Beta Atomic Contacts: Identifying Critical Specific Contacts in Protein Binding Interfaces

PubMed Central

Liu, Qian; Kwoh, Chee Keong; Hoi, Steven C. H.

2013-01-01

Specific binding between proteins plays a crucial role in molecular functions and biological processes. Protein binding interfaces and their atomic contacts are typically defined by simple criteria, such as distance-based definitions that only use some threshold of spatial distance in previous studies. These definitions neglect the nearby atomic organization of contact atoms, and thus detect predominant contacts which are interrupted by other atoms. It is questionable whether such kinds of interrupted contacts are as important as other contacts in protein binding. To tackle this challenge, we propose a new definition called beta (β) atomic contacts. Our definition, founded on the β-skeletons in computational geometry, requires that there is no other atom in the contact spheres defined by two contact atoms; this sphere is similar to the van der Waals spheres of atoms. The statistical analysis on a large dataset shows that β contacts are only a small fraction of conventional distance-based contacts. To empirically quantify the importance of β contacts, we design βACV, an SVM classifier with β contacts as input, to classify homodimers from crystal packing. We found that our βACV is able to achieve the state-of-the-art classification performance superior to SVM classifiers with distance-based contacts as input. Our βACV also outperforms several existing methods when being evaluated on several datasets in previous works. The promising empirical performance suggests that β contacts can truly identify critical specific contacts in protein binding interfaces. β contacts thus provide a new model for more precise description of atomic organization in protein quaternary structures than distance-based contacts. PMID:23630569

Molecular architecture of protein-RNA recognition sites.

PubMed

Barik, Amita; C, Nithin; Pilla, Smita P; Bahadur, Ranjit Prasad

2015-01-01

The molecular architecture of protein-RNA interfaces are analyzed using a non-redundant dataset of 152 protein-RNA complexes. We find that an average protein-RNA interface is smaller than an average protein-DNA interface but larger than an average protein-protein interface. Among the different classes of protein-RNA complexes, interfaces with tRNA are the largest, while the interfaces with the single-stranded RNA are the smallest. Significantly, RNA contributes more to the interface area than its partner protein. Moreover, unlike protein-protein interfaces where the side chain contributes less to the interface area compared to the main chain, the main chain and side chain contributions flipped in protein-RNA interfaces. We find that the protein surface in contact with the RNA in protein-RNA complexes is better packed than that in contact with the DNA in protein-DNA complexes, but loosely packed than that in contact with the protein in protein-protein complexes. Shape complementarity and electrostatic potential are the two major factors that determine the specificity of the protein-RNA interaction. We find that the H-bond density at the protein-RNA interfaces is similar with that of protein-DNA interfaces but higher than the protein-protein interfaces. Unlike protein-DNA interfaces where the deoxyribose has little role in intermolecular H-bonds, due to the presence of an oxygen atom at the 2' position, the ribose in RNA plays significant role in protein-RNA H-bonds. We find that besides H-bonds, salt bridges and stacking interactions also play significant role in stabilizing protein-nucleic acids interfaces; however, their contribution at the protein-protein interfaces is insignificant.

Adhesion of bubbles and drops to solid surfaces, and anisotropic surface tensions studied by capillary meniscus dynamometry.

PubMed

Danov, Krassimir D; Stanimirova, Rumyana D; Kralchevsky, Peter A; Marinova, Krastanka G; Stoyanov, Simeon D; Blijdenstein, Theodorus B J; Cox, Andrew R; Pelan, Eddie G

2016-07-01

Here, we review the principle and applications of two recently developed methods: the capillary meniscus dynamometry (CMD) for measuring the surface tension of bubbles/drops, and the capillary bridge dynamometry (CBD) for quantifying the bubble/drop adhesion to solid surfaces. Both methods are based on a new data analysis protocol, which allows one to decouple the two components of non-isotropic surface tension. For an axisymmetric non-fluid interface (e.g. bubble or drop covered by a protein adsorption layer with shear elasticity), the CMD determines the two different components of the anisotropic surface tension, σs and σφ, which are acting along the "meridians" and "parallels", and vary throughout the interface. The method uses data for the instantaneous bubble (drop) profile and capillary pressure, but the procedure for data processing is essentially different from that of the conventional drop shape analysis (DSA) method. In the case of bubble or drop pressed against a substrate, which forms a capillary bridge, the CBD method allows one to determine also the capillary-bridge force for both isotropic (fluid) and anisotropic (solidified) adsorption layers. The experiments on bubble (drop) detachment from the substrate show the existence of a maximal pulling force, Fmax, that can be resisted by an adherent fluid particle. Fmax can be used to quantify the strength of adhesion of bubbles and drops to solid surfaces. Its value is determined by a competition of attractive transversal tension and repulsive disjoining pressure forces. The greatest Fmax values have been measured for bubbles adherent to glass substrates in pea-protein solutions. The bubble/wall adhesion is lower in solutions containing the protein HFBII hydrophobin, which could be explained with the effect of sandwiched protein aggregates. The applicability of the CBD method to emulsion systems is illustrated by experiments with soybean-oil drops adherent to hydrophilic and hydrophobic substrates in egg yolk solutions. The results reveal how the interfacial rigidity, as well as the bubble/wall and drop/wall adhesion forces, can be quantified and controlled in relation to optimizing the properties of foams and emulsions. Copyright © 2015 Elsevier B.V. All rights reserved.

Formation and organization of protein domains in the immunological synapse

NASA Astrophysics Data System (ADS)

Carlson, Andreas; Mahadevan, L.

2014-11-01

The cellular basis for the adaptive immune response during antigen recognition relies on a specialized protein interface known as the immunological synapse. Here, we propose a minimal mathematical model for the dynamics of the IS that encompass membrane mechanics, hydrodynamics and protein kinetics. Simple scaling laws describe the dynamics of protein clusters as a function of membrane stiffness, rigidity of the adhesive proteins, and fluid flow in the synaptic cleft. Numerical simulations complement the scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment suggests that passive dynamics suffices to describe the short-time formation and organization of protein clusters, while the stabilization and long time dynamics of the synapse is likely determined by active cytoskeleton processes triggered by receptor binding. Our study reveals that the fluid flow generated by the interplay between membrane deformation and protein binding kinetics can assist immune cells in regulating protein sorting.

Kinetics of protein unfolding at interfaces

NASA Astrophysics Data System (ADS)

Yano, Yohko F.

2012-12-01

The conformation of protein molecules is determined by a balance of various forces, including van der Waals attraction, electrostatic interaction, hydrogen bonding, and conformational entropy. When protein molecules encounter an interface, they are often adsorbed on the interface. The conformation of an adsorbed protein molecule strongly depends on the interaction between the protein and the interface. Recent time-resolved investigations have revealed that protein conformation changes during the adsorption process due to the protein-protein interaction increasing with increasing interface coverage. External conditions also affect the protein conformation. This review considers recent dynamic observations of protein adsorption at various interfaces and their implications for the kinetics of protein unfolding at interfaces.

Enhanced Cellular Adhesion on Titanium by Silk Functionalized with titanium binding and RGD peptides

PubMed Central

Vidal, Guillaume; Blanchi, Thomas; Mieszawska, Aneta J.; Calabrese, Rossella; Rossi, Claire; Vigneron, Pascale; Duval, Jean-Luc; Kaplan, David L.; Egles, Christophe

2012-01-01

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. Quartz Crystal Microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived RGD peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by Scanning Electron Microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk-peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required. PMID:22975628

Mass Measurements of Focal Adhesions in Single Cells Using High Resolution Surface Plasmon Resonance Microscopy.

PubMed

Peterson, Alexander W; Halter, Michael; Tona, Alessandro; Plant, Anne L; Elliott, John T

2018-01-01

Surface plasmon resonance microscopy (SPRM) is a powerful label-free imaging technique with spatial resolution approaching the optical diffraction limit. The high sensitivity of SPRM to small changes in index of refraction at an interface allows imaging of dynamic protein structures within a cell. Visualization of subcellular features, such as focal adhesions (FAs), can be performed on live cells using a high numerical aperture objective lens with a digital light projector to precisely position the incident angle of the excitation light. Within the cell-substrate region of the SPRM image, punctate regions of high contrast are putatively identified as the cellular FAs. Optical parameter analysis is achieved by application of the Fresnel model to the SPRM data and resulting refractive index measurements are used to calculate protein density and mass. FAs are known to be regions of high protein density that reside at the cell-substratum interface. Comparing SPRM with fluorescence images of antibody stained for vinculin, a component in FAs, reveals similar measurements of FA size. In addition, a positive correlation between FA size and protein density is revealed by SPRM. Comparing SPRM images for two cell types reveals a distinct difference in the protein density and mass of their respective FAs. Application of SPRM to quantify mass can greatly aid monitoring basic processes that control FA mass and growth and contribute to accurate models that describe cell-extracellular interactions.

Understanding Protein-Interface Interactions of a Fusion Protein at Silicone Oil-Water Interface Probed by Sum Frequency Generation Vibrational Spectroscopy.

PubMed

Li, Yaoxin; Pan, Duohai; Nashine, Vishal; Deshmukh, Smeet; Vig, Balvinder; Chen, Zhan

2018-02-01

Protein adsorbed at the silicone oil-water interface can undergo a conformational change that has the potential to induce protein aggregation on storage. Characterization of the protein structures at interface is therefore critical for understanding the protein-interface interactions. In this article, we have applied sum frequency generation (SFG) spectroscopy for studying the secondary structures of a fusion protein at interface and the surfactant effect on protein adsorption to silicone oil-water interface. SFG and chiral SFG spectra from adsorbed protein in the amide I region were analyzed. The presence of beta-sheet vibrational band at 1635 cm -1 implies the protein secondary structure was likely perturbed when protein adsorbed at silicone oil interface. The time-dependent SFG study showed a significant reduction in the SFG signal of preadsorbed protein when polysorbate 20 was introduced, suggesting surfactant has stronger interaction with the interface leading to desorption of protein from the interface. In the preadsorbed surfactant and a mixture of protein/polysorbate 20, SFG analysis confirmed that surfactant can dramatically prevent the protein adsorption to silicone oil surface. This study has demonstrated the potential of SFG for providing the detailed molecular level understanding of protein conformation at interface and assessing the influence of surfactant on protein adsorption behavior. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

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

Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

PubMed

Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

2016-01-29

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

Quantifying protein interface footprinting by hydroxyl radical oxidation and molecular dynamics simulation: application to galectin-1.

PubMed

Charvátová, Olga; Foley, B Lachele; Bern, Marshall W; Sharp, Joshua S; Orlando, Ron; Woods, Robert J

2008-11-01

Biomolecular surface mapping methods offer an important alternative method for characterizing protein-protein and protein-ligand interactions in cases in which it is not possible to determine high-resolution three-dimensional (3D) structures of complexes. Hydroxyl radical footprinting offers a significant advance in footprint resolution compared with traditional chemical derivatization. Here we present results of footprinting performed with hydroxyl radicals generated on the nanosecond time scale by laser-induced photodissociation of hydrogen peroxide. We applied this emerging method to a carbohydrate-binding protein, galectin-1. Since galectin-1 occurs as a homodimer, footprinting was employed to characterize the interface of the monomeric subunits. Efficient analysis of the mass spectrometry data for the oxidized protein was achieved with the recently developed ByOnic (Palo Alto, CA) software that was altered to handle the large number of modifications arising from side-chain oxidation. Quantification of the level of oxidation has been achieved by employing spectral intensities for all of the observed oxidation states on a per-residue basis. The level of accuracy achievable from spectral intensities was determined by examination of mixtures of synthetic peptides related to those present after oxidation and tryptic digestion of galectin-1. A direct relationship between side-chain solvent accessibility and level of oxidation emerged, which enabled the prediction of the level of oxidation given the 3D structure of the protein. The precision of this relationship was enhanced through the use of average solvent accessibilities computed from 10 ns molecular dynamics simulations of the protein.

A Strategy Based on Protein-Protein Interface Motifs May Help in Identifying Drug Off-Targets

PubMed Central

Engin, H. Billur; Keskin, Ozlem; Nussinov, Ruth; Gursoy, Attila

2014-01-01

Networks are increasingly used to study the impact of drugs at the systems level. From the algorithmic standpoint, a drug can ‘attack’ nodes or edges of a protein-protein interaction network. In this work, we propose a new network strategy, “The Interface Attack”, based on protein-protein interfaces. Similar interface architectures can occur between unrelated proteins. Consequently, in principle, a drug that binds to one has a certain probability of binding others. The interface attack strategy simultaneously removes from the network all interactions that consist of similar interface motifs. This strategy is inspired by network pharmacology and allows inferring potential off-targets. We introduce a network model which we call “Protein Interface and Interaction Network (P2IN)”, which is the integration of protein-protein interface structures and protein interaction networks. This interface-based network organization clarifies which protein pairs have structurally similar interfaces, and which proteins may compete to bind the same surface region. We built the P2IN of p53 signaling network and performed network robustness analysis. We show that (1) ‘hitting’ frequent interfaces (a set of edges distributed around the network) might be as destructive as eleminating high degree proteins (hub nodes); (2) frequent interfaces are not always topologically critical elements in the network; and (3) interface attack may reveal functional changes in the system better than attack of single proteins. In the off-target detection case study, we found that drugs blocking the interface between CDK6 and CDKN2D may also affect the interaction between CDK4 and CDKN2D. PMID:22817115

Protein-Protein Interface Predictions by Data-Driven Methods: A Review

PubMed Central

Xue, Li C; Dobbs, Drena; Bonvin, Alexandre M.J.J.; Honavar, Vasant

2015-01-01

Reliably pinpointing which specific amino acid residues form the interface(s) between a protein and its binding partner(s) is critical for understanding the structural and physicochemical determinants of protein recognition and binding affinity, and has wide applications in modeling and validating protein interactions predicted by high-throughput methods, in engineering proteins, and in prioritizing drug targets. Here, we review the basic concepts, principles and recent advances in computational approaches to the analysis and prediction of protein-protein interfaces. We point out caveats for objectively evaluating interface predictors, and discuss various applications of data-driven interface predictors for improving energy model-driven protein-protein docking. Finally, we stress the importance of exploiting binding partner information in reliably predicting interfaces and highlight recent advances in this emerging direction. PMID:26460190

Protein Frustratometer 2: a tool to localize energetic frustration in protein molecules, now with electrostatics.

PubMed

Parra, R Gonzalo; Schafer, Nicholas P; Radusky, Leandro G; Tsai, Min-Yeh; Guzovsky, A Brenda; Wolynes, Peter G; Ferreiro, Diego U

2016-07-08

The protein frustratometer is an energy landscape theory-inspired algorithm that aims at localizing and quantifying the energetic frustration present in protein molecules. Frustration is a useful concept for analyzing proteins' biological behavior. It compares the energy distributions of the native state with respect to structural decoys. The network of minimally frustrated interactions encompasses the folding core of the molecule. Sites of high local frustration often correlate with functional regions such as binding sites and regions involved in allosteric transitions. We present here an upgraded version of a webserver that measures local frustration. The new implementation that allows the inclusion of electrostatic energy terms, important to the interactions with nucleic acids, is significantly faster than the previous version enabling the analysis of large macromolecular complexes within a user-friendly interface. The webserver is freely available at URL: http://frustratometer.qb.fcen.uba.ar. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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.

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.

Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-κB Essential Modulator (NEMO)/IKKβ Protein-Protein Interface

PubMed Central

Golden, Mary S.; Cote, Shaun M.; Sayeg, Marianna; Zerbe, Brandon S.; Villar, Elizabeth A.; Beglov, Dmitri; Sazinsky, Stephen L.; Georgiadis, Rosina M.; Vajda, Sandor; Kozakov, Dima; Whitty, Adrian

2013-01-01

We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction (PPI) interface between NF-κB Essential Modulator (NEMO) and IκB kinase subunit β (IKKβ), an interaction that is critical for NF-κB pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NBD region of IKKβ contains the highest concentration of hot spot residues, the strongest of which are W739, W741 and L742 (ΔΔG = 4.3, 3.5 and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for ~16 Å to additionally include the regions that bind IKKβ L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot spot regions centered on IKKβ residues L708/V709 and L719/I723. The computational approach successfully identified all three hot spot regions on IKKβ. Moreover, the method was able to accurately quantify the energetic importance of all hot spots residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small molecule inhibitors that target the NEMO/IKKβ interaction. They additionally clarify the structural and energetic complementarity between “pocket-forming” and “pocket occupying” hot spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces. PMID:23506214

Exploring the potential of 3D Zernike descriptors and SVM for protein-protein interface prediction.

PubMed

Daberdaku, Sebastian; Ferrari, Carlo

2018-02-06

The correct determination of protein-protein interaction interfaces is important for understanding disease mechanisms and for rational drug design. To date, several computational methods for the prediction of protein interfaces have been developed, but the interface prediction problem is still not fully understood. Experimental evidence suggests that the location of binding sites is imprinted in the protein structure, but there are major differences among the interfaces of the various protein types: the characterising properties can vary a lot depending on the interaction type and function. The selection of an optimal set of features characterising the protein interface and the development of an effective method to represent and capture the complex protein recognition patterns are of paramount importance for this task. In this work we investigate the potential of a novel local surface descriptor based on 3D Zernike moments for the interface prediction task. Descriptors invariant to roto-translations are extracted from circular patches of the protein surface enriched with physico-chemical properties from the HQI8 amino acid index set, and are used as samples for a binary classification problem. Support Vector Machines are used as a classifier to distinguish interface local surface patches from non-interface ones. The proposed method was validated on 16 classes of proteins extracted from the Protein-Protein Docking Benchmark 5.0 and compared to other state-of-the-art protein interface predictors (SPPIDER, PrISE and NPS-HomPPI). The 3D Zernike descriptors are able to capture the similarity among patterns of physico-chemical and biochemical properties mapped on the protein surface arising from the various spatial arrangements of the underlying residues, and their usage can be easily extended to other sets of amino acid properties. The results suggest that the choice of a proper set of features characterising the protein interface is crucial for the interface prediction task, and that optimality strongly depends on the class of proteins whose interface we want to characterise. We postulate that different protein classes should be treated separately and that it is necessary to identify an optimal set of features for each protein class.

HomPPI: a class of sequence homology based protein-protein interface prediction methods

PubMed Central

2011-01-01

Background Although homology-based methods are among the most widely used methods for predicting the structure and function of proteins, the question as to whether interface sequence conservation can be effectively exploited in predicting protein-protein interfaces has been a subject of debate. Results We studied more than 300,000 pair-wise alignments of protein sequences from structurally characterized protein complexes, including both obligate and transient complexes. We identified sequence similarity criteria required for accurate homology-based inference of interface residues in a query protein sequence. Based on these analyses, we developed HomPPI, a class of sequence homology-based methods for predicting protein-protein interface residues. We present two variants of HomPPI: (i) NPS-HomPPI (Non partner-specific HomPPI), which can be used to predict interface residues of a query protein in the absence of knowledge of the interaction partner; and (ii) PS-HomPPI (Partner-specific HomPPI), which can be used to predict the interface residues of a query protein with a specific target protein. Our experiments on a benchmark dataset of obligate homodimeric complexes show that NPS-HomPPI can reliably predict protein-protein interface residues in a given protein, with an average correlation coefficient (CC) of 0.76, sensitivity of 0.83, and specificity of 0.78, when sequence homologs of the query protein can be reliably identified. NPS-HomPPI also reliably predicts the interface residues of intrinsically disordered proteins. Our experiments suggest that NPS-HomPPI is competitive with several state-of-the-art interface prediction servers including those that exploit the structure of the query proteins. The partner-specific classifier, PS-HomPPI can, on a large dataset of transient complexes, predict the interface residues of a query protein with a specific target, with a CC of 0.65, sensitivity of 0.69, and specificity of 0.70, when homologs of both the query and the target can be reliably identified. The HomPPI web server is available at http://homppi.cs.iastate.edu/. Conclusions Sequence homology-based methods offer a class of computationally efficient and reliable approaches for predicting the protein-protein interface residues that participate in either obligate or transient interactions. For query proteins involved in transient interactions, the reliability of interface residue prediction can be improved by exploiting knowledge of putative interaction partners. PMID:21682895

Evolutionary diversification of protein-protein interactions by interface add-ons.

PubMed

Plach, Maximilian G; Semmelmann, Florian; Busch, Florian; Busch, Markus; Heizinger, Leonhard; Wysocki, Vicki H; Merkl, Rainer; Sterner, Reinhard

2017-10-03

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein-protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein-protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein-protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein-protein interactions.

A PDB-wide, evolution-based assessment of protein-protein interfaces.

PubMed

Baskaran, Kumaran; Duarte, Jose M; Biyani, Nikhil; Bliven, Spencer; Capitani, Guido

2014-10-18

Thanks to the growth in sequence and structure databases, more than 50 million sequences are now available in UniProt and 100,000 structures in the PDB. Rich information about protein-protein interfaces can be obtained by a comprehensive study of protein contacts in the PDB, their sequence conservation and geometric features. An automated computational pipeline was developed to run our Evolutionary Protein-Protein Interface Classifier (EPPIC) software on the entire PDB and store the results in a relational database, currently containing > 800,000 interfaces. This allows the analysis of interface data on a PDB-wide scale. Two large benchmark datasets of biological interfaces and crystal contacts, each containing about 3000 entries, were automatically generated based on criteria thought to be strong indicators of interface type. The BioMany set of biological interfaces includes NMR dimers solved as crystal structures and interfaces that are preserved across diverse crystal forms, as catalogued by the Protein Common Interface Database (ProtCID) from Xu and Dunbrack. The second dataset, XtalMany, is derived from interfaces that would lead to infinite assemblies and are therefore crystal contacts. BioMany and XtalMany were used to benchmark the EPPIC approach. The performance of EPPIC was also compared to classifications from the Protein Interfaces, Surfaces, and Assemblies (PISA) program on a PDB-wide scale, finding that the two approaches give the same call in about 88% of PDB interfaces. By comparing our safest predictions to the PDB author annotations, we provide a lower-bound estimate of the error rate of biological unit annotations in the PDB. Additionally, we developed a PyMOL plugin for direct download and easy visualization of EPPIC interfaces for any PDB entry. Both the datasets and the PyMOL plugin are available at http://www.eppic-web.org/ewui/\\#downloads. Our computational pipeline allows us to analyze protein-protein contacts and their sequence conservation across the entire PDB. Two new benchmark datasets are provided, which are over an order of magnitude larger than existing manually curated ones. These tools enable the comprehensive study of several aspects of protein-protein contacts in the PDB and represent a basis for future, even larger scale studies of protein-protein interactions.

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis

PubMed Central

2018-01-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that ‘leftover’ proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles. PMID:29518071

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis.

PubMed

Holland, David O; Johnson, Margaret E

2018-03-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that 'leftover' proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles.

Protein-protein recognition control by modulating electrostatic interactions.

PubMed

Han, Song; Yin, Shijin; Yi, Hong; Mouhat, Stéphanie; Qiu, Su; Cao, Zhijian; Sabatier, Jean-Marc; Wu, Yingliang; Li, Wenxin

2010-06-04

Protein-protein control recognition remains a huge challenge, and its development depends on understanding the chemical and biological mechanisms by which these interactions occur. Here we describe a protein-protein control recognition technique based on the dominant electrostatic interactions occurring between the proteins. We designed a potassium channel inhibitor, BmP05-T, that was 90.32% identical to wild-type BmP05. Negatively charged residues were translocated from the nonbinding interface to the binding interface of BmP05 inhibitor, such that BmP05-T now used BmP05 nonbinding interface as the binding interface. This switch demonstrated that nonbinding interfaces were able to control the orientation of protein binding interfaces in the process of protein-protein recognition. The novel function findings of BmP05-T peptide suggested that the control recognition technique described here had the potential for use in designing and utilizing functional proteins in many biological scenarios.

Differential Protein Expression in the Hemolymph of Bithynia siamensis goniomphalos Infected with Opisthorchis viverrini

PubMed Central

Suwannatrai, Kulwadee; Suwannatrai, Apiporn; Tabsripair, Pairat; Welbat, Jariya Umka; Tangkawattana, Sirikachorn; Cantacessi, Cinzia; Mulvenna, Jason; Tesana, Smarn; Loukas, Alex

2016-01-01

Bithynia siamensis goniomphalos is a freshwater snail that serves as the first intermediate host of the human liver fluke Opisthorchis viverrini. This parasite is a major public health problem in different countries throughout the Greater Mekong sub-region (Thailand, southern Vietnam, Lao PDR and Cambodia). Chronic O. viverrini infection also results in a gradual increase of fibrotic tissues in the biliary tract that are associated with hepatobiliary diseases and contribute to cholangiocarcinoma (a fatal type of bile duct cancer). Infectivity of the parasite in the snail host is strongly correlated with destruction of helminths by the snail’s innate immune system, composed of cellular (hemocyte) and humoral (plasma) defense factors. To better understand this important host-parasite interface we applied sequential window acquisition of all theoretical spectra mass spectrometry (SWATH-MS) to identify and quantify the proteins from the hemolymph of B. siamensis goniomphalos experimentally infected with O. viverrini and compare them to non-infected snails (control group). A total of 362 and 242 proteins were identified in the hemocytes and plasma, respectively. Of these, 145 and 117 proteins exhibited significant differences in expression upon fluke infection in hemocytes and plasma, respectively. Among the proteins with significantly different expression patterns, we found proteins related to immune response (up-regulated in both hemocyte and plasma of infected snails) and proteins belonging to the structural and motor group (mostly down-regulated in hemocytes but up-regulated in plasma of infected snails). The proteins identified and quantified in this work will provide important information for the understanding of the factors involved in snail defense against O. viverrini and might facilitate the development of new strategies to control O. viverrini infection in endemic areas. PMID:27893749

Blind predictions of protein interfaces by docking calculations in CAPRI.

PubMed

Lensink, Marc F; Wodak, Shoshana J

2010-11-15

Reliable prediction of the amino acid residues involved in protein-protein interfaces can provide valuable insight into protein function, and inform mutagenesis studies, and drug design applications. A fast-growing number of methods are being proposed for predicting protein interfaces, using structural information, energetic criteria, or sequence conservation or by integrating multiple criteria and approaches. Overall however, their performance remains limited, especially when applied to nonobligate protein complexes, where the individual components are also stable on their own. Here, we evaluate interface predictions derived from protein-protein docking calculations. To this end we measure the overlap between the interfaces in models of protein complexes submitted by 76 participants in CAPRI (Critical Assessment of Predicted Interactions) and those of 46 observed interfaces in 20 CAPRI targets corresponding to nonobligate complexes. Our evaluation considers multiple models for each target interface, submitted by different participants, using a variety of docking methods. Although this results in a substantial variability in the prediction performance across participants and targets, clear trends emerge. Docking methods that perform best in our evaluation predict interfaces with average recall and precision levels of about 60%, for a small majority (60%) of the analyzed interfaces. These levels are significantly higher than those obtained for nonobligate complexes by most extant interface prediction methods. We find furthermore that a sizable fraction (24%) of the interfaces in models ranked as incorrect in the CAPRI assessment are actually correctly predicted (recall and precision ≥50%), and that these models contribute to 70% of the correct docking-based interface predictions overall. Our analysis proves that docking methods are much more successful in identifying interfaces than in predicting complexes, and suggests that these methods have an excellent potential of addressing the interface prediction challenge. © 2010 Wiley-Liss, Inc.

Protein interactions in 3D: from interface evolution to drug discovery.

PubMed

Winter, Christof; Henschel, Andreas; Tuukkanen, Anne; Schroeder, Michael

2012-09-01

Over the past 10years, much research has been dedicated to the understanding of protein interactions. Large-scale experiments to elucidate the global structure of protein interaction networks have been complemented by detailed studies of protein interaction interfaces. Understanding the evolution of interfaces allows one to identify convergently evolved interfaces which are evolutionary unrelated but share a few key residues and hence have common binding partners. Understanding interaction interfaces and their evolution is an important basis for pharmaceutical applications in drug discovery. Here, we review the algorithms and databases on 3D protein interactions and discuss in detail applications in interface evolution, drug discovery, and interface prediction. Copyright © 2012 Elsevier Inc. All rights reserved.

Conservation of coevolving protein interfaces bridges prokaryote-eukaryote homologies in the twilight zone.

PubMed

Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

2016-12-27

Protein-protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein-protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein-protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein-protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach.

A web interface for easy flexible protein-protein docking with ATTRACT.

PubMed

de Vries, Sjoerd J; Schindler, Christina E M; Chauvot de Beauchêne, Isaure; Zacharias, Martin

2015-02-03

Protein-protein docking programs can give valuable insights into the structure of protein complexes in the absence of an experimental complex structure. Web interfaces can facilitate the use of docking programs by structural biologists. Here, we present an easy web interface for protein-protein docking with the ATTRACT program. While aimed at nonexpert users, the web interface still covers a considerable range of docking applications. The web interface supports systematic rigid-body protein docking with the ATTRACT coarse-grained force field, as well as various kinds of protein flexibility. The execution of a docking protocol takes up to a few hours on a standard desktop computer. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Shape Complementarity of Protein-Protein Complexes at Multiple Resolutions

PubMed Central

Zhang, Qing; Sanner, Michel; Olson, Arthur J.

2010-01-01

Biological complexes typically exhibit intermolecular interfaces of high shape complementarity. Many computational docking approaches use this surface complementarity as a guide in the search for predicting the structures of protein-protein complexes. Proteins often undergo conformational changes in order to create a highly complementary interface when associating. These conformational changes are a major cause of failure for automated docking procedures when predicting binding modes between proteins using their unbound conformations. Low resolution surfaces in which high frequency geometric details are omitted have been used to address this problem. These smoothed, or blurred, surfaces are expected to minimize the differences between free and bound structures, especially those that are due to side chain conformations or small backbone deviations. In spite of the fact that this approach has been used in many docking protocols, there has yet to be a systematic study of the effects of such surface smoothing on the shape complementarity of the resulting interfaces. Here we investigate this question by computing shape complementarity of a set of 66 protein-protein complexes represented by multi-resolution blurred surfaces. Complexed and unbound structures are available for these protein-protein complexes. They are a subset of complexes from a non-redundant docking benchmark selected for rigidity (i.e. the proteins undergo limited conformational changes between their bound and unbound states). In this work we construct the surfaces by isocontouring a density map obtained by accumulating the densities of Gaussian functions placed at all atom centers of the molecule. The smoothness or resolution is specified by a Gaussian fall-off coefficient, termed “blobbyness”. Shape complementarity is quantified using a histogram of the shortest distances between two proteins' surface mesh vertices for both the crystallographic complexes and the complexes built using the protein structures in their unbound conformation. The histograms calculated for the bound complex structures demonstrate that medium resolution smoothing (blobbyness=−0.9) can reproduce about 88% of the shape complementarity of atomic resolution surfaces. Complexes formed from the free component structures show a partial loss of shape complementarity (more overlaps and gaps) with the atomic resolution surfaces. For surfaces smoothed to low resolution (blobbyness=−0.3), we find more consistency of shape complementarity between the complexed and free cases. To further reduce bad contacts without significantly impacting the good contacts we introduce another blurred surface, in which the Gaussian densities of flexible atoms are reduced. From these results we discuss the use of shape complementarity in protein-protein docking. PMID:18837463

Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces.

PubMed

Bordner, Andrew J; Gorin, Andrey A

2008-05-12

Protein-protein interactions are ubiquitous and essential for all cellular processes. High-resolution X-ray crystallographic structures of protein complexes can reveal the details of their function and provide a basis for many computational and experimental approaches. Differentiation between biological and non-biological contacts and reconstruction of the intact complex is a challenging computational problem. A successful solution can provide additional insights into the fundamental principles of biological recognition and reduce errors in many algorithms and databases utilizing interaction information extracted from the Protein Data Bank (PDB). We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1) comprehensively collecting all protein-protein interfaces; (2) clustering similar protein-protein interfaces together; (3) estimating the probability that each cluster is relevant based on a diverse set of properties; and (4) combining these scores for each PDB entry in order to predict the complex structure. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions. These interfaces, as well as the predicted protein complexes, are available from the Protein Interface Server (PInS) website (see Availability and requirements section). Our method demonstrates an almost two-fold reduction of the annotation error rate as evaluated on a large benchmark set of complexes validated from the literature. We also estimate relative contributions of each interface property to the accurate discrimination of biologically relevant interfaces and discuss possible directions for further improving the prediction method.

Change of the isoelectric point of hemoglobin at the air/water interface probed by the orientational flip-flop of water molecules.

PubMed

Devineau, Stéphanie; Inoue, Ken-Ichi; Kusaka, Ryoji; Urashima, Shu-Hei; Nihonyanagi, Satoshi; Baigl, Damien; Tsuneshige, Antonio; Tahara, Tahei

2017-04-19

Elucidation of the molecular mechanisms of protein adsorption is of essential importance for further development of biotechnology. Here, we use interface-selective nonlinear vibrational spectroscopy to investigate protein charge at the air/water interface by probing the orientation of interfacial water molecules. We measured the Im χ (2) spectra of hemoglobin, myoglobin, serum albumin and lysozyme at the air/water interface in the CH and OH stretching regions using heterodyne-detected vibrational sum frequency generation (HD-VSFG) spectroscopy, and we deduced the isoelectric point of the protein by monitoring the orientational flip-flop of water molecules at the interface. Strikingly, our measurements indicate that the isoelectric point of hemoglobin is significantly lowered (by about one pH unit) at the air/water interface compared to that in the bulk. This can be predominantly attributed to the modifications of the protein structure at the air/water interface. Our results also suggest that a similar mechanism accounts for the modification of myoglobin charge at the air/water interface. This effect has not been reported for other model proteins at interfaces probed by conventional VSFG techniques, and it emphasizes the importance of the structural modifications of proteins at the interface, which can drastically affect their charge profiles in a protein-specific manner. The direct experimental approach using HD-VSFG can unveil the changes of the isoelectric point of adsorbed proteins at various interfaces, which is of major relevance to many biological applications and sheds new light on the effect of interfaces on protein charge.

Role of the Lipid Environment in the Dimerization of Transmembrane Domains of Glycophorin A

PubMed Central

Kuznetsov, A. S.; Volynsky, P. E.; Efremov, R. G.

2015-01-01

An efficient computational approach is developed to quantify the free energy of a spontaneous association of the α-helices of proteins in the membrane environment. The approach is based on the numerical decomposition of the free energy profiles of the transmembrane (TM) helices into components corresponding to protein-protein, protein-lipid, and protein-water interactions. The method was tested for the TM segments of human glycophorin A (GpA) and two mutant forms, Gly83Ala and Thr87Val. It was shown that lipids make a significant negative contribution to the free energy of dimerization, while amino acid residues forming the interface of the helix-helix contact may be unfavorable in terms of free energy. The detailed balance between different energy contributions is highly dependent on the amino acid sequence of the TM protein segment. The results show the dominant role of the environment in the interaction of membrane proteins that is changing our notion of the driving force behind the spontaneous association of TM α-helices. Adequate estimation of the contribution of the water-lipid environment thus becomes an extremely urgent task for a rational design of new molecules targeting bitopic membrane proteins, including receptor tyrosine kinases. PMID:26798499

Adsorption and conformations of lysozyme and α-lactalbumin at a water-octane interface

NASA Astrophysics Data System (ADS)

Cheung, David L.

2017-11-01

As proteins contain both hydrophobic and hydrophilic amino acids, they will readily adsorb onto interfaces between water and hydrophobic fluids such as oil. This adsorption normally causes changes in the protein structure, which can result in loss of protein function and irreversible adsorption, leading to the formation of protein interfacial films. While this can be advantageous in some applications (e.g., food technology), in most cases it limits our ability to exploit protein functionality at interfaces. To understand and control protein interfacial adsorption and function, it is necessary to understand the microscopic conformation of proteins at liquid interfaces. In this paper, molecular dynamics simulations are used to investigate the adsorption and conformation of two similar proteins, lysozyme and α-lactalbumin, at a water-octane interface. While they both adsorb onto the interface, α-lactalbumin does so in a specific orientation, mediated by two amphipathic helices, while lysozyme adsorbs in a non-specific manner. Using replica exchange simulations, both proteins are found to possess a number of distinct interfacial conformations, with compact states similar to the solution conformation being most common for both proteins. Decomposing the different contributions to the protein energy at oil-water interfaces suggests that conformational change for α-lactalbumin, unlike lysozyme, is driven by favourable protein-oil interactions. Revealing these differences between the factors that govern the conformational change at interfaces in otherwise similar proteins can give insight into the control of protein interfacial adsorption, aggregation, and function.

Size-dependent protein segregation at membrane interfaces

NASA Astrophysics Data System (ADS)

Schmid, Eva M.; Bakalar, Matthew H.; Choudhuri, Kaushik; Weichsel, Julian; Ann, Hyoung Sook; Geissler, Phillip L.; Dustin, Michael L.; Fletcher, Daniel A.

2016-07-01

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell-cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

PubMed Central

He, Yi-Ming; Ma, Bin-Guang

2016-01-01

Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions. PMID:27220911

Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

NASA Astrophysics Data System (ADS)

He, Yi-Ming; Ma, Bin-Guang

2016-05-01

Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

Broad-Bandwidth Chiral Sum Frequency Generation Spectroscopy for Probing the Kinetics of Proteins at Interfaces

PubMed Central

2016-01-01

The kinetics of proteins at interfaces plays an important role in biological functions and inspires solutions to fundamental problems in biomedical sciences and engineering. Nonetheless, due to the lack of surface-specific and structural-sensitive biophysical techniques, it still remains challenging to probe protein kinetics in situ and in real time without the use of spectroscopic labels at interfaces. Broad-bandwidth chiral sum frequency generation (SFG) spectroscopy has been recently developed for protein kinetic studies at interfaces by tracking the chiral vibrational signals of proteins. In this article, we review our recent progress in kinetic studies of proteins at interfaces using broad-bandwidth chiral SFG spectroscopy. We illustrate the use of chiral SFG signals of protein side chains in the C–H stretch region to monitor self-assembly processes of proteins at interfaces. We also present the use of chiral SFG signals from the protein backbone in the N–H stretch region to probe the real-time kinetics of proton exchange between protein and water at interfaces. In addition, we demonstrate the applications of spectral features of chiral SFG that are typical of protein secondary structures in both the amide I and the N–H stretch regions for monitoring the kinetics of aggregation of amyloid proteins at membrane surfaces. These studies exhibit the power of broad-bandwidth chiral SFG to study protein kinetics at interfaces and the promise of this technique in research areas of surface science to address fundamental problems in biomedical and material sciences. PMID:26196215

BFEE: A User-Friendly Graphical Interface Facilitating Absolute Binding Free-Energy Calculations.

PubMed

Fu, Haohao; Gumbart, James C; Chen, Haochuan; Shao, Xueguang; Cai, Wensheng; Chipot, Christophe

2018-03-26

Quantifying protein-ligand binding has attracted the attention of both theorists and experimentalists for decades. Many methods for estimating binding free energies in silico have been reported in recent years. Proper use of the proposed strategies requires, however, adequate knowledge of the protein-ligand complex, the mathematical background for deriving the underlying theory, and time for setting up the simulations, bookkeeping, and postprocessing. Here, to minimize human intervention, we propose a toolkit aimed at facilitating the accurate estimation of standard binding free energies using a geometrical route, coined the binding free-energy estimator (BFEE), and introduced it as a plug-in of the popular visualization program VMD. Benefitting from recent developments in new collective variables, BFEE can be used to generate the simulation input files, based solely on the structure of the complex. Once the simulations are completed, BFEE can also be utilized to perform the post-treatment of the free-energy calculations, allowing the absolute binding free energy to be estimated directly from the one-dimensional potentials of mean force in simulation outputs. The minimal amount of human intervention required during the whole process combined with the ergonomic graphical interface makes BFEE a very effective and practical tool for the end-user.

Fibrillization kinetics of insulin solution in an interfacial shearing flow

NASA Astrophysics Data System (ADS)

Balaraj, Vignesh; McBride, Samantha; Hirsa, Amir; Lopez, Juan

2015-11-01

Although the association of fibril plaques with neurodegenerative diseases like Alzheimer's and Parkinson's is well established, in-depth understanding of the roles played by various physical factors in seeding and growth of fibrils is far from well known. Of the numerous factors affecting this complex phenomenon, the effect of fluid flow and shear at interfaces is paramount as it is ubiquitous and the most varying factor in vivo. Many amyloidogenic proteins have been found to denature upon contact at hydrophobic interfaces due to the self-assembling nature of protein in its monomeric state. Here, fibrillization kinetics of insulin solution is studied in an interfacial shearing flow. The transient surface rheological response of the insulin solution to the flow and its effect on the bulk fibrillization process has been quantified. Minute differences in hydrophobic characteristics between two variants of insulin- Human recombinant and Bovine insulin are found to result in very different responses. Results presented will be in the form of fibrillization assays, images of fibril plaques formed, and changes in surface rheological properties of the insulin solution. The interfacial velocity field, measured from images (via Brewster Angle Microscopy), is compared with computations. Supported by NNX13AQ22G, National Aeronautics and Space Administration.

Exploiting three kinds of interface propensities to identify protein binding sites.

PubMed

Liu, Bin; Wang, Xiaolong; Lin, Lei; Dong, Qiwen; Wang, Xuan

2009-08-01

Predicting the binding sites between two interacting proteins provides important clues to the function of a protein. In this study, we present a building block of proteins called order profiles to use the evolutionary information of the protein sequence frequency profiles and apply this building block to produce a class of propensities called order profile interface propensities. For comparisons, we revisit the usage of residue interface propensities and binary profile interface propensities for protein binding site prediction. Each kind of propensities combined with sequence profiles and accessible surface areas are inputted into SVM. When tested on four types of complexes (hetero-permanent complexes, hetero-transient complexes, homo-permanent complexes and homo-transient complexes), experimental results show that the order profile interface propensities are better than residue interface propensities and binary profile interface propensities. Therefore, order profile is a suitable profile-level building block of the protein sequences and can be widely used in many tasks of computational biology, such as the sequence alignment, the prediction of domain boundary, the designation of knowledge-based potentials and the protein remote homology detection.

Size-dependent protein segregation at membrane interfaces

PubMed Central

Schmid, Eva M; Bakalar, Matthew H; Choudhuri, Kaushik; Weichsel, Julian; Ann, HyoungSook; Geissler, Phillip L; Dustin, Michael L; Fletcher, Daniel A

2016-01-01

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane protein organization, such as E-cadherin enrichment in epithelial junctional complexes and CD45 exclusion from the signaling foci of immunological synapses. To isolate the role of protein size in these processes, we reconstituted membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between binding and non-binding proteins can dramatically alter their organization at membrane interfaces in the absence of active contributions from the cytoskeleton, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally-driven membrane height fluctuations that transiently limit access to the interface. This simple, sensitive, and highly effective means of passively segregating proteins has implications for signaling at cell-cell junctions and protein sorting at intracellular contact points between membrane-bound organelles. PMID:27980602

Evolutionary diversification of protein–protein interactions by interface add-ons

PubMed Central

Plach, Maximilian G.; Semmelmann, Florian; Busch, Florian; Busch, Markus; Heizinger, Leonhard; Wysocki, Vicki H.; Sterner, Reinhard

2017-01-01

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein–protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein–protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein–protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein–protein interactions. PMID:28923934

Protein-protein interface detection using the energy centrality relationship (ECR) characteristic of proteins.

