectral properties of the states I and II were identified. Based on these findings the interactions of cytochrome c with phospholipid vesicles as well as with its physiological reaction partner cytocbrome c oxidase were analysed. A systematic study of the cytochmme c/phospholipid system by varying the lipid composition and the temperature revealed mutual structural changes in both the lipid and the protein structure.

Structure and expression of a novel compact myelin protein – Small VCP-interacting protein (SVIP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Jiawen; Peng, Dungeng; Voehler, Markus

2013-10-11

Highlights: •SVIP (small p97/VCP-interacting protein) co-localizes with myelin basic protein (MBP) in compact myelin. •We determined that SVIP is an intrinsically disordered protein (IDP). •The helical content of SVIP increases dramatically during its interaction with negatively charged lipid membrane. •This study provides structural insight into interactions between SVIP and myelin membranes. -- Abstract: SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCPmore » were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes.« less

Discrete structural features among interface residue-level classes.

PubMed

Sowmya, Gopichandran; Ranganathan, Shoba

2015-01-01

Protein-protein interaction (PPI) is essential for molecular functions in biological cells. Investigation on protein interfaces of known complexes is an important step towards deciphering the driving forces of PPIs. Each PPI complex is specific, sensitive and selective to binding. Therefore, we have estimated the relative difference in percentage of polar residues between surface and the interface for each complex in a non-redundant heterodimer dataset of 278 complexes to understand the predominant forces driving binding. Our analysis showed ~60% of protein complexes with surface polarity greater than interface polarity (designated as class A). However, a considerable number of complexes (~40%) have interface polarity greater than surface polarity, (designated as class B), with a significantly different p-value of 1.66E-45 from class A. Comprehensive analyses of protein complexes show that interface features such as interface area, interface polarity abundance, solvation free energy gain upon interface formation, binding energy and the percentage of interface charged residue abundance distinguish among class A and class B complexes, while electrostatic visualization maps also help differentiate interface classes among complexes. Class A complexes are classical with abundant non-polar interactions at the interface; however class B complexes have abundant polar interactions at the interface, similar to protein surface characteristics. Five physicochemical interface features analyzed from the protein heterodimer dataset are discriminatory among the interface residue-level classes. These novel observations find application in developing residue-level models for protein-protein binding prediction, protein-protein docking studies and interface inhibitor design as drugs.

Discrete structural features among interface residue-level classes

PubMed Central

2015-01-01

Background Protein-protein interaction (PPI) is essential for molecular functions in biological cells. Investigation on protein interfaces of known complexes is an important step towards deciphering the driving forces of PPIs. Each PPI complex is specific, sensitive and selective to binding. Therefore, we have estimated the relative difference in percentage of polar residues between surface and the interface for each complex in a non-redundant heterodimer dataset of 278 complexes to understand the predominant forces driving binding. Results Our analysis showed ~60% of protein complexes with surface polarity greater than interface polarity (designated as class A). However, a considerable number of complexes (~40%) have interface polarity greater than surface polarity, (designated as class B), with a significantly different p-value of 1.66E-45 from class A. Comprehensive analyses of protein complexes show that interface features such as interface area, interface polarity abundance, solvation free energy gain upon interface formation, binding energy and the percentage of interface charged residue abundance distinguish among class A and class B complexes, while electrostatic visualization maps also help differentiate interface classes among complexes. Conclusions Class A complexes are classical with abundant non-polar interactions at the interface; however class B complexes have abundant polar interactions at the interface, similar to protein surface characteristics. Five physicochemical interface features analyzed from the protein heterodimer dataset are discriminatory among the interface residue-level classes. These novel observations find application in developing residue-level models for protein-protein binding prediction, protein-protein docking studies and interface inhibitor design as drugs. PMID:26679043

Introduction of a specific binding domain on myoglobin surface by new chemical modification.

PubMed

Hayashi, T; Ando, T; Matsuda, T; Yonemura, H; Yamada, S; Hisaeda, Y

2000-11-01

A new myoglobin, reconstituted with a modified zinc protoporphyrin, having a total of four ammonium groups at the terminal of the two propionate side chains was constructed to introduce a substrate binding site. The protein with a positively charged patch on the surface formed a stable complex with negatively charged substrates, such as hexacyanoferrate(III) and anthraquinonesulfonate via an electrostatic interaction. The complexation was monitored by fluorescence quenching due to singlet electron transfer from the photoexcited reconstituted zinc myoglobin to the substrates. The binding properties were evaluated by Stern-Volmer plots from the fluorescence quenching of the zinc myoglobin by a quencher. Particularly, anthraquinone-2,7-disulfonic acid showed a high affinity with a binding constant of 1.5 x 10(5) M(-1) in 10 mM phosphate buffer, pH 7.0. In contrast, the plots upon the addition of anthraquinone-2-sulfonic acid at different ionic strengths indicated that the complex was formed not only by an electrostatic interaction but also by a hydrophobic contact. The findings from the fluorescence studies conclude that the present system is a useful model for discussion of electron transfer via non-covalently linked donor-acceptor pairing on the protein surface.

Predicting Nonspecific Ion Binding Using DelPhi

PubMed Central

Petukh, Marharyta; Zhenirovskyy, Maxim; Li, Chuan; Li, Lin; Wang, Lin; Alexov, Emil

2012-01-01

Ions are an important component of the cell and affect the corresponding biological macromolecules either via direct binding or as a screening ion cloud. Although some ion binding is highly specific and frequently associated with the function of the macromolecule, other ions bind to the protein surface nonspecifically, presumably because the electrostatic attraction is strong enough to immobilize them. Here, we test such a scenario and demonstrate that experimentally identified surface-bound ions are located at a potential that facilitates binding, which indicates that the major driving force is the electrostatics. Without taking into consideration geometrical factors and structural fluctuations, we show that ions tend to be bound onto the protein surface at positions with strong potential but with polarity opposite to that of the ion. This observation is used to develop a method that uses a DelPhi-calculated potential map in conjunction with an in-house-developed clustering algorithm to predict nonspecific ion-binding sites. Although this approach distinguishes only the polarity of the ions, and not their chemical nature, it can predict nonspecific binding of positively or negatively charged ions with acceptable accuracy. One can use the predictions in the Poisson-Boltzmann approach by placing explicit ions in the predicted positions, which in turn will reduce the magnitude of the local potential and extend the limits of the Poisson-Boltzmann equation. In addition, one can use this approach to place the desired number of ions before conducting molecular-dynamics simulations to neutralize the net charge of the protein, because it was shown to perform better than standard screened Coulomb canned routines, or to predict ion-binding sites in proteins. This latter is especially true for proteins that are involved in ion transport, because such ions are loosely bound and very difficult to detect experimentally. PMID:22735539

Complex coacervation of supercharged proteins with polyelectrolytes.

PubMed

Obermeyer, Allie C; Mills, Carolyn E; Dong, Xue-Hui; Flores, Romeo J; Olsen, Bradley D

2016-04-21

Complexation of proteins with polyelectrolytes or block copolymers can lead to phase separation to generate a coacervate phase or self-assembly of coacervate core micelles. However, many proteins do not coacervate at conditions near neutral pH and physiological ionic strength. Here, protein supercharging is used to systematically explore the effect of protein charge on the complex coacervation with polycations. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry. Proteins phase separated with strong polycations when the ratio of negatively charged residues to positively charged residues on the protein (α) was greater than 1.1-1.2. Efficient partitioning of the protein into the coacervate phase required larger α (1.5-2.0). The preferred charge ratio for coacervation was shifted away from charge symmetry for three of the four model proteins and indicated an excess of positive charge in the coacervate phase. The composition of protein and polymer in the coacervate phase was determined using fluorescently labeled components, revealing that several of the coacervates likely have both induced charging and a macromolecular charge imbalance. The model proteins were also encapsulated in complex coacervate core micelles and micelles formed when the protein charge ratio α was greater than 1.3-1.4. Small angle neutron scattering and transmission electron microscopy showed that the micelles were spherical. The stability of the coacervate phase in both the bulk and micelles improved to increased ionic strength as the net charge on the protein increased. The micelles were also stable to dehydration and elevated temperatures.

A Molecular Dynamics Investigation of the Physical-Chemical Properties of Calicivirus Capsid Protein Adsorption to Fomites

NASA Astrophysics Data System (ADS)

Peeler, David; Matysiak, Silvina

2013-03-01

Any inanimate object with an exposed surface bears the possibility of hosting a virus and may therefore be labeled a fomite. This research hopes to distinguish which chemical-physical differences in fomite surface and virus capsid protein characteristics cause variations in virus adsorption through an alignment of in silico molecular dynamics simulations with in vitro measurements. The impact of surface chemistry on the adsorption of the human norovirus (HNV)-surrogate calicivirus capsid protein 2MS2 has been simulated for monomer and trimer structures and is reported in terms of protein-self assembled monolayer (SAM) binding free energy. The coarse-grained MARTINI forcefield was used to maximize spatial and temporal resolution while minimizing computational load. Future work will investigate the FCVF5 and SMSVS4 calicivirus trimers and will extend beyond hydrophobic and hydrophilic SAM surface chemistry to charged SAM surfaces in varying ionic concentrations. These results will be confirmed by quartz crystal microbalance experiments conducted by Dr. Wigginton at the University of Michigan. This should provide a novel method for predicting the transferability of viruses that cannot be studied in vitro such as dangerous foodborne and nosocomially-acquired viruses like HNV.

Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene.

PubMed

Holmberg, Maria; Hansen, Thomas Steen; Lind, Johan Ulrik; Hjortø, Gertrud Malene

2012-04-01

In this study we compare histidine-tagged and native proteins with regards to adsorption properties. We observe significantly increased adsorption of proteins with an incorporated polyhistidine amino acid motif (HIS-tag) onto tissue culture polystyrene (TCPS) compared to similar proteins without a HIS-tag. The effect is not observed on polystyrene (PS). Adsorption experiments have been performed at physiological pH (7.4) and the effect was only observed for the investigated proteins that have pI values below or around 7.4. Competitive adsorption experiments with imidazole and ethylenediaminetetraacetic acid (EDTA), as well as adsorption performed at different pH and ionic strength indicates that the high adsorption is caused by electrostatic interaction between negatively charged carboxylate groups on the TCPS surface and positively charged histidine residues in the proteins. Pre-adsorption of bovine serum albumin (BSA) does not decrease the adsorption of HIS-tagged proteins onto TCPS. Our findings identify a potential problem in using HIS-tagged signalling molecule in assays with cells cultured on TCPS, since the concentration of the molecule in solution might be affected and this could critically influence the assay outcome. Copyright © 2011 Elsevier B.V. All rights reserved.

Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and cryoEM modeling

PubMed Central

Rizzo, Alessandro A.; Suhanovsky, Margaret M.; Baker, Matthew L.; Fraser, LaTasha C.R.; Jones, Lisa M.; Rempel, Don L.; Gross, Michael L.; Chiu, Wah; Alexandrescu, Andrei T.; Teschke, Carolyn M.

2014-01-01

SUMMARY Some capsid proteins built on the ubiquitous HK97-fold have accessory domains that impart specific functions. Bacteriophage P22 coat protein has a unique inserted I-domain. Two prior I-domain models from sub-nanometer cryoEM reconstructions differed substantially. Therefore, the NMR structure of the I-domain was determined, which also was used to improve cryoEM models of coat protein. The I-domain has an anti-parallel 6-stranded β-barrel fold, previously not observed in HK97-fold accessory domains. The D-loop, which is dynamic both in the isolated I-domain and intact monomeric coat protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. A newly described S-loop is important for capsid size determination, likely through intra-subunit interactions. Ten of eighteen coat protein temperature-sensitive-folding substitutions are in the I-domain, indicating its importance in folding and stability. Several are found on a positively charged face of the β-barrel that anchors the I-domain to a negatively charged surface of the coat protein HK97-core. PMID:24836025

Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling.

PubMed

Rizzo, Alessandro A; Suhanovsky, Margaret M; Baker, Matthew L; Fraser, LaTasha C R; Jones, Lisa M; Rempel, Don L; Gross, Michael L; Chiu, Wah; Alexandrescu, Andrei T; Teschke, Carolyn M

2014-06-10

Some capsid proteins built on the ubiquitous HK97-fold have accessory domains imparting specific functions. Bacteriophage P22 coat protein has a unique insertion domain (I-domain). Two prior I-domain models from subnanometer cryoelectron microscopy (cryoEM) reconstructions differed substantially. Therefore, the I-domain's nuclear magnetic resonance structure was determined and also used to improve cryoEM models of coat protein. The I-domain has an antiparallel six-stranded β-barrel fold, not previously observed in HK97-fold accessory domains. The D-loop, which is dynamic in the isolated I-domain and intact monomeric coat protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. The S-loop is important for capsid size determination, likely through intrasubunit interactions. Ten of 18 coat protein temperature-sensitive-folding substitutions are in the I-domain, indicating its importance in folding and stability. Several are found on a positively charged face of the β-barrel that anchors the I-domain to a negatively charged surface of the coat protein HK97-core. Copyright © 2014 Elsevier Ltd. All rights reserved.

Agglutination of like-charged red blood cells induced by binding of beta2-glycoprotein I to outer cell surface.

PubMed

Lokar, Marusa; Urbanija, Jasna; Frank, Mojca; Hägerstrand, Henry; Rozman, Blaz; Bobrowska-Hägerstrand, Malgorzata; Iglic, Ales; Kralj-Iglic, Veronika

2008-08-01

Plasma protein-mediated attractive interaction between membranes of red blood cells (RBCs) and phospholipid vesicles was studied. It is shown that beta(2)-glycoprotein I (beta(2)-GPI) may induce RBC discocyte-echinocyte-spherocyte shape transformation and subsequent agglutination of RBCs. Based on the observed beta(2)-GPI-induced RBC cell shape transformation it is proposed that the hydrophobic portion of beta(2)-GPI molecule protrudes into the outer lipid layer of the RBC membrane and increases the area of this layer. It is also suggested that the observed agglutination of RBCs is at least partially driven by an attractive force which is of electrostatic origin and depends on the specific molecular shape and internal charge distribution of membrane-bound beta(2)-GPI molecules. The suggested beta(2)-GPI-induced attractive electrostatic interaction between like-charged RBC membrane surfaces is qualitatively explained by using a simple mathematical model within the functional density theory of the electric double layer, where the electrostatic attraction between the positively charged part of the first domains of bound beta(2)-GPI molecules and negatively charged glycocalyx of the adjacent RBC membrane is taken into account.

Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

PubMed

Vieira, Monica L; de Morais, Zenaide M; Gonçales, Amane P; Romero, Eliete C; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-01-01

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis. 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Prediction of potency of protease inhibitors using free energy simulations with polarizable quantum mechanics-based ligand charges and a hybrid water model.

PubMed

Das, Debananda; Koh, Yasuhiro; Tojo, Yasushi; Ghosh, Arun K; Mitsuya, Hiroaki

2009-12-01

Reliable and robust prediction of the binding affinity for drug molecules continues to be a daunting challenge. We simulated the binding interactions and free energy of binding of nine protease inhibitors (PIs) with wild-type and various mutant proteases by performing GBSA simulations in which each PI's partial charge was determined by quantum mechanics (QM) and the partial charge accounts for the polarization induced by the protease environment. We employed a hybrid solvation model that retains selected explicit water molecules in the protein with surface-generalized Born (SGB) implicit solvent. We examined the correlation of the free energy with the antiviral potency of PIs with regard to amino acid substitutions in protease. The GBSA free energy thus simulated showed strong correlations (r > 0.75) with antiviral IC(50) values of PIs when amino acid substitutions were present in the protease active site. We also simulated the binding free energy of PIs with P2-bis-tetrahydrofuranylurethane (bis-THF) or related cores, utilizing a bis-THF-containing protease crystal structure as a template. The free energy showed a strong correlation (r = 0.93) with experimentally determined anti-HIV-1 potency. The present data suggest that the presence of selected explicit water in protein and protein polarization-induced quantum charges for the inhibitor, compared to lack of explicit water and a static force-field-based charge model, can serve as an improved lead optimization tool and warrants further exploration.

Mechanism underlying bioinertness of self-assembled monolayers of oligo(ethyleneglycol)-terminated alkanethiols on gold: protein adsorption, platelet adhesion, and surface forces.

PubMed

Hayashi, Tomohiro; Tanaka, Yusaku; Koide, Yuki; Tanaka, Masaru; Hara, Masahiko

2012-08-07

The mechanism underlying the bioinertness of the self-assembled monolayers of oligo(ethylene glycol)-terminated alkanethiol (OEG-SAM) was investigated with protein adsorption experiments, platelet adhesion tests, and surface force measurements with an atomic force microscope (AFM). In this work, we performed systematic analysis with SAMs having various terminal groups (-OEG, -OH, -COOH, -NH(2), and -CH(3)). The results of the protein adsorption experiment by the quartz crystal microbalance (QCM) method suggested that having one EG unit and the neutrality of total charges of the terminal groups are essential for protein-resistance. In particular, QCM with energy dissipation analyses indicated that proteins absorb onto the OEG-SAM via a very weak interaction compared with other SAMs. Contrary to the protein resistance, at least three EG units as well as the charge neutrality of the SAM are found to be required for anti-platelet adhesion. When the identical SAMs were formed on both AFM probe and substrate, our force measurements revealed that only the OEG-SAMs possessing more than two EG units showed strong repulsion in the range of 4 to 6 nm. In addition, we found that the SAMs with other terminal groups did not exhibit such repulsion. The repulsion between OEG-SAMs was always observed independent of solution conditions [NaCl concentration (between 0 and 1 M) and pH (between 3 and 11)] and was not observed in solution mixed with ethanol, which disrupts the three-dimensional network of the water molecules. We therefore concluded that the repulsion originated from structured interfacial water molecules. Considering the correlation between the above results, we propose that the layer of the structured interfacial water with a thickness of 2 to 3 nm (half of the range of the repulsion observed in the surface force measurements) plays an important role in deterring proteins and platelets from adsorption or adhesion.

Electrostatic Solvation Energy for Two Oppositely Charged Ions in a Solvated Protein System: Salt Bridges Can Stabilize Proteins

PubMed Central

Gong, Haipeng; Freed, Karl F.

2010-01-01

Abstract Born-type electrostatic continuum methods have been an indispensable ingredient in a variety of implicit-solvent methods that reduce computational effort by orders of magnitude compared to explicit-solvent MD simulations and thus enable treatment using larger systems and/or longer times. An analysis of the limitations and failures of the Born approaches serves as a guide for fundamental improvements without diminishing the importance of prior works. One of the major limitations of the Born theory is the lack of a liquidlike description of the response of solvent dipoles to the electrostatic field of the solute and the changes therein, a feature contained in the continuum Langevin-Debye (LD) model applied here to investigate how Coulombic interactions depend on the location of charges relative to the protein/water boundary. This physically more realistic LD model is applied to study the stability of salt bridges. When compared head to head using the same (independently measurable) physical parameters (radii, dielectric constants, etc.), the LD model is in good agreement with observations, whereas the Born model is grossly in error. Our calculations also suggest that a salt bridge on the protein's surface can be stabilizing when the charge separation is ≤4 Å. PMID:20141761

Electrospun nylon 6/zinc doped hydroxyapatite membrane for protein separation: Mechanism of fouling and blocking model.

PubMed

Esfahani, Hamid; Prabhakaran, Molamma P; Salahi, Esmaeil; Tayebifard, Ali; Rahimipour, Mohamad Reza; Keyanpour-Rad, Mansour; Ramakrishna, Seeram

2016-02-01

Development of composite nanofibrous membrane via electrospinning a polymer with ceramic nanoparticles (NPs) for application in protein separation systems is explored during this study. Positively charged zinc doped hydroxyapatite (xZH) NPs were prepared in three different compositions via chemical precipitation method. Herein, we created a positively charged surface containing nanoparticles on electrospun Nylon-6 nanofibers (NFs) to improve the separation and selectivity properties for adsorption of negatively charged protein, namely bovine serum albumin (BSA). The decline in permeate flux was analyzed using the framework of classical blocking models and fitting, demonstrated that the transition of fouling mechanisms was dominated during the filtration process. The standard blocking model provided the best fit of the experimental results during the mid-filtration period. The membrane decorated by NPs containing 4at.% zinc cations not only provided maximum BSA separation but also capable of separating higher amounts of BSA molecules (even after 1h filtration) than the pure Nylon membrane. Protein separation was achieved through this membrane with the incorporation of NPs that had high zeta potential (+5.9±0.2mV) and lower particle area (22,155nm(2)). The developed membrane has great potential to act as a high efficiency membrane for capturing BSA. Copyright © 2015 Elsevier B.V. All rights reserved.