PubMed

Sudarshan, Sanjana; Kodathala, Sasi B; Mahadik, Amruta C; Mehta, Isha; Beck, Brian W

2014-01-01

Specific protein interactions are responsible for most biological functions. Distinguishing Functionally Linked Interfaces of Proteins (FLIPs), from Functionally uncorrelated Contacts (FunCs), is therefore important to characterizing these interactions. To achieve this goal, we have created a database of protein structures called FLIPdb, containing proteins belonging to various functional sub-categories. Here, we use geometric features coupled with Kortemme and Baker's computational alanine scanning method to calculate the energetic sensitivity of each amino acid at the interface to substitution, identify hotspots, and identify other factors that may contribute towards an interface being FLIP or FunC. Using Principal Component Analysis and K-means clustering on a training set of 160 interfaces, we could distinguish FLIPs from FunCs with an accuracy of 76%. When these methods were applied to two test sets of 18 and 170 interfaces, we achieved similar accuracies of 78% and 80%. We have identified that FLIP interfaces have a stronger central organizing tendency than FunCs, due, we suggest, to greater specificity. We also observe that certain functional sub-categories, such as enzymes, antibody-heavy-light, antibody-antigen, and enzyme-inhibitors form distinct sub-clusters. The antibody-antigen and enzyme-inhibitors interfaces have patterns of physical characteristics similar to those of FunCs, which is in agreement with the fact that the selection pressures of these interfaces is differently evolutionarily driven. As such, our ECR model also successfully describes the impact of evolution and natural selection on protein-protein interfaces. Finally, we indicate how our ECR method may be of use in reducing the false positive rate of docking calculations.

Protein-Protein Interface Detection Using the Energy Centrality Relationship (ECR) Characteristic of Proteins

PubMed Central

Sudarshan, Sanjana; Kodathala, Sasi B.; Mahadik, Amruta C.; Mehta, Isha; Beck, Brian W.

2014-01-01

Specific protein interactions are responsible for most biological functions. Distinguishing Functionally Linked Interfaces of Proteins (FLIPs), from Functionally uncorrelated Contacts (FunCs), is therefore important to characterizing these interactions. To achieve this goal, we have created a database of protein structures called FLIPdb, containing proteins belonging to various functional sub-categories. Here, we use geometric features coupled with Kortemme and Baker's computational alanine scanning method to calculate the energetic sensitivity of each amino acid at the interface to substitution, identify hotspots, and identify other factors that may contribute towards an interface being FLIP or FunC. Using Principal Component Analysis and K-means clustering on a training set of 160 interfaces, we could distinguish FLIPs from FunCs with an accuracy of 76%. When these methods were applied to two test sets of 18 and 170 interfaces, we achieved similar accuracies of 78% and 80%. We have identified that FLIP interfaces have a stronger central organizing tendency than FunCs, due, we suggest, to greater specificity. We also observe that certain functional sub-categories, such as enzymes, antibody-heavy-light, antibody-antigen, and enzyme-inhibitors form distinct sub-clusters. The antibody-antigen and enzyme-inhibitors interfaces have patterns of physical characteristics similar to those of FunCs, which is in agreement with the fact that the selection pressures of these interfaces is differently evolutionarily driven. As such, our ECR model also successfully describes the impact of evolution and natural selection on protein-protein interfaces. Finally, we indicate how our ECR method may be of use in reducing the false positive rate of docking calculations. PMID:24830938

Experimentally quantifying anion polarizability at the air/water interface.

PubMed

Tong, Yujin; Zhang, Igor Ying; Campen, R Kramer

2018-04-03

The adsorption of large, polarizable anions from aqueous solution on the air/water interface controls important atmospheric chemistry and is thought to resemble anion adsorption at hydrophobic interfaces generally. While the favourability of adsorption of such ions is clear, quantifying adsorption thermodynamics has proven challenging because it requires accurate description of the structure of the anion and its solvation shell at the interface. In principle anion polarizability offers a structural window, but to the best of our knowledge there has so far been no experimental technique that allowed its characterization with interfacial specificity. Here, we meet this challenge using interface-specific vibrational spectroscopy of Cl-O vibrations of the [Formula: see text] anion at the air/water interface and report that the interface breaks the symmetry of the anion, the anisotropy of [Formula: see text]'s polarizability tensor is more than two times larger than in bulk water and concentration dependent, and concentration-dependent polarizability changes are consistent with correlated changes in surface tension.

Protein docking prediction using predicted protein-protein interface.

PubMed

Li, Bin; Kihara, Daisuke

2012-01-10

Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm), is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

Protein denaturants at aqueous-hydrophobic interfaces: self-consistent correlation between induced interfacial fluctuations and denaturant stability at the interface.

PubMed

Cui, Di; Ou, Shu-Ching; Patel, Sandeep

2015-01-08

The notion of direct interaction between denaturing cosolvent and protein residues has been proposed in dialogue relevant to molecular mechanisms of protein denaturation. Here we consider the correlation between free energetic stability and induced fluctuations of an aqueous-hydrophobic interface between a model hydrophobically associating protein, HFBII, and two common protein denaturants, guanidinium cation (Gdm(+)) and urea. We compute potentials of mean force along an order parameter that brings the solute molecule close to the known hydrophobic region of the protein. We assess potentials of mean force for different relative orientations between the protein and denaturant molecule. We find that in both cases of guanidinium cation and urea relative orientations of the denaturant molecule that are parallel to the local protein-water interface exhibit greater stability compared to edge-on or perpendicular orientations. This behavior has been observed for guanidinium/methylguanidinium cations at the liquid-vapor interface of water, and thus the present results further corroborate earlier findings. Further analysis of the induced fluctuations of the aqueous-hydrophobic interface upon approach of the denaturant molecule indicates that the parallel orientation, displaying a greater stability at the interface, also induces larger fluctuations of the interface compared to the perpendicular orientations. The correlation of interfacial stability and induced interface fluctuation is a recurring theme for interface-stable solutes at hydrophobic interfaces. Moreover, observed correlations between interface stability and induced fluctuations recapitulate connections to local hydration structure and patterns around solutes as evidenced by experiment (Cooper et al., J. Phys. Chem. A 2014, 118, 5657.) and high-level ab initio/DFT calculations (Baer et al., Faraday Discuss 2013, 160, 89).

Peri-Implant Distribution of Polyethylene Debris in Postmortem-Retrieved Knee Arthroplasties: Can Polyethylene Debris Explain Loss of Cement-Bone Interlock in Successful Total Knee Arthroplasties?

PubMed

Cyndari, Karen I; Goodheart, Jacklyn R; Miller, Mark A; Oest, Megan E; Damron, Timothy A; Mann, Kenneth A

2017-07-01

Loss of mechanical interlock between cement and bone with in vivo service has been recently quantified for functioning, nonrevised, cemented total knee arthroplasties (TKAs). The cause of interlocking trabecular resorption is not known. The goal of this study is to quantify the distribution of PE debris at the cement-bone interface and determine if polyethylene (PE) debris is locally associated with loss of interlock. Fresh, nonrevised, postmortem-retrieved TKAs (n = 8) were obtained en bloc. Laboratory-prepared constructs (n = 2) served as negative controls. The intact cement-bone interface of each proximal tibia was embedded in Spurr's resin, sectioned, and imaged under polarized light to identify birefringent PE particles. PE wear particle number density was quantified at the cement-bone interface and distal to the interface, and then compared with local loss of cement-bone interlock. The average PE particle number density for postmortem-retrieved TKAs ranged from 8.6 (1.3) to 24.9 (3.1) particles/mm 2 (standard error) but was weakly correlated with years in service. The average particle number density was twice as high as distal (>5mm) to the interface compared to at the interface. The local loss of interlock at the interface was not related to the presence, absence, or particle density of PE. PE debris can migrate extensively along the cement-bone interface of well-fixed tibial components. However, the amount of local bone loss at the cement-bone interface was not correlated with the amount of PE debris at the interface, suggesting that the observed loss of trabecular interlock in these well-fixed TKAs may be due to alternative factors. Copyright © 2017 Elsevier Inc. All rights reserved.

Ordering Transitions in Liquid Crystals Permit Imaging of Spatial and Temporal Patterns Formed by Proteins Penetrating into Lipid-Laden Interfaces

PubMed Central

Daschner De Tercero, Maren; Abbott, Nicholas L.

2013-01-01

Recent studies have reported that full monolayers of L-α-dilaurylphosphatidylcholine (L-DLPC) and D-α-dipalmitoylphosphatidylcholine (D-DPPC) formed at interfaces between thermotropic liquid crystals (LCs) and aqueous phases lead to homeotropic (perpendicular) orientations of nematic LCs and that specific binding of proteins to these interfaces (such as phospholipase A2 binding to D-DPPC) can trigger orientational ordering transitions in the liquid crystals. We report on the nonspecific interactions of proteins with aqueous-LC interfaces decorated with partial monolayer coverage of L-DLPC. Whereas nonspecific interactions of four proteins (cytochrome c, bovine serum albumin,immunoglobulins, and neutravidin) do not perturb the ordering of the LC when a full monolayer of L-DLPC is assembled at the aqueous-LC interface, we observe patterned orientational transitions in the LC that reflect penetration of proteins into the interface of the LC with partial monolayer coverage of L-DLPC. The spatial patterns formed by the proteins and lipids at the interface are surprisingly complex, and in some cases the protein domains are found to compartmentalize lipid within the interfaces. These results suggest that phospholipid-decorated interfaces between thermotropic liquid crystals and aqueous phases offer the basis of a simple and versatile tool to study the spatial organization and dynamics ofprotein networks formed at mobile, lipid-decorated interfaces. PMID:23671353

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.

Functional structural motifs for protein-ligand, protein-protein, and protein-nucleic acid interactions and their connection to supersecondary structures.

PubMed

Kinjo, Akira R; Nakamura, Haruki

2013-01-01

Protein functions are mediated by interactions between proteins and other molecules. One useful approach to analyze protein functions is to compare and classify the structures of interaction interfaces of proteins. Here, we describe the procedures for compiling a database of interface structures and efficiently comparing the interface structures. To do so requires a good understanding of the data structures of the Protein Data Bank (PDB). Therefore, we also provide a detailed account of the PDB exchange dictionary necessary for extracting data that are relevant for analyzing interaction interfaces and secondary structures. We identify recurring structural motifs by classifying similar interface structures, and we define a coarse-grained representation of supersecondary structures (SSS) which represents a sequence of two or three secondary structure elements including their relative orientations as a string of four to seven letters. By examining the correspondence between structural motifs and SSS strings, we show that no SSS string has particularly high propensity to be found interaction interfaces in general, indicating any SSS can be used as a binding interface. When individual structural motifs are examined, there are some SSS strings that have high propensity for particular groups of structural motifs. In addition, it is shown that while the SSS strings found in particular structural motifs for nonpolymer and protein interfaces are as abundant as in other structural motifs that belong to the same subunit, structural motifs for nucleic acid interfaces exhibit somewhat stronger preference for SSS strings. In regard to protein folds, many motif-specific SSS strings were found across many folds, suggesting that SSS may be a useful description to investigate the universality of ligand binding modes.

Conservation of coevolving protein interfaces bridges prokaryote–eukaryote homologies in the twilight zone

PubMed Central

Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

2016-01-01

Protein–protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein–protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein–protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein–protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach. PMID:27965389

Protein aggregation and particle formation in prefilled glass syringes.

PubMed

Gerhardt, Alana; Mcgraw, Nicole R; Schwartz, Daniel K; Bee, Jared S; Carpenter, John F; Randolph, Theodore W

2014-06-01

The stability of therapeutic proteins formulated in prefilled syringes (PFS) may be negatively impacted by the exposure of protein molecules to silicone oil-water interfaces and air-water interfaces. In addition, agitation, such as that experienced during transportation, may increase the detrimental effects (i.e., protein aggregation and particle formation) of protein interactions with interfaces. In this study, surfactant-free formulations containing either a monoclonal antibody or lysozyme were incubated in PFS, where they were exposed to silicone oil-water interfaces (siliconized syringe walls), air-water interfaces (air bubbles), and agitation stress (occurring during end-over-end rotation). Using flow microscopy, particles (≥2 μm diameter) were detected under all conditions. The highest particle concentrations were found in agitated, siliconized syringes containing an air bubble. The particles formed in this condition consisted of silicone oil droplets and aggregated protein, as well as agglomerates of protein aggregates and silicone oil. We propose an interfacial mechanism of particle generation in PFS in which capillary forces at the three-phase (silicone oil-water-air) contact line remove silicone oil and gelled protein aggregates from the interface and transport them into the bulk. This mechanism explains the synergistic effects of silicone oil-water interfaces, air-water interfaces, and agitation in the generation of particles in protein formulations. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Protein-protein interface analysis and hot spots identification for chemical ligand design.

PubMed

Chen, Jing; Ma, Xiaomin; Yuan, Yaxia; Pei, Jianfeng; Lai, Luhua

2014-01-01

Rational design for chemical compounds targeting protein-protein interactions has grown from a dream to reality after a decade of efforts. There are an increasing number of successful examples, though major challenges remain in the field. In this paper, we will first give a brief review of the available methods that can be used to analyze protein-protein interface and predict hot spots for chemical ligand design. New developments of binding sites detection, ligandability and hot spots prediction from the author's group will also be described. Pocket V.3 is an improved program for identifying hot spots in protein-protein interface using only an apo protein structure. It has been developed based on Pocket V.2 that can derive receptor-based pharmacophore model for ligand binding cavity. Given similarities and differences between the essence of pharmacophore and hot spots for guiding design of chemical compounds, not only energetic but also spatial properties of protein-protein interface are used in Pocket V.3 for dealing with protein-protein interface. In order to illustrate the capability of Pocket V.3, two datasets have been used. One is taken from ASEdb and BID having experimental alanine scanning results for testing hot spots prediction. The other is taken from the 2P2I database containing complex structures of protein-ligand binding at the original protein-protein interface for testing hot spots application in ligand design.

Protein-Protein Interface and Disease: Perspective from Biomolecular Networks.

PubMed

Hu, Guang; Xiao, Fei; Li, Yuqian; Li, Yuan; Vongsangnak, Wanwipa

Protein-protein interactions are involved in many important biological processes and molecular mechanisms of disease association. Structural studies of interfacial residues in protein complexes provide information on protein-protein interactions. Characterizing protein-protein interfaces, including binding sites and allosteric changes, thus pose an imminent challenge. With special focus on protein complexes, approaches based on network theory are proposed to meet this challenge. In this review we pay attention to protein-protein interfaces from the perspective of biomolecular networks and their roles in disease. We first describe the different roles of protein complexes in disease through several structural aspects of interfaces. We then discuss some recent advances in predicting hot spots and communication pathway analysis in terms of amino acid networks. Finally, we highlight possible future aspects of this area with respect to both methodology development and applications for disease treatment.

JAIL: a structure-based interface library for macromolecules.

PubMed

Günther, Stefan; von Eichborn, Joachim; May, Patrick; Preissner, Robert

2009-01-01

The increasing number of solved macromolecules provides a solid number of 3D interfaces, if all types of molecular contacts are being considered. JAIL annotates three different kinds of macromolecular interfaces, those between interacting protein domains, interfaces of different protein chains and interfaces between proteins and nucleic acids. This results in a total number of about 184,000 database entries. All the interfaces can easily be identified by a detailed search form or by a hierarchical tree that describes the protein domain architectures classified by the SCOP database. Visual inspection of the interfaces is possible via an interactive protein viewer. Furthermore, large scale analyses are supported by an implemented sequential and by a structural clustering. Similar interfaces as well as non-redundant interfaces can be easily picked out. Additionally, the sequential conservation of binding sites was also included in the database and is retrievable via Jmol. A comprehensive download section allows the composition of representative data sets with user defined parameters. The huge data set in combination with various search options allow a comprehensive view on all interfaces between macromolecules included in the Protein Data Bank (PDB). The download of the data sets supports numerous further investigations in macromolecular recognition. JAIL is publicly available at http://bioinformatics.charite.de/jail.

Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin.

PubMed

Fuchs, Julian E; Huber, Roland G; Waldner, Birgit J; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R

2015-01-01

Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm "dynamics govern specificity" might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.

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.

FastRNABindR: Fast and Accurate Prediction of Protein-RNA Interface Residues.

PubMed

El-Manzalawy, Yasser; Abbas, Mostafa; Malluhi, Qutaibah; Honavar, Vasant

2016-01-01

A wide range of biological processes, including regulation of gene expression, protein synthesis, and replication and assembly of many viruses are mediated by RNA-protein interactions. However, experimental determination of the structures of protein-RNA complexes is expensive and technically challenging. Hence, a number of computational tools have been developed for predicting protein-RNA interfaces. Some of the state-of-the-art protein-RNA interface predictors rely on position-specific scoring matrix (PSSM)-based encoding of the protein sequences. The computational efforts needed for generating PSSMs severely limits the practical utility of protein-RNA interface prediction servers. In this work, we experiment with two approaches, random sampling and sequence similarity reduction, for extracting a representative reference database of protein sequences from more than 50 million protein sequences in UniRef100. Our results suggest that random sampled databases produce better PSSM profiles (in terms of the number of hits used to generate the profile and the distance of the generated profile to the corresponding profile generated using the entire UniRef100 data as well as the accuracy of the machine learning classifier trained using these profiles). Based on our results, we developed FastRNABindR, an improved version of RNABindR for predicting protein-RNA interface residues using PSSM profiles generated using 1% of the UniRef100 sequences sampled uniformly at random. To the best of our knowledge, FastRNABindR is the only protein-RNA interface residue prediction online server that requires generation of PSSM profiles for query sequences and accepts hundreds of protein sequences per submission. Our approach for determining the optimal BLAST database for a protein-RNA interface residue classification task has the potential of substantially speeding up, and hence increasing the practical utility of, other amino acid sequence based predictors of protein-protein and protein-DNA interfaces.

Proteins at the air-water interface in a lattice model

NASA Astrophysics Data System (ADS)

Zhao, Yani; Cieplak, Marek

2018-03-01

We construct a lattice protein version of the hydrophobic-polar model to study the effects of the air-water interface on the protein and on an interfacial layer formed through aggregation of many proteins. The basic unit of the model is a 14-mer that is known to have a unique ground state in three dimensions. The equilibrium and kinetic properties of the systems with and without the interface are studied through a Monte Carlo process. We find that the proteins at high dilution can be pinned and depinned many times from the air-water interface. When pinned, the proteins undergo deformation. The staying time depends on the strength of the coupling to the interface. For dense protein systems, we observe glassy effects. Thus, the lattice model yields results which are similar to those obtained through molecular dynamics in off-lattice models. In addition, we study dynamical effects induced by local temperature gradients in protein films.

Interface-related attributes of the Maillard reaction-born glycoproteins.

PubMed

Karbasi, Mehri; Madadlou, Ashkan

2017-01-19

Interfacial behavior of proteins which is a chief parameter to their emulsifying and foaming properties can be tailored through the Maillard reaction. The reaction can increase protein solubility at isoelectric point and ought to be controlled for example by high pressure processing to suppress melanoidins formation. Branched and long saccharides bring considerable steric hindrance which is associated with their site of conjugation to proteins. Conjugation with high molecular weight polysaccharides (such as 440 kDa dextran) may indeed increase the thickness of globular proteins interfacial film up to approximately 25 nm. However, an overly long saccharide can shield protein charge and slow down the electrophoretic mobility of conjugate. Maillard conjugation may decrease the diffusion rate of proteins to interface, allowing further unfolding at interface. As well, it can increase desorption iteration of proteins from interface. In addition to tempering proteins adsorption to interface, Maillard conjugation influences the rheology of protein membranes. Oligosaccharides (especially at higher glycation degrees) decrease the elastic modulus and Huggins constant of protein film; whereas, monosaccharides yield a more elastic interface. Accordingly, glycation of random coil proteins has been exploited to stiffen the corresponding interfacial membrane. Partial hydrolysis of proteins accompanied with anti-solvent-triggered nanoparticulation either before or after conjugation is a feasible way to enhance their emulsifying activity.

Osteoinductive recombinant silk fusion proteins for bone regeneration.

PubMed

Dinjaski, Nina; Plowright, Robyn; Zhou, Shun; Belton, David J; Perry, Carole C; Kaplan, David L

2017-02-01

Protein polymers provide a unique opportunity for tunable designs of material systems due to the genetic basis of sequence control. To address the challenge of biomineralization interfaces with protein based materials, we genetically engineered spider silks to design organic-inorganic hybrid systems. The spider silk inspired domain (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT) 15 served as an organic scaffold to control material stability and to allow multiple modes of processing, whereas the hydroxyapatite binding domain VTKHLNQISQSY (VTK), provided control over osteogenesis. The VTK domain was fused either to the N-, C- or both terminals of the spider silk domain to understand the effect of position on material properties and mineralization. The addition of the VTK domain to silk did not affect the physical properties of the silk recombinant constructs, but it had a critical role in the induction of biomineralization. When the VTK domain was placed on both the C- and N-termini the formation of crystalline hydroxyapatite was significantly increased. In addition, all of the recombinant proteins in film format supported the growth and proliferation of human mesenchymal stem cells (hMSCs). Importantly, the presence of the VTK domain enhanced osteoinductive properties up to 3-fold compared to the control (silk alone without VTK). Therefore, silk-VTK fusion proteins have been shown suitable for mineralization and functionalization for specific biomedical applications. Organic-inorganic interfaces are integral to biomaterial functions in many areas of repair and regeneration. Several protein polymers have been investigated for this purpose. Despite their success the limited options to fine-tune their material properties, degradation patterns and functionalize them for each specific biomedical application limits their application. Various studies have shown that the biological performance of such proteins can be improved by genetic engineering. The present study provides data relating protein design parameters and functional outcome quantified by biomineralization and human mesenchymal stem cell differentiation. As such, it helps the design of osteoinductive recombinant biomaterials for bone regeneration. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A comparison of successful and failed protein interface designs highlights the challenges of designing buried hydrogen bonds.

PubMed

Stranges, P Benjamin; Kuhlman, Brian

2013-01-01

The accurate design of new protein-protein interactions is a longstanding goal of computational protein design. However, most computationally designed interfaces fail to form experimentally. This investigation compares five previously described successful de novo interface designs with 158 failures. Both sets of proteins were designed with the molecular modeling program Rosetta. Designs were considered a success if a high-resolution crystal structure of the complex closely matched the design model and the equilibrium dissociation constant for binding was less than 10 μM. The successes and failures represent a wide variety of interface types and design goals including heterodimers, homodimers, peptide-protein interactions, one-sided designs (i.e., where only one of the proteins was mutated) and two-sided designs. The most striking feature of the successful designs is that they have fewer polar atoms at their interfaces than many of the failed designs. Designs that attempted to create extensive sets of interface-spanning hydrogen bonds resulted in no detectable binding. In contrast, polar atoms make up more than 40% of the interface area of many natural dimers, and native interfaces often contain extensive hydrogen bonding networks. These results suggest that Rosetta may not be accurately balancing hydrogen bonding and electrostatic energies against desolvation penalties and that design processes may not include sufficient sampling to identify side chains in preordered conformations that can fully satisfy the hydrogen bonding potential of the interface. Copyright © 2012 The Protein Society.

Hydration of protein–RNA recognition sites

PubMed Central

Barik, Amita; Bahadur, Ranjit Prasad

2014-01-01

We investigate the role of water molecules in 89 protein–RNA complexes taken from the Protein Data Bank. Those with tRNA and single-stranded RNA are less hydrated than with duplex or ribosomal proteins. Protein–RNA interfaces are hydrated less than protein–DNA interfaces, but more than protein–protein interfaces. Majority of the waters at protein–RNA interfaces makes multiple H-bonds; however, a fraction do not make any. Those making H-bonds have preferences for the polar groups of RNA than its partner protein. The spatial distribution of waters makes interfaces with ribosomal proteins and single-stranded RNA relatively ‘dry’ than interfaces with tRNA and duplex RNA. In contrast to protein–DNA interfaces, mainly due to the presence of the 2′OH, the ribose in protein–RNA interfaces is hydrated more than the phosphate or the bases. The minor groove in protein–RNA interfaces is hydrated more than the major groove, while in protein–DNA interfaces it is reverse. The strands make the highest number of water-mediated H-bonds per unit interface area followed by the helices and the non-regular structures. The preserved waters at protein–RNA interfaces make higher number of H-bonds than the other waters. Preserved waters contribute toward the affinity in protein–RNA recognition and should be carefully treated while engineering protein–RNA interfaces. PMID:25114050

Molecular simulation of hydrophobin adsorption at an oil-water interface.

PubMed

Cheung, David L

2012-06-12

Hydrophobins are small, amphiphilic proteins expressed by strains of filamentous fungi. They fulfill a number of biological functions, often related to adsorption at hydrophobic interfaces, and have been investigated for a number of applications in materials science and biotechnology. In order to understand the biological function and applications of these proteins, a microscopic picture of the adsorption of these proteins at interfaces is needed. Using molecular dynamics simulations with a chemically detailed coarse-grained potential, the behavior of typical hydrophobins at the water-octane interface is studied. Calculation of the interfacial adsorption strengths indicates that the adsorption is essentially irreversible, with adsorption strengths of the order of 100 k(B)T (comparable to values determined for synthetic nanoparticles but significantly larger than small molecule surfactants and biomolecules). The protein structure at the interface is unchanged at the interface, which is consistent with the biological function of these proteins. Comparison of native proteins with pseudoproteins that consist of uniform particles shows that the surface structure of these proteins has a large effect on the interfacial adsorption strengths, as does the flexibility of the protein.

Protein Denaturants at Aqueous–Hydrophobic Interfaces: Self-Consistent Correlation between Induced Interfacial Fluctuations and Denaturant Stability at the Interface

PubMed Central

2015-01-01

The notion of direct interaction between denaturing cosolvent and protein residues has been proposed in dialogue relevant to molecular mechanisms of protein denaturation. Here we consider the correlation between free energetic stability and induced fluctuations of an aqueous–hydrophobic interface between a model hydrophobically associating protein, HFBII, and two common protein denaturants, guanidinium cation (Gdm+) and urea. We compute potentials of mean force along an order parameter that brings the solute molecule close to the known hydrophobic region of the protein. We assess potentials of mean force for different relative orientations between the protein and denaturant molecule. We find that in both cases of guanidinium cation and urea relative orientations of the denaturant molecule that are parallel to the local protein–water interface exhibit greater stability compared to edge-on or perpendicular orientations. This behavior has been observed for guanidinium/methylguanidinium cations at the liquid–vapor interface of water, and thus the present results further corroborate earlier findings. Further analysis of the induced fluctuations of the aqueous–hydrophobic interface upon approach of the denaturant molecule indicates that the parallel orientation, displaying a greater stability at the interface, also induces larger fluctuations of the interface compared to the perpendicular orientations. The correlation of interfacial stability and induced interface fluctuation is a recurring theme for interface-stable solutes at hydrophobic interfaces. Moreover, observed correlations between interface stability and induced fluctuations recapitulate connections to local hydration structure and patterns around solutes as evidenced by experiment (Cooper et al., J. Phys. Chem. A2014, 118, 5657.) and high-level ab initio/DFT calculations (Baer et al., Faraday Discuss2013, 160, 89). PMID:25536388

Surface adsorption considerations when working with amyloid fibrils in multiwell plates and Eppendorf tubes.

PubMed

Murray, Amber N; Palhano, Fernando L; Bieschke, Jan; Kelly, Jeffery W

2013-11-01

The accumulation of cross-β-sheet amyloid fibrils is the hallmark of amyloid diseases. Recently, we reported the discovery of amyloid disaggregase activities in extracts from mammalian cells and Caenorhabditis elegans. However, we have discovered a problem with the interpretation of our previous results as Aβ disaggregation in vitro. Here, we show that Aβ fibrils adsorb to the plastic surface of multiwell plates and Eppendorf tubes. This adsorption is markedly increased in the presence of complex biological mixtures subjected to a denaturing air-water interface. The time-dependent loss of thioflavin T fluorescence that we interpreted previously as disaggregation is due to increased adsorption of Aβ amyloid to the surfaces of multiwell plates and Eppendorf tubes in the presence of biological extracts. As the proteins in biological extracts denature over time at the air-water interface due to agitation/shaking, their adsorption increases, in turn promoting adsorption of amyloid fibrils. We delineate important control experiments that quantify the extent of amyloid adsorption to the surface of plastic and quartz containers. Based on the results described in this article, we conclude that our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231-2241 and Murray et al., Protein Sci 2010;19:836-846 is invalid when used as evidence for a disaggregase activity. Thus, we correct the two prior publications reporting that worm or mammalian cell extracts disaggregate Aβ amyloid fibrils in vitro at 37°C (see Corrigenda in this issue of Protein Science). We apologize for misinterpreting our previous data and for any confounding experimental efforts this may have caused. © 2013 The Protein Society.

The protein-protein interface evolution acts in a similar way to antibody affinity maturation.

PubMed

Li, Bohua; Zhao, Lei; Wang, Chong; Guo, Huaizu; Wu, Lan; Zhang, Xunming; Qian, Weizhu; Wang, Hao; Guo, Yajun

2010-02-05

Understanding the evolutionary mechanism that acts at the interfaces of protein-protein complexes is a fundamental issue with high interest for delineating the macromolecular complexes and networks responsible for regulation and complexity in biological systems. To investigate whether the evolution of protein-protein interface acts in a similar way as antibody affinity maturation, we incorporated evolutionary information derived from antibody affinity maturation with common simulation techniques to evaluate prediction success rates of the computational method in affinity improvement in four different systems: antibody-receptor, antibody-peptide, receptor-membrane ligand, and receptor-soluble ligand. It was interesting to find that the same evolutionary information could improve the prediction success rates in all the four protein-protein complexes with an exceptional high accuracy (>57%). One of the most striking findings in our present study is that not only in the antibody-combining site but in other protein-protein interfaces almost all of the affinity-enhancing mutations are located at the germline hotspot sequences (RGYW or WA), indicating that DNA hot spot mechanisms may be widely used in the evolution of protein-protein interfaces. Our data suggest that the evolution of distinct protein-protein interfaces may use the same basic strategy under selection pressure to maintain interactions. Additionally, our data indicate that classical simulation techniques incorporating the evolutionary information derived from in vivo antibody affinity maturation can be utilized as a powerful tool to improve the binding affinity of protein-protein complex with a high accuracy.

Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin

PubMed Central

Fuchs, Julian E.; Huber, Roland G.; Waldner, Birgit J.; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R.

2015-01-01

Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm “dynamics govern specificity” might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design. PMID:26496636

Effects of the conjugation of whey proteins with gellan polysaccharides on surfactant-induced competitive displacement from the air-water interface.

PubMed

Cai, B; Ikeda, S

2016-08-01

Whey proteins can be used to stabilize foams and emulsions against coalescence because of their ability to form viscoelastic films at the interface that resist film rupture on collision between colloidal particles. However, whey proteins are competitively displaced from the interface if small-molecule surfactants are added, leading to destabilization of the entire system. This is because surfactants are more effective in molecular packing at the interface, and they lower interfacial tension to a greater degree than whey proteins do, but their interfacial films are poor in viscoelasticity. We hypothesized that whey proteins would become more resistant to surfactant-induced competitive displacement if they were conjugated with network-forming polysaccharides. The protein moiety of the conjugate would be expected to enable its adsorption to the interface, and the polysaccharide moiety would be expected to form self-assembled networks, strengthening the interfacial film as a whole. In this study, whey proteins were conjugated with gellan polysaccharides using the Maillard reaction. Atomic force microscopy images of interfacial films formed by the whey protein-gellan conjugate at the air-water interface and transferred onto mica sheets using the Langmuir-Blodgett method revealed that gellan did form self-assembled networks at the interface and that interfacial films also contained a large number of unconjugated whey protein molecules. Following the addition of a small-molecule surfactant (Tween 20) to the sub-phase, surface pressure increased, indicating spontaneous adsorption of surfactants to the interface. Atomic force microscopy images showed decreases in interfacial area coverage by whey proteins as surface pressure increased. At a given surface pressure, the interfacial area coverage by whey protein-gellan conjugates was greater than coverage by unconjugated whey proteins, confirming that whey proteins became more resistant to surfactant-induced displacement after conjugation with gellan. Furthermore, gellan molecules added to the sub-phase after the formation of a monolayer of whey proteins at the air-water interface did not adsorb to the interfacial protein film. These results provide a molecular basis for designing interfacial structures to enhance the stability of colloidal systems. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Versatility and Invariance in the Evolution of Homologous Heteromeric Interfaces

PubMed Central

Andreani, Jessica; Faure, Guilhem; Guerois, Raphaël

2012-01-01

Evolutionary pressures act on protein complex interfaces so that they preserve their complementarity. Nonetheless, the elementary interactions which compose the interface are highly versatile throughout evolution. Understanding and characterizing interface plasticity across evolution is a fundamental issue which could provide new insights into protein-protein interaction prediction. Using a database of 1,024 couples of close and remote heteromeric structural interologs, we studied protein-protein interactions from a structural and evolutionary point of view. We systematically and quantitatively analyzed the conservation of different types of interface contacts. Our study highlights astonishing plasticity regarding polar contacts at complex interfaces. It also reveals that up to a quarter of the residues switch out of the interface when comparing two homologous complexes. Despite such versatility, we identify two important interface descriptors which correlate with an increased conservation in the evolution of interfaces: apolar patches and contacts surrounding anchor residues. These observations hold true even when restricting the dataset to transiently formed complexes. We show that a combination of six features related either to sequence or to geometric properties of interfaces can be used to rank positions likely to share similar contacts between two interologs. Altogether, our analysis provides important tracks for extracting meaningful information from multiple sequence alignments of conserved binding partners and for discriminating near-native interfaces using evolutionary information. PMID:22952442

Computational structure analysis of biomacromolecule complexes by interface geometry.

PubMed

Mahdavi, Sedigheh; Salehzadeh-Yazdi, Ali; Mohades, Ali; Masoudi-Nejad, Ali

2013-12-01

The ability to analyze and compare protein-nucleic acid and protein-protein interaction interface has critical importance in understanding the biological function and essential processes occurring in the cells. Since high-resolution three-dimensional (3D) structures of biomacromolecule complexes are available, computational characterizing of the interface geometry become an important research topic in the field of molecular biology. In this study, the interfaces of a set of 180 protein-nucleic acid and protein-protein complexes are computed to understand the principles of their interactions. The weighted Voronoi diagram of the atoms and the Alpha complex has provided an accurate description of the interface atoms. Our method is implemented in the presence and absence of water molecules. A comparison among the three types of interaction interfaces show that RNA-protein complexes have the largest size of an interface. The results show a high correlation coefficient between our method and the PISA server in the presence and absence of water molecules in the Voronoi model and the traditional model based on solvent accessibility and the high validation parameters in comparison to the classical model. Copyright © 2013 Elsevier Ltd. All rights reserved.

Vibrational Stark effect spectroscopy reveals complementary electrostatic fields created by protein-protein binding at the interface of Ras and Ral.

PubMed

Walker, David M; Hayes, Ellen C; Webb, Lauren J

2013-08-07

Electrostatic fields at the interface of the GTPase H-Ras (Ras) docked with the Ras binding domain of the protein Ral guanine nucleoside dissociation stimulator (Ral) were measured with vibrational Stark effect (VSE) spectroscopy. Nine residues on the surface of Ras that participate in the protein-protein interface were systematically mutated to cysteine and subsequently converted to cyanocysteine in order to introduce a nitrile VSE probe into the protein-protein interface. The absorption energy of the nitrile was measured both on the surface of Ras in its monomeric state, then after incubation with the Ras binding domain of Ral to form the docked complex. Boltzmann-weighted structural snapshots of the nitrile-labeled Ras protein were generated both in monomeric and docked configurations from molecular dynamics simulations using enhanced sampling of the cyanocysteine side chain's χ2 dihedral angle. These snapshots were used to determine that on average, most of the nitrile probes were aligned along the Ras surface, parallel to the Ras-Ral interface. The average solvent-accessible surface areas (SASA) of the cyanocysteine side chain were found to be <60 Å(2) for all measured residues, and was not significantly different whether the nitrile was on the surface of the Ras monomer or immersed in the docked complex. Changes in the absorption energy of the nitrile probe at nine positions along the Ras-Ral interface were compared to results of a previous study examining this interface with Ral-based probes, and found a pattern of low electrostatic field in the core of the interface surrounded by a ring of high electrostatic field around the perimeter of the interface. These data are used to rationalize several puzzling features of the Ras-Ral interface.

Molecular dynamics simulation of the cooperative adsorption of barley lipid transfer protein and cis-isocohumulone at the vacuum-water interface.

PubMed

Euston, S R; Hughes, P; Naser, Md A; Westacott, R E

2008-11-01

Molecular dynamic simulations have been carried out on systems containing a mixture of barley lipid transfer protein (LTP) and cis-isocohumulone (a hop derived iso-alpha-acid) in one of its enol forms, in bulk water and at the vacuum-water interface. In solution, the cis-isocohumulone molecules bind to the surface of the LTP molecule. The mechanism of binding appears to be purely hydrophobic in nature via desolvation of the protein surface. Binding of hop acids to the LTP leads to a small change in the 3-D conformation of the protein, but no change in the proportion of secondary structure present in helices, even though there is a significant degree of hop acid binding to the helical regions. At the vacuum-water interface, cis-isocohumulone shows a high surface activity and adsorbs rapidly at the interface. LTP then shows a preference to bind to the preadsorbed hop acid layer at the interface rather than to the bare water-vacuum interface. The free energy of adsorption of LTP at the hop-vacuum-water interface is more favorable than for adsorption at the vacuum-water interface. Our results support the view that hop iso-alpha-acids promote beer foam stability by forming bridges between separate adsorbed protein molecules, thus strengthening the adsorbed protein layer and reducing foam breakdown by lamellar phase drainage. The results also suggest a second mechanism may also occur, whereby the concentration of protein at the interface is increased via enhanced protein adsorption to adsorbed hop acid layers. This too would increase foam stability through its effect on the stabilizing protein layer around the foam bubbles.

Human Apolipoprotein A1 at Solid/Liquid and Liquid/Gas Interfaces.

PubMed

Dogan, Susanne; Paulus, Michael; Forov, Yury; Weis, Christopher; Kampmann, Matthias; Cewe, Christopher; Kiesel, Irena; Degen, Patrick; Salmen, Paul; Rehage, Heinz; Tolan, Metin

2018-04-12

An X-ray reflectivity study on the adsorption behavior of human apolipoprotein A1 (apoA1) at hydrophilic and hydrophobic interfaces is presented. It is shown that the protein interacts via electrostatic and hydrophobic interactions with the interfaces, resulting in the absorption of the protein. pH dependent measurements at the solid/liquid interface between silicon dioxide and aqueous protein solution show that in a small pH range between pH 4 and 6, adsorption is increased due to electrostatic attraction. Here, the native shape of the protein seems to be conserved. In contrast, the adsorption at the liquid/gas interface is mainly driven by hydrophobic effects, presumably by extending the hydrophobic regions of the amphipathic helices, and results in a conformational change of the protein during adsorption. However, the addition of differently charged membrane-forming lipids at the liquid/gas interface illustrates the ability of apoA1 to include lipids, resulting in a depletion of the lipids from the interface.

Mapping a Noncovalent Protein-Peptide Interface by Top-Down FTICR Mass Spectrometry Using Electron Capture Dissociation

NASA Astrophysics Data System (ADS)

Clarke, David J.; Murray, Euan; Hupp, Ted; Mackay, C. Logan; Langridge-Smith, Pat R. R.

2011-08-01

Noncovalent protein-ligand and protein-protein complexes are readily detected using electrospray ionization mass spectrometry (ESI MS). Furthermore, recent reports have demonstrated that careful use of electron capture dissociation (ECD) fragmentation allows covalent backbone bonds of protein complexes to be dissociated without disruption of noncovalent protein-ligand interactions. In this way the site of protein-ligand interfaces can be identified. To date, protein-ligand complexes, which have proven tractable to this technique, have been mediated by ionic electrostatic interactions, i.e., ion pair interactions or salt bridging. Here we extend this methodology by applying ECD to study a protein-peptide complex that contains no electrostatics interactions. We analyzed the complex between the 21 kDa p53-inhibitor protein anterior gradient-2 and its hexapeptide binding ligand (PTTIYY). ECD fragmentation of the 1:1 complex occurs with retention of protein-peptide binding and analysis of the resulting fragments allows the binding interface to be localized to a C-terminal region between residues 109 and 175. These finding are supported by a solution-phase competition assay, which implicates the region between residues 108 and 122 within AGR2 as the PTTIYY binding interface. Our study expands previous findings by demonstrating that top-down ECD mass spectrometry can be used to determine directly the sites of peptide-protein interfaces. This highlights the growing potential of using ECD and related top-down fragmentation techniques for interrogation of protein-protein interfaces.

Incorporating both physical and kinetic limitations in quantifying dissolved oxygen flux to aquatic sediments

USGS Publications Warehouse

O'Connor, B.L.; Hondzo, Miki; Harvey, J.W.

2009-01-01

Traditionally, dissolved oxygen (DO) fluxes have been calculated using the thin-film theory with DO microstructure data in systems characterized by fine sediments and low velocities. However, recent experimental evidence of fluctuating DO concentrations near the sediment-water interface suggests that turbulence and coherent motions control the mass transfer, and the surface renewal theory gives a more mechanistic model for quantifying fluxes. Both models involve quantifying the mass transfer coefficient (k) and the relevant concentration difference (??C). This study compared several empirical models for quantifying k based on both thin-film and surface renewal theories, as well as presents a new method for quantifying ??C (dynamic approach) that is consistent with the observed DO concentration fluctuations near the interface. Data were used from a series of flume experiments that includes both physical and kinetic uptake limitations of the flux. Results indicated that methods for quantifying k and ??C using the surface renewal theory better estimated the DO flux across a range of fluid-flow conditions. ?? 2009 ASCE.

Protein-surfactant interactions at hydrophobic interfaces studied with total internal reflection fluorescence correlation spectroscopy (TIR-FCS).

PubMed

Sonesson, Andreas W; Blom, Hans; Hassler, Kai; Elofsson, Ulla M; Callisen, Thomas H; Widengren, Jerker; Brismar, Hjalmar

2008-01-15

The aim of this work was to study the dynamics of proteins near solid surfaces in the presence or absence of competing surfactants by means of total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Two different proteins were studied, bovine serum albumin (BSA) and Thermomyces lanuginosus lipase (TLL). A nonionic/anionic (C12E6/LAS) surfactant composition was used to mimic a detergent formulation and the surfaces used were C18 terminated glass. It was found that with increasing surfactant concentrations the term in the autocorrelation function (ACF) representing surface binding decreased. This suggested that the proteins were competed off the hydrophobic surface by the surfactant. When fitting the measured ACF to a model for surface kinetics, it was seen that with raised C12E6/LAS concentration, the surface interaction rate increased for both proteins. Under these experimental conditions this meant that the time the protein was bound to the surface decreased. At 10 microM C12E6/LAS the surface interaction was not visible for BSA, whereas it was still distinguishable in the ACF for TLL. This indicated that TLL had a higher affinity than BSA for the C18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption.

Revealing the global map of protein folding space by large-scale simulations

NASA Astrophysics Data System (ADS)

Sinner, Claude; Lutz, Benjamin; Verma, Abhinav; Schug, Alexander

2015-12-01

The full characterization of protein folding is a remarkable long-standing challenge both for experiment and simulation. Working towards a complete understanding of this process, one needs to cover the full diversity of existing folds and identify the general principles driving the process. Here, we want to understand and quantify the diversity in folding routes for a large and representative set of protein topologies covering the full range from all alpha helical topologies towards beta barrels guided by the key question: Does the majority of the observed routes contribute to the folding process or only a particular route? We identified a set of two-state folders among non-homologous proteins with a sequence length of 40-120 residues. For each of these proteins, we ran native-structure based simulations both with homogeneous and heterogeneous contact potentials. For each protein, we simulated dozens of folding transitions in continuous uninterrupted simulations and constructed a large database of kinetic parameters. We investigate folding routes by tracking the formation of tertiary structure interfaces and discuss whether a single specific route exists for a topology or if all routes are equiprobable. These results permit us to characterize the complete folding space for small proteins in terms of folding barrier ΔG‡, number of routes, and the route specificity RT.

Relating the variation of secondary structure of gelatin at fish oil-water interface to adsorption kinetics, dynamic interfacial tension and emulsion stability.

PubMed

Liu, Huihua; Wang, Bo; Barrow, Colin J; Adhikari, Benu

2014-01-15

The objectives of this study were to quantify the relationship between secondary structure of gelatin and its adsorption at the fish-oil/water interface and to quantify the implication of the adsorption on the dynamic interfacial tension (DST) and emulsion stability. The surface hydrophobicity of the gelatin solutions decreased when the pH increased from 4.0 to 6.0, while opposite tend was observed in the viscosity of the solution. The DST values decreased as the pH increased from 4.0 to 6.0, indicating that higher positive charges (measured trough zeta potential) in the gelatin solution tended to result in higher DST values. The adsorption kinetics of the gelatin solution was examined through the calculated diffusion coefficients (Deff). The addition of acid promoted the random coil and β-turn structures at the expense of α-helical structure. The addition of NaOH decreased the β-turn and increased the α-helix and random coil. The decrease in the random coil and triple helix structures in the gelatin solution resulted into increased Deff values. The highest diffusion coefficients, the highest emulsion stability and the lowest amount of random coil and triple helix structures were observed at pH=4.8. The lowest amount of random coil and triple helix structures in the interfacial protein layer correlated with the highest stability of the emulsion (highest ESI value). The lower amount of random coil and triple helix structures allowed higher coverage of the oil-water interface by relatively highly ordered secondary structure of gelatin. Copyright © 2013 Elsevier Ltd. All rights reserved.

Adsorption of the natural protein surfactant Rsn-2 onto liquid interfaces.

PubMed

Brandani, Giovanni B; Vance, Steven J; Schor, Marieke; Cooper, Alan; Kennedy, Malcolm W; Smith, Brian O; MacPhee, Cait E; Cheung, David L

2017-03-22

To stabilize foams, droplets and films at liquid interfaces a range of protein biosurfactants have evolved in nature. Compared to synthetic surfactants, these combine surface activity with biocompatibility and low solution aggregation. One recently studied example is Rsn-2, a component of the foam nest of the frog Engystomops pustulosus, which has been predicted to undergo a clamshell-like opening transition at the air-water interface. Using atomistic molecular dynamics simulations and surface tension measurements we study the adsorption of Rsn-2 onto air-water and cyclohexane-water interfaces. The protein adsorbs readily at both interfaces, with adsorption mediated by the hydrophobic N-terminus. At the cyclohexane-water interface the clamshell opens, due to the favourable interaction between hydrophobic residues and cyclohexane molecules and the penetration of cyclohexane molecules into the protein core. Simulations of deletion mutants showed that removal of the N-terminus inhibits interfacial adsorption, which is consistent with the surface tension measurements. Deletion of the hydrophilic C-terminus also affects adsorption, suggesting that this plays a role in orienting the protein at the interface. The characterisation of the interfacial behaviour gives insight into the factors that control the interfacial adsorption of proteins, which may inform new applications of this and similar proteins in areas including drug delivery and food technology and may also be used in the design of synthetic molecules showing similar changes in conformation at interfaces.

A Graph Approach to Mining Biological Patterns in the Binding Interfaces.

PubMed

Cheng, Wen; Yan, Changhui

2017-01-01

Protein-RNA interactions play important roles in the biological systems. Searching for regular patterns in the Protein-RNA binding interfaces is important for understanding how protein and RNA recognize each other and bind to form a complex. Herein, we present a graph-mining method for discovering biological patterns in the protein-RNA interfaces. We represented known protein-RNA interfaces using graphs and then discovered graph patterns enriched in the interfaces. Comparison of the discovered graph patterns with UniProt annotations showed that the graph patterns had a significant overlap with residue sites that had been proven crucial for the RNA binding by experimental methods. Using 200 patterns as input features, a support vector machine method was able to classify protein surface patches into RNA-binding sites and non-RNA-binding sites with 84.0% accuracy and 88.9% precision. We built a simple scoring function that calculated the total number of the graph patterns that occurred in a protein-RNA interface. That scoring function was able to discriminate near-native protein-RNA complexes from docking decoys with a performance comparable with that of a state-of-the-art complex scoring function. Our work also revealed possible patterns that might be important for binding affinity.

The film tells the story: Physical-chemical characteristics of IgG at the liquid-air interface.

PubMed

Koepf, Ellen; Schroeder, Rudolf; Brezesinski, Gerald; Friess, Wolfgang

2017-10-01

The presence of liquid-air interfaces in protein pharmaceuticals is known to negatively impact product stability. Nevertheless, the mechanisms behind interface-related protein aggregation are not yet fully understood. Little is known about the physical-chemical behavior of proteins adsorbed to the interface. Therefore, the combinatorial use of appropriate surface-sensitive analytical methods such as Langmuir trough experiments, Infrared Reflection-Absorption Spectroscopy (IRRAS), Brewster Angle Microscopy (BAM), and Atomic Force Microscopy (AFM) is highly expedient to uncover structures and events at the liquid-air interface directly. Concentration-dependent adsorption of a human immunoglobulin G (IgG) and characteristic surface-pressure/area isotherms substantiated the amphiphilic nature of the protein molecules as well as the formation of a compressible protein film at the liquid-air interface. Upon compression, the IgG molecules do not readily desorb but form a highly compressible interfacial film. IRRA spectra proved not only the presence of the protein at the interface, but also showed that the secondary structure does not change considerably during adsorption or compression. IRRAS experiments at different angles of incidence indicated that the film thickness and/or packing density increases upon compression. Furthermore, BAM images exposed the presence of a coherent but heterogeneous distribution of the protein at the interface. Topographical differences within the protein film after adsorption, compression and decompression were revealed using underwater AFM. The combinatorial use of physical-chemical, spectroscopic and microscopic methods provided useful insights into the liquid-air interfacial protein behavior and revealed the formation of a continuous but inhomogeneous film of native-like protein molecules whose topographical appearance is affected by compressive forces. Copyright © 2017 Elsevier B.V. All rights reserved.

Surface shapes and surrounding environment analysis of single- and double-stranded DNA-binding proteins in protein-DNA interface.

PubMed

Wang, Wei; Liu, Juan; Sun, Lin

2016-07-01

Protein-DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the residues shape (peak, flat, or valley) and the surrounding environment of double-stranded DNA-binding proteins (DSBs) and single-stranded DNA-binding proteins (SSBs) in protein-DNA interfaces. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins. Built on the investigation results, we constructed a random forest (RF) classifier to distinguish DSBs and SSBs with satisfying performance. In conclusion, we present a novel methodology to characterize protein interfaces, which will deepen our understanding of the specificity of proteins binding to ssDNA (single-stranded DNA) or dsDNA (double-stranded DNA). Proteins 2016; 84:979-989. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Beta-Strand Interfaces of Non-Dimeric Protein Oligomers Are Characterized by Scattered Charged Residue Patterns

PubMed Central

Feverati, Giovanni; Achoch, Mounia; Zrimi, Jihad; Vuillon, Laurent; Lesieur, Claire

2012-01-01

Protein oligomers are formed either permanently, transiently or even by default. The protein chains are associated through intermolecular interactions constituting the protein interface. The protein interfaces of 40 soluble protein oligomers of stœchiometries above two are investigated using a quantitative and qualitative methodology, which analyzes the x-ray structures of the protein oligomers and considers their interfaces as interaction networks. The protein oligomers of the dataset share the same geometry of interface, made by the association of two individual β-strands (β-interfaces), but are otherwise unrelated. The results show that the β-interfaces are made of two interdigitated interaction networks. One of them involves interactions between main chain atoms (backbone network) while the other involves interactions between side chain and backbone atoms or between only side chain atoms (side chain network). Each one has its own characteristics which can be associated to a distinct role. The secondary structure of the β-interfaces is implemented through the backbone networks which are enriched with the hydrophobic amino acids favored in intramolecular β-sheets (MCWIV). The intermolecular specificity is provided by the side chain networks via positioning different types of charged residues at the extremities (arginine) and in the middle (glutamic acid and histidine) of the interface. Such charge distribution helps discriminating between sequences of intermolecular β-strands, of intramolecular β-strands and of β-strands forming β-amyloid fibers. This might open new venues for drug designs and predictive tool developments. Moreover, the β-strands of the cholera toxin B subunit interface, when produced individually as synthetic peptides, are capable of inhibiting the assembly of the toxin into pentamers. Thus, their sequences contain the features necessary for a β-interface formation. Such β-strands could be considered as ‘assemblons’, independent associating units, by homology to the foldons (independent folding unit). Such property would be extremely valuable in term of assembly inhibitory drug development. PMID:22496732

Polysaccharide chemistry regulates kinetics of calcite nucleation through competition of interfacial energies.

PubMed

Giuffre, Anthony J; Hamm, Laura M; Han, Nizhou; De Yoreo, James J; Dove, Patricia M

2013-06-04

Calcified skeletons are produced within complex assemblages of proteins and polysaccharides whose roles in mineralization are not well understood. Here we quantify the kinetics of calcite nucleation onto a suite of high-purity polysaccharide (PS) substrates under controlled conditions. The energy barriers to nucleation are PS-specific by a systematic relationship to PS charge density and substrate structure that is rooted in minimization of the competing substrate-crystal and substrate-liquid interfacial energies. Chitosan presents a low-energy barrier to nucleation because its near-neutral charge favors formation of a substrate-crystal interface, thus reducing substrate interactions with water. Progressively higher barriers are measured for negatively charged alginates and heparin that favor contact with the solution over the formation of new substrate-crystal interfaces. The findings support a directing role for PS in biomineral formation and demonstrate that substrate-crystal interactions are one end-member in a larger continuum of competing forces that regulate heterogeneous crystal nucleation.

Polysaccharide chemistry regulates kinetics of calcite nucleation through competition of interfacial energies

PubMed Central

Hamm, Laura M.; Han, Nizhou; De Yoreo, James J.; Dove, Patricia M.

2013-01-01

Calcified skeletons are produced within complex assemblages of proteins and polysaccharides whose roles in mineralization are not well understood. Here we quantify the kinetics of calcite nucleation onto a suite of high-purity polysaccharide (PS) substrates under controlled conditions. The energy barriers to nucleation are PS-specific by a systematic relationship to PS charge density and substrate structure that is rooted in minimization of the competing substrate–crystal and substrate–liquid interfacial energies. Chitosan presents a low-energy barrier to nucleation because its near-neutral charge favors formation of a substrate–crystal interface, thus reducing substrate interactions with water. Progressively higher barriers are measured for negatively charged alginates and heparin that favor contact with the solution over the formation of new substrate–crystal interfaces. The findings support a directing role for PS in biomineral formation and demonstrate that substrate–crystal interactions are one end-member in a larger continuum of competing forces that regulate heterogeneous crystal nucleation. PMID:23690577

Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

PubMed

Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

2015-03-23

Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

Predicting the Effect of Mutations on Protein-Protein Binding Interactions through Structure-Based Interface Profiles

PubMed Central

Brender, Jeffrey R.; Zhang, Yang

2015-01-01

The formation of protein-protein complexes is essential for proteins to perform their physiological functions in the cell. Mutations that prevent the proper formation of the correct complexes can have serious consequences for the associated cellular processes. Since experimental determination of protein-protein binding affinity remains difficult when performed on a large scale, computational methods for predicting the consequences of mutations on binding affinity are highly desirable. We show that a scoring function based on interface structure profiles collected from analogous protein-protein interactions in the PDB is a powerful predictor of protein binding affinity changes upon mutation. As a standalone feature, the differences between the interface profile score of the mutant and wild-type proteins has an accuracy equivalent to the best all-atom potentials, despite being two orders of magnitude faster once the profile has been constructed. Due to its unique sensitivity in collecting the evolutionary profiles of analogous binding interactions and the high speed of calculation, the interface profile score has additional advantages as a complementary feature to combine with physics-based potentials for improving the accuracy of composite scoring approaches. By incorporating the sequence-derived and residue-level coarse-grained potentials with the interface structure profile score, a composite model was constructed through the random forest training, which generates a Pearson correlation coefficient >0.8 between the predicted and observed binding free-energy changes upon mutation. This accuracy is comparable to, or outperforms in most cases, the current best methods, but does not require high-resolution full-atomic models of the mutant structures. The binding interface profiling approach should find useful application in human-disease mutation recognition and protein interface design studies. PMID:26506533

Protein-protein interfaces are vdW dominant with selective H-bonds and (or) electrostatics towards broad functional specificity.

PubMed

Nilofer, Christina; Sukhwal, Anshul; Mohanapriya, Arumugam; Kangueane, Pandjassarame

2017-01-01

Several catalysis, cellular regulation, immune function, cell wall assembly, transport, signaling and inhibition occur through Protein- Protein Interactions (PPI). This is possible with the formation of specific yet stable protein-protein interfaces. Therefore, it is of interest to understand its molecular principles using structural data in relation to known function. Several interface features have been documented using known X-ray structures of protein complexes since 1975. This has improved our understanding of the interface using structural features such as interface area, binding energy, hydrophobicity, relative hydrophobicity, salt bridges and hydrogen bonds. The strength of binding between two proteins is dependent on interface size (number of residues at the interface) and thus its corresponding interface area. It is known that large interfaces have high binding energy (sum of (van der Waals) vdW, H-bonds, electrostatics). However, the selective role played by each of these energy components and more especially that of vdW is not explicitly known. Therefore, it is important to document their individual role in known protein-protein structural complexes. It is of interest to relate interface size with vdW, H-bonds and electrostatic interactions at the interfaces of protein structural complexes with known function using statistical and multiple linear regression analysis methods to identify the prominent force. We used the manually curated non-redundant dataset of 278 hetero-dimeric protein structural complexes grouped using known functions by Sowmya et al. (2015) to gain additional insight to this phenomenon using a robust inter-atomic non-covalent interaction analyzing tool PPCheck (Anshul and Sowdhamini, 2015). This dataset consists of obligatory (enzymes, regulator, biological assembly), immune and nonobligatory (enzyme and regulator inhibitors) complexes. Results show that the total binding energy is more for large interfaces. However, this is not true for its individual energy factors. Analysis shows that vdW energies contribute to about 75% ± 11% on average among all complexes and it also increases with interface size (r2 ranging from 0.67 to 0.89 with p<0.01) at 95% confidence limit irrespective of molecular function. Thus, vdW is both dominant and proportional at the interface independent of molecular function. Nevertheless, H bond energy contributes to 15% ± 6.5% on average in these complexes. It also moderately increases with interface size (r2 ranging from 0.43 to 0.61 with p<0.01) only among obligatory and immune complexes. Moreover, there is about 11.3% ± 8.7% contribution by electrostatic energy. It increases with interface size specifically among non-obligatory regulator-inhibitors (r2 = 0.44). It is implied that both H-bonds and electrostatics are neither dominant nor proportional at the interface. Nonetheless, their presence cannot be ignored in binding. Therefore, H-bonds and (or) electrostatic energy having specific role for improved stability in complexes is implied. Thus, vdW is common at the interface stabilized further with selective H-bonds and (or) electrostatic interactions at an atomic level in almost all complexes. Comparison of this observation with residue level analysis of the interface is compelling. The role by H-bonds (14.83% ± 6.5% and r2 = 0.61 with p<0.01) among obligatory and electrostatic energy (8.8% ± 4.77% and r2 = 0.63 with p <0.01) among non-obligatory complexes within interfaces (class A) having more non-polar residues than surface is influencing our inference. However, interfaces (class B) having less non-polar residues than surface show 1.5 fold more electrostatic energy on average. The interpretation of the interface using inter-atomic (vdW, H-bonds, electrostatic) interactions combined with inter-residue predominance (class A and class B) in relation to known function is the key to reveal its molecular principles with new challenges.

Structural adaptation of the subunit interface of oligomeric thermophilic and hyperthermophilic enzymes.

PubMed

Maugini, Elisa; Tronelli, Daniele; Bossa, Francesco; Pascarella, Stefano

2009-04-01

Enzymes from thermophilic and, particularly, from hyperthermophilic organisms are surprisingly stable. Understanding of the molecular origin of protein thermostability and thermoactivity attracted the interest of many scientist both for the perspective comprehension of the principles of protein structure and for the possible biotechnological applications through application of protein engineering. Comparative studies at sequence and structure levels were aimed at detecting significant differences of structural parameters related to protein stability between thermophilic and hyperhermophilic structures and their mesophilic homologs. Comparative studies were useful in the identification of a few recurrent themes which the evolution utilized in different combinations in different protein families. These studies were mostly carried out at the monomer level. However, maintenance of a proper quaternary structure is an essential prerequisite for a functional macromolecule. At the environmental temperatures experienced typically by hyper- and thermophiles, the subunit interactions mediated by the interface must be sufficiently stable. Our analysis was therefore aimed at the identification of the molecular strategies adopted by evolution to enhance interface thermostability of oligomeric enzymes. The variation of several structural properties related to protein stability were tested at the subunit interfaces of thermophilic and hyperthermophilic oligomers. The differences of the interface structural features observed between the hyperthermophilic and thermophilic enzymes were compared with the differences of the same properties calculated from pairwise comparisons of oligomeric mesophilic proteins contained in a reference dataset. The significance of the observed differences of structural properties was measured by a t-test. Ion pairs and hydrogen bonds do not vary significantly while hydrophobic contact area increases specially in hyperthermophilic interfaces. Interface compactness also appears to increase in the hyperthermophilic proteins. Variations of amino acid composition at the interfaces reflects the variation of the interface properties.

Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces

DOE PAGES

Bordner, Andrew J.; Gorin, Andrey A.

2008-05-12

Here, protein-protein interactions are ubiquitous and essential for cellular processes. High-resolution X-ray crystallographic structures of protein complexes can elucidate the details of their function and provide a basis for many computational and experimental approaches. Here we demonstrate that existing annotations of protein complexes, including those provided by the Protein Data Bank (PDB) itself, contain a significant fraction of incorrect annotations. Results: We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1) comprehensively collecting all protein-protein interfaces; (2) clustering similar protein-protein interfaces together; (3) estimating the probability that each cluster ismore » relevant based on a diverse set of properties; and (4) finally combining these scores for each entry in order to predict the complex structure. Unlike previous annotation methods, consistent prediction of complexes with identical or almost identical protein content is insured. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions.« less

Hot-spot residues at the E9/Im9 interface help binding via different mechanisms.

PubMed

Wong, Sergio E; Baron, Riccardo; McCammon, J Andrew

2008-11-01

Protein-protein association involves many interface interactions, but they do not contribute equally. Ala scanning experiments reveal that only a few mutations significantly lower binding affinity. These key residues, which appear to drive protein-protein association, are called hot-spot residues. Molecular dynamics simulations of the Colicin E9/Im9 complex show Im9 Glu41 and Im9 Ser50, both hot-spots, bind via different mechanisms. The results suggest that Im9 Ser50 restricts Glu41 in a conformation auspicious for salt-bridge formation across the interface. This type of model may be helpful in engineering hot-spot clusters at protein-protein interfaces and, consequently, the design of specificity.

A Comparison of Structural and Evolutionary Attributes of Escherichia coli and Thermus thermophilus Small Ribosomal Subunits: Signatures of Thermal Adaptation

PubMed Central

Mallik, Saurav; Kundu, Sudip

2013-01-01

Here we compare the structural and evolutionary attributes of Thermus thermophilus and Escherichia coli small ribosomal subunits (SSU). Our results indicate that with few exceptions, thermophilic 16S ribosomal RNA (16S rRNA) is densely packed compared to that of mesophilic at most of the analogous spatial regions. In addition, we have located species-specific cavity clusters (SSCCs) in both species. E. coli SSCCs are numerous and larger compared to T. thermophilus SSCCs, which again indicates densely packed thermophilic 16S rRNA. Thermophilic ribosomal proteins (r-proteins) have longer disordered regions than their mesophilic homologs and they experience larger disorder-to-order transitions during SSU-assembly. This is reflected in the predicted higher conformational changes of thermophilic r-proteins compared to their mesophilic homologs during SSU-assembly. This high conformational change of thermophilic r-proteins may help them to associate with the 16S ribosomal RNA with high complementary interfaces, larger interface areas, and denser molecular contacts, compared to those of mesophilic. Thus, thermophilic protein-rRNA interfaces are tightly associated with 16S rRNA than their mesophilic homologs. Densely packed 16S rRNA interior and tight protein-rRNA binding of T. thermophilus (compared to those of E. coli) are likely the signatures of its thermal adaptation. We have found a linear correlation between the free energy of protein-RNA interface formation, interface size, and square of conformational changes, which is followed in both prokaryotic and eukaryotic SSU. Disorder is associated with high protein-RNA interface polarity. We have found an evolutionary tendency to maintain high polarity (thereby disorder) at protein-rRNA interfaces, than that at rest of the protein structures. However, some proteins exhibit exceptions to this general trend. PMID:23940533

The modular architecture of protein-protein binding interfaces.

PubMed

Reichmann, D; Rahat, O; Albeck, S; Meged, R; Dym, O; Schreiber, G

2005-01-04

Protein-protein interactions are essential for life. Yet, our understanding of the general principles governing binding is not complete. In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules. The boundaries between modules are defined by clustering the contact map of the interface. We show that mutations in one module do not affect residues located in a neighboring module. As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small. To the contrary, within their module, mutations cause complex energetic and structural consequences. Experimentally, this phenomenon is shown on the interaction between TEM1-beta-lactamase and beta-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography. Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface. The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved.

A Dominant Conformational Role for Amino Acid Diversity in Minimalist Protein-Protein Interfaces

PubMed Central

Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko; Sidhu, Sachdev S.; Koide, Shohei

2008-01-01

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies”. One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose binding protein (MBP). The YSX monobody bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution x-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side-chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces. PMID:18602117

Interfacial dilatational deformation accelerates particle formation in monoclonal antibody solutions.

PubMed

Lin, Gigi L; Pathak, Jai A; Kim, Dong Hyun; Carlson, Marcia; Riguero, Valeria; Kim, Yoen Joo; Buff, Jean S; Fuller, Gerald G

2016-04-14

Protein molecules are amphiphilic moieties that spontaneously adsorb at the air/solution (A/S) interface to lower the surface energy. Previous studies have shown that hydrodynamic disruptions to these A/S interfaces can result in the formation of protein aggregates that are of concern to the pharmaceutical industry. Interfacial hydrodynamic stresses encountered by protein therapeutic solutions under typical manufacturing, filling, and shipping conditions will impact protein stability, prompting a need to characterize the contribution of basic fluid kinematics to monoclonal antibody (mAb) destabilization. We demonstrate that dilatational surface deformations are more important to antibody stability when compared to constant-area shear of the A/S interface. We have constructed a dilatational interfacial rheometer that utilizes simultaneous pressure and bubble shape measurements to study the mechanical stability of mAbs under interfacial aging. It has a distinct advantage over methods utilizing the Young-Laplace equation, which incorrectly describes viscoelastic interfaces. We provide visual evidence of particle ejection from dilatated A/S interfaces and spectroscopic data of ejected mAb particles. These rheological studies frame a molecular understanding of the protein-protein interactions at the complex-fluid interface.

A comparison of successful and failed protein interface designs highlights the challenges of designing buried hydrogen bonds

PubMed Central

Stranges, P Benjamin; Kuhlman, Brian

2013-01-01

The accurate design of new protein–protein interactions is a longstanding goal of computational protein design. However, most computationally designed interfaces fail to form experimentally. This investigation compares five previously described successful de novo interface designs with 158 failures. Both sets of proteins were designed with the molecular modeling program Rosetta. Designs were considered a success if a high-resolution crystal structure of the complex closely matched the design model and the equilibrium dissociation constant for binding was less than 10 μM. The successes and failures represent a wide variety of interface types and design goals including heterodimers, homodimers, peptide-protein interactions, one-sided designs (i.e., where only one of the proteins was mutated) and two-sided designs. The most striking feature of the successful designs is that they have fewer polar atoms at their interfaces than many of the failed designs. Designs that attempted to create extensive sets of interface-spanning hydrogen bonds resulted in no detectable binding. In contrast, polar atoms make up more than 40% of the interface area of many natural dimers, and native interfaces often contain extensive hydrogen bonding networks. These results suggest that Rosetta may not be accurately balancing hydrogen bonding and electrostatic energies against desolvation penalties and that design processes may not include sufficient sampling to identify side chains in preordered conformations that can fully satisfy the hydrogen bonding potential of the interface. PMID:23139141

iATTRACT: simultaneous global and local interface optimization for protein-protein docking refinement.

PubMed

Schindler, Christina E M; de Vries, Sjoerd J; Zacharias, Martin

2015-02-01

Protein-protein interactions are abundant in the cell but to date structural data for a large number of complexes is lacking. Computational docking methods can complement experiments by providing structural models of complexes based on structures of the individual partners. A major caveat for docking success is accounting for protein flexibility. Especially, interface residues undergo significant conformational changes upon binding. This limits the performance of docking methods that keep partner structures rigid or allow limited flexibility. A new docking refinement approach, iATTRACT, has been developed which combines simultaneous full interface flexibility and rigid body optimizations during docking energy minimization. It employs an atomistic molecular mechanics force field for intermolecular interface interactions and a structure-based force field for intramolecular contributions. The approach was systematically evaluated on a large protein-protein docking benchmark, starting from an enriched decoy set of rigidly docked protein-protein complexes deviating by up to 15 Å from the native structure at the interface. Large improvements in sampling and slight but significant improvements in scoring/discrimination of near native docking solutions were observed. Complexes with initial deviations at the interface of up to 5.5 Å were refined to significantly better agreement with the native structure. Improvements in the fraction of native contacts were especially favorable, yielding increases of up to 70%. © 2014 Wiley Periodicals, Inc.

A conserved protein interaction interface on the type 5 G protein beta subunit controls proteolytic stability and activity of R7 family regulator of G protein signaling proteins.

PubMed

Porter, Morwenna Y; Xie, Keqiang; Pozharski, Edwin; Koelle, Michael R; Martemyanov, Kirill A

2010-12-24

Regulators of G protein signaling (RGS) proteins of the R7 subfamily limit signaling by neurotransmitters in the brain and by light in the retina. They form obligate complexes with the Gβ5 protein that are subject to proteolysis to control their abundance and alter signaling. The mechanisms that regulate this proteolysis, however, remain unclear. We used genetic screens to find mutations in Gβ5 that selectively destabilize one of the R7 RGS proteins in Caenorhabditis elegans. These mutations cluster at the binding interface between Gβ5 and the N terminus of R7 RGS proteins. Equivalent mutations within mammalian Gβ5 allowed the interface to still bind the N-terminal DEP domain of R7 RGS proteins, and mutant Gβ5-R7 RGS complexes initially formed in cells but were then rapidly degraded by proteolysis. Molecular dynamics simulations suggest the mutations weaken the Gβ5-DEP interface, thus promoting dynamic opening of the complex to expose determinants of proteolysis known to exist on the DEP domain. We propose that conformational rearrangements at the Gβ5-DEP interface are key to controlling the stability of R7 RGS protein complexes.

MDcons: Intermolecular contact maps as a tool to analyze the interface of protein complexes from molecular dynamics trajectories

PubMed Central

2014-01-01

Background Molecular Dynamics (MD) simulations of protein complexes suffer from the lack of specific tools in the analysis step. Analyses of MD trajectories of protein complexes indeed generally rely on classical measures, such as the RMSD, RMSF and gyration radius, conceived and developed for single macromolecules. As a matter of fact, instead, researchers engaged in simulating the dynamics of a protein complex are mainly interested in characterizing the conservation/variation of its biological interface. Results On these bases, herein we propose a novel approach to the analysis of MD trajectories or other conformational ensembles of protein complexes, MDcons, which uses the conservation of inter-residue contacts at the interface as a measure of the similarity between different snapshots. A "consensus contact map" is also provided, where the conservation of the different contacts is drawn in a grey scale. Finally, the interface area of the complex is monitored during the simulations. To show its utility, we used this novel approach to study two protein-protein complexes with interfaces of comparable size and both dominated by hydrophilic interactions, but having binding affinities at the extremes of the experimental range. MDcons is demonstrated to be extremely useful to analyse the MD trajectories of the investigated complexes, adding important insight into the dynamic behavior of their biological interface. Conclusions MDcons specifically allows the user to highlight and characterize the dynamics of the interface in protein complexes and can thus be used as a complementary tool for the analysis of MD simulations of both experimental and predicted structures of protein complexes. PMID:25077693

MDcons: Intermolecular contact maps as a tool to analyze the interface of protein complexes from molecular dynamics trajectories.

PubMed

Abdel-Azeim, Safwat; Chermak, Edrisse; Vangone, Anna; Oliva, Romina; Cavallo, Luigi

2014-01-01

Molecular Dynamics (MD) simulations of protein complexes suffer from the lack of specific tools in the analysis step. Analyses of MD trajectories of protein complexes indeed generally rely on classical measures, such as the RMSD, RMSF and gyration radius, conceived and developed for single macromolecules. As a matter of fact, instead, researchers engaged in simulating the dynamics of a protein complex are mainly interested in characterizing the conservation/variation of its biological interface. On these bases, herein we propose a novel approach to the analysis of MD trajectories or other conformational ensembles of protein complexes, MDcons, which uses the conservation of inter-residue contacts at the interface as a measure of the similarity between different snapshots. A "consensus contact map" is also provided, where the conservation of the different contacts is drawn in a grey scale. Finally, the interface area of the complex is monitored during the simulations. To show its utility, we used this novel approach to study two protein-protein complexes with interfaces of comparable size and both dominated by hydrophilic interactions, but having binding affinities at the extremes of the experimental range. MDcons is demonstrated to be extremely useful to analyse the MD trajectories of the investigated complexes, adding important insight into the dynamic behavior of their biological interface. MDcons specifically allows the user to highlight and characterize the dynamics of the interface in protein complexes and can thus be used as a complementary tool for the analysis of MD simulations of both experimental and predicted structures of protein complexes.

Nano-Bio Interactions of Porous and Nonporous Silica Nanoparticles of Varied Surface Chemistry: A Structural, Kinetic, and Thermodynamic Study of Protein Adsorption from RPMI Culture Medium.

PubMed

Lehman, Sean E; Mudunkotuwa, Imali A; Grassian, Vicki H; Larsen, Sarah C

2016-01-26

Understanding complex chemical changes that take place at nano-bio interfaces is of great concern for being able to sustainably implement nanomaterials in key applications such as drug delivery, imaging, and environmental remediation. Typical in vitro assays use cell viability as a proxy to understanding nanotoxicity but often neglect how the nanomaterial surface can be altered by adsorption of solution-phase components in the medium. Protein coronas form on the nanomaterial surface when incubated in proteinaceous solutions. Herein, we apply a broad array of techniques to characterize and quantify protein corona formation on silica nanoparticle surfaces. The porosity and surface chemistry of the silica nanoparticles have been systematically varied. Using spectroscopic tools such as FTIR and circular dichroism, structural changes and kinetic processes involved in protein adsorption were evaluated. Additionally, by implementing thermogravimetric analysis, quantitative protein adsorption measurements allowed for the direct comparison between samples. Taken together, these measurements enabled the extraction of useful chemical information on protein binding onto nanoparticles in solution. Overall, we demonstrate that small alkylamines can increase protein adsorption and that even large polymeric molecules such as poly(ethylene glycol) (PEG) cannot prevent protein adsorption in these systems. The implications of these results as they relate to further understanding nano-bio interactions are discussed.

Optimizing energy functions for protein-protein interface design.

PubMed

Sharabi, Oz; Yanover, Chen; Dekel, Ayelet; Shifman, Julia M

2011-01-15

Protein design methods have been originally developed for the design of monomeric proteins. When applied to the more challenging task of protein–protein complex design, these methods yield suboptimal results. In particular, they often fail to recapitulate favorable hydrogen bonds and electrostatic interactions across the interface. In this work, we aim to improve the energy function of the protein design program ORBIT to better account for binding interactions between proteins. By using the advanced machine learning framework of conditional random fields, we optimize the relative importance of all the terms in the energy function, attempting to reproduce the native side-chain conformations in protein–protein interfaces. We evaluate the performance of several optimized energy functions, each describes the van der Waals interactions using a different potential. In comparison with the original energy function, our best energy function (a) incorporates a much “softer” repulsive van der Waals potential, suitable for the discrete rotameric representation of amino acid side chains; (b) does not penalize burial of polar atoms, reflecting the frequent occurrence of polar buried residues in protein–protein interfaces; and (c) significantly up-weights the electrostatic term, attesting to the high importance of these interactions for protein–protein complex formation. Using this energy function considerably improves side chain placement accuracy for interface residues in a large test set of protein–protein complexes. Moreover, the optimized energy function recovers the native sequences of protein–protein interface at a higher rate than the default function and performs substantially better in predicting changes in free energy of binding due to mutations.

Surface adsorption considerations when working with amyloid fibrils in multiwell plates and Eppendorf tubes

PubMed Central

Murray, Amber N; Palhano, Fernando L; Bieschke, Jan; Kelly, Jeffery W

2013-01-01

The accumulation of cross-β-sheet amyloid fibrils is the hallmark of amyloid diseases. Recently, we reported the discovery of amyloid disaggregase activities in extracts from mammalian cells and Caenorhabditis elegans. However, we have discovered a problem with the interpretation of our previous results as Aβ disaggregation in vitro. Here, we show that Aβ fibrils adsorb to the plastic surface of multiwell plates and Eppendorf tubes. This adsorption is markedly increased in the presence of complex biological mixtures subjected to a denaturing air-water interface. The time-dependent loss of thioflavin T fluorescence that we interpreted previously as disaggregation is due to increased adsorption of Aβ amyloid to the surfaces of multiwell plates and Eppendorf tubes in the presence of biological extracts. As the proteins in biological extracts denature over time at the air-water interface due to agitation/shaking, their adsorption increases, in turn promoting adsorption of amyloid fibrils. We delineate important control experiments that quantify the extent of amyloid adsorption to the surface of plastic and quartz containers. Based on the results described in this article, we conclude that our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231–2241 and Murray et al., Protein Sci 2010;19:836–846 is invalid when used as evidence for a disaggregase activity. Thus, we correct the two prior publications reporting that worm or mammalian cell extracts disaggregate Aβ amyloid fibrils in vitro at 37°C (see Corrigenda in this issue of Protein Science). We apologize for misinterpreting our previous data and for any confounding experimental efforts this may have caused. PMID:23963844

Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes.

PubMed

Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

2016-10-18

The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains-In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model.

Quantification of the Mass Transfer at Fluid Interfaces in Microfluidic Channels

NASA Astrophysics Data System (ADS)

Wismeth, Carina; Manhart, Michael; Niessner, Reinhard; Baumann, Thomas

2017-04-01

Mass transfer rates at interfaces in a complex porous media are relevant in many environmental applications and control the functions of natural filter systems in subsurface environments. The mass transfer at fluid interfaces is associated with interface convection caused by local inhomogeneities in interface tension and hydrodynamic instabilities at the interface. If there is a surface tension gradient along the surface a shear stress jump is generated that results in fluid motion along the surface that is called Marangoni effect. These spontaneous convection currents can lead to an increased mass transfer of the transition component at the phase boundary and to an increased mixing of the phases. Therefore compensatory currents at the interface can have a significant influence on the subsurface transport of contaminants in the groundwater area, especially in the vadose zone. Using microfluidic channels and advanced experimental techniques it is possible to measure the fluid flow and mass transfer rates directly and to quantify the effect of the Marangoni convection on the mass transfer at interfaces between a non-aqueous liquid and water with high temporal and spatial resolution. The use of fluorescent particles as well as the recording and analysis of their trajectories is intended to visualize interfacial processes and to quantify the mass transfer at fluid phase boundaries. Concentration gradients at the interface are analysed by spectroscopic methods and allow an assessment of the enrichment and depletion at the phase boundaries. Extensive test series provide the experimental basis for quantifying and analysing the impact of the Marangoni effect on the mass transfer rates at interfaces in porous media in subsurface aquatic environments. Within this research project we concentrate on the effect of Marangoni convection on the mass transfer near an 1-octanol-water interface, which serves as a well defined proxy for non-aqueous phase liquids in porous media. Experiments and a numerical simulation are closely coupled to provide a generic data set with high reproducibility and used to obtain highly resolved three-dimensional data of mass transfer in two- and three-phase systems to foster the understanding of subsurface transport, especially in the vadose zone.

Interactome INSIDER: a structural interactome browser for genomic studies.

PubMed

Meyer, Michael J; Beltrán, Juan Felipe; Liang, Siqi; Fragoza, Robert; Rumack, Aaron; Liang, Jin; Wei, Xiaomu; Yu, Haiyuan

2018-01-01

We present Interactome INSIDER, a tool to link genomic variant information with structural protein-protein interactomes. Underlying this tool is the application of machine learning to predict protein interaction interfaces for 185,957 protein interactions with previously unresolved interfaces in human and seven model organisms, including the entire experimentally determined human binary interactome. Predicted interfaces exhibit functional properties similar to those of known interfaces, including enrichment for disease mutations and recurrent cancer mutations. Through 2,164 de novo mutagenesis experiments, we show that mutations of predicted and known interface residues disrupt interactions at a similar rate and much more frequently than mutations outside of predicted interfaces. To spur functional genomic studies, Interactome INSIDER (http://interactomeinsider.yulab.org) enables users to identify whether variants or disease mutations are enriched in known and predicted interaction interfaces at various resolutions. Users may explore known population variants, disease mutations, and somatic cancer mutations, or they may upload their own set of mutations for this purpose.

Low-stringency selection of TEM1 for BLIP shows interface plasticity and selection for faster binders

PubMed Central

Cohen-Khait, Ruth; Schreiber, Gideon

2016-01-01

Protein–protein interactions occur via well-defined interfaces on the protein surface. Whereas the location of homologous interfaces is conserved, their composition varies, suggesting that multiple solutions may support high-affinity binding. In this study, we examined the plasticity of the interface of TEM1 β-lactamase with its protein inhibitor BLIP by low-stringency selection of a random TEM1 library using yeast surface display. Our results show that most interfacial residues could be mutated without a loss in binding affinity, protein stability, or enzymatic activity, suggesting plasticity in the interface composition supporting high-affinity binding. Interestingly, many of the selected mutations promoted faster association. Further selection for faster binders was achieved by drastically decreasing the library–ligand incubation time to 30 s. Preequilibrium selection as suggested here is a novel methodology for specifically selecting faster-associating protein complexes. PMID:27956635

HotRegion: a database of predicted hot spot clusters.

PubMed

Cukuroglu, Engin; Gursoy, Attila; Keskin, Ozlem

2012-01-01

Hot spots are energetically important residues at protein interfaces and they are not randomly distributed across the interface but rather clustered. These clustered hot spots form hot regions. Hot regions are important for the stability of protein complexes, as well as providing specificity to binding sites. We propose a database called HotRegion, which provides the hot region information of the interfaces by using predicted hot spot residues, and structural properties of these interface residues such as pair potentials of interface residues, accessible surface area (ASA) and relative ASA values of interface residues of both monomer and complex forms of proteins. Also, the 3D visualization of the interface and interactions among hot spot residues are provided. HotRegion is accessible at http://prism.ccbb.ku.edu.tr/hotregion.

Protein docking by the interface structure similarity: how much structure is needed?

PubMed

Sinha, Rohita; Kundrotas, Petras J; Vakser, Ilya A

2012-01-01

The increasing availability of co-crystallized protein-protein complexes provides an opportunity to use template-based modeling for protein-protein docking. Structure alignment techniques are useful in detection of remote target-template similarities. The size of the structure involved in the alignment is important for the success in modeling. This paper describes a systematic large-scale study to find the optimal definition/size of the interfaces for the structure alignment-based docking applications. The results showed that structural areas corresponding to the cutoff values <12 Å across the interface inadequately represent structural details of the interfaces. With the increase of the cutoff beyond 12 Å, the success rate for the benchmark set of 99 protein complexes, did not increase significantly for higher accuracy models, and decreased for lower-accuracy models. The 12 Å cutoff was optimal in our interface alignment-based docking, and a likely best choice for the large-scale (e.g., on the scale of the entire genome) applications to protein interaction networks. The results provide guidelines for the docking approaches, including high-throughput applications to modeled structures.

Cortical geometry may influence placement of interface between Par protein domains in early Caenorhabditis elegans embryos.

PubMed

Dawes, Adriana T; Iron, David

2013-09-21

During polarization, proteins and other polarity determinants segregate to the opposite ends of the cell (the poles) creating biochemically and dynamically distinct regions. Embryos of the nematode worm Caenorhabditis elegans (C. elegans) polarize shortly after fertilization, creating distinct regions of Par protein family members. These regions are maintained through to first cleavage when the embryo divides along the plane specified by the interface between regions, creating daughter cells with different protein content. In wild type single cell embryos the interface between these Par protein regions is reliably positioned at approximately 60% egg length, however, it is not known what mechanisms are responsible for specifying the position of the interface. In this investigation, we use two mathematical models to investigate the movement and positioning of the interface: a biologically based reaction-diffusion model of Par protein dynamics, and the analytically tractable perturbed Allen-Cahn equation. When we numerically simulate the models on a static 2D domain with constant thickness, both models exhibit a persistently moving interface that specifies the boundary between distinct regions. When we modify the simulation domain geometry, movement halts and the interface is stably positioned where the domain thickness increases. Using asymptotic analysis with the perturbed Allen-Cahn equation, we show that interface movement depends explicitly on domain geometry. Using a combination of analytic and numeric techniques, we demonstrate that domain geometry, a historically overlooked aspect of cellular simulations, may play a significant role in spatial protein patterning during polarization. Copyright © 2013 Elsevier Ltd. All rights reserved.