Highly sensitive protein detection by combination of atomic force microscopy fishing with charge generation and mass spectrometry analysis.

PubMed

Ivanov, Yuri D; Pleshakova, Tatyana; Malsagova, Krystina; Kozlov, Andrey; Kaysheva, Anna; Kopylov, Arthur; Izotov, Alexander; Andreeva, Elena; Kanashenko, Sergey; Usanov, Sergey; Archakov, Alexander

2014-10-01

An approach combining atomic force microscopy (AFM) fishing and mass spectrometry (MS) analysis to detect proteins at ultra-low concentrations is proposed. Fishing out protein molecules onto a highly oriented pyrolytic graphite surface coated with polytetrafluoroethylene film was carried out with and without application of an external electric field. After that they were visualized by AFM and identified by MS. It was found that injection of solution leads to charge generation in the solution, and an electric potential within the measuring cell is induced. It was demonstrated that without an external electric field in the rapid injection input of diluted protein solution the fishing is efficient, as opposed to slow fluid input. The high sensitivity of this method was demonstrated by detection of human serum albumin and human cytochrome b5 in 10(-17) -10(-18) m water solutions. It was shown that an external negative voltage applied to highly oriented pyrolytic graphite hinders the protein fishing. The efficiency of fishing with an external positive voltage was similar to that obtained without applying any voltage. © 2014 FEBS.

An Investigation of Low Biofouling Copper-charged Membranes

NASA Astrophysics Data System (ADS)

Asapu, Sunitha

Water is essential for the survival of life on Earth, but pollutants in water can cause dangerous diseases and fatalities. The need for purified water has been increasing with increasing world population; however, natural sources of water such as rivers, lakes and streams, are progressively falling shorter and shorter of meeting water needs. The provision of clean, drinkable water to people is a key factor for the development of novel and alternative water purification technologies, such as membrane separations. Nanofiltration (NF) is a membrane separations technology that purifies water from lower quality sources, such as brackish water, seawater and wastewater. During the filtration of such sources, materials that are rejected by the membrane may accumulate on the surface of the membrane to foul it. Such materials include organic and inorganic matter, colloids, salts and microorganisms. The former four can often be controlled via pretreatment; however, the accumulation of microorganisms is more problematic to membranes. Biofouling is the accumulation and growth of microorganisms on the surface of membranes and on feed spacers. After attachment, microorganisms excrete extracellular polymeric substances (EPS), which form a matrix around the organism's outer surface as biofilm. These biofilms are detrimental and result in irreversible membrane fouling. Copper and silver ions inactivate the bacterial cells and prevent the DNA replication in microbial cells. Previous studies using copper-charged feed spacers have shown the ability of copper to control biofouling without a significant amount of copper leaching from copper-charged polypropylene (PP) feed spacers during crossflow filtration. Also, filtration using unmodified speed facers experienced almost 70% flux decline, while filtration using copper-charged feed spacers displayed only 25% flux decline. These intriguing results led to the hypothesis that the polymer chemistry could be extrapolated to produce membranes with increased biofouling resistance. The goal of this project was to develop low-biofouling nanofiltration cellulose acetate (CA) membranes through functionalization with metal chelating ligands charged with biocidal metal ions, i.e. copper ions. To this end, glycidyl methacrylate (GMA), an epoxy, was used to attach a chelating agent, iminodiacetic acid (IDA) to facilitate the charging of copper to the membrane surface. Both CA and CA-GMA membranes were cast using the phase-inversion method. The CA-GMA membranes were then charged with copper ions to make them low biofouling. Pore size distribution analysis of CA and copper charged membranes were conducted using various molecular weights of polyethylene glycol (PEG). CA and copper-charged membranes were characterized using Fourier Transform Infrared (FTIR), contact angle to measure hydrophilicity changes, and using scanning electron microscope (SEM) coupled with X-ray energy dispersive spectroscopy EDS to monitor copper leaching. Permeation experiments were conducted with distilled (DI) water, protein solutions, and synthetic brackish water containing microorganisms. The DI water permeation of the copper-charged membranes was initially lower than the CA membranes. The membranes were then subjected to bovine serum albumin (BSA) and lipase filtration. The copper-charged membranes showed higher pure water flux values for both proteins as compared to CA membranes. The rejection of BSA and lipase was the same for both the copper charged and CA membranes. The filtration with the synthetic brackish water showed that copper-charged membranes had higher flux values as compared to CA membranes, and biofouling analysis showed more bacteria on the CA membranes as compared to copper-charged membranes. Therefore, the copper-charged membranes made here have shown a potential to be used as low-biofouling membranes in the future.

Alterations in nanoparticle protein corona by biological surfactants: impact of bile salts on β-lactoglobulin-coated gold nanoparticles.

PubMed

Winuprasith, Thunnalin; Chantarak, Sirinya; Suphantharika, Manop; He, Lili; McClements, David Julian

2014-07-15

The impact of biological surfactants (bile salts) on the protein (β-lactoglobulin) corona surrounding gold nanoparticles (200 nm) was studied using a variety of analytical techniques at pH 7: dynamic light scattering (DLS); particle electrophoresis (ζ-potential); UV-visible (UV) spectroscopy; transmission electron microscopy (TEM); and surface-enhanced Raman scattering (SERS). The bile salts adsorbed to the protein-coated nanoparticle surfaces and altered their interfacial composition, charge, and structure. SERS spectra of protein-coated nanoparticles after bile salt addition contained bands from both protein and bile salts, indicating that the protein was not fully displaced by the bile salts. UV, DLS and TEM techniques also indicated that the protein coating was not fully displaced from the nanoparticle surfaces. The impact of bile salts could be described by an orogenic mechanism: mixed interfaces were formed that consisted of islands of aggregated proteins surrounded by a sea of bile salts. This knowledge is useful for understanding the interactions of bile salts with protein-coated colloidal particles, which may be important for controlling the fate of colloidal delivery systems in the human gastrointestinal tract, or the gastrointestinal fate of ingested inorganic nanoparticles. Copyright © 2014 Elsevier Inc. All rights reserved.

Essential slow degrees of freedom in protein-surface simulations: A metadynamics investigation.

PubMed

Prakash, Arushi; Sprenger, K G; Pfaendtner, Jim

2018-03-29

Many proteins exhibit strong binding affinities to surfaces, with binding energies much greater than thermal fluctuations. When modelling these protein-surface systems with classical molecular dynamics (MD) simulations, the large forces that exist at the protein/surface interface generally confine the system to a single free energy minimum. Exploring the full conformational space of the protein, especially finding other stable structures, becomes prohibitively expensive. Coupling MD simulations with metadynamics (enhanced sampling) has fast become a common method for sampling the adsorption of such proteins. In this paper, we compare three different flavors of metadynamics, specifically well-tempered, parallel-bias, and parallel-tempering in the well-tempered ensemble, to exhaustively sample the conformational surface-binding landscape of model peptide GGKGG. We investigate the effect of mobile ions and ion charge, as well as the choice of collective variable (CV), on the binding free energy of the peptide. We make the case for explicitly biasing ions to sample the true binding free energy of biomolecules when the ion concentration is high and the binding free energies of the solute and ions are similar. We also make the case for choosing CVs that apply bias to all atoms of the solute to speed up calculations and obtain the maximum possible amount of information about the system. Copyright © 2017 Elsevier Inc. All rights reserved.

Electrical activity of ferroelectric biomaterials and its effects on the adhesion, growth and enzymatic activity of human osteoblast-like cells

NASA Astrophysics Data System (ADS)

Vaněk, P.; Kolská, Z.; Luxbacher, T.; García, J. A. L.; Lehocký, M.; Vandrovcová, M.; Bačáková, L.; Petzelt, J.

2016-05-01

Ferroelectrics have been, among others, studied as electroactive implant materials. Previous investigations have indicated that such implants induce improved bone formation. If a ferroelectric is immersed in a liquid, an electric double layer and a diffusion layer are formed at the interface. This is decisive for protein adsorption and bioactive behaviour, particularly for the adhesion and growth of cells. The charge distribution can be characterized, in a simplified way, by the zeta potential. We measured the zeta potential in dependence on the surface polarity on poled ferroelectric single crystalline LiNbO3 plates. Both our results and recent results of colloidal probe microscopy indicate that the charge distribution at the surface can be influenced by the surface polarity of ferroelectrics under certain ‘ideal’ conditions (low ionic strength, non-contaminated surface, very low roughness). However, suggested ferroelectric coatings on the surface of implants are far from ideal: they are rough, polycrystalline, and the body fluid is complex and has high ionic strength. In real cases, it can therefore be expected that there is rather low influence of the sign of the surface polarity on the electric diffusion layer and thus on the specific adsorption of proteins. This is supported by our results from studies of the adhesion, growth and the activity of alkaline phosphatase of human osteoblast-like Saos-2 cells on ferroelectric LiNbO3 plates in vitro.

Scanning probe microscopy for the analysis of composite Ti/hydrocarbon plasma polymer thin films

NASA Astrophysics Data System (ADS)

Choukourov, A.; Grinevich, A.; Slavinska, D.; Biederman, H.; Saito, N.; Takai, O.

2008-03-01

Composite Ti/hydrocarbon plasma polymer films with different Ti concentration were deposited on silicon by dc magnetron sputtering of titanium in an atmosphere of argon and hexane. As measured by Kelvin force microscopy and visco-elastic atomic force microscopy, respectively, surface potential and hardness increase with increasing Ti content. Adhesion force to silicon and to fibrinogen molecules was stronger for the Ti-rich films as evaluated from the AFM force-distance curves. Fibrinogen forms a very soft layer on these composites with part of the protein molecules embedded in the outermost region of the plasma polymer. An increase of the surface charge due to fibrinogen adsorption has been observed and attributed to positively charged αC domains of fibrinogen molecule.

Crystal structure at 2.8 A of Huntingtin-interacting protein 1 (HIP1) coiled-coil domain reveals a charged surface suitable for HIP1 protein interactor (HIPPI).

PubMed

Niu, Qian; Ybe, Joel A

2008-02-01

Huntington's disease is a genetic neurological disorder that is triggered by the dissociation of the huntingtin protein (htt) from its obligate interaction partner Huntingtin-interacting protein 1 (HIP1). The release of the huntingtin protein permits HIP1 protein interactor (HIPPI) to bind to its recognition site on HIP1 to form a HIPPI/HIP1 complex that recruits procaspase-8 to begin the process of apoptosis. The interaction module between HIPPI and HIP1 was predicted to resemble a death-effector domain. Our 2.8-A crystal structure of the HIP1 371-481 subfragment that includes F432 and K474, which is important for HIPPI binding, is not a death-effector domain but is a partially opened coiled coil. The HIP1 371-481 model reveals a basic surface that we hypothesize to be suitable for binding HIPPI. There is an opened region next to the putative HIPPI site that is highly negatively charged. The acidic residues in this region are highly conserved in HIP1 and a related protein, HIP1R, from different organisms but are not conserved in the yeast homologue of HIP1, sla2p. We have modeled approximately 85% of the coiled-coil domain by joining our new HIP1 371-481 structure to the HIP1 482-586 model (Protein Data Bank code: 2NO2). Finally, the middle of this coiled-coil domain may be intrinsically flexible and suggests a new interaction model where HIPPI binds to a U-shaped HIP1 molecule.

Molecular insight into the counteraction of trehalose on urea-induced protein denaturation using molecular dynamics simulation.

PubMed

Zhang, Na; Liu, Fu-Feng; Dong, Xiao-Yan; Sun, Yan

2012-06-21

Considerable experimental evidence indicates that trehalose can counteract the denaturing effects of urea on proteins. However, its molecular mechanism remains unknown due to the limitations of current experimental techniques. Herein, molecular dynamics simulations were performed to investigate the counteracting effects of trehalose against urea-induced denaturation of chymotrypsin inhibitor 2. The simulations indicate that the protein unfolds in 8 mol/L urea, but at the same condition the protein retains its native structure in the ternary solution of 8 mol/L urea and 1 mol/L trehalose. It is confirmed that the preferential exclusion of trehalose from the protein surface is the origin of its counteracting effects. It is found that trehalose binds urea via hydrogen bonds, so urea molecules are also expelled from the protein surface along with the preferential exclusion of trehalose. The exclusion of urea from the protein surface leads to the alleviation of the Lennard-Jones interactions between urea and the hydrophobic side chains of the protein in the ternary solution. In contrast, the electrostatic interactions between urea and the protein change little in the presence of trehalose because the decrease in the electrostatic interactions between urea and the protein backbone is canceled by the increase in the electrostatic interactions between urea and the charged side chains of the protein. The results have provided molecular explanations for the counteraction of urea-induced protein denaturation by trehalose.

Surface Oxide Net Charge of a Titanium Alloy ; Modulation of Fibronectin-Activated Attachment and Spreading of Osteogenic Cells

PubMed Central

Rapuano, Bruce E.; MacDonald, Daniel E.

2010-01-01

In the current study, we have altered the surface oxide properties of a Ti6Al4V alloy using heat treatment or radiofrequency glow discharge (RFGD) in order to evaluate the relationship between the physico-chemical and biological properties of the alloy's surface oxide. The effects of surface pretreatments on the attachment of cells from two osteogenic cell lines (MG63 and MC3T3) and a mesenchymal stem cell line (C3H10T1/2) to fibronectin adsorbed to the alloy were measured. Both heat and RFGD pretreatments produced a several-fold increase in the number of cells that attached to fibronectin adsorbed to the alloy (0.001 and 10 nM FN) for each cell line tested. An antibody (HFN7.1) directed against the central integrin binding domain of fibronectin produced a 65-70% inhibition of cell attachment to fibronectin-coated disks, incdicating that cell attachment to the metal discs was dependent on fibronectin binding to cell integrin receptors. Both treatments also accelerated the cell spreading response manifested by extensive flattening and an increase in mean cellular area. The treatment-induced increases in the cell attachment activity of adsorbed fibronectin were correlated with previously demonstrated increases in Ti6Al4V oxide negative net surface charge at physiological pH produced by both heat and RFGD pretreatments. Since neither treatment increased the adsorption mass of fibronectin, these findings suggest that negatively charged surface oxide functional groups in Ti6Al4V can modulate fibronectin's integrin receptor activity by altering the adsorbed protein's conformation. Our results further suggest that negatively charged functional groups in the surface oxide can play a prominent role in the osseointegration of metallic implant materials. PMID:20884181

Monolayers of derivatized poly(l-lysine)-grafted poly(ethylene glycol) on metal oxides as a class of biomolecular interfaces

PubMed Central

Ruiz-Taylor, L. A.; Martin, T. L.; Zaugg, F. G.; Witte, K.; Indermuhle, P.; Nock, S.; Wagner, P.

2001-01-01

We report on the design and characterization of a class of biomolecular interfaces based on derivatized poly(l-lysine)-grafted poly(ethylene glycol) copolymers adsorbed on negatively charged surfaces. As a model system, we synthesized biotin-derivatized poly(l-lysine)-grafted poly(ethylene glycol) copolymers, PLL-g-[(PEGm)(1−x) (PEG-biotin)x], where x varies from 0 to 1. Monolayers were produced on titanium dioxide substrates and characterized by x-ray photoelectron spectroscopy. The specific biorecognition properties of these biotinylated surfaces were investigated with the use of radiolabeled streptavidin alone and within complex protein mixtures. The PLL-g-PEG-biotin monolayers specifically capture streptavidin, even from a complex protein mixture, while still preventing nonspecific adsorption of other proteins. This streptavidin layer can subsequently capture biotinylated proteins. Finally, with the use of microfluidic networks and protein arraying, we demonstrate the potential of this class of biomolecular interfaces for applications based on protein patterning. PMID:11158560

Sum-frequency generation analyses of the structure of water at amphoteric SAM-liquid water interfaces.

PubMed

Nomura, Kouji; Nakaji-Hirabayashi, Tadashi; Gemmei-Ide, Makoto; Kitano, Hiromi; Noguchi, Hidenori; Uosaki, Kohei

2014-09-01

Surfaces of both a cover glass and the flat plane of a semi-cylindrical quartz prism were modified with a mixture of positively and negatively charged silane coupling reagents (3-aminopropyltriethoxysilane (APTES) and 3-(trihydroxysilyl)propylmethylphosphonate (THPMP), respectively). The glass surface modified with a self-assembled monolayer (SAM) prepared at a mixing ratio of APTES:THPMP=4:6 was electrically almost neutral and was resistant to non-specific adsorption of proteins, whereas fibroblasts gradually adhered to an amphoteric (mixed) SAM surface probably due to its stiffness, though the number of adhered cells was relatively small. Sum frequency generation (SFG) spectra indicated that total intensity of the OH stretching region (3000-3600cm(-1)) for the amphoteric SAM-modified quartz immersed in liquid water was smaller than those for the positively and negatively charged SAM-modified quartz prisms and a bare quartz prism in contact with liquid water. These results suggested that water molecules at the interface of water and an amphoteric SAM-modified quartz prism are not strongly oriented in comparison with those at the interface of a lopsidedly charged SAM-modified quartz prism and bare quartz. The importance of charge neutralization for the anti-biofouling properties of solid materials was strongly suggested. Copyright © 2014 Elsevier B.V. All rights reserved.

Light-induced charge separation across bio-inorganic interface.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dimitrijevic, N. M.; Rajh, T.; De La Garza, L.

Rational design of hybrid biomolecule - nanoparticulate semiconductor conjugates enables coupling of functionality of biomolecules with the capability of semiconductors for solar energy capture, that can have potential application in energy conversion, sensing and catalysis. The particular challenge is to obtain efficient charge separation analogous to the natural photosynthesis process. The synthesis of axially anisotropic TiO{sub 2} nano-objects such as tubes, rods and bricks, as well as spherical and faceted nanoparticles has been developed in our laboratory. Depending on their size and shape, these nanostructures exhibit different domains of crystallinity, surface areas and aspect ratios. Moreover, in order to accommodatemore » for high curvature in nanoscale regime, the surfaces of TiO{sub 2} nano-objects reconstructs resulting in changes in the coordination of surface Ti atoms from octahedral (D{sub 2d}) to square pyramidal structures (C{sub 4v}). The formation of these coordinatively unsaturated Ti atoms, thus depends strongly on the size and shape of nanocrystallites and affects trapping and reactivity of photogenerated charges. We have exploited these coordinatively unsaturated Ti atoms to coupe electron-donating (such as dopamine) and electron-accepting (pyrroloquinoline quinone) conductive linkers that allow wiring of biomolecules and proteins resulting in enhanced charge separation which increases the yield of ensuing chemical transformations.« less

Adsorbed Layers of Ferritin at Solid and Fluid Interfaces Studied by Atomic Force Microscopy.

PubMed

Johnson; Yuan; Lenhoff

2000-03-15

The adsorption of the iron storage protein ferritin was studied by liquid tapping mode atomic force microscopy in order to obtain molecular resolution in the adsorbed layer within the aqueous environment in which the adsorption was carried out. The surface coverage and the structure of the adsorbed layer were investigated as functions of ionic strength and pH on two different charged surfaces, namely chemically modified glass slides and mixed surfactant films at the air-water interface, which were transferred to graphite substrates after adsorption. Surface coverage trends with both ionic strength and pH indicate the dominance of electrostatic effects, with the balance shifting between intermolecular repulsion and protein-surface attraction. The resulting behavior is more complex than that seen for larger colloidal particles, which appear to follow a modified random sequential adsorption model monotonically. The structure of the adsorbed layers at the solid surfaces is random, but some indication of long-range order is apparent at fluid interfaces, presumably due to the higher protein mobility at the fluid interface. Copyright 2000 Academic Press.

Constructing irregular surfaces to enclose macromolecular complexes for mesoscale modeling using the discrete surface charge optimization (DISCO) algorithm.