Specificity of molecular interactions in transient protein-protein interaction interfaces.

PubMed

Cho, Kyu-il; Lee, KiYoung; Lee, Kwang H; Kim, Dongsup; Lee, Doheon

2006-11-15

In this study, we investigate what types of interactions are specific to their biological function, and what types of interactions are persistent regardless of their functional category in transient protein-protein heterocomplexes. This is the first approach to analyze protein-protein interfaces systematically at the molecular interaction level in the context of protein functions. We perform systematic analysis at the molecular interaction level using classification and feature subset selection technique prevalent in the field of pattern recognition. To represent the physicochemical properties of protein-protein interfaces, we design 18 molecular interaction types using canonical and noncanonical interactions. Then, we construct input vector using the frequency of each interaction type in protein-protein interface. We analyze the 131 interfaces of transient protein-protein heterocomplexes in PDB: 33 protease-inhibitors, 52 antibody-antigens, 46 signaling proteins including 4 cyclin dependent kinase and 26 G-protein. Using kNN classification and feature subset selection technique, we show that there are specific interaction types based on their functional category, and such interaction types are conserved through the common binding mechanism, rather than through the sequence or structure conservation. The extracted interaction types are C(alpha)-- H...O==C interaction, cation...anion interaction, amine...amine interaction, and amine...cation interaction. With these four interaction types, we achieve the classification success rate up to 83.2% with leave-one-out cross-validation at k = 15. Of these four interaction types, C(alpha)--H...O==C shows binding specificity for protease-inhibitor complexes, while cation-anion interaction is predominant in signaling complexes. The amine ... amine and amine...cation interaction give a minor contribution to the classification accuracy. When combined with these two interactions, they increase the accuracy by 3.8%. In the case of antibody-antigen complexes, the sign is somewhat ambiguous. From the evolutionary perspective, while protease-inhibitors and sig-naling proteins have optimized their interfaces to suit their biological functions, antibody-antigen interactions are the happenstance, implying that antibody-antigen complexes do not show distinctive interaction types. Persistent interaction types such as pi...pi, amide-carbonyl, and hydroxyl-carbonyl interaction, are also investigated. Analyzing the structural orientations of the pi...pi stacking interactions, we find that herringbone shape is a major configuration in transient protein-protein interfaces. This result is different from that of protein core, where parallel-displaced configurations are the major configuration. We also analyze overall trend of amide-carbonyl and hydroxyl-carbonyl interactions. It is noticeable that nearly 82% of the interfaces have at least one hydroxyl-carbonyl interactions. (c) 2006 Wiley-Liss, Inc.

NPIDB: Nucleic acid-Protein Interaction DataBase.

PubMed

Kirsanov, Dmitry D; Zanegina, Olga N; Aksianov, Evgeniy A; Spirin, Sergei A; Karyagina, Anna S; Alexeevski, Andrei V

2013-01-01

The Nucleic acid-Protein Interaction DataBase (http://npidb.belozersky.msu.ru/) contains information derived from structures of DNA-protein and RNA-protein complexes extracted from the Protein Data Bank (3846 complexes in October 2012). It provides a web interface and a set of tools for extracting biologically meaningful characteristics of nucleoprotein complexes. The content of the database is updated weekly. The current version of the Nucleic acid-Protein Interaction DataBase is an upgrade of the version published in 2007. The improvements include a new web interface, new tools for calculation of intermolecular interactions, a classification of SCOP families that contains DNA-binding protein domains and data on conserved water molecules on the DNA-protein interface.

Sequence-Specific Recognition of DNA by Proteins: Binding Motifs Discovered Using a Novel Statistical/Computational Analysis

PubMed Central

Jakubec, David; Laskowski, Roman A.; Vondrasek, Jiri

2016-01-01

Decades of intensive experimental studies of the recognition of DNA sequences by proteins have provided us with a view of a diverse and complicated world in which few to no features are shared between individual DNA-binding protein families. The originally conceived direct readout of DNA residue sequences by amino acid side chains offers very limited capacity for sequence recognition, while the effects of the dynamic properties of the interacting partners remain difficult to quantify and almost impossible to generalise. In this work we investigated the energetic characteristics of all DNA residue—amino acid side chain combinations in the conformations found at the interaction interface in a very large set of protein—DNA complexes by the means of empirical potential-based calculations. General specificity-defining criteria were derived and utilised to look beyond the binding motifs considered in previous studies. Linking energetic favourability to the observed geometrical preferences, our approach reveals several additional amino acid motifs which can distinguish between individual DNA bases. Our results remained valid in environments with various dielectric properties. PMID:27384774

A dominant conformational role for amino acid diversity in minimalist protein–protein interfaces

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

Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies.” One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose-binding protein. The YSX monobodymore » bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution X-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces.« less

Computational redesign of a protein-protein interface for high affinity and binding specificity using modular architecture and naturally occurring template fragments.

PubMed

Potapov, V; Reichmann, D; Abramovich, R; Filchtinski, D; Zohar, N; Ben Halevy, D; Edelman, M; Sobolev, V; Schreiber, G

2008-12-05

A new method is presented for the redesign of protein-protein interfaces, resulting in specificity of the designed pair while maintaining high affinity. The design is based on modular interface architecture and was carried out on the interaction between TEM1 beta-lactamase and its inhibitor protein, beta-lactamase inhibitor protein. The interface between these two proteins is composed of several mostly independent modules. We previously showed that it is possible to delete a complete module without affecting the overall structure of the interface. Here, we replace a complete module with structure fragments taken from nonrelated proteins. Nature-optimized fragments were chosen from 10(7) starting templates found in the Protein Data Bank. A procedure was then developed to identify sets of interacting template residues with a backbone arrangement mimicking the original module. This generated a final list of 361 putative replacement modules that were ranked using a novel scoring function based on grouped atom-atom contact surface areas. The top-ranked designed complex exhibited an affinity of at least the wild-type level and a mode of binding that was remarkably specific despite the absence of negative design in the procedure. In retrospect, the combined application of three factors led to the success of the design approach: utilizing the modular construction of the interface, capitalizing on native rather than artificial templates, and ranking with an accurate atom-atom contact surface scoring function.

Prediction of the interaction site on the surface of an isolated protein structure by analysis of side chain energy scores.

PubMed

Liang, Shide; Zhang, Jian; Zhang, Shicui; Guo, Huarong

2004-11-15

We show that residues at the interfaces of protein-protein complexes have higher side-chain energy than other surface residues. Eight different sets of protein complexes were analyzed. For each protein pair, the complex structure was used to identify the interface residues in the unbound monomer structures. Side-chain energy was calculated for each surface residue in the unbound monomer using our previously developed scoring function.1 The mean energy was calculated for the interface residues and the other surface residues. In 15 of the 16 monomers, the mean energy of the interface residues was higher than that of other surface residues. By decomposing the scoring function, we found that the energy term of the buried surface area of non-hydrogen-bonded hydrophilic atoms is the most important factor contributing to the high energy of the interface regions. In spite of lacking hydrophilic residues, the interface regions were found to be rich in buried non-hydrogen-bonded hydrophilic atoms. Although the calculation results could be affected by the inaccuracy of the scoring function, patch analysis of side-chain energy on the surface of an isolated protein may be helpful in identifying the possible protein-protein interface. A patch was defined as 20 residues surrounding the central residue on the protein surface, and patch energy was calculated as the mean value of the side-chain energy of all residues in the patch. In 12 of the studied monomers, the patch with the highest energy overlaps with the observed interface. The results are more remarkable when only three residues with the highest energy in a patch are averaged to derive the patch energy. All three highest-energy residues of the top energy patch belong to interfacial residues in four of the eight small protomers. We also found that the residue with the highest energy score on the surface of a small protomer is very possibly the key interaction residue. (c) 2004 Wiley-Liss, Inc.

Antifreeze Proteins at the Ice/Water Interface: Three Calculated Discriminating Properties for Orientation of Type I Proteins

PubMed Central

Wierzbicki, Andrzej; Dalal, Pranav; Cheatham, Thomas E.; Knickelbein, Jared E.; Haymet, A. D. J.; Madura, Jeffry D.

2007-01-01

Antifreeze proteins (AFPs) protect many plants and organisms from freezing in low temperatures. Of the different AFPs, the most studied AFP Type I from winter flounder is used in the current computational studies to gain molecular insight into its adsorption at the ice/water interface. Employing molecular dynamics simulations, we calculate the free energy difference between the hydrophilic and hydrophobic faces of the protein interacting with ice. Furthermore, we identify three properties of Type I “antifreeze” proteins that discriminate among these two orientations of the protein at the ice/water interface. The three properties are: the “surface area” of the protein; a measure of the interaction of the protein with neighboring water molecules as determined by the number of hydrogen bond count, for example; and the side-chain orientation angles of the threonine residues. All three discriminants are consistent with our free energy results, which clearly show that the hydrophilic protein face orientations toward the ice/water interface, as hypothesized from experimental and ice/vacuum simulations, are incorrect and support the hypothesis that the hydrophobic face is oriented toward the ice/water interface. The adsorption free energy is calculated to be 2–3 kJ/mol. PMID:17526572

Protein painting reveals solvent-excluded drug targets hidden within native protein–protein interfaces

PubMed Central

Luchini, Alessandra; Espina, Virginia; Liotta, Lance A.

2014-01-01

Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein–protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets. PMID:25048602

The Protein Identifier Cross-Referencing (PICR) service: reconciling protein identifiers across multiple source databases.

PubMed

Côté, Richard G; Jones, Philip; Martens, Lennart; Kerrien, Samuel; Reisinger, Florian; Lin, Quan; Leinonen, Rasko; Apweiler, Rolf; Hermjakob, Henning

2007-10-18

Each major protein database uses its own conventions when assigning protein identifiers. Resolving the various, potentially unstable, identifiers that refer to identical proteins is a major challenge. This is a common problem when attempting to unify datasets that have been annotated with proteins from multiple data sources or querying data providers with one flavour of protein identifiers when the source database uses another. Partial solutions for protein identifier mapping exist but they are limited to specific species or techniques and to a very small number of databases. As a result, we have not found a solution that is generic enough and broad enough in mapping scope to suit our needs. We have created the Protein Identifier Cross-Reference (PICR) service, a web application that provides interactive and programmatic (SOAP and REST) access to a mapping algorithm that uses the UniProt Archive (UniParc) as a data warehouse to offer protein cross-references based on 100% sequence identity to proteins from over 70 distinct source databases loaded into UniParc. Mappings can be limited by source database, taxonomic ID and activity status in the source database. Users can copy/paste or upload files containing protein identifiers or sequences in FASTA format to obtain mappings using the interactive interface. Search results can be viewed in simple or detailed HTML tables or downloaded as comma-separated values (CSV) or Microsoft Excel (XLS) files suitable for use in a local database or a spreadsheet. Alternatively, a SOAP interface is available to integrate PICR functionality in other applications, as is a lightweight REST interface. We offer a publicly available service that can interactively map protein identifiers and protein sequences to the majority of commonly used protein databases. Programmatic access is available through a standards-compliant SOAP interface or a lightweight REST interface. The PICR interface, documentation and code examples are available at http://www.ebi.ac.uk/Tools/picr.

The Protein Identifier Cross-Referencing (PICR) service: reconciling protein identifiers across multiple source databases

PubMed Central

Côté, Richard G; Jones, Philip; Martens, Lennart; Kerrien, Samuel; Reisinger, Florian; Lin, Quan; Leinonen, Rasko; Apweiler, Rolf; Hermjakob, Henning

2007-01-01

Background Each major protein database uses its own conventions when assigning protein identifiers. Resolving the various, potentially unstable, identifiers that refer to identical proteins is a major challenge. This is a common problem when attempting to unify datasets that have been annotated with proteins from multiple data sources or querying data providers with one flavour of protein identifiers when the source database uses another. Partial solutions for protein identifier mapping exist but they are limited to specific species or techniques and to a very small number of databases. As a result, we have not found a solution that is generic enough and broad enough in mapping scope to suit our needs. Results We have created the Protein Identifier Cross-Reference (PICR) service, a web application that provides interactive and programmatic (SOAP and REST) access to a mapping algorithm that uses the UniProt Archive (UniParc) as a data warehouse to offer protein cross-references based on 100% sequence identity to proteins from over 70 distinct source databases loaded into UniParc. Mappings can be limited by source database, taxonomic ID and activity status in the source database. Users can copy/paste or upload files containing protein identifiers or sequences in FASTA format to obtain mappings using the interactive interface. Search results can be viewed in simple or detailed HTML tables or downloaded as comma-separated values (CSV) or Microsoft Excel (XLS) files suitable for use in a local database or a spreadsheet. Alternatively, a SOAP interface is available to integrate PICR functionality in other applications, as is a lightweight REST interface. Conclusion We offer a publicly available service that can interactively map protein identifiers and protein sequences to the majority of commonly used protein databases. Programmatic access is available through a standards-compliant SOAP interface or a lightweight REST interface. The PICR interface, documentation and code examples are available at . PMID:17945017

Residue frequencies and pairing preferences at protein-protein interfaces.

PubMed

Glaser, F; Steinberg, D M; Vakser, I A; Ben-Tal, N

2001-05-01

We used a nonredundant set of 621 protein-protein interfaces of known high-resolution structure to derive residue composition and residue-residue contact preferences. The residue composition at the interfaces, in entire proteins and in whole genomes correlates well, indicating the statistical strength of the data set. Differences between amino acid distributions were observed for interfaces with buried surface area of less than 1,000 A(2) versus interfaces with area of more than 5,000 A(2). Hydrophobic residues were abundant in large interfaces while polar residues were more abundant in small interfaces. The largest residue-residue preferences at the interface were recorded for interactions between pairs of large hydrophobic residues, such as Trp and Leu, and the smallest preferences for pairs of small residues, such as Gly and Ala. On average, contacts between pairs of hydrophobic and polar residues were unfavorable, and the charged residues tended to pair subject to charge complementarity, in agreement with previous reports. A bootstrap procedure, lacking from previous studies, was used for error estimation. It showed that the statistical errors in the set of pairing preferences are generally small; the average standard error is approximately 0.2, i.e., about 8% of the average value of the pairwise index (2.9). However, for a few pairs (e.g., Ser-Ser and Glu-Asp) the standard error is larger in magnitude than the pairing index, which makes it impossible to tell whether contact formation is favorable or unfavorable. The results are interpreted using physicochemical factors and their implications for the energetics of complex formation and for protein docking are discussed. Proteins 2001;43:89-102. Copyright 2001 Wiley-Liss, Inc.

The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities.

PubMed

Aydin, Cihan; Mukherjee, Sourav; Hanson, Alicia M; Frick, David N; Schiffer, Celia A

2013-12-01

Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full-length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain-domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that the two domains of NS3/4A are dynamically coupled through the interface. Interestingly, mutations designed to disrupt this interface did not hinder the catalytic activities of either domain. In contrast, substrate cleavage and DNA unwinding by these mutants were mostly enhanced compared with the wild-type protein. Disrupting the interface did not significantly alter RNA unwinding activity; however, the full-length protein was more efficient in RNA unwinding than the isolated protease domain, suggesting a more direct role in RNA processing independent of the interface. Our findings suggest that HCV NS3/4A adopts an "extended" catalytically active conformation, and interface formation acts as a switch to regulate activity. We propose a unifying model connecting HCV NS3/4A conformational states and protease and helicase function, where interface formation and the dynamic interplay between the two enzymatic domains of HCV NS3/4A potentially modulate the protease and helicase activities in vivo. © 2013 The Protein Society.

DockRank: ranking docked conformations using partner-specific sequence homology-based protein interface prediction.

PubMed

Xue, Li C; Jordan, Rafael A; El-Manzalawy, Yasser; Dobbs, Drena; Honavar, Vasant

2014-02-01

Selecting near-native conformations from the immense number of conformations generated by docking programs remains a major challenge in molecular docking. We introduce DockRank, a novel approach to scoring docked conformations based on the degree to which the interface residues of the docked conformation match a set of predicted interface residues. DockRank uses interface residues predicted by partner-specific sequence homology-based protein-protein interface predictor (PS-HomPPI), which predicts the interface residues of a query protein with a specific interaction partner. We compared the performance of DockRank with several state-of-the-art docking scoring functions using Success Rate (the percentage of cases that have at least one near-native conformation among the top m conformations) and Hit Rate (the percentage of near-native conformations that are included among the top m conformations). In cases where it is possible to obtain partner-specific (PS) interface predictions from PS-HomPPI, DockRank consistently outperforms both (i) ZRank and IRAD, two state-of-the-art energy-based scoring functions (improving Success Rate by up to 4-fold); and (ii) Variants of DockRank that use predicted interface residues obtained from several protein interface predictors that do not take into account the binding partner in making interface predictions (improving success rate by up to 39-fold). The latter result underscores the importance of using partner-specific interface residues in scoring docked conformations. We show that DockRank, when used to re-rank the conformations returned by ClusPro, improves upon the original ClusPro rankings in terms of both Success Rate and Hit Rate. DockRank is available as a server at http://einstein.cs.iastate.edu/DockRank/. Copyright © 2013 Wiley Periodicals, Inc.

Monitoring reactive microencapsulation dynamics using microfluidics

PubMed Central

Brosseau, Quentin; Baret, Jean-Christophe

2015-01-01

We use microfluidic polydimethylsiloxane (PDMS) devices to measure the kinetics of reactive encapsulations occurring at the interface of emulsion droplets. The formation of the polymeric shell is inferred from the droplet deformability measured in a series of expansion–constriction chambers along the microfluidic chip. With this tool we quantify the kinetic processes governing the encapsulation at the very early stage of shell formation with a time resolution of the order of the millisecond for overall reactions occurring in less than 0.5 s. We perform a comparison of monomer reactivities used for the encapsulation. We study the formation of polyurea microcapsules (PUMCs); the shell formation proceeds at the water–oil interface by an immediate reaction of amines dissolved in the aqueous phase and isocyanates dissolved in the oil phase. We observe that both monomers contribute differently to the encapsulation kinetics. The kinetics of the shell formation process at the oil-in-water (O/W) experiments significantly differs from the water-in-oil (W/O) systems; the component dissolved in the continuous phase has the largest impact on the kinetics. In addition, we quantified the retarding effect on the encapsulation kinetics by the interface stabilizing agent (surfactant). Our approach is valuable for quantifying in situ reactive encapsulation processes and provides guidelines to generate microcapsules with soft interfaces of tailored and controllable interfacial properties. PMID:25705975

KFC Server: interactive forecasting of protein interaction hot spots.

PubMed

Darnell, Steven J; LeGault, Laura; Mitchell, Julie C

2008-07-01

The KFC Server is a web-based implementation of the KFC (Knowledge-based FADE and Contacts) model-a machine learning approach for the prediction of binding hot spots, or the subset of residues that account for most of a protein interface's; binding free energy. The server facilitates the automated analysis of a user submitted protein-protein or protein-DNA interface and the visualization of its hot spot predictions. For each residue in the interface, the KFC Server characterizes its local structural environment, compares that environment to the environments of experimentally determined hot spots and predicts if the interface residue is a hot spot. After the computational analysis, the user can visualize the results using an interactive job viewer able to quickly highlight predicted hot spots and surrounding structural features within the protein structure. The KFC Server is accessible at http://kfc.mitchell-lab.org.

Optimized hydrophobic interactions and hydrogen bonding at the target-ligand interface leads the pathways of drug-designing.

PubMed

Patil, Rohan; Das, Suranjana; Stanley, Ashley; Yadav, Lumbani; Sudhakar, Akulapalli; Varma, Ashok K

2010-08-16

Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds. In-silico docking studies were performed, using Discovery Studio software modules LigandFit, CDOCKER and ZDOCK, to investigate the role of ligand binding affinity at the hydrophobic pocket of c-Src and c-Abl kinase. Hydrophobic and hydrogen bonding interactions of docked molecules were compared using LigPlot program. Furthermore, 3D-QSAR and MFA calculations were scrutinized to quantify the role of weak interactions in binding affinity and drug efficacy. The in-silico method has enabled us to reveal that a multi-targeted small molecule binds with low affinity to its respective targets. But its binding affinity can be altered by integrating the conformationally favored functional groups at the active site of the ligand-target interface. Docking studies of 4-amino-substituted molecules at the bioactive cascade of the c-Src and c-Abl have concluded that 3D structural folding at the protein-ligand groove is also a hallmark for molecular recognition of multi-targeted compounds and for predicting their biological activity. The results presented here demonstrate that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy.

Optimized Hydrophobic Interactions and Hydrogen Bonding at the Target-Ligand Interface Leads the Pathways of Drug-Designing

PubMed Central

Stanley, Ashley; Yadav, Lumbani; Sudhakar, Akulapalli; Varma, Ashok K.

2010-01-01

Background Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds. Methodology In-silico docking studies were performed, using Discovery Studio software modules LigandFit, CDOCKER and ZDOCK, to investigate the role of ligand binding affinity at the hydrophobic pocket of c-Src and c-Abl kinase. Hydrophobic and hydrogen bonding interactions of docked molecules were compared using LigPlot program. Furthermore, 3D-QSAR and MFA calculations were scrutinized to quantify the role of weak interactions in binding affinity and drug efficacy. Conclusions The in-silico method has enabled us to reveal that a multi-targeted small molecule binds with low affinity to its respective targets. But its binding affinity can be altered by integrating the conformationally favored functional groups at the active site of the ligand-target interface. Docking studies of 4-amino-substituted molecules at the bioactive cascade of the c-Src and c-Abl have concluded that 3D structural folding at the protein-ligand groove is also a hallmark for molecular recognition of multi-targeted compounds and for predicting their biological activity. The results presented here demonstrate that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy. PMID:20808434

Interface investigation of solution processed high- κ ZrO2/Si MOS structure by DLTS

NASA Astrophysics Data System (ADS)

Kumar, Arvind; Mondal, Sandip; Rao, Ksr Koteswara

The interfacial region is dominating due to the continuous downscaling and integration of high- k oxides in CMOS applications. The accurate characterization of high- k oxides/semiconductor interface has the significant importance towards its usage in memory and thin film devices. The interface traps at the high - k /semiconductor interface can be quantified by deep level transient spectroscopy (DLTS) with better accuracy in contrast to capacitance-voltage (CV) and conductance technique. We report the fabrication of high- k ZrO2 films on p-Si substrate by a simple and inexpensive sol-gel spin-coating technique. Further, the ZrO2/Si interface is characterized through DLTS. The flat-band voltage (VFB) and the density of slow interface states (oxide trapped charges) extracted from CV characteristics are 0.37 V and 2x10- 11 C/cm2, respectively. The activation energy, interface state density and capture cross-section quantified by DLTS are EV + 0.42 eV, 3.4x1011 eV- 1 cm- 2 and 5.8x10- 18 cm2, respectively. The high quality ZrO2 films own high dielectric constant 15 with low leakage current density might be an appropriate insulating layer in future electronic application. The low value of interface state density and capture cross-section are the indication of high quality interface and the defect present at the interface may not affect the device performance to a great extent. The DLTS study provides a broad understanding about the traps present at the interface of spin-coated ZrO2/Si.

Maillard Conjugation of Sodium Alginate to Whey Protein for Enhanced Resistance to Surfactant-Induced Competitive Displacement from Air-Water Interfaces.

PubMed

Cai, Bingqing; Saito, Anna; Ikeda, Shinya

2018-01-24

Whey protein adsorbed to an interface forms a viscoelastic interfacial film but is displaced competitively from the interface by a small-molecule surfactant added afterward. The present study evaluated the impact of the covalent conjugation of high- or low-molecular-weight sodium alginate (HA or LA) to whey protein isolate (WPI) via the Maillard reaction on the ability of whey protein to resist surfactant-induced competitive displacement from the air-water interface. Surfactant added after the pre-adsorption of conjugate to the interface increased surface pressure. At a given surface pressure, the WPI-LA conjugate showed a significantly higher interfacial area coverage and lower interfacial film thickness compared to those of the WPI-HA conjugate or unconjugated WPI. The addition of LA to the aqueous phase had little effect on the interfacial area and thickness of pre-adsorbed WPI. These results suggest the importance of the molecular weight of the polysaccharide moiety in determining interfacial properties of whey protein-alginate conjugates.

Structural basis of a rationally rewired protein-protein interface critical to bacterial signaling

PubMed Central

Podgornaia, Anna I.; Casino, Patricia; Marina, Alberto; Laub, Michael T.

2013-01-01

Summary Two-component signal transduction systems typically involve a sensor histidine kinase that specifically phosphorylates a single, cognate response regulator. This protein-protein interaction relies on molecular recognition via a small set of residues in each protein. To better understand how these residues determine the specificity of kinase-substrate interactions, we rationally rewired the interaction interface of a Thermotoga maritima two-component system, HK853-RR468, to match that found in a different two-component system, E. coli PhoR-PhoB. The rewired proteins interacted robustly with each other, but no longer interacted with the parent proteins. Analysis of the crystal structures of the wild-type and mutant protein complexes, along with a systematic mutagenesis study, reveals how individual mutations contribute to the rewiring of interaction specificity. Our approach and conclusions have implications for studies of other protein-protein interactions, protein evolution, and the design of novel protein interfaces. PMID:23954504

Molecular assembly, interfacial rheology and foaming properties of oligofructose fatty acid esters.

PubMed

van Kempen, Silvia E H J; Schols, Henk A; van der Linden, Erik; Sagis, Leonard M C

2014-01-01

Two major types of food-grade surfactants used to stabilize foams are proteins and low molecular weight (LMW) surfactants. Proteins lower the surface tension of interfaces and tend to unfold and stabilize the interface by the formation of a visco-elastic network, which leads to high surface moduli. In contrast, LMW surfactants lower the surface tension more than proteins, but do not form interfaces with a high modulus. Instead, they stabilize the interface through the Gibbs-Marangoni mechanism that relies on rapid diffusion of surfactants, when surface tension gradients develop as a result of deformations of the interface. A molecule than can lower the surface tension considerably, like a LMW surfactant, but also provide the interface with a high modulus, like a protein, would be an excellent foam stabilizer. In this article we will discuss molecules with those properties: oligofructose fatty acid esters, both in pure and mixed systems. First, we will address the synthesis and structural characterization of the esters. Next, we will address self-assembly and rheological properties of air/water interfaces stabilized by the esters. Subsequently, this paper will deal with mixed systems of mono-esters with either di-esters and lauric acid, or proteins. Then, the foaming functionality of the esters is discussed.

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.

Structural principles within the human-virus protein-protein interaction network

PubMed Central

Franzosa, Eric A.; Xia, Yu

2011-01-01

General properties of the antagonistic biomolecular interactions between viruses and their hosts (exogenous interactions) remain poorly understood, and may differ significantly from known principles governing the cooperative interactions within the host (endogenous interactions). Systems biology approaches have been applied to study the combined interaction networks of virus and human proteins, but such efforts have so far revealed only low-resolution patterns of host-virus interaction. Here, we layer curated and predicted 3D structural models of human-virus and human-human protein complexes on top of traditional interaction networks to reconstruct the human-virus structural interaction network. This approach reveals atomic resolution, mechanistic patterns of host-virus interaction, and facilitates systematic comparison with the host’s endogenous interactions. We find that exogenous interfaces tend to overlap with and mimic endogenous interfaces, thereby competing with endogenous binding partners. The endogenous interfaces mimicked by viral proteins tend to participate in multiple endogenous interactions which are transient and regulatory in nature. While interface overlap in the endogenous network results largely from gene duplication followed by divergent evolution, viral proteins frequently achieve interface mimicry without any sequence or structural similarity to an endogenous binding partner. Finally, while endogenous interfaces tend to evolve more slowly than the rest of the protein surface, exogenous interfaces—including many sites of endogenous-exogenous overlap—tend to evolve faster, consistent with an evolutionary “arms race” between host and pathogen. These significant biophysical, functional, and evolutionary differences between host-pathogen and within-host protein-protein interactions highlight the distinct consequences of antagonism versus cooperation in biological networks. PMID:21680884

Improving protein-protein interaction prediction using evolutionary information from low-quality MSAs.

PubMed

Várnai, Csilla; Burkoff, Nikolas S; Wild, David L

2017-01-01

Evolutionary information stored in multiple sequence alignments (MSAs) has been used to identify the interaction interface of protein complexes, by measuring either co-conservation or co-mutation of amino acid residues across the interface. Recently, maximum entropy related correlated mutation measures (CMMs) such as direct information, decoupling direct from indirect interactions, have been developed to identify residue pairs interacting across the protein complex interface. These studies have focussed on carefully selected protein complexes with large, good-quality MSAs. In this work, we study protein complexes with a more typical MSA consisting of fewer than 400 sequences, using a set of 79 intramolecular protein complexes. Using a maximum entropy based CMM at the residue level, we develop an interface level CMM score to be used in re-ranking docking decoys. We demonstrate that our interface level CMM score compares favourably to the complementarity trace score, an evolutionary information-based score measuring co-conservation, when combined with the number of interface residues, a knowledge-based potential and the variability score of individual amino acid sites. We also demonstrate, that, since co-mutation and co-complementarity in the MSA contain orthogonal information, the best prediction performance using evolutionary information can be achieved by combining the co-mutation information of the CMM with co-conservation information of a complementarity trace score, predicting a near-native structure as the top prediction for 41% of the dataset. The method presented is not restricted to small MSAs, and will likely improve interface prediction also for complexes with large and good-quality MSAs.

Study of intermolecular contacts in proteins and oligomer interfaces and preliminary investigations into the design and production of nanomaterials from proteins

NASA Astrophysics Data System (ADS)

Iyer, Ganesh Hariharan

The first part of this research involved a study of the nature and extent of nonbonded interactions at crystal and oligomer interfaces. A survey was compiled of several characteristics of intersubunit contacts in 58 different oligomeric proteins, and of the intermolecular contacts in 223 protein crystal structures. Routines written in "S" language were utilized for the generation of the observed and expected contacts. The information in the Protein Data Bank (PDB) was extracted using the database management system, Protein Knowledge Base (PKB). Potentials of mean force for atom-atom contacts and residue-residue contacts were derived by comparison of the number of observed interactions with the number expected by mass action. Preference association matrices and log-linear analyses were applied to determine the different factors that could contribute to the overall interactions at the interfaces of oligomers and crystals. Surface patches at oligomer and crystal interfaces were also studied to further investigate the origin of the differences in their stabilities. Total number of atoms in contact and the secondary structure elements involved are similar in the two types of interfaces. Crystal contacts result from more numerous interactions by polar residues, compared with a tendency toward nonpolar amino acid prominent in oligomer interfaces. Contact potentials indicate that hydrophobic interactions at oligomer interfaces favor aromatic amino acids and methionine over aliphatic amino acids; and that crystal contacts form in such a way as to avoid inclusion of hydrophobic interactions. The second part involved the development of a new class of biomaterials from two-dimensional arrays of ordered proteins. Point mutations were planned to introduce cysteine residues at appropriate locations to enable cross-linking at the molecular interface within given crystallographic planes. Crystallization and subsequent cross-linking of the modified protein would lead to the formation of arrays on subsequent dissociation of the crystal. Novel protein architectures can be generated from these cross-linked nanostructures. Experiments with model protein, maltose-binding protein (MBP) were performed to develop purification, cross-linking and crystallization techniques. The long-term goal of this project is to apply the experience gained with MBP to the fabrication of nanomaterials from other, application-specific proteins for ultrafiltration and microelectronic devices.

GMR biosensor arrays: a system perspective.

PubMed

Hall, D A; Gaster, R S; Lin, T; Osterfeld, S J; Han, S; Murmann, B; Wang, S X

2010-05-15

Giant magnetoresistive biosensors are becoming more prevalent for sensitive, quantifiable biomolecular detection. However, in order for magnetic biosensing to become competitive with current optical protein microarray technology, there is a need to increase the number of sensors while maintaining the high sensitivity and fast readout time characteristic of smaller arrays (1-8 sensors). In this paper, we present a circuit architecture scalable for larger sensor arrays (64 individually addressable sensors) while maintaining a high readout rate (scanning the entire array in less than 4s). The system utilizes both time domain multiplexing and frequency domain multiplexing in order to achieve this scan rate. For the implementation, we propose a new circuit architecture that does not use a classical Wheatstone bridge to measure the small change in resistance of the sensor. Instead, an architecture designed around a transimpedance amplifier is employed. A detailed analysis of this architecture including the noise, distortion, and potential sources of errors is presented, followed by a global optimization strategy for the entire system comprising the magnetic tags, sensors, and interface electronics. To demonstrate the sensitivity, quantifiable detection of two blindly spiked samples of unknown concentrations has been performed at concentrations below the limit of detection for the enzyme-linked immunosorbent assay. Lastly, the multiplexing capability and reproducibility of the system was demonstrated by simultaneously monitoring sensors functionalized with three unique proteins at different concentrations in real-time. 2010 Elsevier B.V. All rights reserved.

GMR Biosensor Arrays: A System Perspective

PubMed Central

Hall, D. A.; Gaster, R. S.; Lin, T.; Osterfeld, S. J.; Han, S.; Murmann, B.; Wang, S. X.

2010-01-01

Giant magnetoresistive biosensors are becoming more prevalent for sensitive, quantifiable biomolecular detection. However, in order for magnetic biosensing to become competitive with current optical protein microarray technology, there is a need to increase the number of sensors while maintaining the high sensitivity and fast readout time characteristic of smaller arrays (1 – 8 sensors). In this paper, we present a circuit architecture scalable for larger sensor arrays (64 individually addressable sensors) while maintaining a high readout rate (scanning the entire array in less than 4 seconds). The system utilizes both time domain multiplexing and frequency domain multiplexing in order to achieve this scan rate. For the implementation, we propose a new circuit architecture that does not use a classical Wheatstone bridge to measure the small change in resistance of the sensor. Instead, an architecture designed around a transimpedance amplifier is employed. A detailed analysis of this architecture including the noise, distortion, and potential sources of errors is presented, followed by a global optimization strategy for the entire system comprising the magnetic tags, sensors, and interface electronics. To demonstrate the sensitivity, quantifiable detection of two blindly spiked samples of unknown concentrations has been performed at concentrations below the limit of detection for the enzyme-linked immunosorbent assay. Lastly, the multipexability and reproducibility of the system was demonstrated by simultaneously monitoring sensors functionalized with three unique proteins at different concentrations in real-time. PMID:20207130

Interlayer Water Regulates the Bio-nano Interface of a β-sheet Protein stacking on Graphene

PubMed Central

Lv, Wenping; Xu, Guiju; Zhang, Hongyan; Li, Xin; Liu, Shengju; Niu, Huan; Xu, Dongsheng; Wu, Ren'an

2015-01-01

Using molecular dynamics simulations, we investigated an integrated bio-nano interface consisting of a β-sheet protein stacked onto graphene. We found that the stacking assembly of the model protein on graphene could be controlled by water molecules. The interlayer water filled within interstices of the bio-nano interface could suppress the molecular vibration of surface groups on protein, and could impair the CH···π interaction driving the attraction of the protein and graphene. The intermolecular coupling of interlayer water would be relaxed by the relative motion of protein upon graphene due to the interaction between water and protein surface. This effect reduced the hindrance of the interlayer water against the assembly of protein on graphene, resulting an appropriate adsorption status of protein on graphene with a deep free energy trap. Thereby, the confinement and the relative sliding between protein and graphene, the coupling of protein and water, and the interaction between graphene and water all have involved in the modulation of behaviors of water molecules within the bio-nano interface, governing the hindrance of interlayer water against the protein assembly on hydrophobic graphene. These results provide a deep insight into the fundamental mechanism of protein adsorption onto graphene surface in water. PMID:25557857

Interlayer water regulates the bio-nano interface of a β-sheet protein stacking on graphene.

PubMed

Lv, Wenping; Xu, Guiju; Zhang, Hongyan; Li, Xin; Liu, Shengju; Niu, Huan; Xu, Dongsheng; Wu, Ren'an

2015-01-05

Using molecular dynamics simulations, we investigated an integrated bio-nano interface consisting of a β-sheet protein stacked onto graphene. We found that the stacking assembly of the model protein on graphene could be controlled by water molecules. The interlayer water filled within interstices of the bio-nano interface could suppress the molecular vibration of surface groups on protein, and could impair the CH···π interaction driving the attraction of the protein and graphene. The intermolecular coupling of interlayer water would be relaxed by the relative motion of protein upon graphene due to the interaction between water and protein surface. This effect reduced the hindrance of the interlayer water against the assembly of protein on graphene, resulting an appropriate adsorption status of protein on graphene with a deep free energy trap. Thereby, the confinement and the relative sliding between protein and graphene, the coupling of protein and water, and the interaction between graphene and water all have involved in the modulation of behaviors of water molecules within the bio-nano interface, governing the hindrance of interlayer water against the protein assembly on hydrophobic graphene. These results provide a deep insight into the fundamental mechanism of protein adsorption onto graphene surface in water.

Interlayer Water Regulates the Bio-nano Interface of a β-sheet Protein stacking on Graphene

NASA Astrophysics Data System (ADS)

Lv, Wenping; Xu, Guiju; Zhang, Hongyan; Li, Xin; Liu, Shengju; Niu, Huan; Xu, Dongsheng; Wu, Ren'an

2015-01-01

Using molecular dynamics simulations, we investigated an integrated bio-nano interface consisting of a β-sheet protein stacked onto graphene. We found that the stacking assembly of the model protein on graphene could be controlled by water molecules. The interlayer water filled within interstices of the bio-nano interface could suppress the molecular vibration of surface groups on protein, and could impair the CH...π interaction driving the attraction of the protein and graphene. The intermolecular coupling of interlayer water would be relaxed by the relative motion of protein upon graphene due to the interaction between water and protein surface. This effect reduced the hindrance of the interlayer water against the assembly of protein on graphene, resulting an appropriate adsorption status of protein on graphene with a deep free energy trap. Thereby, the confinement and the relative sliding between protein and graphene, the coupling of protein and water, and the interaction between graphene and water all have involved in the modulation of behaviors of water molecules within the bio-nano interface, governing the hindrance of interlayer water against the protein assembly on hydrophobic graphene. These results provide a deep insight into the fundamental mechanism of protein adsorption onto graphene surface in water.

Local and global anatomy of antibody-protein antigen recognition.

PubMed

Wang, Meryl; Zhu, David; Zhu, Jianwei; Nussinov, Ruth; Ma, Buyong

2018-05-01

Deciphering antibody-protein antigen recognition is of fundamental and practical significance. We constructed an antibody structural dataset, partitioned it into human and murine subgroups, and compared it with nonantibody protein-protein complexes. We investigated the physicochemical properties of regions on and away from the antibody-antigen interfaces, including net charge, overall antibody charge distributions, and their potential role in antigen interaction. We observed that amino acid preference in antibody-protein antigen recognition is entropy driven, with residues having low side-chain entropy appearing to compensate for the high backbone entropy in interaction with protein antigens. Antibodies prefer charged and polar antigen residues and bridging water molecules. They also prefer positive net charge, presumably to promote interaction with negatively charged protein antigens, which are common in proteomes. Antibody-antigen interfaces have large percentages of Tyr, Ser, and Asp, but little Lys. Electrostatic and hydrophobic interactions in the Ag binding sites might be coupled with Fab domains through organized charge and residue distributions away from the binding interfaces. Here we describe some features of antibody-antigen interfaces and of Fab domains as compared with nonantibody protein-protein interactions. The distributions of interface residues in human and murine antibodies do not differ significantly. Overall, our results provide not only a local but also a global anatomy of antibody structures. Copyright © 2017 John Wiley & Sons, Ltd.

Catalysis by a de novo zinc-mediated protein interface: implications for natural enzyme evolution and rational enzyme engineering.

PubMed

Der, Bryan S; Edwards, David R; Kuhlman, Brian

2012-05-08

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

Real-time investigation of protein unfolding at an air–water interface at the 1 s time scale

PubMed Central

Yano, Yohko F.; Arakawa, Etsuo; Voegeli, Wolfgang; Matsushita, Tadashi

2013-01-01

Protein unfolding at an air–water interface has been demonstrated such that the X-ray reflectivity can be measured with an acquisition time of 1 s using a recently developed simultaneous multiple-angle–wavelength-dispersive X-ray reflectometer. This has enabled the electron density profile of the adsorbed protein molecules to be obtained in real time. A globular protein, lysozyme, adsorbed at the air–water interface is found to unfold into a flat shape within 1 s. PMID:24121352

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.

Boltzmann Energy-based Image Analysis Demonstrates that Extracellular Domain Size Differences Explain Protein Segregation at Immune Synapses

PubMed Central

Burroughs, Nigel J.; Köhler, Karsten; Miloserdov, Vladimir; Dustin, Michael L.; van der Merwe, P. Anton; Davis, Daniel M.

2011-01-01

Immune synapses formed by T and NK cells both show segregation of the integrin ICAM1 from other proteins such as CD2 (T cell) or KIR (NK cell). However, the mechanism by which these proteins segregate remains unclear; one key hypothesis is a redistribution based on protein size. Simulations of this mechanism qualitatively reproduce observed segregation patterns, but only in certain parameter regimes. Verifying that these parameter constraints in fact hold has not been possible to date, this requiring a quantitative coupling of theory to experimental data. Here, we address this challenge, developing a new methodology for analysing and quantifying image data and its integration with biophysical models. Specifically we fit a binding kinetics model to 2 colour fluorescence data for cytoskeleton independent synapses (2 and 3D) and test whether the observed inverse correlation between fluorophores conforms to size dependent exclusion, and further, whether patterned states are predicted when model parameters are estimated on individual synapses. All synapses analysed satisfy these conditions demonstrating that the mechanisms of protein redistribution have identifiable signatures in their spatial patterns. We conclude that energy processes implicit in protein size based segregation can drive the patternation observed in individual synapses, at least for the specific examples tested, such that no additional processes need to be invoked. This implies that biophysical processes within the membrane interface have a crucial impact on cell∶cell communication and cell signalling, governing protein interactions and protein aggregation. PMID:21829338

Quantifying protein-protein interactions in high throughput using protein domain microarrays.

PubMed

Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

2010-04-01

Protein microarrays provide an efficient way to identify and quantify protein-protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain-peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (K(D)s) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein-ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein-protein interaction networks.

BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

PubMed

Wei, Qing; La, David; Kihara, Daisuke

2017-01-01

Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

The roles of carrier concentration and interface, bulk, and grain-boundary recombination for 25% efficient CdTe solar cells

DOE PAGES

Kanevce, A.; Reese, Matthew O.; Barnes, T. M.; ...

2017-06-06

CdTe devices have reached efficiencies of 22% due to continuing improvements in bulk material properties, including minority carrier lifetime. Device modeling has helped to guide these device improvements by quantifying the impacts of material properties and different device designs on device performance. One of the barriers to truly predictive device modeling is the interdependence of these material properties. For example, interfaces become more critical as bulk properties, particularly, hole density and carrier lifetime, increase. We present device-modeling analyses that describe the effects of recombination at the interfaces and grain boundaries as lifetime and doping of the CdTe layer change. Themore » doping and lifetime should be priorities for maximizing open-circuit voltage (V oc) and efficiency improvements. However, interface and grain boundary recombination become bottlenecks for device performance at increased lifetime and doping levels. In conclusion, this work quantifies and discusses these emerging challenges for next-generation CdTe device efficiency.« less

Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

PubMed Central

Russell, Jason D.; Scalf, Mark; Book, Adam J.; Ladror, Daniel T.; Vierstra, Richard D.; Smith, Lloyd M.; Coon, Joshua J.

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1. PMID:23536786

Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.

PubMed

Russell, Jason D; Scalf, Mark; Book, Adam J; Ladror, Daniel T; Vierstra, Richard D; Smith, Lloyd M; Coon, Joshua J

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.

Steered Molecular Dynamics Simulations Predict Conformational Stability of Glutamate Receptors.

PubMed

Musgaard, Maria; Biggin, Philip C

2016-09-26

The stability of protein-protein interfaces can be essential for protein function. For ionotropic glutamate receptors, a family of ligand-gated ion channels vital for normal function of the central nervous system, such an interface exists between the extracellular ligand binding domains (LBDs). In the full-length protein, the LBDs are arranged as a dimer of dimers. Agonist binding to the LBDs opens the ion channel, and briefly after activation the receptor desensitizes. Several residues at the LBD dimer interface are known to modulate desensitization, and conformational changes around these residues are believed to be involved in the state transition. The general hypothesis is that the interface is disrupted upon desensitization, and structural evidence suggests that the disruption might be substantial. However, when cross-linking the central part of this interface, functional data suggest that the receptor can still undergo desensitization, contradicting the hypothesis of major interface disruption. Here, we illustrate how opening the dimer interface using steered molecular dynamics (SMD) simulations, and analyzing the work values required, provides a quantitative measure for interface stability. For one subtype of glutamate receptors, which is regulated by ion binding to the dimer interface, we show that opening the interface without ions bound requires less work than with ions present, suggesting that ion binding indeed stabilizes the interface. Likewise, for interface mutants with longer-lived active states, the interface is more stable, while the work required to open the interface is reduced for less active mutants. Moreover, a cross-linked mutant can still undergo initial interface opening motions similar to the native receptor and at similar energetic cost. Thus, our results support that interface opening is involved in desensitization. Furthermore, they provide reconciliation of apparently opposing data and demonstrate that SMD simulations can give relevant biological insight into longer time scale processes without the need for expensive calculations.

Atomic analysis of protein-protein interfaces with known inhibitors: the 2P2I database.

PubMed

Bourgeas, Raphaël; Basse, Marie-Jeanne; Morelli, Xavier; Roche, Philippe

2010-03-09

In the last decade, the inhibition of protein-protein interactions (PPIs) has emerged from both academic and private research as a new way to modulate the activity of proteins. Inhibitors of these original interactions are certainly the next generation of highly innovative drugs that will reach the market in the next decade. However, in silico design of such compounds still remains challenging. Here we describe this particular PPI chemical space through the presentation of 2P2I(DB), a hand-curated database dedicated to the structure of PPIs with known inhibitors. We have analyzed protein/protein and protein/inhibitor interfaces in terms of geometrical parameters, atom and residue properties, buried accessible surface area and other biophysical parameters. The interfaces found in 2P2I(DB) were then compared to those of representative datasets of heterodimeric complexes. We propose a new classification of PPIs with known inhibitors into two classes depending on the number of segments present at the interface and corresponding to either a single secondary structure element or to a more globular interacting domain. 2P2I(DB) complexes share global shape properties with standard transient heterodimer complexes, but their accessible surface areas are significantly smaller. No major conformational changes are seen between the different states of the proteins. The interfaces are more hydrophobic than general PPI's interfaces, with less charged residues and more non-polar atoms. Finally, fifty percent of the complexes in the 2P2I(DB) dataset possess more hydrogen bonds than typical protein-protein complexes. Potential areas of study for the future are proposed, which include a new classification system consisting of specific families and the identification of PPI targets with high druggability potential based on key descriptors of the interaction. 2P2I database stores structural information about PPIs with known inhibitors and provides a useful tool for biologists to assess the potential druggability of their interfaces. The database can be accessed at http://2p2idb.cnrs-mrs.fr.

Comparison of the protein-protein interfaces in the p53-DNA crystal structures: towards elucidation of the biological interface.

PubMed

Ma, Buyong; Pan, Yongping; Gunasekaran, K; Venkataraghavan, R Babu; Levine, Arnold J; Nussinov, Ruth

2005-03-15

p53, the tumor suppressor protein, functions as a dimer of dimers. However, how the tetramer binds to the DNA is still an open question. In the crystal structure, three copies of the p53 monomers (containing chains A, B, and C) were crystallized with the DNA-consensus element. Although the structure provides crucial data on the p53-DNA contacts, the active oligomeric state is unclear because the two dimeric (A-B and B-C) interfaces present in the crystal cannot both exist in the tetramer. Here, we address the question of which of these two dimeric interfaces may be more biologically relevant. We analyze the sequence and structural properties of the p53-p53 dimeric interfaces and carry out extensive molecular dynamics simulations of the crystal structures of the human and mouse p53 dimers. We find that the A-B interface residues are more conserved than those of the B-C. Molecular dynamics simulations show that the A-B interface can provide a stable DNA-binding motif in the dimeric state, unlike B-C. Our results indicate that the interface between chains A-B in the p53-DNA complex constitutes a better candidate for a stable biological interface, whereas the B-C interface is more likely to be due to crystal packing. Thus, they have significant implications toward our understanding of DNA binding by p53 as well as p53-mediated interactions with other proteins.

Attomole quantitation of protein separations with accelerator mass spectrometry

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

Vogel, J S; Grant, P G; Buccholz, B A

2000-12-15

Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to sub-attomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5% . Micro-proton-induced-xray-emission quantifies elemental abundancesmore » in separated metalloprotein samples to nanogram amounts and is capable of quantifying phosphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.« less

Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

PubMed

Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

2018-05-01

We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

Milk whey proteins and xanthan gum interactions in solution and at the air-water interface: a rheokinetic study.

PubMed

Perez, Adrián A; Sánchez, Cecilio Carrera; Patino, Juan M Rodríguez; Rubiolo, Amelia C; Santiago, Liliana G

2010-11-01

In this contribution, we present experimental information about the effect of xanthan gum (XG) on the adsorption behaviour of two milk whey protein samples (MWP), beta-lactoglobulin (beta-LG) and whey protein concentrate (WPC), at the air-water interface. The MWP concentration studied corresponded to the protein bulk concentration which is able to saturate the air-water interface (1.0 wt%). Temperature, pH and ionic strength of aqueous systems were kept constant at 20 degrees C, pH 7 and 0.05 M, respectively, while the XG bulk concentration varied in the range 0.00-0.25 wt%. Biopolymer interactions in solution were analyzed by extrinsic fluorescence spectroscopy using 1-anilino-8-naphtalene sulphonic acid (ANS) as a protein fluorescence probe. Interfacial biopolymer interactions were evaluated by dynamic tensiometry and surface dilatational rheology. Adsorption behaviour was discussed from a rheokinetic point of view in terms of molecular diffusion, penetration and conformational rearrangement of adsorbed protein residues at the air-water interface. Differences in the interaction magnitude, both in solution and at the interface vicinity, and in the adsorption rheokinetic parameters were observed in MWP/XG mixed systems depending on the protein type (beta-LG or WPC) and biopolymer relative concentration. beta-LG adsorption in XG presence could be promoted by mechanisms based on biopolymer segregative interactions and thermodynamic incompatibility in the interface vicinity, resulting in better surface and viscoelastic properties. The same mechanism could be responsible of WPC interfacial adsorption in the presence of XG. The interfacial functionality of WPC was improved by the synergistic interactions with XG, although WPC chemical complexity might complicate the elucidation of molecular events that govern adsorption dynamics of WPC/XG mixed systems at the air-water interface. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Energetics of side-chain partitioning of β-signal residues in unassisted folding of a transmembrane β-barrel protein

PubMed Central

Iyer, Bharat Ramasubramanian; Zadafiya, Punit; Vetal, Pallavi Vijay

2017-01-01

The free energy of water-to-interface amino acid partitioning is a major contributing factor in membrane protein folding and stability. The interface residues at the C terminus of transmembrane β-barrels form the β-signal motif required for assisted β-barrel assembly in vivo but are believed to be less important for β-barrel assembly in vitro. Here, we experimentally measured the thermodynamic contribution of all 20 amino acids at the β-signal motif to the unassisted folding of the model β-barrel protein PagP. We obtained the partitioning free energy for all 20 amino acids at the lipid-facing interface (ΔΔG0w,i(φ)) and the protein-facing interface (ΔΔG0w,i(π)) residues and found that hydrophobic amino acids are most favorably transferred to the lipid-facing interface, whereas charged and polar groups display the highest partitioning energy. Furthermore, the change in non-polar surface area correlated directly with the partitioning free energy for the lipid-facing residue and inversely with the protein-facing residue. We also demonstrate that the interface residues of the β-signal motif are vital for in vitro barrel assembly, because they exhibit a side chain–specific energetic contribution determined by the change in nonpolar accessible surface. We further establish that folding cooperativity and hydrophobic collapse are balanced at the membrane interface for optimal stability of the PagP β-barrel scaffold. We conclude that the PagP C-terminal β-signal motif influences the folding cooperativity and stability of the folded β-barrel and that the thermodynamic contributions of the lipid- and protein-facing residues in the transmembrane protein β-signal motif depend on the nature of the amino acid side chain. PMID:28592485

Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers

PubMed Central

Fischer, Audrey; Holden, Matthew A.; Pentelute, Brad L.; Collier, R. John

2011-01-01

Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation. PMID:21949363

Understanding and Manipulating Electrostatic Fields at the Protein-Protein Interface Using Vibrational Spectroscopy and Continuum Electrostatics Calculations.

PubMed

Ritchie, Andrew W; Webb, Lauren J

2015-11-05

Biological function emerges in large part from the interactions of biomacromolecules in the complex and dynamic environment of the living cell. For this reason, macromolecular interactions in biological systems are now a major focus of interest throughout the biochemical and biophysical communities. The affinity and specificity of macromolecular interactions are the result of both structural and electrostatic factors. Significant advances have been made in characterizing structural features of stable protein-protein interfaces through the techniques of modern structural biology, but much less is understood about how electrostatic factors promote and stabilize specific functional macromolecular interactions over all possible choices presented to a given molecule in a crowded environment. In this Feature Article, we describe how vibrational Stark effect (VSE) spectroscopy is being applied to measure electrostatic fields at protein-protein interfaces, focusing on measurements of guanosine triphosphate (GTP)-binding proteins of the Ras superfamily binding with structurally related but functionally distinct downstream effector proteins. In VSE spectroscopy, spectral shifts of a probe oscillator's energy are related directly to that probe's local electrostatic environment. By performing this experiment repeatedly throughout a protein-protein interface, an experimental map of measured electrostatic fields generated at that interface is determined. These data can be used to rationalize selective binding of similarly structured proteins in both in vitro and in vivo environments. Furthermore, these data can be used to compare to computational predictions of electrostatic fields to explore the level of simulation detail that is necessary to accurately predict our experimental findings.

Impact of Cavitation, High Shear Stress and Air/Liquid Interfaces on Protein Aggregation.

PubMed

Duerkop, Mark; Berger, Eva; Dürauer, Astrid; Jungbauer, Alois

2018-03-25

The reported impact of shear stress on protein aggregation has been contradictory. At high shear rates, the occurrence of cavitation or entrapment of air is reasonable and their effects possibly misattributed to shear stress. Nine different proteins (α-lactalbumin, two antibodies, fibroblast growth factor 2, granulocyte colony stimulating factor [GCSF], green fluorescence protein [GFP], hemoglobin, human serum albumin, and lysozyme) are tested for their aggregation behavior on vapor/liquid interfaces generated by cavitation and compared it to the isolated effects of high shear stress and air/liquid interfaces generated by foaming. Cavitation induced the aggregation of GCSF by +68.9%, hemoglobin +4%, and human serum albumin +2.9%, compared to a control, whereas the other proteins do not aggregate. The protein aggregation behaviors of the different proteins at air/liquid interfaces are similar to cavitation, but the effect is more pronounced. Air-liquid interface induced the aggregation of GCSF by +94.5%, hemoglobin +35.5%, and human serum albumin (HSA) +31.1%. The results indicate that the sensitivity of a certain protein toward cavitation is very similar to air/liquid-induced aggregation. Hence, hydroxyl radicals cannot be seen as the driving force for protein aggregation when cavitation occurs. Further, high shear rates of up to 10 8  s -1 do not affect any of the tested proteins. Therefore, also within this study generated extremely high isolated shear rates cannot be considered to harm structural integrity when processing proteins. © 2018 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Atom Probe Tomographic Mapping Directly Reveals the Atomic Distribution of Phosphorus in Resin Embedded Ferritin

NASA Astrophysics Data System (ADS)

Perea, Daniel E.; Liu, Jia; Bartrand, Jonah; Dicken, Quinten; Thevuthasan, S. Theva; Browning, Nigel D.; Evans, James E.

2016-02-01

Here we report the atomic-scale analysis of biological interfaces within the ferritin protein using atom probe tomography that is facilitated by an advanced specimen preparation approach. Embedding ferritin in an organic polymer resin lacking nitrogen provided chemical contrast to visualise atomic distributions and distinguish the inorganic-organic interface of the ferrihydrite mineral core and protein shell, as well as the organic-organic interface between the ferritin protein shell and embedding resin. In addition, we definitively show the atomic-scale distribution of phosphorus as being at the surface of the ferrihydrite mineral with the distribution of sodium mapped within the protein shell environment with an enhanced distribution at the mineral/protein interface. The sample preparation method is robust and can be directly extended to further enhance the study of biological, organic and inorganic nanomaterials relevant to health, energy or the environment.

Atom Probe Tomographic Mapping Directly Reveals the Atomic Distribution of Phosphorus in Resin Embedded Ferritin

PubMed Central

Perea, Daniel E.; Liu, Jia; Bartrand, Jonah; Dicken, Quinten; Thevuthasan, S. Theva; Browning, Nigel D.; Evans, James E.

2016-01-01

Here we report the atomic-scale analysis of biological interfaces within the ferritin protein using atom probe tomography that is facilitated by an advanced specimen preparation approach. Embedding ferritin in an organic polymer resin lacking nitrogen provided chemical contrast to visualise atomic distributions and distinguish the inorganic-organic interface of the ferrihydrite mineral core and protein shell, as well as the organic-organic interface between the ferritin protein shell and embedding resin. In addition, we definitively show the atomic-scale distribution of phosphorus as being at the surface of the ferrihydrite mineral with the distribution of sodium mapped within the protein shell environment with an enhanced distribution at the mineral/protein interface. The sample preparation method is robust and can be directly extended to further enhance the study of biological, organic and inorganic nanomaterials relevant to health, energy or the environment. PMID:26924804

The Protein-DNA Interface database

PubMed Central

2010-01-01

The Protein-DNA Interface database (PDIdb) is a repository containing relevant structural information of Protein-DNA complexes solved by X-ray crystallography and available at the Protein Data Bank. The database includes a simple functional classification of the protein-DNA complexes that consists of three hierarchical levels: Class, Type and Subtype. This classification has been defined and manually curated by humans based on the information gathered from several sources that include PDB, PubMed, CATH, SCOP and COPS. The current version of the database contains only structures with resolution of 2.5 Å or higher, accounting for a total of 922 entries. The major aim of this database is to contribute to the understanding of the main rules that underlie the molecular recognition process between DNA and proteins. To this end, the database is focused on each specific atomic interface rather than on the separated binding partners. Therefore, each entry in this database consists of a single and independent protein-DNA interface. We hope that PDIdb will be useful to many researchers working in fields such as the prediction of transcription factor binding sites in DNA, the study of specificity determinants that mediate enzyme recognition events, engineering and design of new DNA binding proteins with distinct binding specificity and affinity, among others. Finally, due to its friendly and easy-to-use web interface, we hope that PDIdb will also serve educational and teaching purposes. PMID:20482798

The Protein-DNA Interface database.

PubMed

Norambuena, Tomás; Melo, Francisco

2010-05-18

The Protein-DNA Interface database (PDIdb) is a repository containing relevant structural information of Protein-DNA complexes solved by X-ray crystallography and available at the Protein Data Bank. The database includes a simple functional classification of the protein-DNA complexes that consists of three hierarchical levels: Class, Type and Subtype. This classification has been defined and manually curated by humans based on the information gathered from several sources that include PDB, PubMed, CATH, SCOP and COPS. The current version of the database contains only structures with resolution of 2.5 A or higher, accounting for a total of 922 entries. The major aim of this database is to contribute to the understanding of the main rules that underlie the molecular recognition process between DNA and proteins. To this end, the database is focused on each specific atomic interface rather than on the separated binding partners. Therefore, each entry in this database consists of a single and independent protein-DNA interface.We hope that PDIdb will be useful to many researchers working in fields such as the prediction of transcription factor binding sites in DNA, the study of specificity determinants that mediate enzyme recognition events, engineering and design of new DNA binding proteins with distinct binding specificity and affinity, among others. Finally, due to its friendly and easy-to-use web interface, we hope that PDIdb will also serve educational and teaching purposes.

Measuring interactions between polydimethylsiloxane and serum proteins at the air-water interface.

PubMed

Liao, Zhengzheng; Hsieh, Wan-Ting; Baumgart, Tobias; Dmochowski, Ivan J

2013-07-30

The interaction between synthetic polymers and proteins at interfaces is relevant to basic science as well as a wide range of applications in biotechnology and medicine. One particularly common and important interface is the air-water interface (AWI). Due to the special energetics and dynamics of molecules at the AWI, the interplay between synthetic polymer and protein can be very different from that in bulk solution. In this paper, we applied the Langmuir-Blodgett technique and fluorescence microscopy to investigate how the compression state of polydimethylsiloxane (PDMS) film at the AWI affects the subsequent adsorption of serum protein [e.g., human serum albumin (HSA) or immunoglobulin G (IgG)] and the interaction between PDMS and protein. Of particular note is our observation of circular PDMS domains with micrometer diameters that form at the AWI in the highly compressed state of the surface film: proteins were shown to adsorb preferentially to the surface of these circular PDMS domains, accompanied by a greater than 4-fold increase in protein found in the interfacial film. The PDMS-only film and the PDMS-IgG composite film were transferred to cover glass, and platinum-carbon replicas of the transferred films were further characterized by scanning electron microscopy and atomic force microscopy. We conclude that the structure of the PDMS film greatly affects the amount and distribution of protein at the interface.

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.

Dissecting fragment-based lead discovery at the von Hippel-Lindau protein:hypoxia inducible factor 1α protein-protein interface.

PubMed

Van Molle, Inge; Thomann, Andreas; Buckley, Dennis L; So, Ernest C; Lang, Steffen; Crews, Craig M; Ciulli, Alessio

2012-10-26

Fragment screening is widely used to identify attractive starting points for drug design. However, its potential and limitations to assess the tractability of often challenging protein:protein interfaces have been underexplored. Here, we address this question by means of a systematic deconstruction of lead-like inhibitors of the pVHL:HIF-1α interaction into their component fragments. Using biophysical techniques commonly employed for screening, we could only detect binding of fragments that violate the Rule of Three, are more complex than those typically screened against classical druggable targets, and occupy two adjacent binding subsites at the interface rather than just one. Analyses based on ligand and group lipophilicity efficiency of anchored fragments were applied to dissect the individual subsites and probe for binding hot spots. The implications of our findings for targeting protein interfaces by fragment-based approaches are discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Silicon/HfO{sub 2} interface: Effects of gamma irradiation

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

Maurya, Savita

2016-05-23

Quality of MOS devices is a strong function of substrate and oxide interface. In this work we have studied how gamma photon irradiation affects the interface of a 13 nm thick, atomic layer deposited hafnium dioxide deposited on silicon wafer. CV and GV measurements have been done for pristine and irradiated samples to quantify the effect of gamma photon irradiation. Gamma photon irradiation not only introduces positive charge in the oxide and at the interface of Si/HfO{sub 2} interface but also induce phase change of oxide layer. Maximum oxide capacitances are affected by gamma photon irradiation.

Protein interactions and ligand binding: from protein subfamilies to functional specificity.

PubMed

Rausell, Antonio; Juan, David; Pazos, Florencio; Valencia, Alfonso

2010-02-02

The divergence accumulated during the evolution of protein families translates into their internal organization as subfamilies, and it is directly reflected in the characteristic patterns of differentially conserved residues. These specifically conserved positions in protein subfamilies are known as "specificity determining positions" (SDPs). Previous studies have limited their analysis to the study of the relationship between these positions and ligand-binding specificity, demonstrating significant yet limited predictive capacity. We have systematically extended this observation to include the role of differential protein interactions in the segregation of protein subfamilies and explored in detail the structural distribution of SDPs at protein interfaces. Our results show the extensive influence of protein interactions in the evolution of protein families and the widespread association of SDPs with protein interfaces. The combined analysis of SDPs in interfaces and ligand-binding sites provides a more complete picture of the organization of protein families, constituting the necessary framework for a large scale analysis of the evolution of protein function.

Protein interface classification by evolutionary analysis

PubMed Central

2012-01-01

Background Distinguishing biologically relevant interfaces from lattice contacts in protein crystals is a fundamental problem in structural biology. Despite efforts towards the computational prediction of interface character, many issues are still unresolved. Results We present here a protein-protein interface classifier that relies on evolutionary data to detect the biological character of interfaces. The classifier uses a simple geometric measure, number of core residues, and two evolutionary indicators based on the sequence entropy of homolog sequences. Both aim at detecting differential selection pressure between interface core and rim or rest of surface. The core residues, defined as fully buried residues (>95% burial), appear to be fundamental determinants of biological interfaces: their number is in itself a powerful discriminator of interface character and together with the evolutionary measures it is able to clearly distinguish evolved biological contacts from crystal ones. We demonstrate that this definition of core residues leads to distinctively better results than earlier definitions from the literature. The stringent selection and quality filtering of structural and sequence data was key to the success of the method. Most importantly we demonstrate that a more conservative selection of homolog sequences - with relatively high sequence identities to the query - is able to produce a clearer signal than previous attempts. Conclusions An evolutionary approach like the one presented here is key to the advancement of the field, which so far was missing an effective method exploiting the evolutionary character of protein interfaces. Its coverage and performance will only improve over time thanks to the incessant growth of sequence databases. Currently our method reaches an accuracy of 89% in classifying interfaces of the Ponstingl 2003 datasets and it lends itself to a variety of useful applications in structural biology and bioinformatics. We made the corresponding software implementation available to the community as an easy-to-use graphical web interface at http://www.eppic-web.org. PMID:23259833

Effect of environmental conditions on variation in the sediment-water interface created by complex macrofaunal burrows on a tidal flat

NASA Astrophysics Data System (ADS)

Koo, Bon Joo; Kwon, Kae Kyoung; Hyun, Jung-Ho

2007-11-01

We quantified the increase in the sediment-water interface created by the burrowing activities of the resident macrofaunal community and its variation with respect to the physical conditions of the habitat on a tidal fat. We investigated environmental factors and dimensions of macrofaunal burrows with respect to tidal height and vegetation during spring and summer at three sites. A resin-casting method was used to quantify the dimensions of all burrows at each site. The dimensions of macrofaunal burrows varied both temporally and spatially and the increase in the sediment-water interface reached a maximum of 311%, ranging from 20 to 255% under different habitat conditions. The sediment-water interface depended on the duration of exposure resulting from tidal height, increased temperatures resulting from seasonality, and marsh plant density. Burrows were deeper and more expansive at both higher tidal levels and higher temperatures in summer. Burrow dimensions were sharply reduced with the disappearance of adult macrofauna in areas where the roots of the marsh plant Suaeda japonica were dense. The significance of this study lies in quantifying the burrow dimensions of the entire macrofaunal community, rather than just a single population, and confirming their spatial and temporal variation with respect to physical conditions of the habitat. Environmental factors responsible for variation in burrow dimensions are discussed.

Functional correlation of bacterial LuxS with their quaternary associations: interface analysis of the structure networks

PubMed Central

Bhattacharyya, Moitrayee; Vishveshwara, Saraswathi

2009-01-01

Background The genome of a wide variety of prokaryotes contains the luxS gene homologue, which encodes for the protein S-ribosylhomocysteinelyase (LuxS). This protein is responsible for the production of the quorum sensing molecule, AI-2 and has been implicated in a variety of functions such as flagellar motility, metabolic regulation, toxin production and even in pathogenicity. A high structural similarity is present in the LuxS structures determined from a few species. In this study, we have modelled the structures from several other species and have investigated their dimer interfaces. We have attempted to correlate the interface features of LuxS with the phenotypic nature of the organisms. Results The protein structure networks (PSN) are constructed and graph theoretical analysis is performed on the structures obtained from X-ray crystallography and on the modelled ones. The interfaces, which are known to contain the active site, are characterized from the PSNs of these homodimeric proteins. The key features presented by the protein interfaces are investigated for the classification of the proteins in relation to their function. From our analysis, structural interface motifs are identified for each class in our dataset, which showed distinctly different pattern at the interface of LuxS for the probiotics and some extremophiles. Our analysis also reveals potential sites of mutation and geometric patterns at the interface that was not evident from conventional sequence alignment studies. Conclusion The structure network approach employed in this study for the analysis of dimeric interfaces in LuxS has brought out certain structural details at the side-chain interaction level, which were elusive from the conventional structure comparison methods. The results from this study provide a better understanding of the relation between the luxS gene and its functional role in the prokaryotes. This study also makes it possible to explore the potential direction towards the design of inhibitors of LuxS and thus towards a wide range of antimicrobials. PMID:19243584

Interface Problems: Structural Constraints on Interpretation?

ERIC Educational Resources Information Center

Frazier, Lyn; Clifton, Charles; Rayner, Keith; Deevy, Patricia; Koh, Sungryong; Bader, Markus

2005-01-01

Five experiments investigated the interpretation of quantified noun phrases in relation to discourse structure. They demonstrated, using questionnaire and on-line reading techniques, that readers in English prefer to give a quantified noun phrase in (VP-external) subject position a presuppositional interpretation, in which the noun phrase limits…

Local Geometry and Evolutionary Conservation of Protein Surfaces Reveal the Multiple Recognition Patches in Protein-Protein Interactions

PubMed Central

Laine, Elodie; Carbone, Alessandra

2015-01-01

Protein-protein interactions (PPIs) are essential to all biological processes and they represent increasingly important therapeutic targets. Here, we present a new method for accurately predicting protein-protein interfaces, understanding their properties, origins and binding to multiple partners. Contrary to machine learning approaches, our method combines in a rational and very straightforward way three sequence- and structure-based descriptors of protein residues: evolutionary conservation, physico-chemical properties and local geometry. The implemented strategy yields very precise predictions for a wide range of protein-protein interfaces and discriminates them from small-molecule binding sites. Beyond its predictive power, the approach permits to dissect interaction surfaces and unravel their complexity. We show how the analysis of the predicted patches can foster new strategies for PPIs modulation and interaction surface redesign. The approach is implemented in JET2, an automated tool based on the Joint Evolutionary Trees (JET) method for sequence-based protein interface prediction. JET2 is freely available at www.lcqb.upmc.fr/JET2. PMID:26690684

Proteomic Analysis of the Cell Cycle of Procylic Form Trypanosoma brucei.

PubMed

Crozier, Thomas W M; Tinti, Michele; Wheeler, Richard J; Ly, Tony; Ferguson, Michael A J; Lamond, Angus I

2018-06-01

We describe a single-step centrifugal elutriation method to produce synchronous Gap1 (G1)-phase procyclic trypanosomes at a scale amenable for proteomic analysis of the cell cycle. Using ten-plex tandem mass tag (TMT) labeling and mass spectrometry (MS)-based proteomics technology, the expression levels of 5325 proteins were quantified across the cell cycle in this parasite. Of these, 384 proteins were classified as cell-cycle regulated and subdivided into nine clusters with distinct temporal regulation. These groups included many known cell cycle regulators in trypanosomes, which validates the approach. In addition, we identify 40 novel cell cycle regulated proteins that are essential for trypanosome survival and thus represent potential future drug targets for the prevention of trypanosomiasis. Through cross-comparison to the TrypTag endogenous tagging microscopy database, we were able to validate the cell-cycle regulated patterns of expression for many of the proteins of unknown function detected in our proteomic analysis. A convenient interface to access and interrogate these data is also presented, providing a useful resource for the scientific community. Data are available via ProteomeXchange with identifier PXD008741 (https://www.ebi.ac.uk/pride/archive/). © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Freezing-induced phase separation and spatial microheterogeneity in protein solutions.

PubMed

Dong, Jinping; Hubel, Allison; Bischof, John C; Aksan, Alptekin

2009-07-30

Amid decades of research, the basic mechanisms of lyo-/cryostabilization of proteins and more complex organisms have not yet been fully established. One major bottleneck is the inability to probe into and control the molecular level interactions. The molecular interactions are responsible for the significant differences in the outcome of the preservation processes. (1) In this communication, we have utilized confocal Raman microspectroscopy to quantify the freezing-induced microheterogeneity and phase separation (solid and liquid) in a frozen solution composed of a model protein (lysozyme) and a lyo-/cryoprotectant (trehalose), which experienced different degrees of supercooling. Detailed quantitative spectral analysis was performed across the ice, the freeze-concentrated liquid (FCL) phases, and the interface region between them. It was established that the characteristics of the microstructures observed after freezing depended not only on the concentration of trehalose in the solution but also on the degree of supercooling. It was shown that, when samples were frozen after high supercooling, small amounts of lysozyme and trehalose were occluded in the ice phase. Lysozyme preserved its native-like secondary structure in the FCL region but was denatured in the ice phase. Also, it was observed that induction of freezing after a high degree of supercooling of high trehalose concentrations resulted in aggregation of the sugar and the protein.

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

Brandt, Riley E.; Mangan, Niall M.; Li, Jian V.

The development of new thin-film photovoltaic (PV) absorbers is often hindered by the search for an optimal heterojunction contact; an unoptimized contact may be mistaken for poor quality of the underlying absorber, making it difficult to assess the reasons for poor performance. Therefore, quantifying the loss in device efficiency and open-circuit voltage (VOC) as a result of the interface is a critical step in evaluating a new material. In the present work, we fabricate thin-film PV devices using cuprous oxide (Cu2O), with several different n-type heterojunction contacts. Their current-voltage characteristics are measured over a range of temperatures and illumination intensitiesmore » (JVTi). We quantify the loss in VOC due to the interface and determine the effective energy gap at the interface. The effective interface gap measured by JVTi matches the gap measured by X-ray photoelectron spectroscopy, albeit with higher energy resolution and an order of magnitude faster. We discuss potential artifacts in JVTi measurements and areas where analytical models are insufficient. Applying JVTi to complete devices, rather than incomplete material stacks, suggests that it can be a quick, accurate method to assess the loss due to unoptimized interface band offsets in thin-film PV devices.« less

Weak conservation of structural features in the interfaces of homologous transient protein–protein complexes

PubMed Central

Sudha, Govindarajan; Singh, Prashant; Swapna, Lakshmipuram S; Srinivasan, Narayanaswamy

2015-01-01

Residue types at the interface of protein–protein complexes (PPCs) are known to be reasonably well conserved. However, we show, using a dataset of known 3-D structures of homologous transient PPCs, that the 3-D location of interfacial residues and their interaction patterns are only moderately and poorly conserved, respectively. Another surprising observation is that a residue at the interface that is conserved is not necessarily in the interface in the homolog. Such differences in homologous complexes are manifested by substitution of the residues that are spatially proximal to the conserved residue and structural differences at the interfaces as well as differences in spatial orientations of the interacting proteins. Conservation of interface location and the interaction pattern at the core of the interfaces is higher than at the periphery of the interface patch. Extents of variability of various structural features reported here for homologous transient PPCs are higher than the variation in homologous permanent homomers. Our findings suggest that straightforward extrapolation of interfacial nature and inter-residue interaction patterns from template to target could lead to serious errors in the modeled complex structure. Understanding the evolution of interfaces provides insights to improve comparative modeling of PPC structures. PMID:26311309

Multivalent-Ion-Activated Protein Adsorption Reflecting Bulk Reentrant Behavior.

PubMed

Fries, Madeleine R; Stopper, Daniel; Braun, Michal K; Hinderhofer, Alexander; Zhang, Fajun; Jacobs, Robert M J; Skoda, Maximilian W A; Hansen-Goos, Hendrik; Roth, Roland; Schreiber, Frank

2017-12-01

Protein adsorption at the solid-liquid interface is an important phenomenon that often can be observed as a first step in biological processes. Despite its inherent importance, still relatively little is known about the underlying microscopic mechanisms. Here, using multivalent ions, we demonstrate the control of the interactions and the corresponding adsorption of net-negatively charged proteins (bovine serum albumin) at a solid-liquid interface. This is demonstrated by ellipsometry and corroborated by neutron reflectivity and quartz-crystal microbalance experiments. We show that the reentrant condensation observed within the rich bulk phase behavior of the system featuring a nonmonotonic dependence of the second virial coefficient on salt concentration c_{s} is reflected in an intriguing way in the protein adsorption d(c_{s}) at the interface. Our findings are successfully described and understood by a model of ion-activated patchy interactions within the framework of the classical density functional theory. In addition to the general challenge of connecting bulk and interface behavior, our work has implications for, inter alia, nucleation at interfaces.

Multivalent-Ion-Activated Protein Adsorption Reflecting Bulk Reentrant Behavior

NASA Astrophysics Data System (ADS)

Fries, Madeleine R.; Stopper, Daniel; Braun, Michal K.; Hinderhofer, Alexander; Zhang, Fajun; Jacobs, Robert M. J.; Skoda, Maximilian W. A.; Hansen-Goos, Hendrik; Roth, Roland; Schreiber, Frank

2017-12-01

Protein adsorption at the solid-liquid interface is an important phenomenon that often can be observed as a first step in biological processes. Despite its inherent importance, still relatively little is known about the underlying microscopic mechanisms. Here, using multivalent ions, we demonstrate the control of the interactions and the corresponding adsorption of net-negatively charged proteins (bovine serum albumin) at a solid-liquid interface. This is demonstrated by ellipsometry and corroborated by neutron reflectivity and quartz-crystal microbalance experiments. We show that the reentrant condensation observed within the rich bulk phase behavior of the system featuring a nonmonotonic dependence of the second virial coefficient on salt concentration cs is reflected in an intriguing way in the protein adsorption d (cs) at the interface. Our findings are successfully described and understood by a model of ion-activated patchy interactions within the framework of the classical density functional theory. In addition to the general challenge of connecting bulk and interface behavior, our work has implications for, inter alia, nucleation at interfaces.

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.

Predicting permanent and transient protein-protein interfaces.

PubMed

La, David; Kong, Misun; Hoffman, William; Choi, Youn Im; Kihara, Daisuke

2013-05-01

Protein-protein interactions (PPIs) are involved in diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, whereas the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of PPIs. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces (PBIs) in protein surfaces. Without knowledge of the interacting partner, the method uses a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method, BindML+, performs very well in protein interface classification. A very high area under the curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient PBIs. Copyright © 2013 Wiley Periodicals, Inc.

DockRank: Ranking docked conformations using partner-specific sequence homology-based protein interface prediction

PubMed Central

Xue, Li C.; Jordan, Rafael A.; EL-Manzalawy, Yasser; Dobbs, Drena; Honavar, Vasant

2015-01-01

Selecting near-native conformations from the immense number of conformations generated by docking programs remains a major challenge in molecular docking. We introduce DockRank, a novel approach to scoring docked conformations based on the degree to which the interface residues of the docked conformation match a set of predicted interface residues. Dock-Rank uses interface residues predicted by partner-specific sequence homology-based protein–protein interface predictor (PS-HomPPI), which predicts the interface residues of a query protein with a specific interaction partner. We compared the performance of DockRank with several state-of-the-art docking scoring functions using Success Rate (the percentage of cases that have at least one near-native conformation among the top m conformations) and Hit Rate (the percentage of near-native conformations that are included among the top m conformations). In cases where it is possible to obtain partner-specific (PS) interface predictions from PS-HomPPI, DockRank consistently outperforms both (i) ZRank and IRAD, two state-of-the-art energy-based scoring functions (improving Success Rate by up to 4-fold); and (ii) Variants of DockRank that use predicted interface residues obtained from several protein interface predictors that do not take into account the binding partner in making interface predictions (improving success rate by up to 39-fold). The latter result underscores the importance of using partner-specific interface residues in scoring docked conformations. We show that DockRank, when used to re-rank the conformations returned by ClusPro, improves upon the original ClusPro rankings in terms of both Success Rate and Hit Rate. DockRank is available as a server at http://einstein.cs.iastate.edu/DockRank/. PMID:23873600

Assessment of Protein Side-Chain Conformation Prediction Methods in Different Residue Environments

PubMed Central

Peterson, Lenna X.; Kang, Xuejiao; Kihara, Daisuke

2016-01-01

Computational prediction of side-chain conformation is an important component of protein structure prediction. Accurate side-chain prediction is crucial for practical applications of protein structure models that need atomic detailed resolution such as protein and ligand design. We evaluated the accuracy of eight side-chain prediction methods in reproducing the side-chain conformations of experimentally solved structures deposited to the Protein Data Bank. Prediction accuracy was evaluated for a total of four different structural environments (buried, surface, interface, and membrane-spanning) in three different protein types (monomeric, multimeric, and membrane). Overall, the highest accuracy was observed for buried residues in monomeric and multimeric proteins. Notably, side-chains at protein interfaces and membrane-spanning regions were better predicted than surface residues even though the methods did not all use multimeric and membrane proteins for training. Thus, we conclude that the current methods are as practically useful for modeling protein docking interfaces and membrane-spanning regions as for modeling monomers. PMID:24619909

Protein Adsorption and Layer Formation at the Stainless Steel-Solution Interface Mediates Shear-Induced Particle Formation for an IgG1 Monoclonal Antibody.

PubMed

Kalonia, Cavan K; Heinrich, Frank; Curtis, Joseph E; Raman, Sid; Miller, Maria A; Hudson, Steven D

2018-03-05

Passage of specific protein solutions through certain pumps, tubing, and/or filling nozzles can result in the production of unwanted subvisible protein particles (SVPs). In this work, surface-mediated SVP formation was investigated. Specifically, the effects of different solid interface materials, interfacial shear rates, and protein concentrations on SVP formation were measured for the National Institute of Standards and Technology monoclonal antibody (NISTmAb), a reference IgG1 monoclonal antibody (mAb). A stainless steel rotary piston pump was used to identify formulation and process parameters that affect aggregation, and a flow cell (alumina or stainless steel interface) was used to further investigate the effect of different interface materials and/or interfacial shear rates. SVP particles produced were monitored using flow microscopy or flow cytometry. Neutron reflectometry and a quartz crystal microbalance with dissipation monitoring were used to characterize adsorption and properties of NISTmAb at the stainless steel interface. Pump/shear cell experiments showed that the NISTmAb concentration and interface material had a significant effect on SVP formation, while the effects of interfacial shear rate and passage number were less important. At the higher NISTmAb concentrations, the adsorbed protein became structurally altered at the stainless steel interface. The primary adsorbed layer remained largely undisturbed during flow, suggesting that SVP formation at high NISTmAb concentration was caused by the disruption of patches and/or secondary interactions.

Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis.

PubMed

Vedula, Pavan; Cruz, Lissette A; Gutierrez, Natasha; Davis, Justin; Ayee, Brian; Abramczyk, Rachel; Rodriguez, Alexis J

2016-06-30

Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation.

Distinctive glial and neuronal interfacing on nanocrystalline diamond.

PubMed

Bendali, Amel; Agnès, Charles; Meffert, Simone; Forster, Valérie; Bongrain, Alexandre; Arnault, Jean-Charles; Sahel, José-Alain; Offenhäusser, Andreas; Bergonzo, Philippe; Picaud, Serge

2014-01-01

Direct electrode/neuron interfacing is a key challenge to achieve high resolution of neuronal stimulation required for visual prostheses. Neuronal interfacing on biomaterials commonly requires the presence of glial cells and/or protein coating. Nanocrystalline diamond is a highly mechanically stable biomaterial with a remarkably large potential window for the electrical stimulation of tissues. Using adult retinal cell cultures from rats, we found that glial cells and retinal neurons grew equally well on glass and nanocrystalline diamond. The use of a protein coating increased cell survival, particularly for glial cells. However, bipolar neurons appeared to grow even in direct contact with bare diamond. We investigated whether the presence of glial cells contributed to this direct neuron/diamond interface, by using purified adult retinal ganglion cells to seed diamond and glass surfaces with and without protein coatings. Surprisingly, these fully differentiated spiking neurons survived better on nanocrystalline diamond without any protein coating. This greater survival was indicated by larger cell numbers and the presence of longer neurites. When a protein pattern was drawn on diamond, neurons did not grow preferentially on the coated area, by contrast to their behavior on a patterned glass. This study highlights the interesting biocompatibility properties of nanocrystalline diamond, allowing direct neuronal interfacing, whereas a protein coating was required for glial cell growth.

Distinctive Glial and Neuronal Interfacing on Nanocrystalline Diamond

PubMed Central

Bendali, Amel; Agnès, Charles; Meffert, Simone; Forster, Valérie; Bongrain, Alexandre; Arnault, Jean-Charles; Sahel, José-Alain; Offenhäusser, Andreas; Bergonzo, Philippe; Picaud, Serge

2014-01-01

Direct electrode/neuron interfacing is a key challenge to achieve high resolution of neuronal stimulation required for visual prostheses. Neuronal interfacing on biomaterials commonly requires the presence of glial cells and/or protein coating. Nanocrystalline diamond is a highly mechanically stable biomaterial with a remarkably large potential window for the electrical stimulation of tissues. Using adult retinal cell cultures from rats, we found that glial cells and retinal neurons grew equally well on glass and nanocrystalline diamond. The use of a protein coating increased cell survival, particularly for glial cells. However, bipolar neurons appeared to grow even in direct contact with bare diamond. We investigated whether the presence of glial cells contributed to this direct neuron/diamond interface, by using purified adult retinal ganglion cells to seed diamond and glass surfaces with and without protein coatings. Surprisingly, these fully differentiated spiking neurons survived better on nanocrystalline diamond without any protein coating. This greater survival was indicated by larger cell numbers and the presence of longer neurites. When a protein pattern was drawn on diamond, neurons did not grow preferentially on the coated area, by contrast to their behavior on a patterned glass. This study highlights the interesting biocompatibility properties of nanocrystalline diamond, allowing direct neuronal interfacing, whereas a protein coating was required for glial cell growth. PMID:24664111

Mechanical properties of protein adsorption layers at the air/water and oil/water interface: a comparison in light of the thermodynamical stability of proteins.