PubMed

Zhang, Qing; Beard, Daniel A; Schlick, Tamar

2003-12-01

Salt-mediated electrostatics interactions play an essential role in biomolecular structures and dynamics. Because macromolecular systems modeled at atomic resolution contain thousands of solute atoms, the electrostatic computations constitute an expensive part of the force and energy calculations. Implicit solvent models are one way to simplify the model and associated calculations, but they are generally used in combination with standard atomic models for the solute. To approximate electrostatics interactions in models on the polymer level (e.g., supercoiled DNA) that are simulated over long times (e.g., milliseconds) using Brownian dynamics, Beard and Schlick have developed the DiSCO (Discrete Surface Charge Optimization) algorithm. DiSCO represents a macromolecular complex by a few hundred discrete charges on a surface enclosing the system modeled by the Debye-Hückel (screened Coulombic) approximation to the Poisson-Boltzmann equation, and treats the salt solution as continuum solvation. DiSCO can represent the nucleosome core particle (>12,000 atoms), for example, by 353 discrete surface charges distributed on the surfaces of a large disk for the nucleosome core particle and a slender cylinder for the histone tail; the charges are optimized with respect to the Poisson-Boltzmann solution for the electric field, yielding a approximately 5.5% residual. Because regular surfaces enclosing macromolecules are not sufficiently general and may be suboptimal for certain systems, we develop a general method to construct irregular models tailored to the geometry of macromolecules. We also compare charge optimization based on both the electric field and electrostatic potential refinement. Results indicate that irregular surfaces can lead to a more accurate approximation (lower residuals), and the refinement in terms of the electric field is more robust. We also show that surface smoothing for irregular models is important, that the charge optimization (by the TNPACK minimizer) is efficient and does not depend on the initial assigned values, and that the residual is acceptable when the distance to the model surface is close to, or larger than, the Debye length. We illustrate applications of DiSCO's model-building procedure to chromatin folding and supercoiled DNA bound to Hin and Fis proteins. DiSCO is generally applicable to other interesting macromolecular systems for which mesoscale models are appropriate, to yield a resolution between the all-atom representative and the polymer level. Copyright 2003 Wiley Periodicals, Inc. J Comput Chem 24: 2063-2074, 2003

Surface characterization and protein interaction of a series of model poly[acrylonitrile-co-(N-vinyl pyrrolidone)] nanocarriers for drug targeting.

PubMed

Staufenbiel, Sven; Merino, Marian; Li, Wenzhong; Huang, Mao-Dong; Baudis, Stefan; Lendlein, Andreas; Müller, Rainer H; Wischke, Christian

2015-05-15

The surface properties of intravenously injected nanoparticles determine the acquired blood protein adsorption pattern and subsequently the organ distribution and cellular recognition. A series of poly[acrylonitrile-co-(N-vinyl pyrrolidone)] (PANcoNVP) model nanoparticles (133-181 nm) was synthesized, in which the surface properties were altered by changing the molar content of NVP (0-33.8 mol%) as the more hydrophilic repeating unit. The extent of achieved surface property variation was comprehensively characterized. The residual sodium dodecyl sulfate (SDS) content from the synthesis was in the range 0.3-1.6 μgml(-1), potentially contributing to the surface properties. Surface hydrophobicity was determined by Rose Bengal dye adsorption, hydrophobic interaction chromatography (HIC) and aqueous two-phase partitioning (TPP). Particle charge was quantified by zeta potential (ZP) measurements including ZP-pH profiles. The interaction with proteins was analyzed by ZP measurements in serum and by adsorption studies with single proteins. Compared to hydrophobic polystyrene model nanoparticles, all PANcoNVP particles were very hydrophilic. Differences in surface hydrophobicity could be detected, which did not linearly correlate with the systematically altered bulk composition of the PANcoNVP nanoparticles. This proves the high importance of a thorough surface characterization applying a full spectrum of methods, complementing predictions solely based on bulk polymer composition. Copyright © 2015. Published by Elsevier B.V.

Degradable Polymer with Protein Resistance in a Marine Environment.

PubMed

Ma, Jielin; Ma, Chunfeng; Zhang, Guangzhao

2015-06-16

Protein resistance is the central issue in marine antibiofouling. We have prepared poly(ε-caprolactone) (PCL)-based polyurethane with 2-(dimethylamino) ethyl methacrylate (DEM) as pendant groups by a combination of the thiol-ene click reaction and the condensation reaction. By the use of quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR), we have investigated the adsorption of fibrinogen, bovine serum albumin (BSA), and lysozyme on the polymer surface. The polymer exhibits protein resistance in seawater but not in fresh water because DEM pendant groups carry net neutral charges in the former. The evaluation of antibacterial adhesion of the polymer by using Micrococcus luteus demonstrates that the polymer can effectively inhibit the settlement of marine bacteria. Our studies also show that the polymer is degradable in marine environments.

Preferential binding of positive nanoparticles on cell membranes is due to electrostatic interactions: A too simplistic explanation that does not take into account the nanoparticle protein corona.

PubMed

Forest, Valérie; Pourchez, Jérémie

2017-01-01

The internalization of nanoparticles by cells (and more broadly the nanoparticle/cell interaction) is a crucial issue both for biomedical applications (for the design of nanocarriers with enhanced cellular uptake to reach their intracellular therapeutic targets) and in a nanosafety context (as the internalized dose is one of the key factors in cytotoxicity). Many parameters can influence the nanoparticle/cell interaction, among them, the nanoparticle physico-chemical features, and especially the surface charge. It is generally admitted that positive nanoparticles are more uptaken by cells than neutral or negative nanoparticles. It is supposedly due to favorable electrostatic interactions with negatively charged cell membrane. However, this theory seems too simplistic as it does not consider a fundamental element: the nanoparticle protein corona. Indeed, once introduced in a biological medium nanoparticles adsorb proteins at their surface, forming a new interface defining the nanoparticle "biological identity". This adds a new level of complexity in the interactions with biological systems that cannot be any more limited to electrostatic binding. These interactions will then influence cell behavior. Based on a literature review and on an example of our own experience the parameters involved in the nanoparticle protein corona formation as well as in the nanoparticle/cell interactions are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Peptide adsorption on the hydrophobic surface: A free energy perspective

NASA Astrophysics Data System (ADS)

Sheng, Yuebiao; Wang, Wei; Chen, P.

2011-05-01

Protein adsorption is a very attractive topic which relates to many novel applications in biomaterials, biotechnology and nanotechnology. Ionic complementary peptides are a group of novel nano-biomaterials with many biomedical applications. In this work, molecular dynamics simulations of the ionic-complementary peptide EAK16-II on a hydrophobic graphite surface were performed under neutral, acidic and basic solution conditions. Adsorption free energy contour maps were obtained by analyzing the dynamical trajectories. Hydrophobic interactions were found to govern the adsorption of the first peptide molecule, and both hydrophobic and electrostatic interactions contributed to the adsorption of the second peptide molecule. Especially under acidic and basic solution conditions, interplay existed among chain-chain hydrophobic, chain-surface hydrophobic and chain-chain electrostatic interactions during the adsorption of the second peptide molecule. Non-charged residues were found to lie on the graphite surface, while charged residue side-chains oriented towards the solution after the peptide deposited on the surface. These results provide a basis for understanding peptide adsorption on the hydrophobic surface under different solution conditions, which is useful for novel applications such as bioactive implant devices and drug delivery material design.

Release of Native-like Gaseous Proteins from Electrospray Droplets via the Charged Residue Mechanism: Insights from Molecular Dynamics Simulations.

PubMed

McAllister, Robert G; Metwally, Haidy; Sun, Yu; Konermann, Lars

2015-10-07

The mechanism whereby gaseous protein ions are released from charged solvent droplets during electrospray ionization (ESI) remains a matter of debate. Also, it is unclear to what extent electrosprayed proteins retain their solution structure. Molecular dynamics (MD) simulations offer insights into the temporal evolution of protein systems. Surprisingly, there have been no all-atom simulations of the protein ESI process to date. The current work closes this gap by investigating the behavior of protein-containing aqueous nanodroplets that carry excess positive charge. We focus on "native ESI", where proteins initially adopt their biologically active solution structures. ESI proceeds while the protein remains entrapped within the droplet. Protein release into the gas phase occurs upon solvent evaporation to dryness. Droplet shrinkage is accompanied by ejection of charge carriers (Na(+) for the conditions chosen here), keeping the droplet at ∼85% of the Rayleigh limit throughout its life cycle. Any remaining charge carriers bind to the protein as the final solvent molecules evaporate. The outcome of these events is largely independent of the initial protein charge and the mode of charge carrier binding. ESI charge states and collision cross sections of the MD structures agree with experimental data. Our results confirm the Rayleigh/charged residue model (CRM). Field emission of excess Na(+) plays an ancillary role by governing the net charge of the shrinking droplet. Models that envision protein ejection from the droplet are not supported. Most nascent CRM ions retain native-like conformations. For unfolded proteins ESI likely proceeds along routes that are different from the native state mechanism explored here.

A docking study of enhanced intracellular survival protein from Mycobacterium tuberculosis with human DUSP16/MKP-7.

PubMed

Yoon, Hye Jin; Kim, Kyoung Hoon; Yang, Jin Kuk; Suh, Se Won; Kim, Hyunsik; Jang, Soonmin

2013-11-01

The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis, and one of its secreted effector proteins, called enhanced intracellular survival (Eis) protein, enhances its survival in macrophages. Mtb Eis activates JNK-specific dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7) through the acetylation on Lys55, thus inactivating JNK by dephosphorylation. Based on the recently reported crystal structure of Mtb Eis, a docking model for the binding of Mtb Eis to DUSP16/MKP-7 was generated. In the docking model, the substrate helix containing Lys55 of DUSP16/MKP-7 fits nicely into the active-site cleft of Mtb Eis; the twisted β-sheet of Eis domain II embraces the substrate helix from one side. Most importantly, the side-chain of Lys55 is inserted toward acetyl-CoA and the resulting distance is 4.6 Å between the NZ atom of Lys55 and the carbonyl carbon of the acetyl group in acetyl-CoA. The binding of Mtb Eis and DUSP16/MKP-7 is maintained by strong electrostatic interactions. The active-site cleft of Mtb Eis has a negatively charged surface formed by Asp25, Glu138, Asp286, Glu395 and the terminal carboxylic group of Phe396. In contrast, DUSP16/MKP-7 contains five basic residues, Lys52, Lys55, Arg56, Arg57 and Lys62, which point toward the negatively charged surface of the active-site pocket of Mtb Eis. Thus, the current docking model suggests that the binding of DUSP16/MKP-7 to Mtb Eis should be established by charge complementarity in addition to a very favorable geometric arrangement. The suggested mode of binding requires the dissociation of the hexameric Mtb Eis into dimers or monomers. This study may be useful for future studies aiming to develop inhibitors of Mtb Eis as a new anti-tuberculosis drug candidate.

Crystal structure at 2.8Å of Huntingtin-interacting protein 1 (HIP1) coiled-coil domain reveals a charged surface suitable for HIP-protein interactor (HIPPI)

PubMed Central

Niu, Qian; Ybe, Joel A.

2008-01-01

Summary Huntington’s disease is a genetic neurological disorder that is triggered by the dissociation of the huntingtin protein (htt) from its obligate interaction partner Huntingtin-interacting protein 1 (HIP1). The release of htt permits HIP-protein interactor (HIPPI) to bind to its recognition site on HIP1 to form a HIPPI/HIP1 complex that recruits Procaspase-8 to begin the process of apoptosis. The interaction module between HIPPI and HIP1 was predicted to resemble a death-effector domain (DED). Our 2.8 Å crystal structure of the HIP1 371-481 sub-fragment that includes F432 and K474 important for HIPPI binding is not a DED, but is a partially opened coiled-coil. The HIP1 371-481 model reveals a basic surface we hypothesize is suitable for binding HIPPI. There is an opened region next to the putative HIPPI site that is highly negatively charged. The acidic residues in this region are highly conserved in HIP1 and a related protein, HIP1R from different organisms, but are not conserved in the yeast homolog of HIP1, sla2p. We have modeled ∼85% of the coiled-coil domain by joining our new HIP1 371-481 structure to the HIP1 482-586 model (PDB code: 2NO2). Finally, the middle of this coiled-coil domain may be intrinsically flexible and suggests a new interaction model where HIPPI binds to a “U” shaped HIP1 molecule. PMID:18155047

Impact of protein modification on the protein corona on nanoparticles and nanoparticle-cell interactions.

PubMed

Treuel, Lennart; Brandholt, Stefan; Maffre, Pauline; Wiegele, Sarah; Shang, Li; Nienhaus, G Ulrich

2014-01-28

Recent studies have firmly established that cellular uptake of nanoparticles is strongly affected by the presence and the physicochemical properties of a protein adsorption layer around these nanoparticles. Here, we have modified human serum albumin (HSA), a serum protein often used in model studies of protein adsorption onto nanoparticles, to alter its surface charge distribution and investigated the consequences for protein corona formation around small (radius ∼5 nm), dihydrolipoic acid-coated quantum dots (DHLA-QDs) by using fluorescence correlation spectroscopy. HSA modified by succinic anhydride (HSAsuc) to generate additional carboxyl groups on the protein surface showed a 3-fold decreased binding affinity toward the nanoparticles. A 1000-fold enhanced affinity was observed for HSA modified by ethylenediamine (HSAam) to increase the number of amino functions on the protein surface. Remarkably, HSAsuc formed a much thicker protein adsorption layer (8.1 nm) than native HSA (3.3 nm), indicating that it binds in a distinctly different orientation on the nanoparticle, whereas the HSAam corona (4.6 nm) is only slightly thicker. Notably, protein binding to DHLA-QDs was found to be entirely reversible, independent of the modification. We have also measured the extent and kinetics of internalization of these nanoparticles without and with adsorbed native and modified HSA by HeLa cells. Pronounced variations were observed, indicating that even small physicochemical changes of the protein corona may affect biological responses.

Sunitinib: from charge-density studies to interaction with proteins.

PubMed

Malińska, Maura; Jarzembska, Katarzyna N; Goral, Anna M; Kutner, Andrzej; Woźniak, Krzysztof; Dominiak, Paulina M

2014-05-01

Protein kinases are targets for the treatment of a number of diseases. Sunitinib malate is a type I inhibitor of tyrosine kinases and was approved as a drug in 2006. This contribution constitutes the first comprehensive analysis of the crystal structures of sunitinib malate and of complexes of sunitinib with a series of protein kinases. The high-resolution single-crystal X-ray measurement and aspherical atom databank approach served as a basis for reconstruction of the charge-density distribution of sunitinib and its protein complexes. Hirshfeld surface and topological analyses revealed a similar interaction pattern in the sunitinib malate crystal structure to that in the protein binding pockets. Sunitinib forms nine preserved bond paths corresponding to hydrogen bonds and also to the C-H···O and C-H···π contacts common to the VEGRF2, CDK2, G2, KIT and IT kinases. In general, sunitinib interacts with the studied proteins with a similar electrostatic interaction energy and can adjust its conformation to fit the binding pocket in such a way as to enhance the electrostatic interactions, e.g. hydrogen bonds in ligand-kinase complexes. Such behaviour may be responsible for the broad spectrum of action of sunitinib as a kinase inhibitor.

Hydrophobic potential of mean force as a solvation function for protein structure prediction.

PubMed

Lin, Matthew S; Fawzi, Nicolas Lux; Head-Gordon, Teresa

2007-06-01

We have developed a solvation function that combines a Generalized Born model for polarization of protein charge by the high dielectric solvent, with a hydrophobic potential of mean force (HPMF) as a model for hydrophobic interaction, to aid in the discrimination of native structures from other misfolded states in protein structure prediction. We find that our energy function outperforms other reported scoring functions in terms of correct native ranking for 91% of proteins and low Z scores for a variety of decoy sets, including the challenging Rosetta decoys. This work shows that the stabilizing effect of hydrophobic exposure to aqueous solvent that defines the HPMF hydration physics is an apparent improvement over solvent-accessible surface area models that penalize hydrophobic exposure. Decoys generated by thermal sampling around the native-state basin reveal a potentially important role for side-chain entropy in the future development of even more accurate free energy surfaces.

Adsorption and Conformation Change of Helical Peptides on Colloidal Silica

NASA Astrophysics Data System (ADS)

Read, Michael; Zhang, Shuguang; Mayes, Anne; Burkett, Sandra

2001-03-01

Helical conformations of short peptides in solution are partly stabilized by the pattern of electrostatic charge formed by the amino acid sequence. We have studied the role of electrostatics in the adsorption and helix-coil transition of peptides on oxide surfaces. Adsorption isotherms, along with a combination of spectroscopic techniques, show that this is a reversible equilibrium process. Strong electrostatic forces between ionic side chains and charged surface sites increase the adsorbed amount, and promote a loss of helicity in the adsorbed state qualitatively different from that observed upon thermal or chemical perturbation. The electrical dipole of the peptide, arising from the amino acid side chains, serves to orient the molecules on the surface. Effects of adsorption, orientation, and conformation change on the activity of peptides in model biological reactions, as well as the relevance of this simplified system to protein adsorption, are considered.

Protein adsorption and excipient effects on kinetic stability of silicone oil emulsions.

PubMed

Ludwig, D Brett; Carpenter, John F; Hamel, Jean-Bernard; Randolph, Theodore W

2010-04-01

The effect of silicone oil on the stability of therapeutic protein formulations is of concern in the biopharmaceutical industry as more proteins are stored and delivered in prefilled syringes. Prefilled syringes provide convenience for medical professionals and patients, but prolonged exposure of proteins to silicone oil within prefilled syringes may be problematic. In this study, we characterize systems of silicone oil-in-aqueous buffer emulsions and model proteins in formulations containing surfactant, sodium chloride, or sucrose. For each of the formulations studied, silicone oil-induced loss of soluble protein, likely through protein adsorption onto the silicone oil droplet surface. Excipient addition affected both protein adsorption and emulsion stability. Addition of surfactant stabilized emulsions but decreased protein adsorption to silicone oil microdroplets. In contrast, addition of sodium chloride increased protein adsorption and decreased emulsion stability. Silicone oil droplets with adsorbed lysozyme rapidly agglomerated and creamed out of suspension. This decrease in the kinetic stability of the emulsion is ascribed to surface charge neutralization and a bridging flocculation phenomenon and illustrates the need to investigate not only the effects of silicone oil on protein stability, but also the effects of protein formulation variables on emulsion stability. 2009 Wiley-Liss, Inc. and the American Pharmacists Association

The composition of liquid atmospheric pressure matrix-assisted laser desorption/ionization matrices and its effect on ionization in mass spectrometry.

PubMed

Ryumin, Pavel; Cramer, Rainer

2018-07-12

New liquid atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) matrices that produce predominantly multiply charged ions have been developed and evaluated with respect to their performance for peptide and protein analysis by mass spectrometry (MS). Both the chromophore and the viscous support liquid in these matrices were optimized for highest MS signal intensity, S/N values and maximum charge state. The best performance in both protein and peptide analysis was achieved employing light diols as matrix support liquids (e.g. ethylene glycol and propylene glycol). Investigating the influence of the chromophore, it was found that 2,5-dihydroxybenzoic acid resulted in a higher analyte ion signal intensity for the analysis of small peptides; however, larger molecules (>17 kDa) were undetectable. For larger molecules, a sample preparation based on α-cyano-4-hydroxycinnammic acid as the chromophore was developed and multiply protonated analytes with charge states of more than 50 were detected. Thus, for the first time it was possible to detect with MALDI MS proteins as large as ∼80 kDa with a high number of charge states, i.e. m/z values below 2000. Systematic investigations of various matrix support liquids have revealed a linear dependency between laser threshold energy and surface tension of the liquid MALDI sample. Copyright © 2018 Elsevier B.V. All rights reserved.

[Application of Brownian dynamics to the description of transmembrane ion flow as exemplified by the chloride channel of glycine receptor].

PubMed

Boronovskiĭ, S E; Nartsissov, Ia R

2009-01-01

Using the Brownian dynamics of the movement of hydrated ion in a viscous water solution, a mathematical model has been built, which describes the transport of charged particles through a single protein pore in a lipid membrane. The dependences of transmembrane ion currents on ion concentrations in solution have been obtained. It was shown that, if the geometry of a membrane pore is identical to that of the inner part of the glycine receptor channel and there is no ion selectivity, then the values of both chloride and sodium currents are not greater than 0.5 pA at the physiological concentrations of these ions. If local charge heterogeneity caused by charged amino acid residues of transmembrane protein segments is included into the model calculations, the chloride current increases to about 3.7 pA, which exceeds more than seven times the value for sodium ions under the conditions of the complex channel geometry in the range of physiological concentrations of ions in the solution. The model takes changes in the density of charge distribution both inside the channel and near the protein surface into account. The alteration of pore geometry can be also considered as a parameter at the researcher's option. Thus, the model appears as an effective tool for the description of transmembrane currents for other types of membrane channels.

Molecular interactions of mussel protective coating protein, mcfp-1, from Mytilus californianus.

PubMed

Lu, Qingye; Hwang, Dong Soo; Liu, Yang; Zeng, Hongbo

2012-02-01

Protective coating of the byssus of mussels (Mytilus sp.) has been suggested as a new paradigm of medical coating due to its high extensibility and hardness co-existence without their mutual detriment. The only known biomacromolecule in the extensible and tough coating on the byssus is mussel foot protein-1 (mfp-1), which is made up with positively charged residues (~20 mol%) and lack of negatively charged residues. Here, adhesion and molecular interaction mechanisms of Mytilus californianus foot protein-1 (mcfp-1) from California blue mussel were investigated using a surface forces apparatus (SFA) in buffer solutions of different ionic concentrations (0.2-0.7 M) and pHs (3.0-5.5). Strong and reversible cohesion between opposed positively charged mcfp-1 films was measured in 0.1 M sodium acetate buffer with 0.1 M KNO(3). Cohesion of mcfp-1 was gradually reduced with increasing the ionic strength, but was not changed with pH variations. Oxidation of 3,4-dihydroxyphenylalanine (DOPA) residues of mcfp-1, a key residue for adhesive and coating proteins of mussel, didn't change the cohesion strength of mcfp-1 films, but the addition of chemicals with aromatic groups (i.e., aspirin and 4-methylcatechol) increased the cohesion. These results suggest that the cohesion of mcfp-1 films is mainly mediated by cation-π interactions between the positively charged residues and benzene rings of DOPA and other aromatic amino acids (~20 mol% of total amino acids of mcfp-1), and π-π interactions between the phenyl groups in mcfp-1. The adhesion mechanism obtained for the mcfp-1 proteins provides important insight into the design and development of functional biomaterials and coatings mimicking the extensible and robust mussel cuticle coating. Copyright © 2011 Elsevier Ltd. All rights reserved.

Insights in understanding aggregate formation and dissociation in cation exchange chromatography for a structurally unstable Fc-fusion protein.

PubMed

Chen, Zhiqiang; Huang, Chao; Chennamsetty, Naresh; Xu, Xuankuo; Li, Zheng Jian

2016-08-19

Cation-exchange chromatography (CEX) of a structurally unstable Fc-fusion protein exhibited multi-peak elution profile upon a salt-step elution due to protein aggregation during intra-column buffer transition where low pH and high salt coexisted. The protein exhibited a single-peak elution behavior during a pH-step elution; nevertheless, the levels of soluble aggregates (i.e. high molecular weight species, HMW) in the CEX eluate were still found up to 12-fold higher than that for the load material. The amount of the aggregates formed upon the pH-step elution was dependent on column loading with maximum HMW achieved at intermediate loading levels, supporting the hypothesis that the aggregation was the result of both the conformational changes of the bound protein and the solution concentration of the aggregation-susceptible proteins during elution. Factors such as high load pH, short protein/resin contact time, hydrophilic resin surface, and weak ionizable ligand were effective, to some extent, to reduce aggregate formation by improving the structural integrity of the bound protein. An orthogonal technique, differential scanning fluorimetry (DSF) using Sypro Orange dye confirmed that the bound protein exposed more hydrophobic area than the native molecule in free solution, especially in the pH 4-5 range. The Sypro Orange dye study of resin surface property also demonstrated that the poly[styrene-divinylbenzene]-based Poros XS with polyhydroxyl surface coating is more hydrophobic compared to the agarose-based CM Sepharose FF and SP Sepharose FF. The hydrophobic property of Poros XS contributed to stronger interactions with the partially unfolded bound protein and consequently to the higher aggregate levels seen in Poros XS eluate. This work also investigates the aggregation reversibility in CEX eluate where up to 66% of the aggregates were observed to dissociate into native monomers over a period of 120h, and links the aggregate stability to such conditions as resin surface properties and charged ligand type. Experimental data was correlated semi-quantitatively with theoretical protein charge and hydrophobicity calculations using homology modeling within the BIOVIA Discovery Studio software. Finally, an arginine-sulphopropyl (Arg-SP) agarose resin immobilized with multi-functional ligands was prepared to verify the proposed hypothesis and to eliminate the aggregate formation. The findings of this work provide general insights in understanding aggregate formation and dissociation for structurally unstable proteins in the CEX step. Copyright © 2016 Elsevier B.V. All rights reserved.

Intuitive representation of surface properties of biomolecules using BioBlender.

PubMed

Andrei, Raluca Mihaela; Callieri, Marco; Zini, Maria Francesca; Loni, Tiziana; Maraziti, Giuseppe; Pan, Mike Chen; Zoppè, Monica

2012-03-28

In living cells, proteins are in continuous motion and interaction with the surrounding medium and/or other proteins and ligands. These interactions are mediated by protein features such as electrostatic and lipophilic potentials. The availability of protein structures enables the study of their surfaces and surface characteristics, based on atomic contribution. Traditionally, these properties are calculated by physico-chemical programs and visualized as range of colors that vary according to the tool used and imposes the necessity of a legend to decrypt it. The use of color to encode both characteristics makes the simultaneous visualization almost impossible, requiring these features to be visualized in different images. In this work, we describe a novel and intuitive code for the simultaneous visualization of these properties. Recent advances in 3D animation and rendering software have not yet been exploited for the representation of biomolecules in an intuitive, animated form. For our purpose we use Blender, an open-source, free, cross-platform application used professionally for 3D work. On the basis Blender, we developed BioBlender, dedicated to biological work: elaboration of protein motion with simultaneous visualization of their chemical and physical features. Electrostatic and lipophilic potentials are calculated using physico-chemical software and scripts, organized and accessed through BioBlender interface. A new visual code is introduced for molecular lipophilic potential: a range of optical features going from smooth-shiny for hydrophobic regions to rough-dull for hydrophilic ones. Electrostatic potential is represented as animated line particles that flow along field lines, proportional to the total charge of the protein. Our system permits visualization of molecular features and, in the case of moving proteins, their continuous perception, calculated for each conformation during motion. Using real world tactile/sight feelings, the nanoscale world of proteins becomes more understandable, familiar to our everyday life, making it easier to introduce "un-seen" phenomena (concepts) such as hydropathy or charges. Moreover, this representation contributes to gain insight into molecular functions by drawing viewer's attention to the most active regions of the protein. The program, available for Windows, Linux and MacOS, can be downloaded freely from the dedicated website http://www.bioblender.eu.

Intuitive representation of surface properties of biomolecules using BioBlender

PubMed Central

2012-01-01

Background In living cells, proteins are in continuous motion and interaction with the surrounding medium and/or other proteins and ligands. These interactions are mediated by protein features such as electrostatic and lipophilic potentials. The availability of protein structures enables the study of their surfaces and surface characteristics, based on atomic contribution. Traditionally, these properties are calculated by physico-chemical programs and visualized as range of colors that vary according to the tool used and imposes the necessity of a legend to decrypt it. The use of color to encode both characteristics makes the simultaneous visualization almost impossible, requiring these features to be visualized in different images. In this work, we describe a novel and intuitive code for the simultaneous visualization of these properties. Methods Recent advances in 3D animation and rendering software have not yet been exploited for the representation of biomolecules in an intuitive, animated form. For our purpose we use Blender, an open-source, free, cross-platform application used professionally for 3D work. On the basis Blender, we developed BioBlender, dedicated to biological work: elaboration of protein motion with simultaneous visualization of their chemical and physical features. Electrostatic and lipophilic potentials are calculated using physico-chemical software and scripts, organized and accessed through BioBlender interface. Results A new visual code is introduced for molecular lipophilic potential: a range of optical features going from smooth-shiny for hydrophobic regions to rough-dull for hydrophilic ones. Electrostatic potential is represented as animated line particles that flow along field lines, proportional to the total charge of the protein. Conclusions Our system permits visualization of molecular features and, in the case of moving proteins, their continuous perception, calculated for each conformation during motion. Using real world tactile/sight feelings, the nanoscale world of proteins becomes more understandable, familiar to our everyday life, making it easier to introduce "un-seen" phenomena (concepts) such as hydropathy or charges. Moreover, this representation contributes to gain insight into molecular functions by drawing viewer's attention to the most active regions of the protein. The program, available for Windows, Linux and MacOS, can be downloaded freely from the dedicated website http://www.bioblender.eu PMID:22536962

Conformational relaxation and water penetration coupled to ionization of internal groups in proteins.

PubMed

Damjanović, Ana; Brooks, Bernard R; García-Moreno, Bertrand

2011-04-28

Molecular dynamics simulations were used to examine the effects of ionization of internal groups on the structures of eighteen variants of staphylococcal nuclease (SNase) with internal Lys, Asp, or Glu. In most cases the RMSD values of internal ionizable side chains were larger when the ionizable moieties were charged than when they were neutral. Calculations of solvent-accessible surface area showed that the internal ionizable side chains were buried in the protein interior when they were neutral and moved toward crevices and toward the protein-water interface when they were charged. The only exceptions are Lys-36, Lys-62, and Lys-103, which remained buried even after charging. With the exception of Lys-38, the number of internal water molecules surrounding the ionizable group increased upon charging: the average number of water oxygen atoms within the first hydration shell increased by 1.7 for Lys residues, by 5.2 for Asp residues, and by 3.2 for Glu residues. The polarity of the microenvironment of the ionizable group also increased when the groups were charged: the average number of polar atoms of any kind within the first hydration shell increased by 2.7 for Lys residues, by 4.8 for Asp residues, and by 4.0 for Glu residues. An unexpected correlation was observed between the absolute value of the shifts in pK(a) values measured experimentally, and several parameters of structural relaxation: the net difference in the polarity of the microenvironment of the charged and neutral forms of the ionizable groups, the net difference in hydration of the charged and neutral forms of the ionizable groups, and the difference in RMSD values of the charged and neutral forms of the ionizable groups. The effects of ionization of internal groups on the conformation of the backbone were noticeable but mostly small and localized to the area immediately next to the internal ionizable moiety. Some variants did exhibit local unfolding.

Target-specific cellular uptake of PLGA nanoparticles coated with poly(L-lysine)-poly(ethylene glycol)-folate conjugate.

PubMed

Kim, Sun Hwa; Jeong, Ji Hoon; Chun, Ki Woo; Park, Tae Gwan

2005-09-13

Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with anionic surface charge were surface coated with cationic di-block copolymer, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL) conjugate, for enhancing their site-specific intracellular delivery against folate receptor overexpressing cancer cells. The PLGA nanoparticles coated with the conjugate were characterized in terms of size, surface charge, and change in surface composition by XPS. By employing the flow cytometry method and confocal image analysis, the extent of cellular uptake was comparatively evaluated under various conditions. PLL-PEG-FOL coated PLGA nanoparticles demonstrated far greater extent of cellular uptake to KB cells, suggesting that they were mainly taken up by folate receptor-mediated endocytosis. The enhanced cellular uptake was also observed even in the presence of serum proteins, possibly due to the densely seeded PEG chains. The PLL-PEG-FOL coated PLGA nanoparticles could be potentially applied for cancer cell targeted delivery of various therapeutic agents.

Molecular Effects of Concentrated Solutes on Protein Hydration, Dynamics, and Electrostatics.

PubMed

Abriata, Luciano A; Spiga, Enrico; Peraro, Matteo Dal

2016-08-23

Most studies of protein structure and function are performed in dilute conditions, but proteins typically experience high solute concentrations in their physiological scenarios and biotechnological applications. High solute concentrations have well-known effects on coarse protein traits like stability, diffusion, and shape, but likely also perturb other traits through finer effects pertinent at the residue and atomic levels. Here, NMR and molecular dynamics investigations on ubiquitin disclose variable interactions with concentrated solutes that lead to localized perturbations of the protein's surface, hydration, electrostatics, and dynamics, all dependent on solute size and chemical properties. Most strikingly, small polar uncharged molecules are sticky on the protein surface, whereas charged small molecules are not, but the latter still perturb the internal protein electrostatics as they diffuse nearby. Meanwhile, interactions with macromolecular crowders are favored mainly through hydrophobic, but not through polar, surface patches. All the tested small solutes strongly slow down water exchange at the protein surface, whereas macromolecular crowders do not exert such strong perturbation. Finally, molecular dynamics simulations predict that unspecific interactions slow down microsecond- to millisecond-timescale protein dynamics despite having only mild effects on pico- to nanosecond fluctuations as corroborated by NMR. We discuss our results in the light of recent advances in understanding proteins inside living cells, focusing on the physical chemistry of quinary structure and cellular organization, and we reinforce the idea that proteins should be studied in native-like media to achieve a faithful description of their function. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

PubMed Central

Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

2012-01-01

Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering. PMID:22792305

New insight in the structural features of haloadaptation in α-amylases from halophilic Archaea following homology modeling strategy: folded and stable conformation maintained through low hydrophobicity and highly negative charged surface

NASA Astrophysics Data System (ADS)

Zorgani, Mohamed Amine; Patron, Kevin; Desvaux, Mickaël

2014-07-01

Proteins from halophilic archaea, which live in extreme saline conditions, have evolved to remain folded, active and stable at very high ionic strengths. Understanding the mechanism of haloadaptation is the first step toward engineering of halostable biomolecules. Amylases are one of the main enzymes used in industry. Yet, no three-dimensional structure has been experimentally resolved for α-amylases from halophilic archaea. In this study, homology structure modeling of α-amylases from the halophilic archaea Haloarcula marismortui, Haloarcula hispanica, and Halalkalicoccus jeotgali were performed. The resulting models were subjected to energy minimization, evaluation, and structural analysis. Calculations of the amino acid composition, salt bridges and hydrophobic interactions were also performed and compared to a set of non-halophilic counterparts. It clearly appeared that haloarchaeal α-amylases exhibited lower propensities for helix formation and higher propensities for coil-forming regions. Furthermore, they could maintain a folded and stable conformation in high salt concentration through highly negative charged surface with over representation of acidic residues, especially Asp, and low hydrophobicity with increase of salt bridges and decrease in hydrophobic interactions on the protein surface. This study sheds some light on the stability of α-amylases from halophilic archaea and provides strong basis not only to understand haloadaptation mechanisms of proteins in microorganisms from hypersalines environments but also for biotechnological applications.

New insight in the structural features of haloadaptation in α-amylases from halophilic Archaea following homology modeling strategy: folded and stable conformation maintained through low hydrophobicity and highly negative charged surface.

PubMed

Zorgani, Mohamed Amine; Patron, Kevin; Desvaux, Mickaël

2014-07-01

Proteins from halophilic archaea, which live in extreme saline conditions, have evolved to remain folded, active and stable at very high ionic strengths. Understanding the mechanism of haloadaptation is the first step toward engineering of halostable biomolecules. Amylases are one of the main enzymes used in industry. Yet, no three-dimensional structure has been experimentally resolved for α-amylases from halophilic archaea. In this study, homology structure modeling of α-amylases from the halophilic archaea Haloarcula marismortui, Haloarcula hispanica, and Halalkalicoccus jeotgali were performed. The resulting models were subjected to energy minimization, evaluation, and structural analysis. Calculations of the amino acid composition, salt bridges and hydrophobic interactions were also performed and compared to a set of non-halophilic counterparts. It clearly appeared that haloarchaeal α-amylases exhibited lower propensities for helix formation and higher propensities for coil-forming regions. Furthermore, they could maintain a folded and stable conformation in high salt concentration through highly negative charged surface with over representation of acidic residues, especially Asp, and low hydrophobicity with increase of salt bridges and decrease in hydrophobic interactions on the protein surface. This study sheds some light on the stability of α-amylases from halophilic archaea and provides strong basis not only to understand haloadaptation mechanisms of proteins in microorganisms from hypersalines environments but also for biotechnological applications.

Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

PubMed Central

Ganguly, Debabani; Zhang, Weihong; Chen, Jianhan

2013-01-01

Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for cellular signaling and regulation. PMID:24278008

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain.

PubMed

Speranskiy, Kirill; Kurnikova, Maria

2005-08-30

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.

Electrodiffusion of lipids on membrane surfaces.

PubMed

Zhou, Y C

2012-05-28

Lateral translocation of lipids and proteins is a universal process on membrane surfaces. Local aggregation or organization of lipids and proteins can be induced when the random lateral motion is mediated by the electrostatic interactions and membrane curvature. Although the lateral diffusion rates of lipids on membranes of various compositions are measured and the electrostatic free energies of predetermined protein-membrane-lipid systems can be computed, the process of the aggregation and the evolution to the electrostatically favorable states remain largely undetermined. Here we propose an electrodiffusion model, based on the variational principle of the free energy functional, for the self-consistent lateral drift-diffusion of multiple species of charged lipids on membrane surfaces. Finite sizes of lipids are modeled to enforce the geometrical constraint of the lipid concentration on membrane surfaces. A surface finite element method is developed to appropriate the Laplace-Beltrami operators in the partial differential equations of the model. Our model properly describes the saturation of lipids on membrane surfaces, and correctly predicts that the MARCKS peptide can consistently sequester three multivalent phosphatidylinositol 4,5-bisphosphate lipids through its basic amino acid residues, regardless of a wide range of the percentage of monovalent phosphatidylserine in the membrane.

Electrodiffusion of lipids on membrane surfaces

NASA Astrophysics Data System (ADS)

Zhou, Y. C.

2012-05-01

Lateral translocation of lipids and proteins is a universal process on membrane surfaces. Local aggregation or organization of lipids and proteins can be induced when the random lateral motion is mediated by the electrostatic interactions and membrane curvature. Although the lateral diffusion rates of lipids on membranes of various compositions are measured and the electrostatic free energies of predetermined protein-membrane-lipid systems can be computed, the process of the aggregation and the evolution to the electrostatically favorable states remain largely undetermined. Here we propose an electrodiffusion model, based on the variational principle of the free energy functional, for the self-consistent lateral drift-diffusion of multiple species of charged lipids on membrane surfaces. Finite sizes of lipids are modeled to enforce the geometrical constraint of the lipid concentration on membrane surfaces. A surface finite element method is developed to appropriate the Laplace-Beltrami operators in the partial differential equations of the model. Our model properly describes the saturation of lipids on membrane surfaces, and correctly predicts that the MARCKS peptide can consistently sequester three multivalent phosphatidylinositol 4,5-bisphosphate lipids through its basic amino acid residues, regardless of a wide range of the percentage of monovalent phosphatidylserine in the membrane.

Protein charge distribution in proteomes and its impact on translation

PubMed Central

Requião, Rodrigo D.; Fernandes, Luiza; de Souza, Henrique José Araujo; Rossetto, Silvana; Domitrovic, Tatiana

2017-01-01

As proteins are synthesized, the nascent polypeptide must pass through a negatively charged exit tunnel. During this stage, positively charged stretches can interact with the ribosome walls and slow the translation. Therefore, charged polypeptides may be important factors that affect protein expression. To determine the frequency and distribution of positively and negatively charged stretches in different proteomes, the net charge was calculated for every 30 consecutive amino acid residues, which corresponds to the length of the ribosome exit tunnel. The following annotated and reviewed proteins in the UniProt database (Swiss-Prot) were analyzed: 551,705 proteins from different organisms and a total of 180 million protein segments. We observed that there were more negative than positive stretches and that super-charged positive sequences (i.e., net charges ≥ 14) were underrepresented in the proteomes. Overall, the proteins were more positively charged at their N-termini and C-termini, and this feature was present in most organisms and subcellular localizations. To investigate whether the N-terminal charges affect the elongation rates, previously published ribosomal profiling data obtained from S. cerevisiae, without translation-interfering drugs, were analyzed. We observed a nonlinear effect of the charge on the ribosome occupancy in which values ≥ +5 and ≤ -6 showed increased and reduced ribosome densities, respectively. These groups also showed different distributions across 80S monosomes and polysomes. Basic polypeptides are more common within short proteins that are translated by monosomes, whereas negative stretches are more abundant in polysome-translated proteins. These findings suggest that the nascent peptide charge impacts translation and can be one of the factors that regulate translation efficiency and protein expression. PMID:28531225

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library.

PubMed

Traxlmayr, Michael W; Kiefer, Jonathan D; Srinivas, Raja R; Lobner, Elisabeth; Tisdale, Alison W; Mehta, Naveen K; Yang, Nicole J; Tidor, Bruce; Wittrup, K Dane

2016-10-21

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (T m of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability.