PubMed

Mitropoulos, Varvara; Mütze, Annekathrin; Fischer, Peter

2014-04-01

Over the last decades numerous studies on the interfacial rheological response of protein adsorption layers have been published. The comparison of these studies and the retrieval of a common parameter to compare protein interfacial activity are hampered by the fact that different boundary conditions (e.g. physico-chemical, instrumental, interfacial) were used. In the present work we review previous studies and attempt a unifying approach for the comparison between bulk protein properties and their adsorption films. Among many common food grade proteins we chose bovine serum albumin, β-lactoglobulin and lysozyme for their difference in thermodynamic stability and studied their adsorption at the air/water and limonene/water interface. In order to achieve this we have i) systematically analyzed protein adsorption kinetics in terms of surface pressure rise using a drop profile analysis tensiometer and ii) we addressed the interfacial layer properties under shear stress using an interfacial shear rheometer under the same experimental conditions. We could show that thermodynamically less stable proteins adsorb generally faster and yield films with higher shear rheological properties at air/water interface. The same proteins showed an analog behavior when adsorbing at the limonene/water interface but at slower rates. Copyright © 2013 Elsevier B.V. All rights reserved.

Specific RNA-protein interactions detected with saturation transfer difference NMR.

PubMed

Harris, Kimberly A; Shekhtman, Alexander; Agris, Paul F

2013-08-01

RNA, at the forefront of biochemical research due to its central role in biology, is recognized by proteins through various mechanisms. Analysis of the RNA-protein interface provides insight into the recognition determinants and function. As such, there is a demand for developing new methods to characterize RNA-protein interactions. Saturation transfer difference (STD) NMR can identify binding ligands for proteins in a rather short period of time, with data acquisitions of just a few hours. Two RNA-protein systems involved in RNA modification were studied using STD NMR. The N (6)-threonylcarbamoyltransferase, YrdC, with nucleoside-specific recognition, was shown to bind the anticodon stem-loop of tRNA(Lys)UUU. The points of contact on the RNA were assigned and a binding interface was identified. STD NMR was also applied to the interaction of the archaeal ribosomal protein, L7Ae, with the box C/D K-turn RNA. The distinctiveness of the two RNA-protein interfaces was evident. Both RNAs exhibited strong STD signals indicative of direct contact with the respective protein, but reflected the nature of recognition. Characterization of nucleic acid recognition determinants traditionally involves cost and time prohibitive methods. This approach offers significant insight into interaction interfaces fairly rapidly, and complements existing structural methods.

Dynamics of the BH3-Only Protein Binding Interface of Bcl-xL.

PubMed

Liu, Xiaorong; Beugelsdijk, Alex; Chen, Jianhan

2015-09-01

The balance and interplay between pro-death and pro-survival members of the B-cell lymphoma-2 (Bcl-2) family proteins play key roles in regulation of the mitochondrial pathway of programmed cell death. Recent NMR and biochemical studies have revealed that binding of the proapoptotic BH3-only protein PUMA induces significant unfolding of antiapoptotic Bcl-xL at the interface, which in turn disrupts the Bcl-xL/p53 interaction to activate apoptosis. However, the molecular mechanism of such regulated unfolding of Bcl-xL is not fully understood. Analysis of the existing Protein Data Bank structures of Bcl-xL in both bound and unbound states reveal substantial intrinsic heterogeneity at its BH3-only protein binding interface. Large-scale atomistic simulations were performed in explicit solvent for six representative structures to further investigate the intrinsic conformational dynamics of Bcl-xL. The results support that the BH3-only protein binding interface of Bcl-xL is much more dynamic compared to the rest of the protein, both unbound and when bound to various BH3-only proteins. Such intrinsic interfacial conformational dynamics likely provides a physical basis that allows Bcl-xL to respond sensitively to detailed biophysical properties of the ligand. The ability of Bcl-xL to retain or even enhance dynamics at the interface in bound states could further facilitate the regulation of its interactions with various BH3-only proteins such as through posttranslational modifications. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

InterProSurf: a web server for predicting interacting sites on protein surfaces

PubMed Central

Negi, Surendra S.; Schein, Catherine H.; Oezguen, Numan; Power, Trevor D.; Braun, Werner

2009-01-01

Summary A new web server, InterProSurf, predicts interacting amino acid residues in proteins that are most likely to interact with other proteins, given the 3D structures of subunits of a protein complex. The prediction method is based on solvent accessible surface area of residues in the isolated subunits, a propensity scale for interface residues and a clustering algorithm to identify surface regions with residues of high interface propensities. Here we illustrate the application of InterProSurf to determine which areas of Bacillus anthracis toxins and measles virus hemagglutinin protein interact with their respective cell surface receptors. The computationally predicted regions overlap with those regions previously identified as interface regions by sequence analysis and mutagenesis experiments. PMID:17933856

Improving predictions of protein-protein interfaces by combining amino acid-specific classifiers based on structural and physicochemical descriptors with their weighted neighbor averages.

PubMed

de Moraes, Fábio R; Neshich, Izabella A P; Mazoni, Ivan; Yano, Inácio H; Pereira, José G C; Salim, José A; Jardine, José G; Neshich, Goran

2014-01-01

Protein-protein interactions are involved in nearly all regulatory processes in the cell and are considered one of the most important issues in molecular biology and pharmaceutical sciences but are still not fully understood. Structural and computational biology contributed greatly to the elucidation of the mechanism of protein interactions. In this paper, we present a collection of the physicochemical and structural characteristics that distinguish interface-forming residues (IFR) from free surface residues (FSR). We formulated a linear discriminative analysis (LDA) classifier to assess whether chosen descriptors from the BlueStar STING database (http://www.cbi.cnptia.embrapa.br/SMS/) are suitable for such a task. Receiver operating characteristic (ROC) analysis indicates that the particular physicochemical and structural descriptors used for building the linear classifier perform much better than a random classifier and in fact, successfully outperform some of the previously published procedures, whose performance indicators were recently compared by other research groups. The results presented here show that the selected set of descriptors can be utilized to predict IFRs, even when homologue proteins are missing (particularly important for orphan proteins where no homologue is available for comparative analysis/indication) or, when certain conformational changes accompany interface formation. The development of amino acid type specific classifiers is shown to increase IFR classification performance. Also, we found that the addition of an amino acid conservation attribute did not improve the classification prediction. This result indicates that the increase in predictive power associated with amino acid conservation is exhausted by adequate use of an extensive list of independent physicochemical and structural parameters that, by themselves, fully describe the nano-environment at protein-protein interfaces. The IFR classifier developed in this study is now integrated into the BlueStar STING suite of programs. Consequently, the prediction of protein-protein interfaces for all proteins available in the PDB is possible through STING_interfaces module, accessible at the following website: (http://www.cbi.cnptia.embrapa.br/SMS/predictions/index.html).

Improving Predictions of Protein-Protein Interfaces by Combining Amino Acid-Specific Classifiers Based on Structural and Physicochemical Descriptors with Their Weighted Neighbor Averages

PubMed Central

de Moraes, Fábio R.; Neshich, Izabella A. P.; Mazoni, Ivan; Yano, Inácio H.; Pereira, José G. C.; Salim, José A.; Jardine, José G.; Neshich, Goran

2014-01-01

Protein-protein interactions are involved in nearly all regulatory processes in the cell and are considered one of the most important issues in molecular biology and pharmaceutical sciences but are still not fully understood. Structural and computational biology contributed greatly to the elucidation of the mechanism of protein interactions. In this paper, we present a collection of the physicochemical and structural characteristics that distinguish interface-forming residues (IFR) from free surface residues (FSR). We formulated a linear discriminative analysis (LDA) classifier to assess whether chosen descriptors from the BlueStar STING database (http://www.cbi.cnptia.embrapa.br/SMS/) are suitable for such a task. Receiver operating characteristic (ROC) analysis indicates that the particular physicochemical and structural descriptors used for building the linear classifier perform much better than a random classifier and in fact, successfully outperform some of the previously published procedures, whose performance indicators were recently compared by other research groups. The results presented here show that the selected set of descriptors can be utilized to predict IFRs, even when homologue proteins are missing (particularly important for orphan proteins where no homologue is available for comparative analysis/indication) or, when certain conformational changes accompany interface formation. The development of amino acid type specific classifiers is shown to increase IFR classification performance. Also, we found that the addition of an amino acid conservation attribute did not improve the classification prediction. This result indicates that the increase in predictive power associated with amino acid conservation is exhausted by adequate use of an extensive list of independent physicochemical and structural parameters that, by themselves, fully describe the nano-environment at protein-protein interfaces. The IFR classifier developed in this study is now integrated into the BlueStar STING suite of programs. Consequently, the prediction of protein-protein interfaces for all proteins available in the PDB is possible through STING_interfaces module, accessible at the following website: (http://www.cbi.cnptia.embrapa.br/SMS/predictions/index.html). PMID:24489849

Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces.

PubMed

Engin, H Billur; Kreisberg, Jason F; Carter, Hannah

2016-01-01

Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10(-4)) and oncogenes (Odds Ratio 1.17, P-value < 10(-3)). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10(-8)). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer genes that encode proteins participating in interactions that are perturbed recurrently across tumors. In summary, mutation of specific protein interactions is an important contributor to tumor heterogeneity and may have important implications for clinical outcomes.

Detection of amide I signals of interfacial proteins in situ using SFG.

PubMed

Wang, Jie; Even, Mark A; Chen, Xiaoyun; Schmaier, Alvin H; Waite, J Herbert; Chen, Zhan

2003-08-20

In this Communication, we demonstrate the novel observation that it is feasible to collect amide signals from polymer/protein solution interfaces in situ using sum frequency generation (SFG) vibrational spectroscopy. Such SFG amide signals allow for acquisition of more detailed molecular level information of entire interfacial protein structures. Proteins investigated include bovine serum albumin, mussel protein mefp-2, factor XIIa, and ubiquitin. Our studies indicate that different proteins generate different SFG amide signals at the polystyrene/protein solution interface, showing that they have different interfacial coverage, secondary structure, or orientation.

Effects of Intercellular Junction Protein Expression on Intracellular Ice Formation in Mouse Insulinoma Cells

PubMed Central

Higgins, Adam Z.; Karlsson, Jens O.M.

2013-01-01

The development of cryopreservation procedures for tissues has proven to be difficult in part because cells within tissue are more susceptible to intracellular ice formation (IIF) than are isolated cells. In particular, previous studies suggest that cell-cell interactions increase the likelihood of IIF by enabling propagation of ice between neighboring cells, a process thought to be mediated by gap junction channels. In this study, we investigated the effects of cell-cell interactions on IIF using three genetically modified strains of the mouse insulinoma cell line MIN6, each of which expressed key intercellular junction proteins (connexin-36, E-cadherin, and occludin) at different levels. High-speed video cryomicroscopy was used to visualize the freezing process in pairs of adherent cells, revealing that the initial IIF event in a given cell pair was correlated with a hitherto unrecognized precursor phenomenon: penetration of extracellular ice into paracellular spaces at the cell-cell interface. Such paracellular ice penetration occurred in the majority of cell pairs observed, and typically preceded and colocalized with the IIF initiation events. Paracellular ice penetration was generally not observed at temperatures >−5.65°C, which is consistent with a penetration mechanism via defects in tight-junction barriers at the cell-cell interface. Although the maximum temperature of paracellular penetration was similar for all four cell strains, genetically modified cells exhibited a significantly higher frequency of ice penetration and a higher mean IIF temperature than did wild-type cells. A four-state Markov chain model was used to quantify the rate constants of the paracellular ice penetration process, the penetration-associated IIF initiation process, and the intercellular ice propagation process. In the initial stages of freezing (>−15°C), junction protein expression appeared to only have a modest effect on the kinetics of propagative IIF, and even cell strains lacking the gap junction protein connexin-36 exhibited nonnegligible ice propagation rates. PMID:24209845

Influence of processing factors on the stability of model mayonnaise with whole egg during long-term storage.

PubMed

Ariizumi, Masahiro; Kubo, Megumi; Handa, Akihiro; Hayakawa, Takashi; Matsumiya, Kentaro; Matsumura, Yasuki

2017-04-01

Mayonnaise-like oil-in-water emulsions with different stabilities-evaluated from the degree of macroscopic defects, e.g., syneresis-were prepared by different formulations and processing conditions (egg yolk weight, homogenizer speed, and vegetable oil temperature). Emulsions prepared with lower egg yolk content were destabilized for shorter periods. The long-term stability of emulsions was weakly related to initial properties, e.g., oil droplet distribution and protein coverage at the interface. Protein aggregation between oil droplets was observed and would be responsible for the instability of emulsions exhibited by the appearance defects. SDS-PAGE results for adsorbed and unadsorbed proteins at the O/W interface suggested that predominant constituents adsorbed onto the interface were egg white proteins as compared with egg yolk components when the amount of added egg yolk was low. In present condition, egg white proteins adsorbed at the O/W interface could be a bridge of neighboring oil droplets thereby causing flocculation in emulsions.

Recent experimental advances on hydrophobic interactions at solid/water and fluid/water interfaces.

PubMed

Zeng, Hongbo; Shi, Chen; Huang, Jun; Li, Lin; Liu, Guangyi; Zhong, Hong

2015-03-15

Hydrophobic effects play important roles in a wide range of natural phenomena and engineering processes such as coalescence of oil droplets in water, air flotation of mineral particles, and folding and assembly of proteins and biomembranes. In this work, the authors highlight recent experimental attempts to reveal the physical origin of hydrophobic effects by directly quantifying the hydrophobic interaction on both solid/water and fluid/water interfaces using state-of-art nanomechanical techniques such as surface forces apparatus and atomic force microscopy (AFM). For solid hydrophobic surfaces of different hydrophobicity, the range of hydrophobic interaction was reported to vary from ∼10 to >100 nm. With various characterization techniques, the very long-ranged attraction (>100 nm) has been demonstrated to be mainly attributed to nonhydrophobic interaction mechanisms such as pre-existing nanobubbles and molecular rearrangement. By ruling out these factors, intrinsic hydrophobic interaction was measured to follow an exponential law with decay length of 1-2 nm with effective range less than 20 nm. On the other hand, hydrophobic interaction measured at fluid interfaces using AFM droplet/bubble probe technique was found to decay with a much shorter length of ∼0.3 nm. This discrepancy of measured decay lengths is proposed to be attributed to inherent physical distinction between solid and fluid interfaces, which impacts the structure of interface-adjacent water molecules. Direct measurement of hydrophobic interaction on a broader range of interfaces and characterization of interfacial water molecular structure using spectroscopic techniques are anticipated to help unravel the origin of this rigidity-related mismatch of hydrophobic interaction and hold promise to uncover the physical nature of hydrophobic effects. With improved understanding of hydrophobic interaction, intrinsic interaction mechanisms of many biological and chemical pathways can be better elucidated, and novel devices/processes can be developed with capacity to modulate and control the hydrophobic effects from the molecular to the macroscopic scale.

Water promotes the sealing of nanoscale packing defects in folding proteins.

PubMed

Fernández, Ariel

2014-05-21

A net dipole moment is shown to arise from a non-Debye component of water polarization created by nanoscale packing defects on the protein surface. Accordingly, the protein electrostatic field exerts a torque on the induced dipole, locally impeding the nucleation of ice at the protein-water interface. We evaluate the solvent orientation steering (SOS) as the reversible work needed to align the induced dipoles with the Debye electrostatic field and computed the SOS for the variable interface of a folding protein. The minimization of the SOS is shown to drive protein folding as evidenced by the entrainment of the total free energy by the SOS energy along trajectories that approach a Debye limit state where no torque arises. This result suggests that the minimization of anomalous water polarization at the interface promotes the sealing of packing defects, thereby maintaining structural integrity and committing the protein chain to fold.

Emulsifying properties and oil/water (O/W) interface adsorption behavior of heated soy proteins: effects of heating concentration, homogenizer rotating speed, and salt addition level.

PubMed

Cui, Zhumei; Chen, Yeming; Kong, Xiangzhen; Zhang, Caimeng; Hua, Yufei

2014-02-19

The adsorption of heat-denatured soy proteins at the oil/water (O/W) interface during emulsification was studied. Protein samples were prepared by heating protein solutions at concentrations of 1-5% (w/v) and were then diluted to 0.3% (w/v). The results showed that soy proteins that had been heated at higher concentrations generated smaller droplet size of emulsion. Increase in homogenizer rotating speed resulted in higher protein adsorption percentages and lower surface loads at the O/W interface. Surface loads for both unheated and heated soy proteins were linearly correlated with the unadsorbed proteins' equilibrium concentration at various rotating speeds. With the rise in NaCl addition level, protein adsorption percentage and surface loads of emulsions increased, whereas lower droplet sizes were obtained at the ionic strength of 0.1 M. The aggregates and non-aggregates displayed different adsorption behaviors when rotating speed or NaCl concentration was varied.

Protein-RNA interface residue prediction using machine learning: an assessment of the state of the art.

PubMed

Walia, Rasna R; Caragea, Cornelia; Lewis, Benjamin A; Towfic, Fadi; Terribilini, Michael; El-Manzalawy, Yasser; Dobbs, Drena; Honavar, Vasant

2012-05-10

RNA molecules play diverse functional and structural roles in cells. They function as messengers for transferring genetic information from DNA to proteins, as the primary genetic material in many viruses, as catalysts (ribozymes) important for protein synthesis and RNA processing, and as essential and ubiquitous regulators of gene expression in living organisms. Many of these functions depend on precisely orchestrated interactions between RNA molecules and specific proteins in cells. Understanding the molecular mechanisms by which proteins recognize and bind RNA is essential for comprehending the functional implications of these interactions, but the recognition 'code' that mediates interactions between proteins and RNA is not yet understood. Success in deciphering this code would dramatically impact the development of new therapeutic strategies for intervening in devastating diseases such as AIDS and cancer. Because of the high cost of experimental determination of protein-RNA interfaces, there is an increasing reliance on statistical machine learning methods for training predictors of RNA-binding residues in proteins. However, because of differences in the choice of datasets, performance measures, and data representations used, it has been difficult to obtain an accurate assessment of the current state of the art in protein-RNA interface prediction. We provide a review of published approaches for predicting RNA-binding residues in proteins and a systematic comparison and critical assessment of protein-RNA interface residue predictors trained using these approaches on three carefully curated non-redundant datasets. We directly compare two widely used machine learning algorithms (Naïve Bayes (NB) and Support Vector Machine (SVM)) using three different data representations in which features are encoded using either sequence- or structure-based windows. Our results show that (i) Sequence-based classifiers that use a position-specific scoring matrix (PSSM)-based representation (PSSMSeq) outperform those that use an amino acid identity based representation (IDSeq) or a smoothed PSSM (SmoPSSMSeq); (ii) Structure-based classifiers that use smoothed PSSM representation (SmoPSSMStr) outperform those that use PSSM (PSSMStr) as well as sequence identity based representation (IDStr). PSSMSeq classifiers, when tested on an independent test set of 44 proteins, achieve performance that is comparable to that of three state-of-the-art structure-based predictors (including those that exploit geometric features) in terms of Matthews Correlation Coefficient (MCC), although the structure-based methods achieve substantially higher Specificity (albeit at the expense of Sensitivity) compared to sequence-based methods. We also find that the expected performance of the classifiers on a residue level can be markedly different from that on a protein level. Our experiments show that the classifiers trained on three different non-redundant protein-RNA interface datasets achieve comparable cross-validation performance. However, we find that the results are significantly affected by differences in the distance threshold used to define interface residues. Our results demonstrate that protein-RNA interface residue predictors that use a PSSM-based encoding of sequence windows outperform classifiers that use other encodings of sequence windows. While structure-based methods that exploit geometric features can yield significant increases in the Specificity of protein-RNA interface residue predictions, such increases are offset by decreases in Sensitivity. These results underscore the importance of comparing alternative methods using rigorous statistical procedures, multiple performance measures, and datasets that are constructed based on several alternative definitions of interface residues and redundancy cutoffs as well as including evaluations on independent test sets into the comparisons.

Modulating in vitro gastric digestion of emulsions using composite whey protein-cellulose nanocrystal interfaces.

PubMed

Sarkar, Anwesha; Zhang, Shuning; Murray, Brent; Russell, Jessica A; Boxal, Sally

2017-10-01

In this study, we designed emulsions with an oil-water interface consisting of a composite layer of whey protein isolate (WPI, 1wt%) and cellulose nanocrystals (CNCs) (1-3wt%). The hypothesis was that a secondary layer of CNCs at the WPI-stabilized oil-water interface could protect the interfacial protein layer against in vitro gastric digestion by pepsin at 37°C. A combination of transmission electron microscopy, ζ-potential measurements, interfacial shear viscosity measurements and theoretical surface coverage considerations suggested the presence of CNCs and WPI together at the O/W interface, owing to the electrostatic attraction between complementarily charged WPI and CNCs at pH 3. Microstructural analysis and droplet sizing revealed that the presence of CNCs increased the resistance of the interfacial protein film to rupture by pepsin, thus inhibiting droplet coalescence in the gastric phase, which occurs rapidly in an emulsion stabilized by WPI alone. It appeared that there was an optimum concentration of CNCs at the interface for such barrier effects. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results further confirmed that the presence of 3wt% of CNCs reduced the rate and extent of proteolysis of protein at the interface. Besides, evidence of adsorption of CNCs to the protein-coated droplets to form more rigid layers, there is also the possibility that network formation by the CNCs in the bulk (continuous) phase reduced the kinetics of proteolysis. Nevertheless, structuring emulsions with mixed protein-particle layers could be an effective strategy to tune and control interfacial barrier properties during gastric passage of emulsions. Copyright © 2017 Elsevier B.V. All rights reserved.

KFC Server: interactive forecasting of protein interaction hot spots

PubMed Central

Darnell, Steven J.; LeGault, Laura; Mitchell, Julie C.

2008-01-01

The KFC Server is a web-based implementation of the KFC (Knowledge-based FADE and Contacts) model—a machine learning approach for the prediction of binding hot spots, or the subset of residues that account for most of a protein interface's; binding free energy. The server facilitates the automated analysis of a user submitted protein–protein or protein–DNA interface and the visualization of its hot spot predictions. For each residue in the interface, the KFC Server characterizes its local structural environment, compares that environment to the environments of experimentally determined hot spots and predicts if the interface residue is a hot spot. After the computational analysis, the user can visualize the results using an interactive job viewer able to quickly highlight predicted hot spots and surrounding structural features within the protein structure. The KFC Server is accessible at http://kfc.mitchell-lab.org. PMID:18539611

Water Dynamics at Protein-Protein Interfaces: Molecular Dynamics Study of Virus-Host Receptor Complexes.

PubMed

Dutta, Priyanka; Botlani, Mohsen; Varma, Sameer

2014-12-26

The dynamical properties of water at protein-water interfaces are unlike those in the bulk. Here we utilize molecular dynamics simulations to study water dynamics in interstitial regions between two proteins. We consider two natural protein-protein complexes, one in which the Nipah virus G protein binds to cellular ephrin B2 and the other in which the same G protein binds to ephrin B3. While the two complexes are structurally similar, the two ephrins share only a modest sequence identity of ∼50%. X-ray crystallography also suggests that these interfaces are fairly extensive and contain exceptionally large amounts of waters. We find that while the interstitial waters tend to occupy crystallographic sites, almost all waters exhibit residence times of less than hundred picoseconds in the interstitial region. We also find that while the differences in the sequence of the two ephrins result in quantitative differences in the dynamics of interstitial waters, the trends in the shifts with respect to bulk values are similar. Despite the high wetness of the protein-protein interfaces, the dynamics of interstitial waters are considerably slower compared to the bulk-the interstitial waters diffuse an order of magnitude slower and have 2-3 fold longer hydrogen bond lifetimes and 2-1000 fold slower dipole relaxation rates. To understand the role of interstitial waters, we examine how implicit solvent models compare against explicit solvent models in producing ephrin-induced shifts in the G conformational density. Ephrin-induced shifts in the G conformational density are critical to the allosteric activation of another viral protein that mediates fusion. We find that in comparison with the explicit solvent model, the implicit solvent model predicts a more compact G-B2 interface, presumably because of the absence of discrete waters at the G-B2 interface. Simultaneously, we find that the two models yield strikingly different induced changes in the G conformational density, even for those residues whose conformational densities in the apo state are unaffected by the treatment of the bulk solvent. Together, these results show that the explicit treatment of interstitial water molecules is necessary for a proper description of allosteric transitions.

Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface.

PubMed

Lajnaf, Roua; Picart-Palmade, Laetitia; Attia, Hamadi; Marchesseau, Sylvie; Ayadi, M A

2017-03-01

The aim of this work was to examine foaming and interfacial behavior of three milk protein mixtures, bovine α-lactalbumin-β-casein (M1), camel α-lactalbumin-β-casein (M2) and β-lactoglobulin-β-casein (M3), alone and in binary mixtures, at the air/water interface in order to better understand the foaming properties of bovine and camel milks. Different mixture ratios (100:0; 75:25; 50:50; 25:75; 0:100) were used during foaming tests and interfacial protein interactions were studied with a pendant drop tensiometer. Experimental results evidenced that the greatest foam was obtained with a higher β-casein amount in all camel and bovine mixtures. Good correlation was observed with the adsorption and the interfacial rheological properties of camel and bovine protein mixtures. The proteins adsorbed layers are mainly affected by the presence of β-casein molecules, which are probably the most abundant protein at interface and the most efficient in reducing the interfacial properties. In contrast of, the globular proteins, α-lactalbumin and β-lactoglobulin that are involved in the protein layer composition, but could not compact well at the interface to ensure foams creation and stabilization because of their rigid molecular structure. Copyright © 2016 Elsevier B.V. All rights reserved.

From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces.

PubMed

Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

2011-09-01

Protein nucleic acid interactions play a critical role in all steps of the gene expression pathway. Nucleic acid (NA) binding proteins interact with their partners, DNA or RNA, via distinct regions on their surface that are characterized by an ensemble of chemical, physical and geometrical properties. In this study, we introduce a novel methodology based on differential geometry, commonly used in face recognition, to characterize and predict NA binding surfaces on proteins. Applying the method on experimentally solved three-dimensional structures of proteins we successfully classify double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) binding proteins, with 83% accuracy. We show that the method is insensitive to conformational changes that occur upon binding and can be applicable for de novo protein-function prediction. Remarkably, when concentrating on the zinc finger motif, we distinguish successfully between RNA and DNA binding interfaces possessing the same binding motif even within the same protein, as demonstrated for the RNA polymerase transcription-factor, TFIIIA. In conclusion, we present a novel methodology to characterize protein surfaces, which can accurately tell apart dsDNA from an ssRNA binding interfaces. The strength of our method in recognizing fine-tuned differences on NA binding interfaces make it applicable for many other molecular recognition problems, with potential implications for drug design.

PRince: a web server for structural and physicochemical analysis of protein-RNA interface.

PubMed

Barik, Amita; Mishra, Abhishek; Bahadur, Ranjit Prasad

2012-07-01

We have developed a web server, PRince, which analyzes the structural features and physicochemical properties of the protein-RNA interface. Users need to submit a PDB file containing the atomic coordinates of both the protein and the RNA molecules in complex form (in '.pdb' format). They should also mention the chain identifiers of interacting protein and RNA molecules. The size of the protein-RNA interface is estimated by measuring the solvent accessible surface area buried in contact. For a given protein-RNA complex, PRince calculates structural, physicochemical and hydration properties of the interacting surfaces. All these parameters generated by the server are presented in a tabular format. The interacting surfaces can also be visualized with software plug-in like Jmol. In addition, the output files containing the list of the atomic coordinates of the interacting protein, RNA and interface water molecules can be downloaded. The parameters generated by PRince are novel, and users can correlate them with the experimentally determined biophysical and biochemical parameters for better understanding the specificity of the protein-RNA recognition process. This server will be continuously upgraded to include more parameters. PRince is publicly accessible and free for use. Available at http://www.facweb.iitkgp.ernet.in/~rbahadur/prince/home.html.

Blocking and Blending: Different Assembly Models of Cyclodextrin and Sodium Caseinate at the Oil/Water Interface.

PubMed

Xu, Hua-Neng; Liu, Huan-Huan; Zhang, Lianfu

2015-08-25

The stability of cyclodextrin (CD)-based emulsions is attributed to the formation of a solid film of oil-CD complexes at the oil/water interface. However, competitive interactions between CDs and other components at the interface still need to be understood. Here we develop two different routes that allow the incorporation of a model protein (sodium caseinate, SC) into emulsions based on β-CD. One route is the components adsorbed simultaneously from a mixed solution to the oil/water interface (route I), and the other is SC was added to a previously established CD-stabilized interface (route II). The adsorption mechanism of β-CD modified by SC at the oil/water interface is investigated by rheological and optical methods. Strong sensitivity of the rheological behavior to the routes is indicated by both steady-state and small-deformation oscillatory experiments. Possible β-CD/SC interaction models at the interface are proposed. In route I, the protein, due to its higher affinity for the interface, adsorbs strongly at the interface with blocking of the adsorption of β-CD and formation of oil-CD complexes. In route II, the protein penetrates and blends into the preadsorbed layer of oil-CD complexes already formed at the interface. The revelation of interfacial assembly is expected to help better understand CD-based emulsions in natural systems and improve their designs in engineering applications.

Large-Scale Validation of Mixed-Solvent Simulations to Assess Hotspots at Protein-Protein Interaction Interfaces.

PubMed

Ghanakota, Phani; van Vlijmen, Herman; Sherman, Woody; Beuming, Thijs

2018-04-23

The ability to target protein-protein interactions (PPIs) with small molecule inhibitors offers great promise in expanding the druggable target space and addressing a broad range of untreated diseases. However, due to their nature and function of interacting with protein partners, PPI interfaces tend to extend over large surfaces without the typical pockets of enzymes and receptors. These features present unique challenges for small molecule inhibitor design. As such, determining whether a particular PPI of interest could be pursued with a small molecule discovery strategy requires an understanding of the characteristics of the PPI interface and whether it has hotspots that can be leveraged by small molecules to achieve desired potency. Here, we assess the ability of mixed-solvent molecular dynamic (MSMD) simulations to detect hotspots at PPI interfaces. MSMD simulations using three cosolvents (acetonitrile, isopropanol, and pyrimidine) were performed on a large test set of 21 PPI targets that have been experimentally validated by small molecule inhibitors. We compare MSMD, which includes explicit solvent and full protein flexibility, to a simpler approach that does not include dynamics or explicit solvent (SiteMap) and find that MSMD simulations reveal additional information about the characteristics of these targets and the ability for small molecules to inhibit the PPI interface. In the few cases were MSMD simulations did not detect hotspots, we explore the shortcomings of this technique and propose future improvements. Finally, using Interleukin-2 as an example, we highlight the advantage of the MSMD approach for detecting transient cryptic druggable pockets that exists at PPI interfaces.

Computational design of protein antigens that interact with the CDR H3 loop of HIV broadly neutralizing antibody 2F5

PubMed Central

Azoitei, M.L.; Ban, Y.A.; Kalyuzhny, O.; Guenaga, J.; Schroeter, A.; Porter, J.; Wyatt, R.; Schief, W.R.

2015-01-01

Rational design of proteins with novel binding specificities and increased affinity is one of the major goals of computational protein design. Epitope-scaffolds are a new class of antigens engineered by transplanting viral epitopes of pre-defined structure to protein scaffolds, or by building protein scaffolds around such epitopes. Epitope-scaffolds are of interest as vaccine components to attempt to elicit neutralizing antibodies targeting the specified epitope. In this study we developed a new computational protocol, MultiGraft Interface, that transplants epitopes but also designs additional scaffold features outside the epitope to enhance antibody-binding specificity and potentially influence the specificity of elicited antibodies. We employed MultiGraft Interface to engineer novel epitope-scaffolds that display the known epitope of HIV-1 neutralizing antibody 2F5 and that also interact with the functionally important CDR H3 antibody loop. MultiGraft Interface generated an epitope-scaffold that bound 2F5 with sub-nanomolar affinity (KD = 400 pM) and that interacted with the antibody CDR H3 loop through computationally designed contacts. Substantial structural modifications were necessary to engineer this antigen, with the 2F5 epitope replacing a helix in the native scaffold and with 15% of the native scaffold sequence being modified in the design stage. This epitope-scaffold represents a successful example of rational protein backbone engineering and protein-protein interface design and could prove useful in the field of HIV vaccine design. MultiGraft Interface can be generally applied to engineer novel binding partners with altered specificity and optimized affinity. PMID:25043744

Influence of antifreeze proteins on the ice/water interface.

PubMed

Todde, Guido; Hovmöller, Sven; Laaksonen, Aatto

2015-02-26

Antifreeze proteins (AFP) are responsible for the survival of several species, ranging from bacteria to fish, that encounter subzero temperatures in their living environment. AFPs have been divided into two main families, moderately and hyperactive, depending on their thermal hysteresis activity. We have studied one protein from both families, the AFP from the snow flea (sfAFP) and from the winter flounder (wfAFP), which belong to the hyperactive and moderately active family, respectively. On the basis of molecular dynamics simulations, we have estimated the thickness of the water/ice interface for systems both with and without the AFPs attached onto the ice surface. The calculation of the diffusion profiles along the simulation box allowed us to measure the interface width for different ice planes. The obtained widths clearly show a different influence of the two AFPs on the ice/water interface. The different impact of the AFPs here studied on the interface thickness can be related to two AFPs properties: the protein hydrophobic surface and the number of hydrogen bonds that the two AFPs faces form with water molecules.

On the Importance of Polar Interactions for Complexes Containing Intrinsically Disordered Proteins

PubMed Central

Wong, Eric T. C.; Na, Dokyun; Gsponer, Jörg

2013-01-01

There is a growing recognition for the importance of proteins with large intrinsically disordered (ID) segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions. PMID:23990768

Binding free energy analysis of protein-protein docking model structures by evERdock.

PubMed

Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

2018-03-14

To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

Binding free energy analysis of protein-protein docking model structures by evERdock

NASA Astrophysics Data System (ADS)

Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

2018-03-01

To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

Predicting nucleic acid binding interfaces from structural models of proteins

PubMed Central

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2011-01-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared to patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. PMID:22086767

Structural deformation upon protein-protein interaction: A structural alphabet approach

PubMed Central

Martin, Juliette; Regad, Leslie; Lecornet, Hélène; Camproux, Anne-Claude

2008-01-01

Background In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. Results In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%). This proportion is even greater in the interface regions (41%). Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Conclusion Our study provides qualitative information about induced fit. These results could be of help for flexible docking. PMID:18307769

Structural deformation upon protein-protein interaction: a structural alphabet approach.

PubMed

Martin, Juliette; Regad, Leslie; Lecornet, Hélène; Camproux, Anne-Claude

2008-02-28

In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%). This proportion is even greater in the interface regions (41%). Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Our study provides qualitative information about induced fit. These results could be of help for flexible docking.

Elucidating the druggable interface of protein-protein interactions using fragment docking and coevolutionary analysis.

PubMed

Bai, Fang; Morcos, Faruck; Cheng, Ryan R; Jiang, Hualiang; Onuchic, José N

2016-12-13

Protein-protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein-protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

Ecohydrological Interfaces as Dynamic Hotspots of Biogeochemical Cycling

NASA Astrophysics Data System (ADS)

Krause, Stefan; Lewandowski, Joerg; Hannah, David; McDonald, Karlie; Folegot, Silvia; Baranov, Victor

2016-04-01

Ecohydrological interfaces, represent the boundaries between water-dependent ecosystems that can alter substantially the fluxes of energy and matter. There is still a critical gap of understanding the organisational principles of the drivers and controls of spatially and temporally variable ecohydrological interface functions. This knowledge gap limits our capacity to efficiently quantify, predict and manage the services provided by complex ecosystems. Many ecohydrological interfaces are characterized by step changes in microbial metabolic activity, steep redox gradients and often even thermodynamic phase shifts, for instance at the interfaces between atmosphere and water or soil matrix and macro-pores interfaces. This paper integrates investigations from point scale laboratory microcosm experiments with reach and subcatchment scale tracer experiments and numerical modeling studies to elaborate similarities in the drivers and controls that constitute the enhanced biogeochemical activity of different types of ecohydrologica interfaces across a range of spatial and temporal scales. We therefore combine smart metabolic activity tracers to quantify the impact of bioturbating benthic fauna onto ecosystem respiration and oxygen consumption and investigate at larger scale, how microbial metabolic activity and carbon turnover at the water-sediment interface are controlled by sediment physical and chemical properties as well as water temperatures. Numerical modeling confirmed that experimentally identified hotspots of streambed biogeochemical cycling were controlled by patterns of physical properties such as hydraulic conductivities or bioavailability of organic matter, impacting on residence time distributions and hence reaction times. In contrast to previous research, our investigations thus confirmed that small-scale variability of physical and chemical interface properties had a major impact on biogeochemical processing at the investigated ecohydrological interfaces. Our results furthermore indicate that to fully understand spatial patterns and temporal dynamics of ecohydrological interface functioning, including hotspots and hot moments, detailed knowledge of the impacts of biological behavior on the physic-chemical ecosystem conditions, and vice-versa, is required.

Ecohydrological Interfaces as Dynamic Hotspots of Biogeochemical Cycling

NASA Astrophysics Data System (ADS)

Krause, S.

2015-12-01

Ecohydrological interfaces, represent the boundaries between water-dependent ecosystems that can alter substantially the fluxes of energy and matter. There is still a critical gap of understanding the organisational principles of the drivers and controls of spatially and temporally variable ecohydrological interface functions. This knowledge gap limits our capacity to efficiently quantify, predict and manage the services provided by complex ecosystems. Many ecohydrological interfaces are characterized by step changes in microbial metabolic activity, steep redox gradients and often even thermodynamic phase shifts, for instance at the interfaces between atmosphere and water or soil matrix and macro-pores interfaces. This paper integrates investigations from point scale microcosm experiments with reach and subcatchment scale tracer experiments and numerical modeling studies to elaborate similarities in the drivers and controls that constitute the enhanced biogeochemical activity of different types of ecohydrologica interfaces across a range of spatial and temporal scales. We therefore combine smart metabolic activity tracers to quantify the impact of bioturbating benthic fauna onto ecosystem respiration and oxygen consumption and investigate at larger scale, how microbial metabolic activity and carbon turnover at the water-sediment interface are controlled by sediment physical and chemical properties as well as water temperatures. Numerical modeling confirmed that experimentally identified hotspots of streambed biogeochemical cycling were controlled by patterns of physical properties such as hydraulic conductivities or bioavailability of organic matter, impacting on residence time distributions and hence reaction times. In contrast to previous research, our investigations thus confirmed that small-scale variability of physical and chemical interface properties had a major impact on biogeochemical processing at the investigated ecohydrological interfaces. Our results furthermore indicate that to fully understand spatial patterns and temporal dynamics of ecohydrological interface functioning, including hotspots and hot moments, detailed knowledge of the impacts of biological behavior on the physic-chemical ecosystem conditions, and vice-versa, is required.

Hot-spot analysis for drug discovery targeting protein-protein interactions.

PubMed

Rosell, Mireia; Fernández-Recio, Juan

2018-04-01

Protein-protein interactions are important for biological processes and pathological situations, and are attractive targets for drug discovery. However, rational drug design targeting protein-protein interactions is still highly challenging. Hot-spot residues are seen as the best option to target such interactions, but their identification requires detailed structural and energetic characterization, which is only available for a tiny fraction of protein interactions. Areas covered: In this review, the authors cover a variety of computational methods that have been reported for the energetic analysis of protein-protein interfaces in search of hot-spots, and the structural modeling of protein-protein complexes by docking. This can help to rationalize the discovery of small-molecule inhibitors of protein-protein interfaces of therapeutic interest. Computational analysis and docking can help to locate the interface, molecular dynamics can be used to find suitable cavities, and hot-spot predictions can focus the search for inhibitors of protein-protein interactions. Expert opinion: A major difficulty for applying rational drug design methods to protein-protein interactions is that in the majority of cases the complex structure is not available. Fortunately, computational docking can complement experimental data. An interesting aspect to explore in the future is the integration of these strategies for targeting PPIs with large-scale mutational analysis.

Wear particles from studded tires and granite pavement induce pro-inflammatory alterations in human monocyte-derived macrophages: a proteomic study.

PubMed

Karlsson, Helen; Lindbom, John; Ghafouri, Bijar; Lindahl, Mats; Tagesson, Christer; Gustafsson, Mats; Ljungman, Anders G

2011-01-14

Airborne particulate matter is considered to be one of the environmental contributors to the mortality in cancer, respiratory, and cardiovascular diseases. For future preventive actions, it is of major concern to investigate the toxicity of defined groups of airborne particles and to clarify their pathways in biological tissues. To expand the knowledge beyond general inflammatory markers, this study examined the toxicoproteomic effects on human monocyte derived macrophages after exposure to wear particles generated from the interface of studded tires and a granite-containing pavement. As comparison, the effect of endotoxin was also investigated. The macrophage proteome was separated using two-dimensional gel electrophoresis. Detected proteins were quantified, and selected proteins were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Among analyzed proteins, seven were significantly decreased and three were increased by exposure to wear particles as compared to unexposed control cells. Endotoxin exposure resulted in significant changes in the expression of six proteins: four decreased and two increased. For example, macrophage capping protein was significantly increased after wear particle exposure only, whereas calgizzarin and galectin-3 were increased by both wear particle and endotoxin exposure. Overall, proteins associated with inflammatory response were increased and proteins involved in cellular functions such as redox balance, anti-inflammatory response, and glycolysis were decreased. Investigating the effects of characterized wear particles on human macrophages with a toxicoproteomic approach has shown to be useful in the search for more detailed information about specific pathways and possible biological markers.

Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

PubMed Central

Castillo, Virginia; Ventura, Salvador

2009-01-01

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins. PMID:19696882

Complementarity of stability patches at the interfaces of protein complexes: Implication for the structural organization of energetic hot spots.