PubMed

Merlino, Antonello; Russo Krauss, Irene; Castellano, Immacolata; Ruocco, Maria Rosaria; Capasso, Alessandra; De Vendittis, Emmanuele; Rossi, Bianca; Sica, Filomena

2014-03-01

A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge-charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme. Copyright © 2014 Elsevier B.V. All rights reserved.

BSA adsorption onto nanospheres: Influence of surface curvature as probed by electrophoretic light scattering and UV/vis spectroscopy

NASA Astrophysics Data System (ADS)

Sánchez-Pérez, Julio A.; Gallardo-Moreno, Amparo M.; González-Martín, M. Luisa; Vadillo-Rodríguez, Virginia

2015-10-01

The influence of surface curvature on the adsorption of bovine serum albumin (BSA) was evaluated through the combination of two fairly simple techniques: electrophoretic light scattering and UV/vis spectroscopy. Measurements were carried out for a range of protein concentrations (0-320 μg/ml) at pH 3.5, 4.5 and 7 using hydrophobic polystyrene nanospheres of 38.8, 82 and 220 nm in diameter. The results obtained demonstrate that the charge of the BSA molecules in solution dictates the pH-dependent behavior of the protein-coated nanospheres, indicating in all cases a significant adsorption of BSA molecules. At a fixed pH, however, it is the zeta potential that characterizes the uncoated nanospheres normalized by their surface area that primarily controls protein adsorption. In particular, it is found that the rate at which BSA interact with the different nanospheres increases as their negative zeta potential per unit area (or diameter) increases (decreases) regardless of the pH. Moreover, provided that adsorption occurs away from the isoelectric point of the protein, highly curved surfaces are found to stabilize the native-like conformation of BSA upon adsorption by likely reducing lateral interactions between adsorbed molecules.

Rational design of a conformation-switchable Ca2+- and Tb3+-binding protein without the use of multiple coupled metal-binding sites.

PubMed

Li, Shunyi; Yang, Wei; Maniccia, Anna W; Barrow, Doyle; Tjong, Harianto; Zhou, Huan-Xiang; Yang, Jenny J

2008-10-01

Ca2+, as a messenger of signal transduction, regulates numerous target molecules via Ca2+-induced conformational changes. Investigation into the determinants for Ca2+-induced conformational change is often impeded by cooperativity between multiple metal-binding sites or protein oligomerization in naturally occurring proteins. To dissect the relative contributions of key determinants for Ca2+-dependent conformational changes, we report the design of a single-site Ca2+-binding protein (CD2.trigger) created by altering charged residues at an electrostatically sensitive location on the surface of the host protein rat Cluster of Differentiation 2 (CD2).CD2.trigger binds to Tb3+ and Ca2+ with dissociation constants of 0.3 +/- 0.1 and 90 +/- 25 microM, respectively. This protein is largely unfolded in the absence of metal ions at physiological pH, but Tb3+ or Ca2+ binding results in folding of the native-like conformation. Neutralization of the charged coordination residues, either by mutation or protonation, similarly induces folding of the protein. The control of a major conformational change by a single Ca2+ ion, achieved on a protein designed without reliance on sequence similarity to known Ca2+-dependent proteins and coupled metal-binding sites, represents an important step in the design of trigger proteins.

E. coli Surface Properties Differ between Stream Water and Sediment Environments.

PubMed

Liang, Xiao; Liao, Chunyu; Thompson, Michael L; Soupir, Michelle L; Jarboe, Laura R; Dixon, Philip M

2016-01-01

The importance of E. coli as an indicator organism in fresh water has led to numerous studies focusing on cell properties and transport behavior. However, previous studies have been unable to assess if differences in E. coli cell surface properties and genomic variation are associated with different environmental habitats. In this study, we investigated the variation in characteristics of E. coli obtained from stream water and stream bottom sediments. Cell properties were measured for 77 genomically different E. coli strains (44 strains isolated from sediments and 33 strains isolated from water) under common stream conditions in the Upper Midwestern United States: pH 8.0, ionic strength 10 mM and 22°C. Measured cell properties include hydrophobicity, zeta potential, net charge, total acidity, and extracellular polymeric substance (EPS) composition. Our results indicate that stream sediment E. coli had significantly greater hydrophobicity, greater EPS protein content and EPS sugar content, less negative net charge, and higher point of zero charge than stream water E. coli . A significant positive correlation was observed between hydrophobicity and EPS protein for stream sediment E. coli but not for stream water E. coli . Additionally, E. coli surviving in the same habitat tended to have significantly larger (GTG) 5 genome similarity. After accounting for the intrinsic impact from the genome, environmental habitat was determined to be a factor influencing some cell surface properties, such as hydrophobicity. The diversity of cell properties and its resulting impact on particle interactions should be considered for environmental fate and transport modeling of aquatic indicator organisms such as E. coli .

Unique surface adsorption behaviors of serum proteins on chemically uniform and alternating surfaces

NASA Astrophysics Data System (ADS)

Song, Sheng

With increasing interests of studying proteins adsorption on the surfaces with nanoscale features in biomedical field, it is crucial to have fundamental understandings on how the proteins are adsorbed on such a surface and what factors contribute to the driving forces of adsorption. Besides, exploring more available nanoscale templates would greatly offer more possibilities one could design surface bio-detection methods with favorable protein-surface interactions. Thus, to fulfill the purpose, the work in this dissertation has been made into three major sections. First, to probe the intermediate states which possibly exist between stable and unstable phases described in mean-field theory diagram, a solvent vapor annealing method is chosen to slowly induce the copolymer polystyrene-block-polyvinylpyridine (PS-b-PVP)'s both blocks undergoing micro-phase separations from initial spherical nanodomains into terminal cylindrical nanodomains. During this process, real time atomic force microscopy (AFM) has been conducted to capture other six intermediate states with different morphologies on the polymeric film surfaces. Secondly, upon recognizing each intermediate state, the solution of immunoglobulin gamma (IgG) proteins has been deposited on the surface and been rinsed off with buffer solution before the protein-bounded surface is imaged by AFM. It has been found IgG showing a strong adsorption preference on PS over P4VP block. Among all the six intermediate states, the proteins are almost exclusively adsorbed on PS nanodomains regardless the concentration and deposition time. Thirdly, a trinodular shape protein fibrinogen (Fg) is selected for investigating how geometry and surface charge of proteins would interplay with cylindrical nanodomains on a surface developed from Polystyrene -block-Poly-(methyl methacrylate) PS-b-PMMA. Also, Fg adsorptions on chemically homogeneous surfaces are included here to have a better contrast of showing how much difference it can make by using it on a nanoscale surface. Interestingly, higher concentration of protein solution promotes the occurrences of single phase packed Fg on the PS domain. The densely packed network has formed where each Fg keeps its main body in PS domain and leaves its two alpha C chains on nearby PMMA domain. We believe this conformation and orientation would maximize both the hydrophobic and electrostatic interactions between Fg and the underlying surface.

Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.

PubMed

Peretti, Leandro E; Gonzalez, Verónica D G; Marcipar, Iván S; Gugliotta, Luis M

2014-08-01

The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease. Copyright © 2014 Elsevier B.V. All rights reserved.

Electron tunnelling through single azurin molecules can be on/off switched by voltage pulses

NASA Astrophysics Data System (ADS)

Baldacchini, Chiara; Kumar, Vivek; Bizzarri, Anna Rita; Cannistraro, Salvatore

2015-05-01

Redox metalloproteins are emerging as promising candidates for future bio-optoelectronic and nano-biomemory devices, and the control of their electron transfer properties through external signals is still a crucial task. Here, we show that a reversible on/off switching of the electron current tunnelling through a single protein can be achieved in azurin protein molecules adsorbed on gold surfaces, by applying appropriate voltage pulses through a scanning tunnelling microscope tip. The observed changes in the hybrid system tunnelling properties are discussed in terms of long-sustained charging of the protein milieu.

Molecular evidence of stereo-specific lactoferrin dimers in solution.

PubMed

Persson, Björn A; Lund, Mikael; Forsman, Jan; Chatterton, Dereck E W; Akesson, Torbjörn

2010-10-01

Gathering experimental evidence suggests that bovine as well as human lactoferrin self-associate in aqueous solution. Still, a molecular level explanation is unavailable. Using force field based molecular modeling of the protein-protein interaction free energy we demonstrate (1) that lactoferrin forms highly stereo-specific dimers at neutral pH and (2) that the self-association is driven by a high charge complementarity across the contact surface of the proteins. Our theoretical predictions of dimer formation are verified by electrophoretic mobility and N-terminal sequence analysis on bovine lactoferrin. 2010 Elsevier B.V. All rights reserved.

Mapping Hydrophobicity on the Protein Molecular Surface at Atom-Level Resolution

PubMed Central

Nicolau Jr., Dan V.; Paszek, Ewa; Fulga, Florin; Nicolau, Dan V.

2014-01-01

A precise representation of the spatial distribution of hydrophobicity, hydrophilicity and charges on the molecular surface of proteins is critical for the understanding of the interaction with small molecules and larger systems. The representation of hydrophobicity is rarely done at atom-level, as this property is generally assigned to residues. A new methodology for the derivation of atomic hydrophobicity from any amino acid-based hydrophobicity scale was used to derive 8 sets of atomic hydrophobicities, one of which was used to generate the molecular surfaces for 35 proteins with convex structures, 5 of which, i.e., lysozyme, ribonuclease, hemoglobin, albumin and IgG, have been analyzed in more detail. Sets of the molecular surfaces of the model proteins have been constructed using spherical probes with increasingly large radii, from 1.4 to 20 Å, followed by the quantification of (i) the surface hydrophobicity; (ii) their respective molecular surface areas, i.e., total, hydrophilic and hydrophobic area; and (iii) their relative densities, i.e., divided by the total molecular area; or specific densities, i.e., divided by property-specific area. Compared with the amino acid-based formalism, the atom-level description reveals molecular surfaces which (i) present an approximately two times more hydrophilic areas; with (ii) less extended, but between 2 to 5 times more intense hydrophilic patches; and (iii) 3 to 20 times more extended hydrophobic areas. The hydrophobic areas are also approximately 2 times more hydrophobicity-intense. This, more pronounced “leopard skin”-like, design of the protein molecular surface has been confirmed by comparing the results for a restricted set of homologous proteins, i.e., hemoglobins diverging by only one residue (Trp37). These results suggest that the representation of hydrophobicity on the protein molecular surfaces at atom-level resolution, coupled with the probing of the molecular surface at different geometric resolutions, can capture processes that are otherwise obscured to the amino acid-based formalism. PMID:25462574

Fabrication of enzyme-degradable and size-controlled protein nanowires using single particle nano-fabrication technique

PubMed Central

Omichi, Masaaki; Asano, Atsushi; Tsukuda, Satoshi; Takano, Katsuyoshi; Sugimoto, Masaki; Saeki, Akinori; Sakamaki, Daisuke; Onoda, Akira; Hayashi, Takashi; Seki, Shu

2014-01-01

Protein nanowires exhibiting specific biological activities hold promise for interacting with living cells and controlling and predicting biological responses such as apoptosis, endocytosis and cell adhesion. Here we report the result of the interaction of a single high-energy charged particle with protein molecules, giving size-controlled protein nanowires with an ultra-high aspect ratio of over 1,000. Degradation of the human serum albumin nanowires was examined using trypsin. The biotinylated human serum albumin nanowires bound avidin, demonstrating the high affinity of the nanowires. Human serum albumin–avidin hybrid nanowires were also fabricated from a solid state mixture and exhibited good mechanical strength in phosphate-buffered saline. The biotinylated human serum albumin nanowires can be transformed into nanowires exhibiting a biological function such as avidin–biotinyl interactions and peroxidase activity. The present technique is a versatile platform for functionalizing the surface of any protein molecule with an extremely large surface area. PMID:24770668

Heterogeneous Nucleation of Protein Crystals on Fluorinated Layered Silicate

PubMed Central

Ino, Keita; Udagawa, Itsumi; Iwabata, Kazuki; Takakusagi, Yoichi; Kubota, Munehiro; Kurosaka, Keiichi; Arai, Kazuhito; Seki, Yasutaka; Nogawa, Masaya; Tsunoda, Tatsuo; Mizukami, Fujio; Taguchi, Hayao; Sakaguchi, Kengo

2011-01-01

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface. PMID:21818343

Modifications of nano-titania surface for in vitro evaluations of hemolysis, cytotoxicity, and nonspecific protein binding

NASA Astrophysics Data System (ADS)

Datta, Aparna; Dasgupta, Sayantan; Mukherjee, Siddhartha

2017-04-01

In the past decade, a variety of drug carriers based on mesoporous silica nanoparticles has been extensively reported. However, their biocompatibility still remains debatable, which motivated us to explore the porous nanostructures of other metal oxides, for example titanium dioxide (TiO2), as potential drug delivery vehicles. Herein, we report the in vitro hemolysis, cytotoxicity, and protein binding of TiO2 nanoparticles, synthesized by a sol-gel method. The surface of the TiO2 nanoparticles was modified with hydroxyl, amine, or thiol containing moieties to examine the influence of surface functional groups on the toxicity and protein binding aspects of the nanoparticles. Our study revealed the superior hemocompatibility of pristine, as well as functionalized TiO2 nanoparticles, compared to that of mesoporous silica, the present gold standard. Among the functional groups studied, aminosilane moieties on the TiO2 surface substantially reduced the degree of hemolysis (down to 5%). Further, cytotoxicity studies by MTT assay suggested that surface functional moieties play a crucial role in determining the biocompatibility of the nanoparticles. The presence of NH2- functional groups on the TiO2 nanoparticle surface enhanced the cell viability by almost 28% as compared to its native counterpart (at 100 μg/ml), which was in agreement with the hemolysis assay. Finally, nonspecific protein adsorption on functionalized TiO2 surfaces was examined using human serum albumin and it was found that negatively charged surface moieties, like -OH and -SH, could mitigate protein adsorption to a significant extent.

MM-ISMSA: An Ultrafast and Accurate Scoring Function for Protein-Protein Docking.

PubMed

Klett, Javier; Núñez-Salgado, Alfonso; Dos Santos, Helena G; Cortés-Cabrera, Álvaro; Perona, Almudena; Gil-Redondo, Rubén; Abia, David; Gago, Federico; Morreale, Antonio

2012-09-11

An ultrafast and accurate scoring function for protein-protein docking is presented. It includes (1) a molecular mechanics (MM) part based on a 12-6 Lennard-Jones potential; (2) an electrostatic component based on an implicit solvent model (ISM) with individual desolvation penalties for each partner in the protein-protein complex plus a hydrogen bonding term; and (3) a surface area (SA) contribution to account for the loss of water contacts upon protein-protein complex formation. The accuracy and performance of the scoring function, termed MM-ISMSA, have been assessed by (1) comparing the total binding energies, the electrostatic term, and its components (charge-charge and individual desolvation energies), as well as the per residue contributions, to results obtained with well-established methods such as APBSA or MM-PB(GB)SA for a set of 1242 decoy protein-protein complexes and (2) testing its ability to recognize the docking solution closest to the experimental structure as that providing the most favorable total binding energy. For this purpose, a test set consisting of 15 protein-protein complexes with known 3D structure mixed with 10 decoys for each complex was used. The correlation between the values afforded by MM-ISMSA and those from the other methods is quite remarkable (r(2) ∼ 0.9), and only 0.2-5.0 s (depending on the number of residues) are spent on a single calculation including an all vs all pairwise energy decomposition. On the other hand, MM-ISMSA correctly identifies the best docking solution as that closest to the experimental structure in 80% of the cases. Finally, MM-ISMSA can process molecular dynamics trajectories and reports the results as averaged values with their standard deviations. MM-ISMSA has been implemented as a plugin to the widely used molecular graphics program PyMOL, although it can also be executed in command-line mode. MM-ISMSA is distributed free of charge to nonprofit organizations.

Coarse-Grained Model for Colloidal Protein Interactions, B22, and Protein Cluster Formation

PubMed Central

Blanco, Marco A.; Sahin, Eric; Robinson, Anne S.; Roberts, Christopher J.

2014-01-01

Reversible protein cluster formation is an important initial step in the processes of native and non-native protein aggregation, but involves relatively long time and length scales for detailed atomistic simulations and extensive mapping of free energy landscapes. A coarse-grained (CG) model is presented to semi-quantitatively characterize the thermodynamics and key configurations involved in the landscape for protein oligomerization, as well as experimental measures of interactions such as the osmotic second virial coefficient (B22). Based on earlier work, this CG model treats proteins as rigid bodies composed of one bead per amino acid, with each amino acid having specific parameters for its size, hydrophobicity, and charge. The net interactions are a combination of steric repulsions, short-range attractions, and screened long-range charge-charge interactions. Model parametrization was done by fitting simulation results against experimental values of the B22 as a function of solution ionic strength for α-chymotrypsinogen A and γD-crystallin (gD-Crys). The CG model is applied to characterize the pairwise interactions and dimerization of gD-Crys and the dependance on temperature, protein concentration, and ionic strength. The results illustrate that at experimentally relevant conditions where stable dimers do not form, the entropic contributions are predominant in the free-energy of protein cluster formation and colloidal protein interactions, arguing against interpretations that treat B22 primarily from energetic considerations alone. Additionally, the results suggest that electrostatic interactions help to modulate the population of the different stable configurations for protein nearest-neighbor pairs, while short-range attractions determine the relative orientations of proteins within these configurations. Finally, simulation results are combined with Principal Component Analysis to identify those amino-acids / surface patches that form inter-protein contacts at conditions that favor dimerization of gD-Crys. The resulting regions agree with previously found aggregation-prone sites, as well as suggesting new ones that may be important. PMID:24289039

Molecular-level understanding of protein adsorption at the interface between water and a strongly interacting uncharged solid surface.

PubMed

Penna, Matthew J; Mijajlovic, Milan; Biggs, Mark J

2014-04-09

Although protein adsorption on solids is of immense relevance, experimental limitations mean there is still a remarkable lack of understanding of the adsorption mechanism, particularly at a molecular level. By subjecting 240+ molecular dynamics simulations of two peptide/water/solid surface systems to statistical analysis, a generalized molecular level mechanism for peptide adsorption has been identified for uncharged surfaces that interact strongly with the solution phase. This mechanism is composed of three phases: (1) biased diffusion of the peptide from the bulk phase toward the surface; (2) anchoring of the peptide to the water/solid interface via interaction of a hydrophilic group with the water adjacent to the surface or a strongly interacting hydrophobic group with the surface; and (3) lockdown of the peptide on the surface via a slow, stepwise and largely sequential adsorption of its residues, which we term 'statistical zippering'. The adsorption mechanism is dictated by the existence of water layers adjacent to the solid and orientational ordering therein. By extending the solid into the solution by ~8 Å and endowing it with a charged character, the water layers ensure the peptide feels the effect of the solid at a range well beyond the dispersion force that arises from it, thus inducing biased diffusion from afar. The charging of the interface also facilitates anchoring of the peptide near the surface via one of its hydrophilic groups, allowing it time it would otherwise not have to rearrange and lockdown. Finally, the slowness of the lockdown process is dictated by the need for the peptide groups to replace adjacent tightly bound interfacial water.

SH3-like motif-containing C-terminal domain of staphylococcal teichoic acid transporter suggests possible function.

PubMed

Ko, Tzu-Ping; Tseng, Shih-Ting; Lai, Shu-Jung; Chen, Sheng-Chia; Guan, Hong-Hsiang; Shin Yang, Chia; Jung Chen, Chun; Chen, Yeh

2016-09-01

The negatively charged bacterial polysaccharides-wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising a transmembrane subunit TagG and an ATPase subunit TagH. We determined the crystal structure of the C-terminal domain of TagH (TagH-C) to investigate its function. The structure shows an N-terminal SH3-like subdomain wrapped by a C-terminal subdomain with an anti-parallel β-sheet and an outer shell of α-helices. A stretch of positively charged surface across the subdomain interface is flanked by two negatively charged regions, suggesting a potential binding site for negatively charged polymers, such as WTAs or acidic peptide chains. Proteins 2016; 84:1328-1332. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Protein corona – from molecular adsorption to physiological complexity

PubMed Central

Docter, Dominic; Maskos, Michael

2015-01-01

Summary In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP–protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs. PMID:25977856

Protein corona - from molecular adsorption to physiological complexity.

PubMed

Treuel, Lennart; Docter, Dominic; Maskos, Michael; Stauber, Roland H

2015-01-01

In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP-protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs.

A molecular modeling based method to predict elution behavior and binding patches of proteins in multimodal chromatography.

PubMed

Banerjee, Suvrajit; Parimal, Siddharth; Cramer, Steven M

2017-08-18

Multimodal (MM) chromatography provides a powerful means to enhance the selectivity of protein separations by taking advantage of multiple weak interactions that include electrostatic, hydrophobic and van der Waals interactions. In order to increase our understanding of such phenomena, a computationally efficient approach was developed that combines short molecular dynamics simulations and continuum solvent based coarse-grained free energy calculations in order to study the binding of proteins to Self Assembled Monolayers (SAM) presenting MM ligands. Using this method, the free energies of protein-MM SAM binding over a range of incident orientations of the protein can be determined. The resulting free energies were then examined to identify the more "strongly bound" orientations of different proteins with two multimodal surfaces. The overall free energy of protein-MM surface binding was then determined and correlated to retention factors from isocratic chromatography. This correlation, combined with analytical expressions from the literature, was then employed to predict protein gradient elution salt concentrations as well as selectivity reversals with different MM resin systems. Patches on protein surfaces that interacted strongly with MM surfaces were also identified by determining the frequency of heavy atom contacts with the atoms of the MM SAMs. A comparison of these patches to Electrostatic Potential and hydrophobicity maps indicated that while all of these patches contained significant positive charge, only the highest frequency sites also possessed hydrophobicity. The ability to identify key binding patches on proteins may have significant impact on process development for the separation of bioproduct related impurities. Copyright © 2017 Elsevier B.V. All rights reserved.

Co-Immobilization of Proteins and DNA Origami Nanoplates to Produce High-Contrast Biomolecular Nanoarrays.

PubMed

Hager, Roland; Burns, Jonathan R; Grydlik, Martyna J; Halilovic, Alma; Haselgrübler, Thomas; Schäffler, Friedrich; Howorka, Stefan

2016-06-01

The biofunctionalization of nanopatterned surfaces with DNA origami nanostructures is an important topic in nanobiotechnology. An unexplored challenge is, however, to co-immobilize proteins with DNA origami at pre-determined substrate sites in high contrast relative to the nontarget areas. The immobilization should, in addition, preferably be achieved on a transparent substrate to allow ultrasensitive optical detection. If successful, specific co-binding would be a step towards stoichiometrically defined arrays with few to individual protein molecules per site. Here, we successfully immobilize with high specificity positively charged avidin proteins and negatively charged DNA origami nanoplates on 100 nm-wide carbon nanoislands while suppressing undesired adsorption to surrounding nontarget areas. The arrays on glass slides achieve unprecedented selectivity factors of up to 4000 and allow ultrasensitive fluorescence read-out. The co-immobilization onto the nanoislands leads to layered biomolecular architectures, which are functional because bound DNA origami influences the number of capturing sites on the nanopatches for other proteins. The novel hybrid DNA origami-protein nanoarrays allow the fabrication of versatile research platforms for applications in biosensing, biophysics, and cell biology, and, in addition, represent an important step towards single-molecule protein arrays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrostatic interactions of colicin E1 with the surface of Escherichia coli total lipid.

PubMed

Tian, Chunhong; Tétreault, Elaine; Huang, Christopher K; Dahms, Tanya E S

2006-06-01

The surface properties of colicin E1, a 522-amino acid protein, and its interaction with monolayers of Escherichia coli (E. coli) total lipid and 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DOPC) were studied using the Langmuir-Blodgett (LB) technique. Colicin E1 is amphiphilic, forming a protein monolayer at the air/buffer interface. The protein is thought to interact with the E. coli total lipid head groups through electrostatic interactions, followed by its insertion into the lipid monolayers. Supported lipid bilayers (SLBs) of E. coli total lipid and DOPC, deposited onto mica at the cell membrane equivalence pressure for E. coli and incubated with colicin E1, were imaged by contact mode atomic force microscopy (CM-AFM). Colicin E1 formed protein aggregates on DOPC SLBs, while E. coli total lipid SLB was deformed following its incubation with colicin E1. Corresponding lateral force images, along with electrostatic surface potentials for colicin E1 P190, imply a direct interaction of colicin E1 with lipid head groups facilitating their charge neutralization.

Ice-like water supports hydration forces and eases sliding friction

PubMed Central

Dhopatkar, Nishad; Defante, Adrian P.; Dhinojwala, Ali

2016-01-01

The nature of interfacial water is critical in several natural processes, including the aggregation of lipids into the bilayer, protein folding, lubrication of synovial joints, and underwater gecko adhesion. The nanometer-thin water layer trapped between two surfaces has been identified to have properties that are very different from those of bulk water, but the molecular cause of such discrepancy is often undetermined. Using surface-sensitive sum frequency generation (SFG) spectroscopy, we discover a strongly coordinated water layer confined between two charged surfaces, formed by the adsorption of a cationic surfactant on the hydrophobic surfaces. By varying the adsorbed surfactant coverage and hence the surface charge density, we observe a progressively evolving water structure that minimizes the sliding friction only beyond the surfactant concentration needed for monolayer formation. At complete surfactant coverage, the strongly coordinated confined water results in hydration forces, sustains confinement and sliding pressures, and reduces dynamic friction. Observing SFG signals requires breakdown in centrosymmetry, and the SFG signal from two oppositely oriented surfactant monolayers cancels out due to symmetry. Surprisingly, we observe the SFG signal for the water confined between the two charged surfactant monolayers, suggesting that this interfacial water layer is noncentrosymmetric. The structure of molecules under confinement and its macroscopic manifestation on adhesion and friction have significance in many complicated interfacial processes prevalent in biology, chemistry, and engineering. PMID:27574706

Lipid rafts sense and direct electric field-induced migration

PubMed Central

Lin, Bo-jian; Tsao, Shun-hao; Chen, Alex; Hu, Shu-Kai; Chao, Ling

2017-01-01

Endogenous electric fields (EFs) are involved in developmental regulation and wound healing. Although the phenomenon is known for more than a century, it is not clear how cells perceive the external EF. Membrane proteins, responding to electrophoretic and electroosmotic forces, have long been proposed as the sensing molecules. However, specific charge modification of surface proteins did not change cell migration motility nor directionality in EFs. Moreover, symmetric alternating current (AC) EF directs cell migration in a frequency-dependent manner. Due to their charge and ability to coalesce, glycolipids are therefore the likely primary EF sensor driving polarization of membrane proteins and intracellular signaling. We demonstrate that detergent-resistant membrane nanodomains, also known as lipid rafts, are the primary response element in EF sensing. The clustering and activation of caveolin and signaling proteins further stabilize raft structure and feed-forward downstream signaling events, such as rho and PI3K activation. Theoretical modeling supports the experimental results and predicts AC frequency-dependent cell and raft migration. Our results establish a fundamental mechanism for cell electrosensing and provide a role in lipid raft mechanotransduction. PMID:28739955

Lipid rafts sense and direct electric field-induced migration.

PubMed

Lin, Bo-Jian; Tsao, Shun-Hao; Chen, Alex; Hu, Shu-Kai; Chao, Ling; Chao, Pen-Hsiu Grace

2017-08-08

Endogenous electric fields (EFs) are involved in developmental regulation and wound healing. Although the phenomenon is known for more than a century, it is not clear how cells perceive the external EF. Membrane proteins, responding to electrophoretic and electroosmotic forces, have long been proposed as the sensing molecules. However, specific charge modification of surface proteins did not change cell migration motility nor directionality in EFs. Moreover, symmetric alternating current (AC) EF directs cell migration in a frequency-dependent manner. Due to their charge and ability to coalesce, glycolipids are therefore the likely primary EF sensor driving polarization of membrane proteins and intracellular signaling. We demonstrate that detergent-resistant membrane nanodomains, also known as lipid rafts, are the primary response element in EF sensing. The clustering and activation of caveolin and signaling proteins further stabilize raft structure and feed-forward downstream signaling events, such as rho and PI3K activation. Theoretical modeling supports the experimental results and predicts AC frequency-dependent cell and raft migration. Our results establish a fundamental mechanism for cell electrosensing and provide a role in lipid raft mechanotransduction.

Comparison of calculation and experiment implicates significant electrostatic contributions to the binding stability of barnase and barstar.

PubMed

Dong, Feng; Vijayakumar, M; Zhou, Huan-Xiang

2003-07-01

The contributions of electrostatic interactions to the binding stability of barnase and barstar were studied by the Poisson-Boltzmann model with three different protocols: a), the dielectric boundary specified as the van der Waals (vdW) surface of the protein along with a protein dielectric constant (epsilon (p)) of 4; b), the dielectric boundary specified as the molecular (i.e., solvent-exclusion (SE)) surface along with epsilon (p) = 4; and c), "SE + epsilon (p) = 20." The "vdW + epsilon (p) = 4" and "SE + epsilon (p) = 20" protocols predicted an overall electrostatic stabilization whereas the "SE + epsilon (p) = 4" protocol predicted an overall electrostatic destabilization. The "vdW + epsilon (p) = 4" protocol was most consistent with experiment. It quantitatively reproduced the observed effects of 17 mutations neutralizing charged residues lining the binding interface and the measured coupling energies of six charge pairs across the interface and reasonably rationalized the experimental ionic strength and pH dependences of the binding constant. In contrast, the "SE + epsilon (p) = 4" protocol predicted significantly larger coupling energies of charge pairs whereas the "SE + epsilon (p) = 20" protocol did not predict any pH dependence. This study calls for further scrutiny of the different Poisson-Boltzmann protocols and demonstrates potential danger in drawing conclusions on electrostatic contributions based on a particular calculation protocol.

Structure-function analysis of rotavirus NSP2 octamer by using a novel complementation system.

PubMed

Taraporewala, Zenobia F; Jiang, Xiaofang; Vasquez-Del Carpio, Rodrigo; Jayaram, Hariharan; Prasad, B V Venkataram; Patton, John T

2006-08-01

Viral inclusion bodies, or viroplasms, that form in rotavirus-infected cells direct replication and packaging of the segmented double-stranded RNA (dsRNA) genome. NSP2, one of two rotavirus proteins needed for viroplasm assembly, possesses NTPase, RNA-binding, and helix-unwinding activities. NSP2 of the rotavirus group causing endemic infantile diarrhea (group A) was shown to self-assemble into large doughnut-shaped octamers with circumferential grooves and deep clefts containing nucleotide-binding histidine triad (HIT)-like motifs. Here, we demonstrate that NSP2 of group C rotavirus, a group that fails to reassort with group A viruses, retains the unique architecture of the group A octamer but differs in surface charge distribution. By using an NSP2-dependent complementation system, we show that the HIT-dependent NTPase activity of NSP2 is necessary for dsRNA synthesis, but not for viroplasm formation. The complementation system also showed that despite the retention of the octamer structure and the HIT-like fold, group C NSP2 failed to rescue replication and viroplasm formation in NSP2-deficient cells infected with group A rotavirus. The distinct differences in the surface charges on the Bristol and SA11 NSP2 octamers suggest that charge complementarity of the viroplasm-forming proteins guides the specificity of viroplasm formation and, possibly, reassortment restriction between rotavirus groups.

The Pseudomonas aeruginosa Catabolite Repression Control Protein Crc Is Devoid of RNA Binding Activity

PubMed Central

Djinovic-Carugo, Kristina; Bläsi, Udo

2013-01-01

The Crc protein has been shown to mediate catabolite repression control in Pseudomonas, leading to a preferential assimilation of carbon sources. It has been suggested that Crc acts as a translational repressor of mRNAs, encoding functions involved in uptake and breakdown of different carbon sources. Moreover, the regulatory RNA CrcZ, the level of which is increased in the presence of less preferred carbon sources, was suggested to bind to and sequester Crc, resulting in a relief of catabolite repression. Here, we determined the crystal structure of Pseudomonas aeruginosa Crc, a member of apurinic/apyrimidinic (AP) endonuclease family, at 1.8 Å. Although Crc displays high sequence similarity with its orthologs, there are amino acid alterations in the area corresponding to the active site in AP proteins. Unlike typical AP endonuclease family proteins, Crc has a reduced overall positive charge and the conserved positively charged amino-acid residues of the DNA-binding surface of AP proteins are partially substituted by negatively charged, polar and hydrophobic residues. Crc protein purified to homogeneity from P. aeruginosa did neither display DNase activity, nor did it bind to previously identified RNA substrates. Rather, the RNA chaperone Hfq was identified as a contaminant in His-tagged Crc preparations purified by one step Ni-affinity chromatography from Escherichia coli, and was shown to account for the RNA binding activity observed with the His-Crc preparations. Taken together, these data challenge a role of Crc as a direct translational repressor in carbon catabolite repression in P. aeruginosa. PMID:23717639

The Pseudomonas aeruginosa catabolite repression control protein Crc is devoid of RNA binding activity.

PubMed

Milojevic, Tetyana; Grishkovskaya, Irina; Sonnleitner, Elisabeth; Djinovic-Carugo, Kristina; Bläsi, Udo

2013-01-01

The Crc protein has been shown to mediate catabolite repression control in Pseudomonas, leading to a preferential assimilation of carbon sources. It has been suggested that Crc acts as a translational repressor of mRNAs, encoding functions involved in uptake and breakdown of different carbon sources. Moreover, the regulatory RNA CrcZ, the level of which is increased in the presence of less preferred carbon sources, was suggested to bind to and sequester Crc, resulting in a relief of catabolite repression. Here, we determined the crystal structure of Pseudomonas aeruginosa Crc, a member of apurinic/apyrimidinic (AP) endonuclease family, at 1.8 Å. Although Crc displays high sequence similarity with its orthologs, there are amino acid alterations in the area corresponding to the active site in AP proteins. Unlike typical AP endonuclease family proteins, Crc has a reduced overall positive charge and the conserved positively charged amino-acid residues of the DNA-binding surface of AP proteins are partially substituted by negatively charged, polar and hydrophobic residues. Crc protein purified to homogeneity from P. aeruginosa did neither display DNase activity, nor did it bind to previously identified RNA substrates. Rather, the RNA chaperone Hfq was identified as a contaminant in His-tagged Crc preparations purified by one step Ni-affinity chromatography from Escherichia coli, and was shown to account for the RNA binding activity observed with the His-Crc preparations. Taken together, these data challenge a role of Crc as a direct translational repressor in carbon catabolite repression in P. aeruginosa.

Template mediated protein self-assembly as a valuable tool in regenerative therapy.

PubMed

Kundu, B; Eltohamy, M; Yadavalli, V K; Reis, R L; Kim, H W

2018-04-11

The assembly of natural proteinaceous biopolymers into macro-scale architectures is of great importance in synthetic biology, soft-material science and regenerative therapy. The self-assembly of protein tends to be limited due to anisotropic interactions among protein molecules, poor solubility and stability. Here, we introduce a unique platform to self-immobilize diverse proteins (fibrous and globular, positively and negatively charged, low and high molecular weight) using silicon surfaces with pendant -NH 2 groups via a facile one step diffusion limited aggregation (DLA) method. All the experimental proteins (type I collagen, bovine serum albumin and cytochrome C) self-assemble into seaweed-like branched dendritic architectures via classical DLA in the absence of any electrolytes. The notable differences in branching architectures are due to dissimilarities in protein colloidal sub-units, which is typical for each protein type, along with the heterogeneous distribution of surface -NH 2 groups. Fractal analysis of assembled structures is used to explain the underlying route of fractal deposition; which concludes how proteins with different functionality can yield similar assembly. Further, the nano-micro-structured surfaces can be used to provide functional topographical cues to study cellular responses, as demonstrated using rat bone marrow stem cells. The results indicate that the immobilization of proteins via DLA does not affect functionality, instead serving as topographical cues to guide cell morphology. This indicates a promising design strategy at the tissue-material interface and is anticipated to guide future surface modifications. A cost-effective standard templating strategy is therefore proposed for fundamental and applied particle aggregation studies, which can be used at multiple length scales for biomaterial design and surface reformation.

Charging of Proteins in Native Mass Spectrometry

DOE PAGES

Susa, Anna C.; Xia, Zijie; Tang, Henry Y. H.; ...

2016-10-12

Factors that influence the charging of protein ions formed by electrospray ionization from aqueous solutions in which proteins have native structures and function were investigated. Protein ions ranging in molecular weight from 12.3 to 79.7 kDa and pI values from 5.4 to 9.6 were formed from different solutions and reacted with volatile bases of gas-phase basicities higher than that of ammonia in the cell of a Fourier-transform ion cyclotron resonance mass spectrometer. The charge-state distribution of cytochrome c ions formed from aqueous ammonium or potassium acetate is the same. Moreover, ions formed from these two solutions do not undergo protonmore » transfer to 2-fluoropyridine, which is 8 kcal/mol more basic than ammonia. These results provide compelling evidence that proton transfer between ammonia and protein ions does not limit protein ion charge in native electrospray ionization. Both circular dichroism and ion mobility measurements indicate that there are differences in conformations of proteins in pure water and aqueous ammonium acetate, and these differences can account for the difference in the extent of charging and proton-transfer reactivities of protein ions formed from these solutions. The extent of proton transfer of the protein ions with higher gas-phase basicity bases trends with how closely the protein ions are charged to the value predicted by the Rayleigh limit for spherical water droplets approximately the same size as the proteins. These results indicate that droplet charge limits protein ion charge in native mass spectrometry and are consistent with these ions being formed by the charged residue mechanism.« less

Charging of Proteins in Native Mass Spectrometry

NASA Astrophysics Data System (ADS)

Susa, Anna C.; Xia, Zijie; Tang, Henry Y. H.; Tainer, John A.; Williams, Evan R.

2017-02-01

Factors that influence the charging of protein ions formed by electrospray ionization from aqueous solutions in which proteins have native structures and function were investigated. Protein ions ranging in molecular weight from 12.3 to 79.7 kDa and pI values from 5.4 to 9.6 were formed from different solutions and reacted with volatile bases of gas-phase basicities higher than that of ammonia in the cell of a Fourier-transform ion cyclotron resonance mass spectrometer. The charge-state distribution of cytochrome c ions formed from aqueous ammonium or potassium acetate is the same. Moreover, ions formed from these two solutions do not undergo proton transfer to 2-fluoropyridine, which is 8 kcal/mol more basic than ammonia. These results provide compelling evidence that proton transfer between ammonia and protein ions does not limit protein ion charge in native electrospray ionization. Both circular dichroism and ion mobility measurements indicate that there are differences in conformations of proteins in pure water and aqueous ammonium acetate, and these differences can account for the difference in the extent of charging and proton-transfer reactivities of protein ions formed from these solutions. The extent of proton transfer of the protein ions with higher gas-phase basicity bases trends with how closely the protein ions are charged to the value predicted by the Rayleigh limit for spherical water droplets approximately the same size as the proteins. These results indicate that droplet charge limits protein ion charge in native mass spectrometry and are consistent with these ions being formed by the charged residue mechanism.

Protein thermal stabilization by charged compatible solutes: Computational studies in rubredoxin from Desulfovibrio gigas.

PubMed

Micaelo, Nuno M; Victor, Bruno L; Soares, Cláudio M

2008-08-01

Molecular dynamics simulation studies of rubredoxin from Desulfovibrio gigas (RDG) were used to characterize the molecular mechanism of thermal stabilization by the compatible solute (CS) diglycerol-phospate (DGP). DGP is a negatively charged CS that accumulates under salt stress in Archaeoglobus fulgidus. Experimental results show that a 100 mM DGP solution exerts a strong protection effect in the half-life of RDG at 363 K (Lamosa et al., Appl Environ Microbiol 2000;66:1974-1979). RDG was simulated in four aqueous solutions at 300 and 363 K: pure aqueous media, 100 mM DGP, 100 mM NaCl, and 500 mM DGP. Our work shows that the 100 mM DGP solution is able to maintain the average protein structure when the temperature is increased, preventing the occurrence of large-scale deviation of a mobile loop involved in the first steps of RDG unfolding. The molecular mechanism of thermal denaturation protection by DGP seems to involve the direct interaction between the protein and the CS by hydrogen bond interactions near the mobile loop. Several clusters of DGP molecules are formed and preferentially localized at neutral electrostatic regions of the surface. The increase of DGP concentration to 500 mM did not yield better stabilization of the protein suggesting that the thermal protective role of this charged CS is achieved at low concentrations, as shown experimentally. (c) 2008 Wiley-Liss, Inc.