PubMed

Kuttner, Yosef Y; Engel, Stanislav

2018-02-01

A rational design of protein complexes with defined functionalities and of drugs aimed at disrupting protein-protein interactions requires fundamental understanding of the mechanisms underlying the formation of specific protein complexes. Efforts to develop efficient small-molecule or protein-based binders often exploit energetic hot spots on protein surfaces, namely, the interfacial residues that provide most of the binding free energy in the complex. The molecular basis underlying the unusually high energy contribution of the hot spots remains obscure, and its elucidation would facilitate the design of interface-targeted drugs. To study the nature of the energetic hot spots, we analyzed the backbone dynamic properties of contact surfaces in several protein complexes. We demonstrate that, in most complexes, the backbone dynamic landscapes of interacting surfaces form complementary "stability patches," in which static areas from the opposing surfaces superimpose, and that these areas are predominantly located near the geometric center of the interface. We propose that a diminished enthalpy-entropy compensation effect augments the degree to which residues positioned within the complementary stability patches contribute to complex affinity, thereby giving rise to the energetic hot spots. These findings offer new insights into the nature of energetic hot spots and the role that backbone dynamics play in facilitating intermolecular recognition. Mapping the interfacial stability patches may provide guidance for protein engineering approaches aimed at improving the stability of protein complexes and could facilitate the design of ligands that target complex interfaces. © 2017 Wiley Periodicals, Inc.

"Quenchbodies": quench-based antibody probes that show antigen-dependent fluorescence.

PubMed

Abe, Ryoji; Ohashi, Hiroyuki; Iijima, Issei; Ihara, Masaki; Takagi, Hiroaki; Hohsaka, Takahiro; Ueda, Hiroshi

2011-11-02

Here, we describe a novel reagentless fluorescent biosensor strategy based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. Using a cell-free translation-mediated position-specific protein labeling system, we found that an antibody single chain variable region (scFv) that had been fluorolabeled at the N-terminal region showed a significant antigen-dependent fluorescence enhancement. Investigation of the enhancement mechanism by mutagenesis of the carboxytetramethylrhodamine (TAMRA)-labeled anti-osteocalcin scFv showed that antigen-dependency was dependent on semiconserved tryptophan residues near the V(H)/V(L) interface. This suggested that the binding of the antigen led to the interruption of a quenching effect caused by the proximity of tryptophan residues to the linker-tagged fluorophore. Using TAMRA-scFv, many targets including peptides, proteins, and haptens including morphine-related drugs could be quantified. Similar or higher sensitivities to those observed in competitive ELISA were obtained, even in human plasma. Because of its versatility, this "quenchbody" is expected to have a range of applications, from in vitro diagnostics, to imaging of various targets in situ.

SNAPPI-DB: a database and API of Structures, iNterfaces and Alignments for Protein–Protein Interactions

PubMed Central

Jefferson, Emily R.; Walsh, Thomas P.; Roberts, Timothy J.; Barton, Geoffrey J.

2007-01-01

SNAPPI-DB, a high performance database of Structures, iNterfaces and Alignments of Protein–Protein Interactions, and its associated Java Application Programming Interface (API) is described. SNAPPI-DB contains structural data, down to the level of atom co-ordinates, for each structure in the Protein Data Bank (PDB) together with associated data including SCOP, CATH, Pfam, SWISSPROT, InterPro, GO terms, Protein Quaternary Structures (PQS) and secondary structure information. Domain–domain interactions are stored for multiple domain definitions and are classified by their Superfamily/Family pair and interaction interface. Each set of classified domain–domain interactions has an associated multiple structure alignment for each partner. The API facilitates data access via PDB entries, domains and domain–domain interactions. Rapid development, fast database access and the ability to perform advanced queries without the requirement for complex SQL statements are provided via an object oriented database and the Java Data Objects (JDO) API. SNAPPI-DB contains many features which are not available in other databases of structural protein–protein interactions. It has been applied in three studies on the properties of protein–protein interactions and is currently being employed to train a protein–protein interaction predictor and a functional residue predictor. The database, API and manual are available for download at: . PMID:17202171

Quantitative first-principles theory of interface absorption in multilayer heterostructures

DOE PAGES

Hachtel, Jordan A.; Sachan, Ritesh; Mishra, Rohan; ...

2015-09-03

The unique chemical bonds and electronic states of interfaces result in optical properties that are different from those of the constituting bulk materials. In the nanoscale regime, the interface effects can be dominant and impact the optical response of devices. Using density functional theory (DFT), the interface effects can be calculated, but DFT is computationally limited to small systems. In this paper, we describe a method to combine DFT with macroscopic methodologies to extract the interface effect on absorption in a consistent and quantifiable manner. The extracted interface effects are an independent parameter and can be applied to more complicatedmore » systems. Finally, we demonstrate, using NiSi 2/Si heterostructures, that by varying the relative volume fractions of interface and bulk, we can tune the spectral range of the heterostructure absorption.« less

Protein-Protein Docking in Drug Design and Discovery.

PubMed

Kaczor, Agnieszka A; Bartuzi, Damian; Stępniewski, Tomasz Maciej; Matosiuk, Dariusz; Selent, Jana

2018-01-01

Protein-protein interactions (PPIs) are responsible for a number of key physiological processes in the living cells and underlie the pathomechanism of many diseases. Nowadays, along with the concept of so-called "hot spots" in protein-protein interactions, which are well-defined interface regions responsible for most of the binding energy, these interfaces can be targeted with modulators. In order to apply structure-based design techniques to design PPIs modulators, a three-dimensional structure of protein complex has to be available. In this context in silico approaches, in particular protein-protein docking, are a valuable complement to experimental methods for elucidating 3D structure of protein complexes. Protein-protein docking is easy to use and does not require significant computer resources and time (in contrast to molecular dynamics) and it results in 3D structure of a protein complex (in contrast to sequence-based methods of predicting binding interfaces). However, protein-protein docking cannot address all the aspects of protein dynamics, in particular the global conformational changes during protein complex formation. In spite of this fact, protein-protein docking is widely used to model complexes of water-soluble proteins and less commonly to predict structures of transmembrane protein assemblies, including dimers and oligomers of G protein-coupled receptors (GPCRs). In this chapter we review the principles of protein-protein docking, available algorithms and software and discuss the recent examples, benefits, and drawbacks of protein-protein docking application to water-soluble proteins, membrane anchoring and transmembrane proteins, including GPCRs.

Unlocking the secrets to protein–protein interface drug targets using structural mass spectrometry techniques

PubMed Central

Dailing, Angela; Luchini, Alessandra; Liotta, Lance

2016-01-01

Protein–protein interactions (PPIs) drive all biologic systems at the subcellular and extracellular level. Changes in the specificity and affinity of these interactions can lead to cellular malfunctions and disease. Consequently, the binding interfaces between interacting protein partners are important drug targets for the next generation of therapies that block such interactions. Unfortunately, protein–protein contact points have proven to be very difficult pharmacological targets because they are hidden within complex 3D interfaces. For the vast majority of characterized binary PPIs, the specific amino acid sequence of their close contact regions remains unknown. There has been an important need for an experimental technology that can rapidly reveal the functionally important contact points of native protein complexes in solution. In this review, experimental techniques employing mass spectrometry to explore protein interaction binding sites are discussed. Hydrogen–deuterium exchange, hydroxyl radical footprinting, crosslinking and the newest technology protein painting, are compared and contrasted. PMID:26400464

Mixed layers of sodium caseinate + dextran sulfate: influence of order of addition to oil-water interface.

PubMed

Jourdain, Laureline S; Schmitt, Christophe; Leser, Martin E; Murray, Brent S; Dickinson, Eric

2009-09-01

We report on the interfacial properties of electrostatic complexes of protein (sodium caseinate) with a highly sulfated polysaccharide (dextran sulfate). Two routes were investigated for preparation of adsorbed layers at the n-tetradecane-water interface at pH = 6. Bilayers were made by the layer-by-layer deposition technique whereby polysaccharide was added to a previously established protein-stabilized interface. Mixed layers were made by the conventional one-step method in which soluble protein-polysaccharide complexes were adsorbed directly at the interface. Protein + polysaccharide systems gave a slower decay of interfacial tension and stronger dilatational viscoelastic properties than the protein alone, but there was no significant difference in dilatational properties between mixed layers and bilayers. Conversely, shear rheology experiments exhibited significant differences between the two kinds of interfacial layers, with the mixed system giving much stronger interfacial films than the bilayer system, i.e., shear viscosities and moduli at least an order of magnitude higher. The film shear viscoelasticity was further enhanced by acidification of the biopolymer mixture to pH = 2 prior to interface formation. Taken together, these measurements provide insight into the origin of previously reported differences in stability properties of oil-in-water emulsions made by the bilayer and mixed layer approaches. Addition of a proteolytic enzyme (trypsin) to both types of interfaces led to a significant increase in the elastic modulus of the film, suggesting that the enzyme was adsorbed at the interface via complexation with dextran sulfate. Overall, this study has confirmed the potential of shear rheology as a highly sensitive probe of associative electrostatic interactions and interfacial structure in mixed biopolymer layers.

From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces

PubMed Central

Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

2011-01-01

Protein nucleic acid interactions play a critical role in all steps of the gene expression pathway. Nucleic acid (NA) binding proteins interact with their partners, DNA or RNA, via distinct regions on their surface that are characterized by an ensemble of chemical, physical and geometrical properties. In this study, we introduce a novel methodology based on differential geometry, commonly used in face recognition, to characterize and predict NA binding surfaces on proteins. Applying the method on experimentally solved three-dimensional structures of proteins we successfully classify double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) binding proteins, with 83% accuracy. We show that the method is insensitive to conformational changes that occur upon binding and can be applicable for de novo protein-function prediction. Remarkably, when concentrating on the zinc finger motif, we distinguish successfully between RNA and DNA binding interfaces possessing the same binding motif even within the same protein, as demonstrated for the RNA polymerase transcription-factor, TFIIIA. In conclusion, we present a novel methodology to characterize protein surfaces, which can accurately tell apart dsDNA from an ssRNA binding interfaces. The strength of our method in recognizing fine-tuned differences on NA binding interfaces make it applicable for many other molecular recognition problems, with potential implications for drug design. PMID:21693557

Computational design of protein antigens that interact with the CDR H3 loop of HIV broadly neutralizing antibody 2F5.

PubMed

Azoitei, M L; Ban, Y A; Kalyuzhny, O; Guenaga, J; Schroeter, A; Porter, J; Wyatt, R; Schief, William R

2014-10-01

Rational design of proteins with novel binding specificities and increased affinity is one of the major goals of computational protein design. Epitope-scaffolds are a new class of antigens engineered by transplanting viral epitopes of predefined structure to protein scaffolds, or by building protein scaffolds around such epitopes. Epitope-scaffolds are of interest as vaccine components to attempt to elicit neutralizing antibodies targeting the specified epitope. In this study we developed a new computational protocol, MultiGraft Interface, that transplants epitopes but also designs additional scaffold features outside the epitope to enhance antibody-binding specificity and potentially influence the specificity of elicited antibodies. We employed MultiGraft Interface to engineer novel epitope-scaffolds that display the known epitope of human immunodeficiency virus 1 (HIV-1) neutralizing antibody 2F5 and that also interact with the functionally important CDR H3 antibody loop. MultiGraft Interface generated an epitope-scaffold that bound 2F5 with subnanomolar affinity (K(D) = 400 pM) and that interacted with the antibody CDR H3 loop through computationally designed contacts. Substantial structural modifications were necessary to engineer this antigen, with the 2F5 epitope replacing a helix in the native scaffold and with 15% of the native scaffold sequence being modified in the design stage. This epitope-scaffold represents a successful example of rational protein backbone engineering and protein-protein interface design and could prove useful in the field of HIV vaccine design. MultiGraft Interface can be generally applied to engineer novel binding partners with altered specificity and optimized affinity. © 2014 Wiley Periodicals, Inc.

CNA web server: rigidity theory-based thermal unfolding simulations of proteins for linking structure, (thermo-)stability, and function.

PubMed

Krüger, Dennis M; Rathi, Prakash Chandra; Pfleger, Christopher; Gohlke, Holger

2013-07-01

The Constraint Network Analysis (CNA) web server provides a user-friendly interface to the CNA approach developed in our laboratory for linking results from rigidity analyses to biologically relevant characteristics of a biomolecular structure. The CNA web server provides a refined modeling of thermal unfolding simulations that considers the temperature dependence of hydrophobic tethers and computes a set of global and local indices for quantifying biomacromolecular stability. From the global indices, phase transition points are identified where the structure switches from a rigid to a floppy state; these phase transition points can be related to a protein's (thermo-)stability. Structural weak spots (unfolding nuclei) are automatically identified, too; this knowledge can be exploited in data-driven protein engineering. The local indices are useful in linking flexibility and function and to understand the impact of ligand binding on protein flexibility. The CNA web server robustly handles small-molecule ligands in general. To overcome issues of sensitivity with respect to the input structure, the CNA web server allows performing two ensemble-based variants of thermal unfolding simulations. The web server output is provided as raw data, plots and/or Jmol representations. The CNA web server, accessible at http://cpclab.uni-duesseldorf.de/cna or http://www.cnanalysis.de, is free and open to all users with no login requirement.

Energetics of protein homodimerization: effects of water sequestering on the formation of beta-lactoglobulin dimer.

PubMed

Bello, Martiniano; Pérez-Hernández, Gerardo; Fernández-Velasco, D Alejandro; Arreguín-Espinosa, Roberto; García-Hernández, Enrique

2008-03-01

Transient protein-protein interactions are functionally relevant as a control mechanism in a variety of biological processes. Analysis of the 3D structure of protein-protein complexes indicates that water molecules trapped at the interface are very common; however, their role in the stability and specificity of protein homodimer interactions has been not addressed yet. To provide new insights into the energetic bases that govern the formation of highly hydrated interfaces, the dissociation process of bovine beta lg variant A at a neutral pH was characterized here thermodynamically by conducting dilution experiments with an isothermal titration calorimeter. Association was enthalpically driven throughout the temperature range spanned. DeltaH and deltaC(p) were significantly more negative than estimates based on surface area changes, suggesting the occurrence of effects additional to the dehydration of the contact surfaces between subunits. Near-UV CD spectra proved to be independent of protein concentration, indicating a rigid body-like association. Furthermore, the process proved not to be coupled to significant changes in the protonation state of ionizable groups or counterion exchange. In contrast, both osmotic stress experiments and a computational analysis of the dimer's 3D structure indicated that a large number of water molecules are incorporated into the interface upon association. Numerical estimates considering the contributions of interface area desolvation and water immobilization accounted satisfactorily for the experimental deltaC(p). Thus, our study highlights the importance of explicitly considering the effects of water sequestering to perform a proper quantitative analysis of the formation of homodimers with highly hydrated interfaces. 2007 Wiley-Liss, Inc.

Standard atomic volumes in double-stranded DNA and packing in protein–DNA interfaces

PubMed Central

Nadassy, Katalin; Tomás-Oliveira, Isabel; Alberts, Ian; Janin, Joël; Wodak, Shoshana J.

2001-01-01

Standard volumes for atoms in double-stranded B-DNA are derived using high resolution crystal structures from the Nucleic Acid Database (NDB) and compared with corresponding values derived from crystal structures of small organic compounds in the Cambridge Structural Database (CSD). Two different methods are used to compute these volumes: the classical Voronoi method, which does not depend on the size of atoms, and the related Radical Planes method which does. Results show that atomic groups buried in the interior of double-stranded DNA are, on average, more tightly packed than in related small molecules in the CSD. The packing efficiency of DNA atoms at the interfaces of 25 high resolution protein–DNA complexes is determined by computing the ratios between the volumes of interfacial DNA atoms and the corresponding standard volumes. These ratios are found to be close to unity, indicating that the DNA atoms at protein–DNA interfaces are as closely packed as in crystals of B-DNA. Analogous volume ratios, computed for buried protein atoms, are also near unity, confirming our earlier conclusions that the packing efficiency of these atoms is similar to that in the protein interior. In addition, we examine the number, volume and solvent occupation of cavities located at the protein–DNA interfaces and compared them with those in the protein interior. Cavities are found to be ubiquitous in the interfaces as well as inside the protein moieties. The frequency of solvent occupation of cavities is however higher in the interfaces, indicating that those are more hydrated than protein interiors. Lastly, we compare our results with those obtained using two different measures of shape complementarity of the analysed interfaces, and find that the correlation between our volume ratios and these measures, as well as between the measures themselves, is weak. Our results indicate that a tightly packed environment made up of DNA, protein and solvent atoms plays a significant role in protein–DNA recognition. PMID:11504874

Mitigating the Hook Effect in Lateral Flow Sandwich Immunoassays Using Real-Time Reaction Kinetics.

PubMed

Rey, Elizabeth G; O'Dell, Dakota; Mehta, Saurabh; Erickson, David

2017-05-02

The quantification of analyte concentrations using lateral flow assays is a low-cost and user-friendly alternative to traditional lab-based assays. However, sandwich-type immunoassays are often limited by the high-dose hook effect, which causes falsely low results when analytes are present at very high concentrations. In this paper, we present a reaction kinetics-based technique that solves this problem, significantly increasing the dynamic range of these devices. With the use of a traditional sandwich lateral flow immunoassay, a portable imaging device, and a mobile interface, we demonstrate the technique by quantifying C-reactive protein concentrations in human serum over a large portion of the physiological range. The technique could be applied to any hook effect-limited sandwich lateral flow assay and has a high level of accuracy even in the hook effect range.

Stable isotope labeling tandem mass spectrometry (SILT) to quantify protein production and clearance rates

PubMed Central

Bateman, Randall J.; Munsell, Ling Y.; Chen, Xianghong; Holtzman, David M.; Yarasheski, Kevin E.

2007-01-01

In all biological systems, protein amount is a function of the rate of production and clearance. The speed of a response to a disturbance in protein homeostasis is determined by turnover rate. Quantifying alterations in protein synthesis and clearance rates is vital to understanding disease pathogenesis (e.g., aging, inflammation). No methods exist for quantifying production and clearance rates of low abundance (femtomole) proteins in vivo. We describe a novel, mass spectrometry-based method for quantitating low abundance protein synthesis and clearance rates in vitro and in vivo in animals and humans. The utility of this method is demonstrated with amyloid-beta (Aß), an important low abundance protein involved in Alzheimer's disease pathogenesis. We used in vivo stable isotope labeling, immunoprecipitation of Aß from cerebrospinal fluid, and quantitative liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-tandem MS) to quantify human Aß protein production and clearance rates. The method is sensitive and specific for stable isotope labeled amino acid incorporation into CNS (± 1% accuracy). This in vivo method can be used to identify pathophysiologic changes in protein metabolism; and may serve as a biomarker for monitoring disease risk, progression, or response to novel therapeutic agents. The technique is adaptable to other macromolecules, such as carbohydrates or lipids. PMID:17383190

Evaluating the Role of the Air-Solution Interface on the Mechanism of Subvisible Particle Formation Caused by Mechanical Agitation for an IgG1 mAb.

PubMed

Ghazvini, Saba; Kalonia, Cavan; Volkin, David B; Dhar, Prajnaparamita

2016-05-01

Mechanical agitation of monoclonal antibody (mAb) solutions often leads to protein particle formation. In this study, various formulations of an immunoglobulin G (IgG) 1 mAb were subjected to different controlled interfacial stresses using a Langmuir trough, and protein particles formed at the interface and measured in bulk solution were characterized using atomic force microscopy and flow digital imaging. Results were compared to mAb solutions agitated in glass vials and unstressed controls. At lower pH, mAb solutions exhibited larger hysteresis in their surface pressure versus area isotherms and increased number of particles in bulk solution, when subjected to interfacial stresses. mAb samples subjected to 750-1000 interfacial compression-expansion cycles in 6 h contained high particle numbers in bulk solution, and displayed similar particulation trends when agitated in vials. At compression rates of 50 cycles in 6 h, however, particle levels in mAb solutions were comparable to unstressed controls, despite protein aggregates being present at the air-solution interface. These results suggest that while the air-solution interface serves as a nucleation site for initiating protein aggregation, the number of protein particles measured in bulk mAb solutions depends on the total number of compression cycles that proteins at the air-solution interface are subjected to within a fixed time. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Protein Adsorption Alters Hydrophobic Surfaces Used for Suspension Culture of Pluripotent Stem Cells.

PubMed

Jonas, Steven J; Stieg, Adam Z; Richardson, Wade; Guo, Shuling; Powers, David N; Wohlschlegel, James; Dunn, Bruce

2015-02-05

This Letter examines the physical and chemical changes that occur at the interface of methyl-terminated alkanethiol self-assembled monolayers (SAMs) after exposure to cell culture media used to derive embryoid bodies (EBs) from pluripotent stem cells. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy analysis of the SAMs indicates that protein components within the EB cell culture medium preferentially adsorb at the hydrophobic interface. In addition, we examined the adsorption process using surface plasmon resonance and atomic force microscopy. These studies identify the formation of a porous, mat-like adsorbed protein film with an approximate thickness of 2.5 nm. Captive bubble contact angle analysis reveals a shift toward superhydrophilic wetting behavior at the cell culture interface due to adsorption of these proteins. These results show how EBs are able to remain in suspension when derived on hydrophobic materials, which carries implications for the rational design of suspension culture interfaces for lineage specific stem-cell differentiation.

Predicting nucleic acid binding interfaces from structural models of proteins.

PubMed

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2012-02-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

Binding regularities in complexes of transcription factors with operator DNA: homeodomain family.

PubMed

Chirgadze, Yu N; Zheltukhin, E I; Polozov, R V; Sivozhelezov, V S; Ivanov, V V

2009-06-01

In order to disclose general regularities of binding in homeodomain-DNA complexes we considered five of them and extended the observed regularities over the entire homeodomain family. The five complexes have been selected by similarity of protein structures and patterns of contacting residues. Their long range interactions and interfaces were compared. The long-range stage of the recognition process was characterized by electrostatic potentials about 5 Angstrom away from molecular surfaces of protein or DNA. For proteins, clear positive potential is displayed only at the side contacting the DNA. The double-chained DNA molecule displays a rather strong negative potential, especially in their grooves. Thus, a functional role of electrostatics is a guiding of the protein into the DNA major groove, so the protein and DNA could form a loose non-specific complex. At the close-range stage, neutralization of the phosphate charges by positively charged residues is necessary for decreasing the strong electrostatic potential of DNA, allowing nucleotide bases to participate in the formation of protein-DNA atomic contacts in the interface. The recognizing alpha-helix of protein was shown to form both invariant and variable groups of contacts with DNA by means of certain specific side groups. The invariant contacts included highly specific protein-DNA hydrogen bonds between asparagine and adenine, nonpolar contacts of hydrophobic amino acids serving as a stereochemical barrier for fixing the protein factor on DNA, and an interface cluster of water molecules providing local conformational mobility necessary for the dissociation process. There is a unique water molecule within the interface that is conservative and located at the interface center. Invariant contacts of the proteins are mostly formed with the TAAT motif of the promoter DNA forward strand. While the invariant contacts specify the family of homeodomains, the variable contacts that are formed with the reverse strand of DNA provide specificity of individual complexes within the homeodomain family.

Comparison of the adsorbed conformation of barley lipid transfer protein at the decane-water and vacuum-water interface: a molecular dynamics simulation.

PubMed

Euston, S R; Hughes, P; Naser, Md A; Westacott, R E

2008-05-01

Molecular dynamics simulation is used to model the adsorption of the barley lipid transfer protein (LTP) at the decane-water and vacuum-water interfaces. Adsorption at both surfaces is driven by displacement of water molecules from the interfacial region. LTP adsorbed at the decane surface exhibits significant changes in its tertiary structure, and penetrates a considerable distance into the decane phase. At the vacuum-water interface LTP shows small conformational changes away from its native structure and does not penetrate into the vacuum space. Modification of the conformational stability of LTP by reduction of its four disulphide bonds leads to an increase in conformational entropy of the molecules, which reduces the driving force for adsorption. Evidence for changes in the secondary structure are also observed for native LTP at the decane-water interface and reduced LTP at the vacuum-water interface. In particular, intermittent formation of short (six-residue) regions of beta-sheet is found in these two systems. Formation of interfacial beta-sheet in adsorbed proteins has been observed experimentally, notably in the globular milk protein beta-lactoglobulin and lysozyme.

IL-4 and IL-13 Compromise the Sinonasal Epithelial Barrier and Perturb Intercellular Junction Protein Expression

PubMed Central

Wise, Sarah K.; Laury, Adrienne M.; Katz, Elizabeth H.; Den Beste, Kyle A.; Parkos, Charles A.; Nusrat, Asma

2014-01-01

Introduction Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a “leaky” epithelial barrier phenotype. We hypothesize that Th2 cytokines IL-4 and IL-13 modulate epithelial junction proteins thereby contributing to the leaky epithelial barrier. Methods Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. Results IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n=6) and 68.6% (n=8) of baseline, respectively. Tight junction protein JAM-A expression decreased 42.2% with IL-4 exposure (n=9) and 37.5% with IL-13 exposure (n=9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n=9) and 32.9% with IL-13 exposure (n=9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or ZO-1 with IL-4 or IL-13 exposure. Conclusion Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity. PMID:24510479

Force transduction and lipid binding in MscL: A continuum-molecular approach

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

Vanegas, Juan M.; Arroyo, Marino; Fotiadis, Dimitrios

2014-12-01

The bacterial mechanosensitive channel MscL, a small protein mainly activated by membrane tension, is a central model system to study the transduction of mechanical stimuli into chemical signals. Mutagenic studies suggest that MscL gating strongly depends on both intra-protein and interfacial lipid-protein interactions. However, there is a gap between this detailed chemical information and current mechanical models of MscL gating. Here, we investigate the MscL bilayer-protein interface through molecular dynamics simulations, and take a combined continuum-molecular approach to connect chemistry and mechanics. We quantify the effect of membrane tension on the forces acting on the surface of the channel, andmore » identify interactions that may be critical in the force transduction between the membrane and MscL. We find that the local stress distribution on the protein surface is largely asymmetric, particularly under tension, with the cytoplasmic side showing significantly larger and more localized forces, which pull the protein radially outward. The molecular interactions that mediate this behavior arise from hydrogen bonds between the electronegative oxygens in the lipid headgroup and a cluster of positively charged lysine residues on the amphipathic S1 domain and the C-terminal end of the second trans-membrane helix. We take advantage of this strong interaction (estimated to be 10–13 kT per lipid) to actuate the channel (by applying forces on protein-bound lipids) and explore its sensitivity to the pulling magnitude and direction. We conclude by highlighting the simple motif that confers MscL with strong anchoring to the bilayer, and its presence in various integral membrane proteins including the human mechanosensitive channel K2P1 and bovine rhodopsin.« less

GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking.

PubMed

Heo, Lim; Lee, Hasup; Seok, Chaok

2016-08-18

Protein-protein docking methods have been widely used to gain an atomic-level understanding of protein interactions. However, docking methods that employ low-resolution energy functions are popular because of computational efficiency. Low-resolution docking tends to generate protein complex structures that are not fully optimized. GalaxyRefineComplex takes such low-resolution docking structures and refines them to improve model accuracy in terms of both interface contact and inter-protein orientation. This refinement method allows flexibility at the protein interface and in the overall docking structure to capture conformational changes that occur upon binding. Symmetric refinement is also provided for symmetric homo-complexes. This method was validated by refining models produced by available docking programs, including ZDOCK and M-ZDOCK, and was successfully applied to CAPRI targets in a blind fashion. An example of using the refinement method with an existing docking method for ligand binding mode prediction of a drug target is also presented. A web server that implements the method is freely available at http://galaxy.seoklab.org/refinecomplex.

Influence of lipids on the interfacial disposition of respiratory syncytical virus matrix protein.

PubMed

McPhee, Helen K; Carlisle, Jennifer L; Beeby, Andrew; Money, Victoria A; Watson, Scott M D; Yeo, R Paul; Sanderson, John M

2011-01-04

The propensity of a matrix protein from an enveloped virus of the Mononegavirales family to associate with lipids representative of the viral envelope has been determined using label-free methods, including tensiometry and Brewster angle microscopy on lipid films at the air-water interface and atomic force microscopy on monolayers transferred to OTS-treated silicon wafers. This has enabled factors that influence the disposition of the protein with respect to the lipid interface to be characterized. In the absence of sphingomyelin, respiratory syncytial virus matrix protein penetrates monolayers composed of mixtures of phosphocholines with phosphoethanolamines or cholesterol at the air-water interface. In ternary mixtures composed of sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol, the protein exhibits two separate behaviors: (1) peripheral association with the surface of sphingomyelin-rich domains and (2) penetration of sphingomyelin-poor domains. Prolonged incubation of the protein with mixtures of phosphocholines and phosphoethanolamines leads to the formation of helical protein assemblies of uniform diameter that demonstrate an inherent propensity of the protein to assemble into a filamentous form.

QuantFusion: Novel Unified Methodology for Enhanced Coverage and Precision in Quantifying Global Proteomic Changes in Whole Tissues.

PubMed

Gunawardena, Harsha P; O'Brien, Jonathon; Wrobel, John A; Xie, Ling; Davies, Sherri R; Li, Shunqiang; Ellis, Matthew J; Qaqish, Bahjat F; Chen, Xian

2016-02-01

Single quantitative platforms such as label-based or label-free quantitation (LFQ) present compromises in accuracy, precision, protein sequence coverage, and speed of quantifiable proteomic measurements. To maximize the quantitative precision and the number of quantifiable proteins or the quantifiable coverage of tissue proteomes, we have developed a unified approach, termed QuantFusion, that combines the quantitative ratios of all peptides measured by both LFQ and label-based methodologies. Here, we demonstrate the use of QuantFusion in determining the proteins differentially expressed in a pair of patient-derived tumor xenografts (PDXs) representing two major breast cancer (BC) subtypes, basal and luminal. Label-based in-spectra quantitative peptides derived from amino acid-coded tagging (AACT, also known as SILAC) of a non-malignant mammary cell line were uniformly added to each xenograft with a constant predefined ratio, from which Ratio-of-Ratio estimates were obtained for the label-free peptides paired with AACT peptides in each PDX tumor. A mixed model statistical analysis was used to determine global differential protein expression by combining complementary quantifiable peptide ratios measured by LFQ and Ratio-of-Ratios, respectively. With minimum number of replicates required for obtaining the statistically significant ratios, QuantFusion uses the distinct mechanisms to "rescue" the missing data inherent to both LFQ and label-based quantitation. Combined quantifiable peptide data from both quantitative schemes increased the overall number of peptide level measurements and protein level estimates. In our analysis of the PDX tumor proteomes, QuantFusion increased the number of distinct peptide ratios by 65%, representing differentially expressed proteins between the BC subtypes. This quantifiable coverage improvement, in turn, not only increased the number of measurable protein fold-changes by 8% but also increased the average precision of quantitative estimates by 181% so that some BC subtypically expressed proteins were rescued by QuantFusion. Thus, incorporating data from multiple quantitative approaches while accounting for measurement variability at both the peptide and global protein levels make QuantFusion unique for obtaining increased coverage and quantitative precision for tissue proteomes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Sugary interfaces mitigate contact damage where stiff meets soft

PubMed Central

Yoo, Hee Young; Iordachescu, Mihaela; Huang, Jun; Hennebert, Elise; Kim, Sangsik; Rho, Sangchul; Foo, Mathias; Flammang, Patrick; Zeng, Hongbo; Hwang, Daehee; Waite, J. Herbert; Hwang, Dong Soo

2016-01-01

The byssal threads of the fan shell Atrina pectinata are non-living functional materials intimately associated with living tissue, which provide an intriguing paradigm of bionic interface for robust load-bearing device. An interfacial load-bearing protein (A. pectinata foot protein-1, apfp-1) with L-3,4-dihydroxyphenylalanine (DOPA)-containing and mannose-binding domains has been characterized from Atrina's foot. apfp-1 was localized at the interface between stiff byssus and the soft tissue by immunochemical staining and confocal Raman imaging, implying that apfp-1 is an interfacial linker between the byssus and soft tissue, that is, the DOPA-containing domain interacts with itself and other byssal proteins via Fe3+–DOPA complexes, and the mannose-binding domain interacts with the soft tissue and cell membranes. Both DOPA- and sugar-mediated bindings are reversible and robust under wet conditions. This work shows the combination of DOPA and sugar chemistry at asymmetric interfaces is unprecedented and highly relevant to bionic interface design for tissue engineering and bionic devices. PMID:27305949

Sugary interfaces mitigate contact damage where stiff meets soft

NASA Astrophysics Data System (ADS)

Yoo, Hee Young; Iordachescu, Mihaela; Huang, Jun; Hennebert, Elise; Kim, Sangsik; Rho, Sangchul; Foo, Mathias; Flammang, Patrick; Zeng, Hongbo; Hwang, Daehee; Waite, J. Herbert; Hwang, Dong Soo

2016-06-01

The byssal threads of the fan shell Atrina pectinata are non-living functional materials intimately associated with living tissue, which provide an intriguing paradigm of bionic interface for robust load-bearing device. An interfacial load-bearing protein (A. pectinata foot protein-1, apfp-1) with L-3,4-dihydroxyphenylalanine (DOPA)-containing and mannose-binding domains has been characterized from Atrina's foot. apfp-1 was localized at the interface between stiff byssus and the soft tissue by immunochemical staining and confocal Raman imaging, implying that apfp-1 is an interfacial linker between the byssus and soft tissue, that is, the DOPA-containing domain interacts with itself and other byssal proteins via Fe3+-DOPA complexes, and the mannose-binding domain interacts with the soft tissue and cell membranes. Both DOPA- and sugar-mediated bindings are reversible and robust under wet conditions. This work shows the combination of DOPA and sugar chemistry at asymmetric interfaces is unprecedented and highly relevant to bionic interface design for tissue engineering and bionic devices.

Nanopore-CMOS Interfaces for DNA Sequencing

PubMed Central

Magierowski, Sebastian; Huang, Yiyun; Wang, Chengjie; Ghafar-Zadeh, Ebrahim

2016-01-01

DNA sequencers based on nanopore sensors present an opportunity for a significant break from the template-based incumbents of the last forty years. Key advantages ushered by nanopore technology include a simplified chemistry and the ability to interface to CMOS technology. The latter opportunity offers substantial promise for improvement in sequencing speed, size and cost. This paper reviews existing and emerging means of interfacing nanopores to CMOS technology with an emphasis on massively-arrayed structures. It presents this in the context of incumbent DNA sequencing techniques, reviews and quantifies nanopore characteristics and models and presents CMOS circuit methods for the amplification of low-current nanopore signals in such interfaces. PMID:27509529

Nanopore-CMOS Interfaces for DNA Sequencing.

PubMed

Magierowski, Sebastian; Huang, Yiyun; Wang, Chengjie; Ghafar-Zadeh, Ebrahim

2016-08-06

DNA sequencers based on nanopore sensors present an opportunity for a significant break from the template-based incumbents of the last forty years. Key advantages ushered by nanopore technology include a simplified chemistry and the ability to interface to CMOS technology. The latter opportunity offers substantial promise for improvement in sequencing speed, size and cost. This paper reviews existing and emerging means of interfacing nanopores to CMOS technology with an emphasis on massively-arrayed structures. It presents this in the context of incumbent DNA sequencing techniques, reviews and quantifies nanopore characteristics and models and presents CMOS circuit methods for the amplification of low-current nanopore signals in such interfaces.

Spatial modeling of the membrane-cytosolic interface in protein kinase signal transduction

PubMed Central

Schröder, Andreas

2018-01-01

The spatial architecture of signaling pathways and the interaction with cell size and morphology are complex, but little understood. With the advances of single cell imaging and single cell biology, it becomes crucial to understand intracellular processes in time and space. Activation of cell surface receptors often triggers a signaling cascade including the activation of membrane-attached and cytosolic signaling components, which eventually transmit the signal to the cell nucleus. Signaling proteins can form steep gradients in the cytosol, which cause strong cell size dependence. We show that the kinetics at the membrane-cytosolic interface and the ratio of cell membrane area to the enclosed cytosolic volume change the behavior of signaling cascades significantly. We suggest an estimate of average concentration for arbitrary cell shapes depending on the cell volume and cell surface area. The normalized variance, known from image analysis, is suggested as an alternative measure to quantify the deviation from the average concentration. A mathematical analysis of signal transduction in time and space is presented, providing analytical solutions for different spatial arrangements of linear signaling cascades. Quantification of signaling time scales reveals that signal propagation is faster at the membrane than at the nucleus, while this time difference decreases with the number of signaling components in the cytosol. Our investigations are complemented by numerical simulations of non-linear cascades with feedback and asymmetric cell shapes. We conclude that intracellular signal propagation is highly dependent on cell geometry and, thereby, conveys information on cell size and shape to the nucleus. PMID:29630597

Analysis of Structural Features Contributing to Weak Affinities of Ubiquitin/Protein Interactions.

PubMed

Cohen, Ariel; Rosenthal, Eran; Shifman, Julia M

2017-11-10

Ubiquitin is a small protein that enables one of the most common post-translational modifications, where the whole ubiquitin molecule is attached to various target proteins, forming mono- or polyubiquitin conjugations. As a prototypical multispecific protein, ubiquitin interacts non-covalently with a variety of proteins in the cell, including ubiquitin-modifying enzymes and ubiquitin receptors that recognize signals from ubiquitin-conjugated substrates. To enable recognition of multiple targets and to support fast dissociation from the ubiquitin modifying enzymes, ubiquitin/protein interactions are characterized with low affinities, frequently in the higher μM and lower mM range. To determine how structure encodes low binding affinity of ubiquitin/protein complexes, we analyzed structures of more than a hundred such complexes compiled in the Ubiquitin Structural Relational Database. We calculated various structure-based features of ubiquitin/protein binding interfaces and compared them to the same features of general protein-protein interactions (PPIs) with various functions and generally higher affinities. Our analysis shows that ubiquitin/protein binding interfaces on average do not differ in size and shape complementarity from interfaces of higher-affinity PPIs. However, they contain fewer favorable hydrogen bonds and more unfavorable hydrophobic/charge interactions. We further analyzed how binding interfaces change upon affinity maturation of ubiquitin toward its target proteins. We demonstrate that while different features are improved in different experiments, the majority of the evolved complexes exhibit better shape complementarity and hydrogen bond pattern compared to wild-type complexes. Our analysis helps to understand how low-affinity PPIs have evolved and how they could be converted into high-affinity PPIs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantitative Analysis of Apisin, a Major Protein Unique to Royal Jelly

PubMed Central

Arai, Yasuko; Kato, Kenji

2016-01-01

Apisin, a protein that is unique to royal jelly (RJ), is known to compose the greater part of the RJ proteins and to exist as a heterooligomer containing major royal jelly protein 1 and apisimin. However, few reports on the methods for quantifying apisin have been published. Thus, we attempted to quantify apisin using HPLC, a widely used analytical technique, as described below. Isoelectric precipitation and size-exclusion chromatography were used to obtain the purified protein, which was identified as apisin by SDS-PAGE and LC-MS analyses. The purified apisin was lyophilized and then used to generate a calibration curve to quantify apisin in RJ. The apisin content was fairly constant (i.e., 3.93 to 4.67 w/w%) in natural RJ. This study is the first to describe a simple, standardized method for quantifying apisin using HPLC and suggests that apisin can be used as a benchmark for future evaluations of RJ quality. PMID:27721892

A force-based, parallel assay for the quantification of protein-DNA interactions.

PubMed

Limmer, Katja; Pippig, Diana A; Aschenbrenner, Daniela; Gaub, Hermann E

2014-01-01

Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

C-terminal interactions mediate the quaternary dynamics of αB-crystallin

PubMed Central

Hilton, Gillian R.; Hochberg, Georg K. A.; Laganowsky, Arthur; McGinnigle, Scott I.; Baldwin, Andrew J.; Benesch, Justin L. P.

2013-01-01

αB-crystallin is a highly dynamic, polydisperse small heat-shock protein that can form oligomers ranging in mass from 200 to 800 kDa. Here we use a multifaceted mass spectrometry approach to assess the role of the C-terminal tail in the self-assembly of αB-crystallin. Titration experiments allow us to monitor the binding of peptides representing the C-terminus to the αB-crystallin core domain, and observe individual affinities to both monomeric and dimeric forms. Notably, we find that binding the second peptide equivalent to the core domain dimer is considerably more difficult than the first, suggesting a role of the C-terminus in regulating assembly. This finding motivates us to examine the effect of point mutations in the C-terminus in the full-length protein, by quantifying the changes in oligomeric distribution and corresponding subunit exchange rates. Our results combine to demonstrate that alterations in the C-terminal tail have a significant impact on the thermodynamics and kinetics of αB-crystallin. Remarkably, we find that there is energy compensation between the inter- and intra-dimer interfaces: when one interaction is weakened, the other is strengthened. This allosteric communication between binding sites on αB-crystallin is likely important for its role in binding target proteins. PMID:23530258

Computational fluid dynamics (CFD) insights into agitation stress methods in biopharmaceutical development.

PubMed

Bai, Ge; Bee, Jared S; Biddlecombe, James G; Chen, Quanmin; Leach, W Thomas

2012-02-28

Agitation of small amounts of liquid is performed routinely in biopharmaceutical process, formulation, and packaging development. Protein degradation commonly results from agitation, but the specific stress responsible or degradation mechanism is usually not well understood. Characterization of the agitation stress methods is critical to identifying protein degradation mechanisms or specific sensitivities. In this study, computational fluid dynamics (CFD) was used to model agitation of 1 mL of fluid by four types of common laboratory agitation instruments, including a rotator, orbital shaker, magnetic stirrer and vortex mixer. Fluid stresses in the bulk liquid and near interfaces were identified, quantified and compared. The vortex mixer provides the most intense stresses overall, while the stir bar system presented locally intense shear proximal to the hydrophobic stir bar surface. The rotator provides gentler fluid stresses, but the air-water interfacial area and surface stresses are relatively high given its low rotational frequency. The orbital shaker provides intermediate-level stresses but with the advantage of a large stable platform for consistent vial-to-vial homogeneity. Selection of experimental agitation methods with targeted types and intensities of stresses can facilitate better understanding of protein degradation mechanisms and predictability for "real world" applications. Copyright © 2011 Elsevier B.V. All rights reserved.

Physical interface dynamics alter how robotic exosuits augment human movement: implications for optimizing wearable assistive devices.

PubMed

Yandell, Matthew B; Quinlivan, Brendan T; Popov, Dmitry; Walsh, Conor; Zelik, Karl E

2017-05-18

Wearable assistive devices have demonstrated the potential to improve mobility outcomes for individuals with disabilities, and to augment healthy human performance; however, these benefits depend on how effectively power is transmitted from the device to the human user. Quantifying and understanding this power transmission is challenging due to complex human-device interface dynamics that occur as biological tissues and physical interface materials deform and displace under load, absorbing and returning power. Here we introduce a new methodology for quickly estimating interface power dynamics during movement tasks using common motion capture and force measurements, and then apply this method to quantify how a soft robotic ankle exosuit interacts with and transfers power to the human body during walking. We partition exosuit end-effector power (i.e., power output from the device) into power that augments ankle plantarflexion (termed augmentation power) vs. power that goes into deformation and motion of interface materials and underlying soft tissues (termed interface power). We provide empirical evidence of how human-exosuit interfaces absorb and return energy, reshaping exosuit-to-human power flow and resulting in three key consequences: (i) During exosuit loading (as applied forces increased), about 55% of exosuit end-effector power was absorbed into the interfaces. (ii) However, during subsequent exosuit unloading (as applied forces decreased) most of the absorbed interface power was returned viscoelastically. Consequently, the majority (about 75%) of exosuit end-effector work over each stride contributed to augmenting ankle plantarflexion. (iii) Ankle augmentation power (and work) was delayed relative to exosuit end-effector power, due to these interface energy absorption and return dynamics. Our findings elucidate the complexities of human-exosuit interface dynamics during transmission of power from assistive devices to the human body, and provide insight into improving the design and control of wearable robots. We conclude that in order to optimize the performance of wearable assistive devices it is important, throughout design and evaluation phases, to account for human-device interface dynamics that affect power transmission and thus human augmentation benefits.