Collagen peptide-based biomaterials for protein delivery and peptide-promoted self-assembly of gold nanoparticles

NASA Astrophysics Data System (ADS)

Ernenwein, Dawn M.

2011-12-01

Bottom-up self-assembly of peptides has driven the research progress for the following two projects: protein delivery vehicles of collagen microflorettes and the assembly of gold nanoparticles with coiled-coil peptides. Collagen is the most abundant protein in the mammals yet due to immunogenic responses, batch-to-batch variability and lack of sequence modifications, synthetic collagen has been designed to self-assemble into native collagen-like structures. In particular with this research, metal binding ligands were incorporated on the termini of collagen-like peptides to generate micron-sized particles, microflorettes. The over-arching goal of the first research project is to engineer MRI-active microflorettes, loaded with His-tagged growth factors with differential release rates while bound to stem cells that can be implemented toward regenerative cell-based therapies. His-tagged proteins, such as green fluorescent protein, have successfully been incorporated on the surface and throughout the microflorettes. Protein release was monitored under physiological conditions and was related to particle degradation. In human plasma full release was obtained within six days. Stability of the microflorettes under physiological conditions was also examined for the development of a therapeutically relevant delivery agent. Additionally, MRI active microflorettes have been generated through the incorporation of a gadolinium binding ligand, DOTA within the collagen-based peptide sequence. To probe peptide-promoted self-assemblies of gold nanoparticles (GNPs) by non-covalent, charge complementary interactions, a highly anionic coiled-coil peptide was designed and synthesized. Upon formation of peptide-GNP interactions, the hydrophobic domain of the coiled-coil were shown to promote the self-assembly of peptide-GNPs clustering. Hydrophobic forces were found to play an important role in the assembly process, as a peptide with an equally overall negative charge, but lacking an ordered hydrophobic face had no effect on GNP assembly. The self-assembly system herein is advantageous due to its reversible nature upon addition of high salt concentrations which masks the surface charge. There is great potential for using this uniquely designed self-assembled peptide-gold nanoparticle system for exploring the interplay between peptide ligation and GNP self-assembly.

Structure-function analysis of the auxilin J-domain reveals an extended Hsc70 interaction interface.

PubMed

Jiang, Jianwen; Taylor, Alexander B; Prasad, Kondury; Ishikawa-Brush, Yumiko; Hart, P John; Lafer, Eileen M; Sousa, Rui

2003-05-20

J-domains are widespread protein interaction modules involved in recruiting and stimulating the activity of Hsp70 family chaperones. We have determined the crystal structure of the J-domain of auxilin, a protein which is involved in uncoating clathrin-coated vesicles. Comparison to the known structures of J-domains from four other proteins reveals that the auxilin J-domain is the most divergent of all J-domain structures described to date. In addition to the canonical J-domain features described previously, the auxilin J-domain contains an extra N-terminal helix and a long loop inserted between helices I and II. The latter loop extends the positively charged surface which forms the Hsc70 binding site, and is shown by directed mutagenesis and surface plasmon resonance to contain side chains important for binding to Hsc70.

Interplay of polyelectrolytes with different adsorbing surfaces

NASA Astrophysics Data System (ADS)

Xie, Feng

We study the adsorption of polyelectrolytes from solution onto different adsorbing surfaces, focusing on the electrostatic interactions. Measurements of the surface excess, fractional ionization of chargeable groups, segmental orientation, and adsorption kinetics were made using Fourier transform infrared spectroscopy in the mode of attenuated total reflection. Different adsorbing surfaces, from single solid surfaces, solid surfaces modified with adsorbed polymer layer, to fluid-like surfaces-biomembranes were adopted. Both atomic force microscopy (AFM) and fluorescent techniques were employed to investigate the fluid-like surfaces in the absence and in the presence of polyelectrolytes. The work focuses on three primary issues: (i) the charge regulation of weak polyelectrolytes on both homogeneous and heterogeneous surfaces, (ii) the dynamics of adsorption when the surface possesses reciprocal mobility, i.e., biomembrane surface, and (iii) the structural and dynamical properties of the fluid-like surfaces interacting with polyelectrolytes. We find that the ionization of chargeable groups in weak polyelectrolytes is controlled by the charge balance between the adsorbates and the surfaces. A new interpretation of ionization in the adsorbed layer provides a new insight into the fundamental problem of whether ions of opposite charge associate or remain separate. Bjerrum length is found to be a criterion for the onset of surface ionization suppression, which helps to predict and control the conformation transition of proteins. In addition to the effect of different surfaces on the adsorption behavior of polyelectrolytes, we also focused on the response of the surfaces to the adsorbates. Chains that encountered sparsely-covered surfaces spread to maximize the number of segment-surface contacts at rates independent of the molar mass. Surface reconstruction rather than molar mass of the adsorbing molecules appeared to determine the rate of spreading. This contrasts starkly with traditional polymer adsorption onto surfaces whose structure is "frozen" and unresponsive. Finally, preliminary studies on dynamical properties of biomembrane surfaces interacting with polyelectrolytes are presented, using fluorescence correlation spectroscopy (FCS). The significance is to characterize domains induced by polyelectrolyte binding.

Coarse-grained model for colloidal protein interactions, B(22), and protein cluster formation.

PubMed

Blanco, Marco A; Sahin, Erinc; Robinson, Anne S; Roberts, Christopher J

2013-12-19

Reversible protein cluster formation is an important initial step in the processes of native and non-native protein aggregation, but involves relatively long time and length scales for detailed atomistic simulations and extensive mapping of free energy landscapes. A coarse-grained (CG) model is presented to semiquantitatively characterize the thermodynamics and key configurations involved in the landscape for protein oligomerization, as well as experimental measures of interactions such as the osmotic second virial coefficient (B22). Based on earlier work (Grüenberger et al., J. Phys. Chem. B 2013, 117, 763), this CG model treats proteins as rigid bodies composed of one bead per amino acid, with each amino acid having specific parameters for its size, hydrophobicity, and charge. The net interactions are a combination of steric repulsions, short-range attractions, and screened long-range charge-charge interactions. Model parametrization was done by fitting simulation results against experimental value of B22 as a function of solution ionic strength for α-chymotrypsinogen A and γD-Crystallin (gD-Crys). The CG model is applied to characterize the pairwise interactions and dimerization of gD-Crys and the dependence on temperature, protein concentration, and ionic strength. The results illustrate that at experimentally relevant conditions where stable dimers do not form, the entropic contributions are predominant in the free-energy of protein cluster formation and colloidal protein interactions, arguing against interpretations that treat B22 primarily from energetic considerations alone. Additionally, the results suggest that electrostatic interactions help to modulate the population of the different stable configurations for protein nearest-neighbor pairs, while short-range attractions determine the relative orientations of proteins within these configurations. Finally, simulation results are combined with Principal Component Analysis to identify those amino-acids/surface patches that form interprotein contacts at conditions that favor dimerization of gD-Crys. The resulting regions agree with previously found aggregation-prone sites, as well as suggesting new ones that may be important.

Stabilization of model beverage cloud emulsions using protein-polysaccharide electrostatic complexes formed at the oil-water interface.

PubMed

Harnsilawat, Thepkunya; Pongsawatmanit, Rungnaphar; McClements, David J

2006-07-26

The potential of utilizing interfacial complexes, formed through the electrostatic interactions of proteins and polysaccharides at oil-water interfaces, to stabilize model beverage cloud emulsions has been examined. These interfacial complexes were formed by mixing charged polysaccharides with oil-in-water emulsions containing oppositely charged protein-coated oil droplets. Model beverage emulsions were prepared that consisted of 0.1 wt % corn oil droplets coated by beta-lactoglobulin (beta-Lg), beta-Lg/alginate, beta-Lg/iota-carrageenan, or beta-Lg/gum arabic interfacial layers (pH 3 or 4). Stable emulsions were formed when the polysaccharide concentration was sufficient to saturate the protein-coated droplets. The emulsions were subjected to variations in pH (from 3 to 7), ionic strength (from 0 to 250 mM NaCl), and thermal processing (from 30 or 90 degrees C), and the influence on their stability was determined. The emulsions containing alginate and carrageenan had the best stability to ionic strength and thermal processing. This study shows that the controlled formation of protein-polysaccharide complexes at droplet surfaces may be used to produce stable beverage emulsions, which may have important implications for industrial applications.

Biogenic and Synthetic Peptides with Oppositely Charged Amino Acids as Binding Sites for Mineralization.

PubMed

Lemloh, Marie-Louise; Altintoprak, Klara; Wege, Christina; Weiss, Ingrid M; Rothenstein, Dirk

2017-01-28

Proteins regulate diverse biological processes by the specific interaction with, e.g., nucleic acids, proteins and inorganic molecules. The generation of inorganic hybrid materials, such as shell formation in mollusks, is a protein-controlled mineralization process. Moreover, inorganic-binding peptides are attractive for the bioinspired mineralization of non-natural inorganic functional materials for technical applications. However, it is still challenging to identify mineral-binding peptide motifs from biological systems as well as for technical systems. Here, three complementary approaches were combined to analyze protein motifs consisting of alternating positively and negatively charged amino acids: (i) the screening of natural biomineralization proteins; (ii) the selection of inorganic-binding peptides derived from phage display; and (iii) the mineralization of tobacco mosaic virus (TMV)-based templates. A respective peptide motif displayed on the TMV surface had a major impact on the SiO₂ mineralization. In addition, similar motifs were found in zinc oxide- and zirconia-binding peptides indicating a general binding feature. The comparative analysis presented here raises new questions regarding whether or not there is a common design principle based on acidic and basic amino acids for peptides interacting with minerals.

The effect of geometrical presentation of multimodal cation-exchange ligands on selective recognition of hydrophobic regions on protein surfaces.

PubMed

Woo, James; Parimal, Siddharth; Brown, Matthew R; Heden, Ryan; Cramer, Steven M

2015-09-18

The effects of spatial organization of hydrophobic and charged moieties on multimodal (MM) cation-exchange ligands were examined by studying protein retention behavior on two commercial chromatographic media, Capto™ MMC and Nuvia™ cPrime™. Proteins with extended regions of surface-exposed aliphatic residues were found to have enhanced retention on the Capto MMC system as compared to the Nuvia cPrime resin. The results further indicated that while the Nuvia cPrime ligand had a strong preference for interactions with aromatic groups, the Capto MMC ligand appeared to interact with both aliphatic and aromatic clusters on the protein surfaces. These observations were formalized into a new set of protein surface property descriptors, which quantified the local distribution of electrostatic and hydrophobic potentials as well as distinguishing between aromatic and aliphatic properties. Using these descriptors, high-performing quantitative structure-activity relationship (QSAR) models (R(2)>0.88) were generated for both the Capto MMC and Nuvia cPrime datasets at pH 5 and pH 6. Descriptors of electrostatic properties were generally common across the four models; however both Capto MMC models included descriptors that quantified regions of aliphatic-based hydrophobicity in addition to aromatic descriptors. Retention was generally reduced by lowering the ligand densities on both MM resins. Notably, elution order was largely unaffected by the change in surface density, but smaller and more aliphatic proteins tended to be more affected by this drop in ligand density. This suggests that modulating the exposure, shape and density of the hydrophobic moieties in multimodal chromatographic systems can alter the preference for surface exposed aliphatic or aromatic residues, thus providing an additional dimension for modulating the selectivity of MM protein separation systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterizing hydrophobicity of amino acid side chains in a protein environment via measuring contact angle of a water nanodroplet on planar peptide network

PubMed Central

Zhu, Chongqin; Gao, Yurui; Li, Hui; Meng, Sheng; Li, Lei; Francisco, Joseph S.; Zeng, Xiao Cheng

2016-01-01

Hydrophobicity of macroscopic planar surface is conventionally characterized by the contact angle of water droplets. However, this engineering measurement cannot be directly extended to surfaces of proteins, due to the nanometer scale of amino acids and inherent nonplanar structures. To measure the hydrophobicity of side chains of proteins quantitatively, numerous parameters were developed to characterize behavior of hydrophobic solvation. However, consistency among these parameters is not always apparent. Herein, we demonstrate an alternative way of characterizing hydrophobicity of amino acid side chains in a protein environment by constructing a monolayer of amino acids (i.e., artificial planar peptide network) according to the primary and the β-sheet secondary structures of protein so that the conventional engineering measurement of the contact angle of a water droplet can be brought to bear. Using molecular dynamics simulations, contact angles θ of a water nanodroplet on the planar peptide network, together with excess chemical potentials of purely repulsive methane-sized Weeks−Chandler−Andersen solute, are computed. All of the 20 types of amino acids and the corresponding planar peptide networks are studied. Expectedly, all of the planar peptide networks with nonpolar amino acids are hydrophobic due to θ > 90°, whereas all of the planar peptide networks of the polar and charged amino acids are hydrophilic due to θ < 90°. Planar peptide networks of the charged amino acids exhibit complete-wetting behavior due to θ = 0°. This computational approach for characterization of hydrophobicity can be extended to artificial planar networks of other soft matter. PMID:27803319

Impact of polymer surface characteristics on the microrheological measurement quality of protein solutions - A tracer particle screening.

PubMed

Bauer, Katharina Christin; Schermeyer, Marie-Therese; Seidel, Jonathan; Hubbuch, Jürgen

2016-05-30

Microrheological measurements prove to be suitable to identify rheological parameters of biopharmaceutical solutions. These give information about the flow characteristics but also about the interactions and network structures in protein solutions. For the microrheological measurement tracer particles are required. Due to their specific surface characteristic not all are suitable for reliable measurement results in biopharmaceutical systems. In the present work a screening of melamine, PMMA, polystyrene and surface modified polystyrene as tracer particles were investigated at various protein solution conditions. The surface characteristics of the screened tracer particles were evaluated by zeta potential measurements. Furthermore each tracer particle was used to determine the dynamic viscosity of lysozyme solutions by microrheology and compared to a standard. The results indicate that the selection of the tracer particle had a strong impact on the quality of the microrheological measurement dependent on pH and additive type. Surface modified polystyrene was the only tracer particle that yielded good microrheological results for all tested conditions. The study indicated that the electrostatic surface charge of the tracer particle had a minor impact than its hydrophobicity. This characteristic was the crucial surface property that needs to be considered for the selection of a suitable tracer particle to achieve high measurement accuracy. Copyright © 2016 Elsevier B.V. All rights reserved.

Surface charge accumulation of solid insulator under nanosecond pulse in vacuum: 3D distribution features and mechanism

NASA Astrophysics Data System (ADS)

Qi, Bo; Gao, Chunjia; Sun, Zelai; Li, Chengrong

2017-11-01

Surface charge accumulation can incur changes in electric field distribution, involved in the electron propagation process, and result in a significant decrease in the surface flashover voltage. The existing 2D surface charge measurement fails to meet the actual needs in real engineering applications that usually adopt the 45° conical frustum insulators. The present research developed a novel 3D measurement platform to capture surface charge distribution on solid insulation under nanosecond pulse in a vacuum. The results indicate that all surface charges are positive under a positive pulse and negative under a negative pulse. Surface charges tend to accumulate more near the upper electrode. Surface charge density increases significantly with the increase in pulse counts and amplitudes. Accumulation of surface charge results in a certain decrease of flashover voltage. Taking consideration of the secondary electron emission for the surface charge accumulation, four materials were obtained to demonstrate the effects on surface charge. Combining the effect incurred by secondary electron emission and the weighty action taken by surface charge accumulation on the flashover phenomena, the discharge mechanism along the insulator surface under nanosecond pulse voltage was proposed.

Control of microtubule trajectory within an electric field by altering surface charge density

PubMed Central

Isozaki, Naoto; Ando, Suguru; Nakahara, Tasuku; Shintaku, Hirofumi; Kotera, Hidetoshi; Meyhöfer, Edgar; Yokokawa, Ryuji

2015-01-01

One of challenges for using microtubules (MTs) driven by kinesin motors in microfluidic environments is to control their direction of movement. Although applying physical biases to rectify MTs is prevalent, it has not been established as a design methodology in conjunction with microfluidic devices. In the future, the methodology is expected to achieve functional motor-driven nanosystems. Here, we propose a method to guide kinesin-propelled MTs in multiple directions under an electric field by designing a charged surface of MT minus ends labeled with dsDNA via a streptavidin-biotin interaction. MTs labeled with 20-bp or 50-bp dsDNA molecules showed significantly different trajectories according to the DNA length, which were in good agreement with values predicted from electrophoretic mobilities measured for their minus ends. Since the effective charge of labeled DNA molecules was equal to that of freely dispersed DNA molecules in a buffer solution, MT trajectory could be estimated by selecting labeling molecules with known charges. Moreover, the estimated trajectory enables to define geometrical sizes of a microfluidic device. This rational molecular design and prediction methodology allows MTs to be guided in multiple directions, demonstrating the feasibility of using molecular sorters driven by motor proteins. PMID:25567007

Control of microtubule trajectory within an electric field by altering surface charge density.

PubMed

Isozaki, Naoto; Ando, Suguru; Nakahara, Tasuku; Shintaku, Hirofumi; Kotera, Hidetoshi; Meyhöfer, Edgar; Yokokawa, Ryuji

2015-01-08

One of challenges for using microtubules (MTs) driven by kinesin motors in microfluidic environments is to control their direction of movement. Although applying physical biases to rectify MTs is prevalent, it has not been established as a design methodology in conjunction with microfluidic devices. In the future, the methodology is expected to achieve functional motor-driven nanosystems. Here, we propose a method to guide kinesin-propelled MTs in multiple directions under an electric field by designing a charged surface of MT minus ends labeled with dsDNA via a streptavidin-biotin interaction. MTs labeled with 20-bp or 50-bp dsDNA molecules showed significantly different trajectories according to the DNA length, which were in good agreement with values predicted from electrophoretic mobilities measured for their minus ends. Since the effective charge of labeled DNA molecules was equal to that of freely dispersed DNA molecules in a buffer solution, MT trajectory could be estimated by selecting labeling molecules with known charges. Moreover, the estimated trajectory enables to define geometrical sizes of a microfluidic device. This rational molecular design and prediction methodology allows MTs to be guided in multiple directions, demonstrating the feasibility of using molecular sorters driven by motor proteins.

Surfactant Functionalization Induces Robust, Differential Adhesion of Tumor Cells and Blood Cells to Charged Nanotube-Coated Biomaterials Under Flow

PubMed Central

Mitchell, Michael J.; Castellanos, Carlos A.; King, Michael R.

2015-01-01

The metastatic spread of cancer cells from the primary tumor to distant sites leads to a poor prognosis in cancers originating from multiple organs. Increasing evidence has linked selectin-based adhesion between circulating tumor cells (CTCs) and endothelial cells of the microvasculature to metastatic dissemination, in a manner similar to leukocyte adhesion during inflammation. Functionalized biomaterial surfaces hold promise as a diagnostic tool to separate CTCs and potentially treat metastasis, utilizing antibody and selectin-mediated interactions for cell capture under flow. However, capture at high purity levels is challenged by the fact that CTCs and leukocytes both possess selectin ligands. Here, a straightforward technique to functionalize and alter the charge of naturally occurring halloysite nanotubes using surfactants is reported to induce robust, differential adhesion of tumor cells and blood cells to nanotube-coated surfaces under flow. Negatively charged sodium dodecanoate-functionalized nanotubes simultaneously enhanced tumor cell capture while negating leukocyte adhesion, both in the presence and absence of adhesion proteins, and can be utilized to isolate circulating tumor cells regardless of biomarker expression. Conversely, diminishing nanotube charge via functionalization with decyltrimethylammonium bromide both abolished tumor cell capture while promoting leukocyte adhesion. PMID:25934290

Nanoparticles-cell association predicted by protein corona fingerprints

NASA Astrophysics Data System (ADS)

Palchetti, S.; Digiacomo, L.; Pozzi, D.; Peruzzi, G.; Micarelli, E.; Mahmoudi, M.; Caracciolo, G.

2016-06-01

In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells. Electronic supplementary information (ESI) available: Table S1. Cell viability (%) and cell association of the different nanoparticles used. Table S2. Total number of identified proteins on the different nanoparticles used. Tables S3-S18. Top 25 most abundant corona proteins identified in the protein corona of nanoparticles NP2-NP16 following 1 hour incubation with HP. Table S19. List of descriptors used. Table S20. Potential targets of protein corona fingerprints with its own interaction score (mentha) and the expression median value in Hela cells. Fig. S1 and S2. Effect of exposure to human plasma on size and zeta potential of NPs. Fig. S3. Predictive modeling of nanoparticle-cell association. See DOI: 10.1039/c6nr03898k

The role of surface charge in the desolvation process of gelatin: implications in nanoparticle synthesis and modulation of drug release

PubMed Central

Ahsan, Saad M; Rao, Chintalagiri Mohan

2017-01-01

The process of moving hydrophobic amino acids into the core of a protein by desolvation is important in protein folding. However, a rapid and forced desolvation can lead to precipitation of proteins. Desolvation of proteins under controlled conditions generates nanoparticles – homogeneous aggregates with a narrow size distribution. The protein nanoparticles, under physiological conditions, undergo surface erosion due to the action of proteases, releasing the entrapped drug/gene. The packing density of protein nanoparticles significantly influences the release kinetics. We have investigated the desolvation process of gelatin, exploring the role of pH and desolvating agent in nanoparticle synthesis. Our results show that the desolvation process, initiated by the addition of acetone, follows distinct pathways for gelatin incubated at different pH values and results in the generation of nanoparticles with varying matrix densities. The nanoparticles synthesized with varying matrix densities show variations in drug loading and protease-dependent extra- and intracellular drug release. These results will be useful in fine-tuning the synthesis of nanoparticles with desirable drug release profiles. PMID:28182126

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

PubMed Central

Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

2016-01-01

Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

PubMed

Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

2015-07-01

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

Free energy calculations of glycosaminoglycan-protein interactions.

PubMed

Gandhi, Neha S; Mancera, Ricardo L

2009-10-01

Glycosaminoglycans (GAGs) are complex highly charged linear polysaccharides that have a variety of roles in biological processes. We report the first use of molecular dynamics (MD) free energy calculations using the MM/PBSA method to investigate the binding of GAGs to protein molecules, namely the platelet endothelial cell adhesion molecule 1 (PECAM-1) and annexin A2. Calculations of the free energy of the binding of heparin fragments of different sizes reveal the existence of a region of low GAG-binding affinity in domains 5-6 of PECAM-1 and a region of high affinity in domains 2-3, consistent with experimental data and ligand-protein docking studies. A conformational hinge movement between domains 2 and 3 was observed, which allows the binding of heparin fragments of increasing size (pentasaccharides to octasaccharides) with an increasingly higher binding affinity. Similar simulations of the binding of a heparin fragment to annexin A2 reveal the optimization of electrostatic and hydrogen bonding interactions with the protein and protein-bound calcium ions. In general, these free energy calculations reveal that the binding of heparin to protein surfaces is dominated by strong electrostatic interactions for longer fragments, with equally important contributions from van der Waals interactions and vibrational entropy changes, against a large unfavorable desolvation penalty due to the high charge density of these molecules.

Molecular insight into the electrostatic membrane surface potential by 14n/31p MAS NMR spectroscopy: nociceptin-lipid association.

PubMed

Lindström, Fredrick; Williamson, Philip T F; Gröbner, Gerhard

2005-05-11

Exploiting naturally abundant (14)N and (31)P nuclei by high-resolution MAS NMR (magic angle spinning nuclear magnetic resonance) provides a molecular view of the electrostatic potential present at the surface of biological model membranes, the electrostatic charge distribution across the membrane interface, and changes that occur upon peptide association. The spectral resolution in (31)P and (14)N MAS NMR spectra is sufficient to probe directly the negatively charged phosphate and positively charged choline segment of the electrostatic P(-)-O-CH(2)-CH(2)-N(+)(CH(3))(3) headgroup dipole of zwitterionic DMPC (dimyristoylphosphatidylcholine) in mixed-lipid systems. The isotropic shifts report on the size of the potential existing at the phosphate and ammonium group within the lipid headgroup while the chemical shielding anisotropy ((31)P) and anisotropic quadrupolar interaction ((14)N) characterize changes in headgroup orientation in response to surface potential. The (31)P/(14)N isotropic chemical shifts for DMPC show opposing systematic changes in response to changing membrane potential, reflecting the size of the electrostatic potential at opposing ends of the P(-)-N(+) dipole. The orientational response of the DMPC lipid headgroup to electrostatic surface variations is visible in the anisotropic features of (14)N and (31)P NMR spectra. These features are analyzed in terms of a modified "molecular voltmeter" model, with changes in dynamic averaging reflecting the tilt of the C(beta)-N(+)(CH)(3) choline and PO(4)(-) segment. These properties have been exploited to characterize the changes in surface potential upon the binding of nociceptin to negatively charged membranes, a process assumed to proceed its agonistic binding to its opoid G-protein coupled receptor.

Material Science Smart Coatings

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rubinstein, A. I.; Sabirianov, R. F.; Namavar, Fereydoon

2014-07-01

The contribution of electrostatic interactions to the free energy of binding between model protein and a ceramic implant surface in the aqueous solvent, considered in the framework of the nonlocal electrostatic model, is calculated as a function of the implant low-frequency dielectric constant. We show that the existence of a dynamically ordered (low-dielectric) interfacial solvent layer at the protein-solvent and ceramic-solvent interface markedly increases charging energy of the protein and ceramic implant, and consequently makes the electrostatic contribution to the protein-ceramic binding energy more favorable (attractive). Our analysis shows that the corresponding electrostatic energy between protein and oxide ceramics dependsmore » nonmonotonically on the dielectric constant of ceramic, ε C. Obtained results indicate that protein can attract electrostatically to the surface if ceramic material has a moderate ε C below or about 35 (in particularly ZrO 2 or Ta 2O 5). This is in contrast to classical (local) consideration of the solvent, which demonstrates an unfavorable electrostatic interaction of protein with typical metal oxide ceramic materials (ε C>10). Thus, a solid implant coated by combining oxide ceramic with a reduced dielectric constant can be beneficial to strengthen the electrostatic binding of the protein-implant complex.« less

Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer

PubMed Central

Guinn, Emily J.; Pegram, Laurel M.; Capp, Michael W.; Pollock, Michelle N.; Record, M. Thomas

2011-01-01

To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients Kp for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. Kp values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH—amide O and amide NH—amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or Kp values. PMID:21930943

Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer.

PubMed

Guinn, Emily J; Pegram, Laurel M; Capp, Michael W; Pollock, Michelle N; Record, M Thomas

2011-10-11

To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients K(p) for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. K(p) values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH--amide O and amide NH--amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or K(p) values.

Templating Biomineralization: Surface Directed Protein Self-assembly and External Magnetic Field Stimulation of Osteoblasts

NASA Astrophysics Data System (ADS)

Ba, Xiaolan

Biomineralization is a wide-spread phenomenon in the biological systems, which is the process of mineral formation by organisms through interaction between its organic contents and the inorganic minerals. The process is essential in a broad spectrum of biological phenomena ranging from bone and tooth formation to pathological mineralization under hypoxic conditions or cancerous formations. In this thesis I studied biomineralization at the earliest stages in order to obtain a better understanding of the fundamental principals involved. This knowledge is essential if we want to engineer devices which will increase bone regeneration or prevent unwanted mineral deposits. Extracellular matrix (ECM) proteins play an essential role during biomineralization in bone and engineered tissues. In this dissertation, I present an approach to mimic the ECM in vitro to probe the interactions of these proteins with calcium phosphate mineral and with each other. Early stage of mineralization is investigated by mechanical properties of the protein fibers using Scanning Probe Microscopy (SPM) and Shear Modulation Force Microscopy (SMFM). The development of mineral crystals on the protein matrices is also characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Grazing Incidence X-ray Diffraction (GIXRD). The results demonstrate complementary actions of the two ECM proteins to collect cations and template calcium phosphate mineral, respectively. Magnets have been clinically used as an "induction source" in various bone or orthodontic treatments. However, the mechanism and effects of magnetic fields remain unclear. In this dissertation, I also undertake the present investigation to study the effects of 150 mT static magnetic fields (SMF) on ECM development and cell biomineralization using MC3T3-E1 osteobalst-like cells. Early stage of biomineralization is characterized by SPM, SMFM and confocal laser scanning microscopy (CSLM). Late stage of biomineralization is investigated by SEM, GIXRD and energy dispersive X-ray spectroscopy (EDXS). Gene expression during the exposure of SMF is also studies by RT-PCR. The results indicated that exposure to SMF induces osteoblasts to produce larger quantities of HA, with higher degree of crystalline order. The controlling and understanding of protein on the surface is of great interest in biomedical application such as implant medicine, biosensor design, food processing, and chromatographic separations. The adsorbed protein onto the surface significantly determines the performance of biomaterials in a biological environment. Recent studies have suggested that the preservation of the native secondary structure of protein adsorbed is essential for biological application. In order to manipulate protein adsorption and design biocompatible materials, the mechanisms underlying protein-surface interactions, especially how surface properties of materials induce conformational changes of adsorbed proteins, needs to be well understood. Here we demonstrated that even though SPS is a necessary condition, it is not sufficient. We show that low substrate conductivity as well as proper salt concentration are also critical in sustained protein adsorption continuously. These factors allow one to pattern regions of different conducting properties and for the first time patterns physiologically relevant protein structures. Here we show that we can achieve patterned biomineralized regimes, both with plasma proteins in a simple and robust manner without additional functionalization or application of electrochemical gradients. Since the data indicate that the patterns just need to differ in electrical conductivity, rather than surface chemistry, we propose that the creation of transient image charges, due to incomplete charge screening, may be responsible for sustain the driving force for continual protein absorption.

The Influence of Fluorocarbon and Hydrocarbon Acyl Groups at the Surface of Bovine Carbonic Anhydrase II on the Kinetics of Denaturation by Sodium Dodecyl Sulfate

PubMed Central

Lee, Andrew; Mirica, Katherine A.; Whitesides, George M.

2011-01-01

This paper examines the influence of acylation of the Lys-ε-NH3+ groups of bovine carbonic anhydrase (BCA, E.C. 4.2.1.1) to Lys-ε-NHCOR (R = -CH3, -CH2CH3, and -CH(CH3)2, -CF3) on the rate of denaturation of this protein in buffer containing sodium dodecyl sulfate (SDS). Analysis of the rates suggested separate effects due to electrostatic charge and hydrophobic interactions. Rates of denaturation (kAc,n) of each series of acylated derivatives depended on the number of acylations (n). Plots of log kAc,n vs. n followed U-shaped curves. Within each series of derivatives, rates of denaturation decreased as n increased to ~7; this decrease was compatible with increasingly unfavorable electrostatic interactions between SDS and protein. In this range of n, rates of denaturation also depended on the choice of the acyl group as n increased to ~7, in a manner compatible with favorable hydrophobic interactions between SDS and the -NHCOR groups. As n increased in the range 7 < n < 14 however, rates of denaturation stayed approximately constant; analysis suggested these rates were compatible with an increasingly important contribution to denaturation that depended both on the net negative charge of the protein and on the hydrophobicity of the R group. The mechanism of denaturation thus seems to change with the extent of acylation of the protein. For derivatives with the same net electrostatic charge, rates of denaturation increased with the acyl group (by a factor of ~3 for n ~ 14) in the order CH3CONH- < CH3CH2CONH- < (CH3)2CHCONH- < CF3CONH-. These results suggested that the hydrophobicity of CF3CONH- is slightly greater (by a factor of < 2) than that of RHCONH- similar in surface area. PMID:21182314

Influence of fluorocarbon and hydrocarbon acyl groups at the surface of bovine carbonic anhydrase II on the kinetics of denaturation by sodium dodecyl sulfate.

PubMed

Lee, Andrew; Mirica, Katherine A; Whitesides, George M

2011-02-10

This paper examines the influence of acylation of the Lys-ε-NH(3)(+) groups of bovine carbonic anhydrase (BCA, EC 4.2.1.1) to Lys-ε-NHCOR (R = -CH(3), -CH(2)CH(3), and -CH(CH(3))(2), -CF(3)) on the rate of denaturation of this protein in buffer containing sodium dodecyl sulfate (SDS). Analysis of the rates suggested separate effects due to electrostatic charge and hydrophobic interactions. Rates of denaturation (k(Ac,n)) of each series of acylated derivatives depended on the number of acylations (n). Plots of log k(Ac,n) vs n followed U-shaped curves. Within each series of derivatives, rates of denaturation decreased as n increased to ∼7; this decrease was compatible with increasingly unfavorable electrostatic interactions between SDS and protein. In this range of n, rates of denaturation also depended on the choice of the acyl group as n increased to ∼7, in a manner compatible with favorable hydrophobic interactions between SDS and the -NHCOR groups. As n increased in the range 7 < n < 14, however, rates of denaturation stayed approximately constant; analysis suggested that these rates were compatible with an increasingly important contribution to denaturation that depended both on the net negative charge of the protein and on the hydrophobicity of the R group. The mechanism of denaturation thus seems to change with the extent of acylation of the protein. For derivatives with the same net electrostatic charge, rates of denaturation increased with the acyl group (by a factor of ∼3 for n ∼ 14) in the order CH(3)CONH- < CH(3)CH(2)CONH- < (CH(3))(2)CHCONH- < CF(3)CONH-. These results suggested that the hydrophobicity of CF(3)CONH- is slightly greater (by a factor of <2) than that of RHCONH- with similar surface area.

Quantification of surface charge density and its effect on boundary slip.

PubMed

Jing, Dalei; Bhushan, Bharat

2013-06-11

Reduction of fluid drag is important in the micro-/nanofluidic systems. Surface charge and boundary slip can affect the fluid drag, and surface charge is also believed to affect boundary slip. The quantification of surface charge and boundary slip at a solid-liquid interface has been widely studied, but there is a lack of understanding of the effect of surface charge on boundary slip. In this paper, the surface charge density of borosilicate glass and octadecyltrichlorosilane (OTS) surfaces immersed in saline solutions with two ionic concentrations and deionized (DI) water with different pH values and electric field values is quantified by fitting experimental atomic force microscopy (AFM) electrostatic force data using a theoretical model relating the surface charge density and electrostatic force. Results show that pH and electric field can affect the surface charge density of glass and OTS surfaces immersed in saline solutions and DI water. The mechanisms of the effect of pH and electric field on the surface charge density are discussed. The slip length of the OTS surface immersed in saline solutions with two ionic concentrations and DI water with different pH values and electric field values is measured, and their effects on the slip length are analyzed from the point of surface charge. Results show that a larger absolute value of surface charge density leads to a smaller slip length for the OTS surface.

Ligand solvation in molecular docking.

PubMed

Shoichet, B K; Leach, A R; Kuntz, I D

1999-01-01

Solvation plays an important role in ligand-protein association and has a strong impact on comparisons of binding energies for dissimilar molecules. When databases of such molecules are screened for complementarity to receptors of known structure, as often occurs in structure-based inhibitor discovery, failure to consider ligand solvation often leads to putative ligands that are too highly charged or too large. To correct for the different charge states and sizes of the ligands, we calculated electrostatic and non-polar solvation free energies for molecules in a widely used molecular database, the Available Chemicals Directory (ACD). A modified Born equation treatment was used to calculate the electrostatic component of ligand solvation. The non-polar component of ligand solvation was calculated based on the surface area of the ligand and parameters derived from the hydration energies of apolar ligands. These solvation energies were subtracted from the ligand-receptor interaction energies. We tested the usefulness of these corrections by screening the ACD for molecules that complemented three proteins of known structure, using a molecular docking program. Correcting for ligand solvation improved the rankings of known ligands and discriminated against molecules with inappropriate charge states and sizes.

Electrostatic roles in electron transfer from [NiFe] hydrogenase to cytochrome c3 from Desulfovibrio vulgaris Miyazaki F.

PubMed

Sugimoto, Yu; Kitazumi, Yuki; Shirai, Osamu; Nishikawa, Koji; Higuchi, Yoshiki; Yamamoto, Masahiro; Kano, Kenji

2017-05-01

Electrostatic interactions between proteins are key factors that govern the association and reaction rate. We spectroscopically determine the second-order reaction rate constant (k) of electron transfer from [NiFe] hydrogenase (H 2 ase) to cytochrome (cyt) c 3 at various ionic strengths (I). The k value decreases with I. To analyze the results, we develop a semi-analytical formula for I dependence of k based on the assumptions that molecules are spherical and the reaction proceeds via a transition state. Fitting of the formula to the experimental data reveals that the interaction occurs in limited regions with opposite charges and with radii much smaller than those estimated from crystal structures. This suggests that local charges in H 2 ase and cyt c 3 play important roles in the reaction. Although the crystallographic data indicate a positive electrostatic potential over almost the entire surface of the proteins, there exists a small region with negative potential on H 2 ase at which the electron transfer from H 2 ase to cyt c 3 may occur. This local negative potential region is identical to the hypothetical interaction sphere predicted by the analysis. Furthermore, I dependence of k is predicted by the Adaptive Poisson-Boltzmann Solver considering all charges of the amino acids in the proteins and the configuration of H 2 ase/cyt c 3 complex. The calculation reproduces the experimental results except at extremely low I. These results indicate that the stabilization derived from the local electrostatic interaction in the H 2 ase/cyt c 3 complex overcomes the destabilization derived from the electrostatic repulsion of the overall positive charge of both proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrostatic complementarity between proteins and ligands. 1. Charge disposition, dielectric and interface effects

NASA Astrophysics Data System (ADS)

Chau, P.-L.; Dean, P. M.

1994-10-01

Electrostatic interactions have always been considered an important factor governing ligand-receptor interactions. Previous work in this field has established the existence of electrostatic complementarity between the ligand and its receptor site. However, this property has not been treated rigorously, and the description remains largely qualitative. In this work, 34 data sets of high quality were chosen from the Brookhaven Protein Databank. The electrostatic complementarity has been calculated between the surface potentials; complementarity is absent between adjacent or neighbouring atoms of the ligand and the receptor. There is little difference between complementarities on the total ligand surface and the interfacial region. Altering the homogeneous dielectric to distance-dependent dielectrics reduces the complementarity slightly, but does not affect the pattern of complementarity.

Electrostatic complementarity between proteins and ligands. 1. Charge disposition, dielectric and interface effects.

PubMed

Chau, P L; Dean, P M

1994-10-01

Electrostatic interactions have always been considered an important factor governing ligand-receptor interactions. Previous work in this field has established the existence of electrostatic complementarity between the ligand and its receptor site. However, this property has not been treated rigorously, and the description remains largely qualitative. In this work, 34 data sets of high quality were chosen from the Brookhaven Protein Databank. The electrostatic complementary has been calculated between the surface potentials; complementarity is absent between adjacent or neighbouring atoms of the ligand and the receptor. There is little difference between complementarities on the total ligand surface and the interfacial region. Altering the homogeneous dielectric to distance-dependent dielectrics reduces the complementarity slightly, but does not affect the pattern of complementarity.

Comparison of all atom, continuum, and linear fitting empirical models for charge screening effect of aqueous medium surrounding a protein molecule

NASA Astrophysics Data System (ADS)

Takahashi, Takuya; Sugiura, Junnnosuke; Nagayama, Kuniaki

2002-05-01

To investigate the role hydration plays in the electrostatic interactions of proteins, the time-averaged electrostatic potential of the B1 domain of protein G in an aqueous solution was calculated with full atomic molecular dynamics simulations that explicitly considers every atom (i.e., an all atom model). This all atom calculated potential was compared with the potential obtained from an electrostatic continuum model calculation. In both cases, the charge-screening effect was fairly well formulated with an effective relative dielectric constant which increased linearly with increasing charge-charge distance. This simulated linear dependence agrees with the experimentally determined linear relation proposed by Pickersgill. Cut-off approximations for Coulomb interactions failed to reproduce this linear relation. Correlation between the all atom model and the continuum models was found to be better than the respective correlation calculated for linear fitting to the two models. This confirms that the continuum model is better at treating the complicated shapes of protein conformations than the simple linear fitting empirical model. We have tried a sigmoid fitting empirical model in addition to the linear one. When weights of all data were treated equally, the sigmoid model, which requires two fitting parameters, fits results of both the all atom and the continuum models less accurately than the linear model which requires only one fitting parameter. When potential values are chosen as weighting factors, the fitting error of the sigmoid model became smaller, and the slope of both linear fitting curves became smaller. This suggests the screening effect of an aqueous medium within a short range, where potential values are relatively large, is smaller than that expected from the linear fitting curve whose slope is almost 4. To investigate the linear increase of the effective relative dielectric constant, the Poisson equation of a low-dielectric sphere in a high-dielectric medium was solved and charges distributed near the molecular surface were indicated as leading to the apparent linearity.