Utilizing knowledge base of amino acids structural neighborhoods to predict protein-protein interaction sites.

PubMed

Jelínek, Jan; Škoda, Petr; Hoksza, David

2017-12-06

Protein-protein interactions (PPI) play a key role in an investigation of various biochemical processes, and their identification is thus of great importance. Although computational prediction of which amino acids take part in a PPI has been an active field of research for some time, the quality of in-silico methods is still far from perfect. We have developed a novel prediction method called INSPiRE which benefits from a knowledge base built from data available in Protein Data Bank. All proteins involved in PPIs were converted into labeled graphs with nodes corresponding to amino acids and edges to pairs of neighboring amino acids. A structural neighborhood of each node was then encoded into a bit string and stored in the knowledge base. When predicting PPIs, INSPiRE labels amino acids of unknown proteins as interface or non-interface based on how often their structural neighborhood appears as interface or non-interface in the knowledge base. We evaluated INSPiRE's behavior with respect to different types and sizes of the structural neighborhood. Furthermore, we examined the suitability of several different features for labeling the nodes. Our evaluations showed that INSPiRE clearly outperforms existing methods with respect to Matthews correlation coefficient. In this paper we introduce a new knowledge-based method for identification of protein-protein interaction sites called INSPiRE. Its knowledge base utilizes structural patterns of known interaction sites in the Protein Data Bank which are then used for PPI prediction. Extensive experiments on several well-established datasets show that INSPiRE significantly surpasses existing PPI approaches.

Integrating Structure to Protein-Protein Interaction Networks That Drive Metastasis to Brain and Lung in Breast Cancer

PubMed Central

Engin, H. Billur; Guney, Emre; Keskin, Ozlem; Oliva, Baldo; Gursoy, Attila

2013-01-01

Blocking specific protein interactions can lead to human diseases. Accordingly, protein interactions and the structural knowledge on interacting surfaces of proteins (interfaces) have an important role in predicting the genotype-phenotype relationship. We have built the phenotype specific sub-networks of protein-protein interactions (PPIs) involving the relevant genes responsible for lung and brain metastasis from primary tumor in breast cancer. First, we selected the PPIs most relevant to metastasis causing genes (seed genes), by using the “guilt-by-association” principle. Then, we modeled structures of the interactions whose complex forms are not available in Protein Databank (PDB). Finally, we mapped mutations to interface structures (real and modeled), in order to spot the interactions that might be manipulated by these mutations. Functional analyses performed on these sub-networks revealed the potential relationship between immune system-infectious diseases and lung metastasis progression, but this connection was not observed significantly in the brain metastasis. Besides, structural analyses showed that some PPI interfaces in both metastasis sub-networks are originating from microbial proteins, which in turn were mostly related with cell adhesion. Cell adhesion is a key mechanism in metastasis, therefore these PPIs may be involved in similar molecular pathways that are shared by infectious disease and metastasis. Finally, by mapping the mutations and amino acid variations on the interface regions of the proteins in the metastasis sub-networks we found evidence for some mutations to be involved in the mechanisms differentiating the type of the metastasis. PMID:24278371

Crystal Structure of a Ube2S-Ubiquitin Conjugate

PubMed Central

Lorenz, Sonja; Bhattacharyya, Moitrayee; Feiler, Christian; Rape, Michael; Kuriyan, John

2016-01-01

Protein ubiquitination occurs through the sequential formation and reorganization of specific protein-protein interfaces. Ubiquitin-conjugating (E2) enzymes, such as Ube2S, catalyze the formation of an isopeptide linkage between the C-terminus of a “donor” ubiquitin and a primary amino group of an “acceptor” ubiquitin molecule. This reaction involves an intermediate, in which the C-terminus of the donor ubiquitin is thioester-bound to the active site cysteine of the E2 and a functionally important interface is formed between the two proteins. A docked model of a Ube2S-donor ubiquitin complex was generated previously, based on chemical shift mapping by NMR, and predicted contacts were validated in functional studies. We now present the crystal structure of a covalent Ube2S-ubiquitin complex. The structure contains an interface between Ube2S and ubiquitin in trans that resembles the earlier model in general terms, but differs in detail. The crystallographic interface is more hydrophobic than the earlier model and is stable in molecular dynamics (MD) simulations. Remarkably, the docked Ube2S-donor complex converges readily to the configuration seen in the crystal structure in 3 out of 8 MD trajectories. Since the crystallographic interface is fully consistent with mutational effects, this indicates that the structure provides an energetically favorable representation of the functionally critical Ube2S-donor interface. PMID:26828794

Multiple protein–protein interactions converging on the Prp38 protein during activation of the human spliceosome

PubMed Central

Schütze, Tonio; Ulrich, Alexander K.C.; Apelt, Luise; Will, Cindy L.; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C.

2016-01-01

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein–protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein–protein interaction platform that might organize the relative positioning of other proteins during splicing. PMID:26673105

Protein hydration in solution: Experimental observation by x-ray and neutron scattering

PubMed Central

Svergun, D. I.; Richard, S.; Koch, M. H. J.; Sayers, Z.; Kuprin, S.; Zaccai, G.

1998-01-01

The structure of the protein–solvent interface is the subject of controversy in theoretical studies and requires direct experimental characterization. Three proteins with known atomic resolution crystal structure (lysozyme, Escherichia coli thioredoxin reductase, and protein R1 of E. coli ribonucleotide reductase) were investigated in parallel by x-ray and neutron scattering in H2O and D2O solutions. The analysis of the protein–solvent interface is based on the significantly different contrasts for the protein and for the hydration shell. The results point to the existence of a first hydration shell with an average density ≈10% larger than that of the bulk solvent in the conditions studied. Comparisons with the results of other studies suggest that this may be a general property of aqueous interfaces. PMID:9482874

Structural rearrangement of β-lactoglobulin at different oil-water interfaces and its effect on emulsion stability.

PubMed

Zhai, Jiali; Wooster, Tim J; Hoffmann, Søren V; Lee, Tzong-Hsien; Augustin, Mary Ann; Aguilar, Marie-Isabel

2011-08-02

Understanding the factors that control protein structure and stability at the oil-water interface continues to be a major focus to optimize the formulation of protein-stabilized emulsions. In this study, a combination of synchrotron radiation circular dichroism spectroscopy, front-face fluorescence spectroscopy, and dual polarization interferometry (DPI) was used to characterize the conformation and geometric structure of β-lactoglobulin (β-Lg) upon adsorption to two oil-water interfaces: a hexadecane-water interface and a tricaprylin-water interface. The results show that, upon adsorption to both oil-water interfaces, β-Lg went through a β-sheet to α-helix transition with a corresponding loss of its globular tertiary structure. The degree of conformational change was also a function of the oil phase polarity. The hexadecane oil induced a much higher degree of non-native α-helix compared to the tricaprylin oil. In contrast to the β-Lg conformation in solution, the non-native α-helical-rich conformation of β-Lg at the interface was resistant to further conformational change upon heating. DPI measurements suggest that β-Lg formed a thin dense layer at emulsion droplet surfaces. The effects of high temperature and the presence of salt on these β-Lg emulsions were then investigated by monitoring changes in the ζ-potential and particle size. In the absence of salt, high electrostatic repulsion meant β-Lg-stabilized emulsions were resistant to heating to 90 °C. Adding salt (120 mM NaCl) before or after heating led to emulsion flocculation due to the screening of the electrostatic repulsion between colloidal particles. This study has provided insight into the structural properties of proteins adsorbed at the oil-water interface and has implications in the formulation and production of emulsions stabilized by globular proteins.

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

Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

PubMed

Schütze, Tonio; Ulrich, Alexander K C; Apelt, Luise; Will, Cindy L; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C

2016-02-01

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing. © 2016 Schütze et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Solid-support immobilization of a "swing" fusion protein for enhanced glucose oxidase catalytic activity.

PubMed

Takatsuji, Yoshiyuki; Yamasaki, Ryota; Iwanaga, Atsushi; Lienemann, Michael; Linder, Markus B; Haruyama, Tetsuya

2013-12-01

The strategic surface immobilization of a protein can add new functionality to a solid substrate; however, protein activity, e.g., enzymatic activity, can be drastically decreased on immobilization onto a solid surface. The concept of a designed and optimized "molecular interface" is herein introduced in order to address this problem. In this study, molecular interface was designed and constructed with the aim of attaining high enzymatic activity of a solid-surface-immobilized a using the hydrophobin HFBI protein in conjunction with a fusion protein of HFBI attached to glucose oxidase (GOx). The ability of HFBI to form a self-organized membrane on a solid surface in addition to its adhesion properties makes it an ideal candidate for immobilization. The developed fusion protein was also able to form an organized membrane, and its structure and immobilized state on a solid surface were investigated using QCM-D measurements. This method of immobilization showed retention of high enzymatic activity and the ability to control the density of the immobilized enzyme. In this study, we demonstrated the importance of the design and construction of molecular interface for numerous purposes. This method of protein immobilization could be utilized for preparation of high throughput products requiring structurally ordered molecular interfaces, in addition to many other applications. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Faraday Discussion 160 Introductory Lecture: Interpreting and Predicting Hofmeister Salt Ion and Solute Effects on Biopolymer and Model Processes Using the Solute Partitioning Model

PubMed Central

Record, M. Thomas; Guinn, Emily; Pegram, Laurel; Capp, Michael

2013-01-01

Understanding how Hofmeister salt ions and other solutes interact with proteins, nucleic acids, other biopolymers and water and thereby affect protein and nucleic acid processes as well as model processes (e.g solubility of model compounds) in aqueous solution is a longstanding goal of biophysical research. Empirical Hofmeister salt and solute “m-values” (derivatives of the observed standard free energy change for a model or biopolymer process with respect to solute or salt concentration m3) are equal to differences in chemical potential derivatives: m-value = Δ(dμ2/dm3) = Δμ23 which quantify the preferential interactions of the solute or salt with the surface of the biopolymer or model system (component 2) exposed or buried in the process. Using the SPM, we dissect μ23 values for interactions of a solute or Hofmeister salt with a set of model compounds displaying the key functional groups of biopolymers to obtain interaction potentials (called α-values) that quantify the interaction of the solute or salt per unit area of each functional group or type of surface. Interpreted using the SPM, these α-values provide quantitative information about both the hydration of functional groups and the competitive interaction of water and the solute or salt with functional groups. The analysis corroborates and quantifies previous proposals that the Hofmeister anion and cation series for biopolymer processes are determined by ion-specific, mostly unfavorable interactions with hydrocarbon surfaces; the balance between these unfavorable nonpolar interactions and often-favorable interactions of ions with polar functional groups determine the series null points. The placement of urea and glycine betaine (GB) at opposite ends of the corresponding series of nonelectrolytes results from the favorable interactions of urea, and unfavorable interactions of GB, with many (but not all) biopolymer functional groups. Interaction potentials and local-bulk partition coefficients quantifying the distribution of solutes (e.g. urea, glycine betaine) and Hofmeister salt ions in the vicinity of each functional group make good chemical sense when interpreted in terms of competitive noncovalent interactions. These interaction potentials allow solute and Hofmeister (noncoulombic) salt effects on protein and nucleic acid processes to be interpreted or predicted, and allow the use of solutes and salts as probes of interface formation and large-scale conformational changes in the steps of a biopolymer mechanism. PMID:23795491

Interfacial Properties of NTAIL, an Intrinsically Disordered Protein.

PubMed

Bénarouche, Anaïs; Habchi, Johnny; Cagna, Alain; Maniti, Ofelia; Girard-Egrot, Agnès; Cavalier, Jean-François; Longhi, Sonia; Carrière, Frédéric

2017-12-19

Intrinsically disordered proteins (IDPs) lack stable secondary and tertiary structure under physiological conditions in the absence of their biological partners and thus exist as dynamic ensembles of interconverting conformers, often highly soluble in water. However, in some cases, IDPs such as the ones involved in neurodegenerative diseases can form protein aggregates and their aggregation process may be triggered by the interaction with membranes. Although the interfacial behavior of globular proteins has been extensively studied, experimental data on IDPs at the air/water (A/W) and water/lipid interfaces are scarce. We studied here the intrinsically disordered C-terminal domain of the Hendra virus nucleoprotein (N TAIL ) and compared its interfacial properties to those of lysozyme that is taken as a model globular protein of similar molecular mass. Adsorption of N TAIL at the A/W interface was studied in the absence and presence of phospholipids using Langmuir films, polarization modulated-infrared reflection-absorption spectroscopy, and an automated drop tensiometer for interfacial tension and elastic modulus determination with oscillating bubbles. N TAIL showed a significant surface activity, with a higher adsorption capacity at the A/W interface and penetration into egg phosphatidylcholine monolayer compared to lysozyme. Whereas lysozyme remains folded upon compression of the protein layer at the A/W interface and shows a quasi-pure elastic behavior, N TAIL shows a much higher molecular area and forms a highly viscoelastic film with a high dilational modulus. To our knowledge, a new disorder-to-order transition is thus observed for the N TAIL protein that folds into an antiparallel β-sheet at the A/W interface and presents strong intermolecular interactions. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Prediction of hot spots in protein interfaces using a random forest model with hybrid features.

PubMed

Wang, Lin; Liu, Zhi-Ping; Zhang, Xiang-Sun; Chen, Luonan

2012-03-01

Prediction of hot spots in protein interfaces provides crucial information for the research on protein-protein interaction and drug design. Existing machine learning methods generally judge whether a given residue is likely to be a hot spot by extracting features only from the target residue. However, hot spots usually form a small cluster of residues which are tightly packed together at the center of protein interface. With this in mind, we present a novel method to extract hybrid features which incorporate a wide range of information of the target residue and its spatially neighboring residues, i.e. the nearest contact residue in the other face (mirror-contact residue) and the nearest contact residue in the same face (intra-contact residue). We provide a novel random forest (RF) model to effectively integrate these hybrid features for predicting hot spots in protein interfaces. Our method can achieve accuracy (ACC) of 82.4% and Matthew's correlation coefficient (MCC) of 0.482 in Alanine Scanning Energetics Database, and ACC of 77.6% and MCC of 0.429 in Binding Interface Database. In a comparison study, performance of our RF model exceeds other existing methods, such as Robetta, FOLDEF, KFC, KFC2, MINERVA and HotPoint. Of our hybrid features, three physicochemical features of target residues (mass, polarizability and isoelectric point), the relative side-chain accessible surface area and the average depth index of mirror-contact residues are found to be the main discriminative features in hot spots prediction. We also confirm that hot spots tend to form large contact surface areas between two interacting proteins. Source data and code are available at: http://www.aporc.org/doc/wiki/HotSpot.

Predicting the tolerated sequences for proteins and protein interfaces using RosettaBackrub flexible backbone design.

PubMed

Smith, Colin A; Kortemme, Tanja

2011-01-01

Predicting the set of sequences that are tolerated by a protein or protein interface, while maintaining a desired function, is useful for characterizing protein interaction specificity and for computationally designing sequence libraries to engineer proteins with new functions. Here we provide a general method, a detailed set of protocols, and several benchmarks and analyses for estimating tolerated sequences using flexible backbone protein design implemented in the Rosetta molecular modeling software suite. The input to the method is at least one experimentally determined three-dimensional protein structure or high-quality model. The starting structure(s) are expanded or refined into a conformational ensemble using Monte Carlo simulations consisting of backrub backbone and side chain moves in Rosetta. The method then uses a combination of simulated annealing and genetic algorithm optimization methods to enrich for low-energy sequences for the individual members of the ensemble. To emphasize certain functional requirements (e.g. forming a binding interface), interactions between and within parts of the structure (e.g. domains) can be reweighted in the scoring function. Results from each backbone structure are merged together to create a single estimate for the tolerated sequence space. We provide an extensive description of the protocol and its parameters, all source code, example analysis scripts and three tests applying this method to finding sequences predicted to stabilize proteins or protein interfaces. The generality of this method makes many other applications possible, for example stabilizing interactions with small molecules, DNA, or RNA. Through the use of within-domain reweighting and/or multistate design, it may also be possible to use this method to find sequences that stabilize particular protein conformations or binding interactions over others.

The influence of protein concentration on the biotribological properties of the stem-cement interface.

PubMed

Zhang, Hong-Yu; Zhou, Ming

2014-01-01

The stem-cement interface in total hip replacement experiences fretting wear following debonding under cyclical physiological loading. However, the influence of protein concentration on the biotribological properties of this interface has not been well taken into consideration. In the present study, a series of fretting frictional tests were performed using polished Ti6Al4V and bone cement, lubricated by bovine serum albumin solutions of different concentrations (5%, 30%, and 75%). Surface characterizations of Ti6Al4V pins were conducted by optical interferometer, scanning electron microscope, and Raman spectroscopy. The results show that the friction coefficient decreases with the increase of protein concentration, although the difference is not significant. In addition, bovine serum albumin is adsorbed onto Ti6Al4V surface, forming a protective film to prevent the metal substrate from wear. The elemental and spectroscopic analyses of the film confirm the presence of protein molecules adsorbed on Ti6Al4V surface, with a thickness of 2.5 μm. It is indicated from this study that fretting wear at the stem-cement interface can be postponed by promotion of protein adsorption on the metal surface.

Evaluation of the influence of the internal aqueous solvent structure on electrostatic interactions at the protein-solvent interface by nonlocal continuum electrostatic approach.

PubMed

Rubinstein, Alexander; Sherman, Simon

The dielectric properties of the polar solvent on the protein-solvent interface at small intercharge distances are still poorly explored. To deconvolute this problem and to evaluate the pair-wise electrostatic interaction (PEI) energies of the point charges located at the protein-solvent interface we used a nonlocal (NL) electrostatic approach along with a static NL dielectric response function of water. The influence of the aqueous solvent microstructure (determined by a strong nonelectrostatic correlation effect between water dipoles within the orientational Debye polarization mode) on electrostatic interactions at the interface was studied in our work. It was shown that the PEI energies can be significantly higher than the energies evaluated by the classical (local) consideration, treating water molecules as belonging to the bulk solvent with a high dielectric constant. Our analysis points to the existence of a rather extended, effective low-dielectric interfacial water shell on the protein surface. The main dielectric properties of this shell (effective thickness together with distance- and orientation-dependent dielectric permittivity function) were evaluated. The dramatic role of this shell was demonstrated when estimating the protein association rate constants.

Structural changes of homodimers in the PDB.

PubMed

Koike, Ryotaro; Amemiya, Takayuki; Horii, Tatsuya; Ota, Motonori

2018-04-01

Protein complexes are involved in various biological phenomena. These complexes are intrinsically flexible, and structural changes are essential to their functions. To perform a large-scale automated analysis of the structural changes of complexes, we combined two original methods. An application, SCPC, compares two structures of protein complexes and decides the match of binding mode. Another application, Motion Tree, identifies rigid-body motions in various sizes and magnitude from the two structural complexes with the same binding mode. This approach was applied to all available homodimers in the Protein Data Bank (PDB). We defined two complex-specific motions: interface motion and subunit-spanning motion. In the former, each subunit of a complex constitutes a rigid body, and the relative movement between subunits occurs at the interface. In the latter, structural parts from distinct subunits constitute a rigid body, providing the relative movement spanning subunits. All structural changes were classified and examined. It was revealed that the complex-specific motions were common in the homodimers, detected in around 40% of families. The dimeric interfaces were likely to be small and flat for interface motion, while large and rugged for subunit-spanning motion. Interface motion was accompanied by a drastic change in contacts at the interface, while the change in the subunit-spanning motion was moderate. These results indicate that the interface properties of homodimers correlated with the type of complex-specific motion. The study demonstrates that the pipeline of SCPC and Motion Tree is useful for the massive analysis of structural change of protein complexes. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessing land clearing potential in the Canadian agriculture–forestry interface with a multi-attribute frontier approach

Treesearch

Denys Yemshanov; Frank Koch; Kurt H. Riitters; Brian McConkey; Ted Huffmand; Stephen Smithe

2015-01-01

The pattern of forest land clearing in a region can be viewed as a gauge of sustainable (or unsustain-able) use of agricultural and forest resources. In this study we examine the geographical distribution ofland clearing potential in the Canadian agriculture–forestry interface and propose a new landscape-scaleindicator that quantifies this potential. We consider the...

Biotic and abiotic effects of human settlements in the wildland-urban interface

Treesearch

Avi Bar-Massada; Volker C. Radeloff; Susan I. Stewart

2014-01-01

The wildland-urban interface (WUI) is the area in which human settlements adjoin or intermix with ecosystems. Although research on the WUI has been focused on wildfire risk to settlements, we argue here that there is a need to quantify the extent of areas in which human settlements interact with adjoining ecosystems, regardless of their ability to support fire spread....

Protein-protein interaction specificity is captured by contact preferences and interface composition.

PubMed

Nadalin, Francesca; Carbone, Alessandra

2018-02-01

Large-scale computational docking will be increasingly used in future years to discriminate protein-protein interactions at the residue resolution. Complete cross-docking experiments make in silico reconstruction of protein-protein interaction networks a feasible goal. They ask for efficient and accurate screening of the millions structural conformations issued by the calculations. We propose CIPS (Combined Interface Propensity for decoy Scoring), a new pair potential combining interface composition with residue-residue contact preference. CIPS outperforms several other methods on screening docking solutions obtained either with all-atom or with coarse-grain rigid docking. Further testing on 28 CAPRI targets corroborates CIPS predictive power over existing methods. By combining CIPS with atomic potentials, discrimination of correct conformations in all-atom structures reaches optimal accuracy. The drastic reduction of candidate solutions produced by thousands of proteins docked against each other makes large-scale docking accessible to analysis. CIPS source code is freely available at http://www.lcqb.upmc.fr/CIPS. alessandra.carbone@lip6.fr. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

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).

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.

Insights into positive and negative requirements for protein-protein interactions by crystallographic analysis of the beta-lactamase inhibitory proteins BLIP, BLIP-I, and BLP.

PubMed

Gretes, Michael; Lim, Daniel C; de Castro, Liza; Jensen, Susan E; Kang, Sung Gyun; Lee, Kye Joon; Strynadka, Natalie C J

2009-06-05

Beta-lactamase inhibitory protein (BLIP) binds a variety of beta-lactamase enzymes with wide-ranging specificity. Its binding mechanism and interface interactions are a well-established model system for the characterization of protein-protein interactions. Published studies have examined the binding of BLIP to diverse target beta-lactamases (e.g., TEM-1, SME-1, and SHV-1). However, apart from point mutations of amino acid residues, variability on the inhibitor side of this enzyme-inhibitor interface has remained unexplored. Thus, we present crystal structures of two likely BLIP relatives: (1) BLIP-I (solved alone and in complex with TEM-1), which has beta-lactamase inhibitory activity very similar to that of BLIP; and (2) beta-lactamase-inhibitory-protein-like protein (BLP) (in two apo forms, including an ultra-high-resolution structure), which is unable to inhibit any tested beta-lactamase. Despite categorical differences in species of origin and function, BLIP-I and BLP share nearly identical backbone conformations, even at loop regions differing in BLIP. We describe interacting residues and provide a comparative structural analysis of the interactions formed at the interface of BLIP-I.TEM-1 versus those formed at the interface of BLIP.TEM-1. Along with initial attempts to functionally characterize BLP, we examine its amino acid residues that structurally correspond to BLIP/BLIP-I binding hotspots to explain its inability to bind and inhibit TEM-1. We conclude that the BLIP family fold is a robust and flexible scaffold that permits the formation of high-affinity protein-protein interactions while remaining highly selective. Comparison of the two naturally occurring, distinct binding interfaces built upon this scaffold (BLIP and BLIP-I) shows that there is substantial variation possible in the subnanomolar binding interaction with TEM-1. The corresponding (non-TEM-1-binding) BLP surface shows that numerous favorable backbone-backbone/backbone-side-chain interactions with a protein partner can be negated by the presence of a few, strongly unfavorable interactions, especially electrostatic repulsions.

Large-scale recrystallization of the S-layer of Bacillus coagulans E38-66 at the air/water interface and on lipid films.

PubMed Central

Pum, D; Weinhandl, M; Hödl, C; Sleytr, U B

1993-01-01

S-layer protein isolated from Bacillus coagulans E38-66 could be recrystallized into large-scale coherent monolayers at an air/water interface and on phospholipid films spread on a Langmuir-Blodgett trough. Because of the asymmetry in the physiochemical surface properties of the S-layer protein, the subunits were associated with their more hydrophobic outer face with the air/water interface and oriented with their negatively charged inner face to the zwitterionic head groups of the dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine (DPPE) monolayer films. The dynamic crystal growth at both types of interfaces was first initiated at several distant nucleation points. The individual monocrystalline areas grew isotropically in all directions until the front edge of neighboring crystals was met. The recrystallized S-layer protein and the S-layer-DPPE layer could be chemically cross-linked from the subphase with glutaraldehyde. Images PMID:8478338

SIMAP—the database of all-against-all protein sequence similarities and annotations with new interfaces and increased coverage

PubMed Central

Arnold, Roland; Goldenberg, Florian; Mewes, Hans-Werner; Rattei, Thomas

2014-01-01

The Similarity Matrix of Proteins (SIMAP, http://mips.gsf.de/simap/) database has been designed to massively accelerate computationally expensive protein sequence analysis tasks in bioinformatics. It provides pre-calculated sequence similarities interconnecting the entire known protein sequence universe, complemented by pre-calculated protein features and domains, similarity clusters and functional annotations. SIMAP covers all major public protein databases as well as many consistently re-annotated metagenomes from different repositories. As of September 2013, SIMAP contains >163 million proteins corresponding to ∼70 million non-redundant sequences. SIMAP uses the sensitive FASTA search heuristics, the Smith–Waterman alignment algorithm, the InterPro database of protein domain models and the BLAST2GO functional annotation algorithm. SIMAP assists biologists by facilitating the interactive exploration of the protein sequence universe. Web-Service and DAS interfaces allow connecting SIMAP with any other bioinformatic tool and resource. All-against-all protein sequence similarity matrices of project-specific protein collections are generated on request. Recent improvements allow SIMAP to cover the rapidly growing sequenced protein sequence universe. New Web-Service interfaces enhance the connectivity of SIMAP. Novel tools for interactive extraction of protein similarity networks have been added. Open access to SIMAP is provided through the web portal; the portal also contains instructions and links for software access and flat file downloads. PMID:24165881

Engineered Bi-Histidine Metal Chelation Sites Map the Structure of the Mechanical Unfolding Transition State of an Elastomeric Protein Domain GB1

PubMed Central

Shen, Tao; Cao, Yi; Zhuang, Shulin; Li, Hongbin

2012-01-01

Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional ψ-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The ϕM2+U-value is defined as ΔΔG‡-N/ΔΔGU-N, which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (ΔG‡-N) relative to its thermodynamic stability (ΔGU-N) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify ϕM2+U-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1’s mechanical unfolding transition state, the interface between force-bearing β-strands 1 and 4 is largely disrupted, and the first β-hairpin is partially disordered while the second β-hairpin and the α-helix remain structured. Our results demonstrate the unique application of ψ-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins. PMID:22947942

Engineered bi-histidine metal chelation sites map the structure of the mechanical unfolding transition state of an elastomeric protein domain GB1.

PubMed

Shen, Tao; Cao, Yi; Zhuang, Shulin; Li, Hongbin

2012-08-22

Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional ψ-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The φ(M2+)(U)-value is defined as ΔΔG(‡-N)/ΔΔG(U-N), which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (ΔG(‡-N)) relative to its thermodynamic stability (ΔG(U-N)) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify φ(M2+)(U)-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1's mechanical unfolding transition state, the interface between force-bearing β-strands 1 and 4 is largely disrupted, and the first β-hairpin is partially disordered while the second β-hairpin and the α-helix remain structured. Our results demonstrate the unique application of ψ-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

New methods for the analysis of the protein-solvent interface

NASA Astrophysics Data System (ADS)

Goodfellow, Julia M.; Pitt, William R.; Smart, Oliver S.; Williams, Mark A.

1995-09-01

The protein-solvent interface is complex and may include solvent channels and cavities as well as the normal surface water molecules. We describe several algorithms for investigating the intra- and inter-molecular interactions of proteins in general but with the aim of developing methods to accurately and definitively characterise the interactions of water and other small ligands with proteins. Specifically, we present the methods which underlie three programs (AQUARIUS2, HOLE and PRO_ACT) which can be used to to look at different aspects of these interactions.

SPLINTS: small-molecule protein ligand interface stabilizers.

PubMed

Fischer, Eric S; Park, Eunyoung; Eck, Michael J; Thomä, Nicolas H

2016-04-01

Regulatory protein-protein interactions are ubiquitous in biology, and small molecule protein-protein interaction inhibitors are an important focus in drug discovery. Remarkably little attention has been given to the opposite strategy-stabilization of protein-protein interactions, despite the fact that several well-known therapeutics act through this mechanism. From a structural perspective, we consider representative examples of small molecules that induce or stabilize the association of protein domains to inhibit, or alter, signaling for nuclear hormone, GTPase, kinase, phosphatase, and ubiquitin ligase pathways. These SPLINTS (small-molecule protein ligand interface stabilizers) drive interactions that are in some cases physiologically relevant, and in others entirely adventitious. The diverse structural mechanisms employed suggest approaches for a broader and systematic search for such compounds in drug discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Do Clustering Monoclonal Antibody Solutions Really Have a Concentration Dependence of Viscosity?

PubMed Central

Pathak, Jai A.; Sologuren, Rumi R.; Narwal, Rojaramani

2013-01-01

Protein solution rheology data in the biophysics literature have incompletely identified factors that govern hydrodynamics. Whereas spontaneous protein adsorption at the air/water (A/W) interface increases the apparent viscosity of surfactant-free globular protein solutions, it is demonstrated here that irreversible clusters also increase system viscosity in the zero shear limit. Solution rheology measured with double gap geometry in a stress-controlled rheometer on a surfactant-free Immunoglobulin solution demonstrated that both irreversible clusters and the A/W interface increased the apparent low shear rate viscosity. Interfacial shear rheology data showed that the A/W interface yields, i.e., shows solid-like behavior. The A/W interface contribution was smaller, yet nonnegligible, in double gap compared to cone-plate geometry. Apparent nonmonotonic composition dependence of viscosity at low shear rates due to irreversible (nonequilibrium) clusters was resolved by filtration to recover a monotonically increasing viscosity-concentration curve, as expected. Although smaller equilibrium clusters also existed, their size and effective volume fraction were unaffected by filtration, rendering their contribution to viscosity invariant. Surfactant-free antibody systems containing clusters have complex hydrodynamic response, reflecting distinct bulk and interface-adsorbed protein as well as irreversible cluster contributions. Literature models for solution viscosity lack the appropriate physics to describe the bulk shear viscosity of unstable surfactant-free antibody solutions. PMID:23442970

Ion-Specific Induced Fluctuations and Free Energetics of Aqueous Protein Hydrophobic Interfaces: Toward Connecting to Specific-Ion Behaviors at Aqueous Liquid–Vapor Interfaces

PubMed Central

2015-01-01

We explore anion-induced interface fluctuations near protein–water interfaces using coarse-grained representations of interfaces as proposed by Willard and Chandler (J. Phys. Chem. B2010, 114, 1954−195820055377). We use umbrella sampling molecular dynamics to compute potentials of mean force along a reaction coordinate bridging the state where the anion is fully solvated and one where it is biased via harmonic restraints to remain at the protein–water interface. Specifically, we focus on fluctuations of an interface between water and a hydrophobic region of hydrophobin-II (HFBII), a 71 amino acid residue protein expressed by filamentous fungi and known for its ability to form hydrophobically mediated self-assemblies at interfaces such as a water/air interface. We consider the anions chloride and iodide that have been shown previously by simulations as displaying specific-ion behaviors at aqueous liquid–vapor interfaces. We find that as in the case of a pure liquid–vapor interface, at the hydrophobic protein–water interface, the larger, less charge-dense iodide anion displays a marginal interfacial stability compared with that of the smaller, more charge-dense chloride anion. Furthermore, consistent with the results at aqueous liquid–vapor interfaces, we find that iodide induces larger fluctuations of the protein–water interface than chloride. PMID:24701961

DOCKSCORE: a webserver for ranking protein-protein docked poses.

PubMed

Malhotra, Sony; Mathew, Oommen K; Sowdhamini, Ramanathan

2015-04-24

Proteins interact with a variety of other molecules such as nucleic acids, small molecules and other proteins inside the cell. Structure-determination of protein-protein complexes is challenging due to several reasons such as the large molecular weights of these macromolecular complexes, their dynamic nature, difficulty in purification and sample preparation. Computational docking permits an early understanding of the feasibility and mode of protein-protein interactions. However, docking algorithms propose a number of solutions and it is a challenging task to select the native or near native pose(s) from this pool. DockScore is an objective scoring scheme that can be used to rank protein-protein docked poses. It considers several interface parameters, namely, surface area, evolutionary conservation, hydrophobicity, short contacts and spatial clustering at the interface for scoring. We have implemented DockScore in form of a webserver for its use by the scientific community. DockScore webserver can be employed, subsequent to docking, to perform scoring of the docked solutions, starting from multiple poses as inputs. The results, on scores and ranks for all the poses, can be downloaded as a csv file and graphical view of the interface of best ranking poses is possible. The webserver for DockScore is made freely available for the scientific community at: http://caps.ncbs.res.in/dockscore/ .

Operation of Kelvin Effect in the Activities of an Antifreeze Protein: A Molecular Dynamics Simulation Study.

PubMed

Midya, Uday Sankar; Bandyopadhyay, Sanjoy

2018-03-29

Ice growth and melting inhibition activities of antifreeze proteins (AFPs) are better explained by the adsorption-inhibition mechanism. Inhibition occurs as a result of the Kelvin effect induced by adsorbed protein molecules onto the surface of seed ice crystal. However, the Kelvin effect has not been explored by the state-of-the-art experimental techniques. In this work, atomistic molecular dynamics simulations have been carried out with Tenebrio molitor antifreeze protein ( TmAFP) placed at ice-water interface to probe the Kelvin effect in the mechanism of AFPs. Simulations show that, below equilibrium melting temperature, ice growth is inhibited through the convex ice-water interface formation toward the water phase and, above equilibrium melting temperature, ice melting is inhibited through the concave ice-water interface formation inward to ice phase. Simulations further reveal that the radius of curvature of the interface formed to stop the ice growth increases with decrease in the degree of supercooling. Our results are in qualitative agreement with the theoretical prediction of the Kelvin effect and thus reveal its operation in the activities of AFPs.

An automated decision-tree approach to predicting protein interaction hot spots.

PubMed

Darnell, Steven J; Page, David; Mitchell, Julie C

2007-09-01

Protein-protein interactions can be altered by mutating one or more "hot spots," the subset of residues that account for most of the interface's binding free energy. The identification of hot spots requires a significant experimental effort, highlighting the practical value of hot spot predictions. We present two knowledge-based models that improve the ability to predict hot spots: K-FADE uses shape specificity features calculated by the Fast Atomic Density Evaluation (FADE) program, and K-CON uses biochemical contact features. The combined K-FADE/CON (KFC) model displays better overall predictive accuracy than computational alanine scanning (Robetta-Ala). In addition, because these methods predict different subsets of known hot spots, a large and significant increase in accuracy is achieved by combining KFC and Robetta-Ala. The KFC analysis is applied to the calmodulin (CaM)/smooth muscle myosin light chain kinase (smMLCK) interface, and to the bone morphogenetic protein-2 (BMP-2)/BMP receptor-type I (BMPR-IA) interface. The results indicate a strong correlation between KFC hot spot predictions and mutations that significantly reduce the binding affinity of the interface. 2007 Wiley-Liss, Inc.

Two-dimensional capillary electrophoresis: capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection

PubMed Central

Dickerson, Jane A.; Ramsay, Lauren M.; Dada, Oluwatosin O.; Cermak, Nathan

2011-01-01

Capillary isoelectric focusing and capillary zone electrophoresis are coupled with laser-induced fluorescence detection to create an ultrasensitive two-dimensional separation method for proteins. In this method, two capillaries are joined through a buffer filled interface. Separate power supplies control the potential at the injection end of the first capillary and at the interface; the detector is held at ground potential. Proteins are labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The labeled proteins were mixed with ampholytes and injected into the first dimension capillary. A focusing step was performed with the injection end of the capillary at high pH and the interface at low pH. To mobilize components, the interface was filled with a high pH buffer, which was compatible with the second dimension separation. A fraction was transferred to the second dimension capillary for separation. The process of fraction transfer and second dimension separation was repeated two dozen times. The separation produced a spot capacity of 125. PMID:20603830

Role of anisotropy in determining stability of electrodeposition at solid-solid interfaces

NASA Astrophysics Data System (ADS)

Ahmad, Zeeshan; Viswanathan, Venkatasubramanian

2017-10-01

We investigate the stability of electrodeposition at solid-solid interfaces for materials exhibiting an anisotropic mechanical response. The stability of electrodeposition or resistance to the formation of dendrites is studied within a linear stability analysis. The deformation and stress equations are solved using the Stroh formalism and faithfully recover the boundary conditions at the interface. The stability parameter is used to quantify the stability of different solid-solid interfaces incorporating the full anisotropy of the elastic tensor of the two materials. Results show a high degree of variability in the stability parameter depending on the crystallographic orientation of the solids in contact, and point to opportunities for exploiting this effect in developing Li metal anodes.

A capillary electrophoresis-tandem mass spectrometry methodology for the determination of non-protein amino acids in vegetable oils as novel markers for the detection of adulterations in olive oils.

PubMed

Sánchez-Hernández, Laura; Marina, Maria Luisa; Crego, Antonio L

2011-07-29

A new analytical methodology based on capillary electrophoresis-mass spectrometry (CE-MS(2)) is presented in this work, enabling the identification and determination of six non-protein amino acids (ornithine, β-alanine, GABA, alloisoleucine, citrulline and pyroglutamic acid) in vegetable oils. This methodology is based on a previous derivatization with butanol and subsequent separation using acidic conditions followed by on-line coupling to an ion trap analyzer for MS(2) detection established through an electrospray-coaxial sheath flow interface. The electrophoretic and interface parameters were optimized obtaining the separation of all compounds in less than 15 min and with resolutions higher than 5. The proposed method was validated by assessing its accuracy, precision (RSD<7% for corrected peak areas), LODs and LOQs (between 0.04-0.19 ng/g and 0.06-0.31 ng/g, respectively) and linearity range (R(2)>0.99), and it was used in order to identify the selected non-protein amino acids in soybean oils, sunflower oils, corn oils and extra virgin olive oils. MS(2) experiments performed the fingerprint fragmentation of these compounds allowing to corroborate ornithine and alloisoleucine in seed oils but not in olive oils. The method was applied to identify and quantify olive oil adulterations with soybean oil detecting in a single run the amino acids in mixtures up to 2% (w/w). The results showed a high potential in using these compounds as novel markers for the detection of adulterations of extra virgin olive oils with seed oils. Thus, the developed method could be considered a simple, rapid and reliable method for the quality evaluation of extra virgin olive oil permitting its authentication. Copyright © 2011 Elsevier B.V. All rights reserved.

The influence of size, structure and hydrophilicity of model surfactants on the adsorption of lysozyme to oil-water interface--interfacial shear measurements.

PubMed

Baldursdottir, Stefania G; Jorgensen, Lene

2011-10-01

The flexibility and aggregation of proteins can cause adsorption to oil-water interfaces and thereby create challenges during formulation and processing. Protein adsorption is a complex process and the presence of surfactants further complicates the system, in which additional parameters need to be considered. The purpose of this study is to scrutinize the influence of surfactants on protein adsorption to interfaces, using lysozyme as a model protein and sorbitan monooleate 80 (S80), polysorbate 80 (T80), polyethylene-block-poly(ethylene glycol) (PE-PEG) and polyglycerol polyricinoleate (PG-PR) as model surfactants. Rheological properties, measured using a TA AR-G2 rheometer equipped with a double wall ring (DWR) geometry, were used to compare the efficacy of the surfactant in hindering lysozyme adsorption. The system consists of a ring and a Delrin® trough with a circular channel (interfacial area=1882.6 mm(2)). Oscillatory shear measurements were conducted at a constant frequency of 0.1 Hz, a temperature of 25°C, and with strain set to 1%. The adsorption of lysozyme to the oil-water interface results in the formation of a viscoelastic film. This can be prevented by addition of surfactants, in a manner depending on the concentration and the type of surfactant. The more hydrophilic surfactants are more effective in hindering lysozyme adsorption to oil-water interfaces. Additionally, the larger surfactants are more persistent in preventing film formation, whereas the smaller ones eventually give space for the lysozyme on the interface. The addition of a mixture of two different surfactants was only beneficial when the two hydrophilic surfactants were mixed, in which case a delay in the multilayer formation was detected. The method is able to detect the interfacial adsorption of lysozyme and thus the hindering of film formation by model surfactants. It can therefore aid in processing of any delivery systems for proteins in which the protein is introduced to oil-water interfaces. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

PubMed Central

Szent-Gyorgyi, Chris; Stanfield, Robyn L.; Andreko, Susan; Dempsey, Alison; Ahmed, Mushtaq; Capek, Sara; Waggoner, Alan; Wilson, Ian A.; Bruchez, Marcel P.

2013-01-01

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents. PMID:23978698

Imaging the molecular dimensions and oligomerization of protein molecules at the solid-liquid interface by surface oriented molecular sizing (SOMS) microscopy

NASA Astrophysics Data System (ADS)

Waner, Mark Joseph

The structure and behavior of proteins at the solid/liquid interface is of great scientific interest. It has application both to fundamental biochemical understanding, as well as to biotechnological purposes. Interfaces play a critical role in many physiological processes. The mechanism of protein adsorption to surfaces is not very well understood. The current model put forth in much of the literature assumes a two step model. In the first step of this model the protein collides with the surface and adsorbs if its energy is sufficient to overcome the free energy of desorption of surface adsorbed solvent. The second step is often assumed to involve significant conformational change of the secondary and tertiary structure of the protein or enzyme, akin to denaturation. This unfolding of the protein would tend to indicate that loss of function would occur concomitantly, but studies have found very little loss in activity upon adsorption for a number of different protein systems. The recent development of the atomic force microscope (AFM) offers another tool for the examination of protein structure at liquid/solid interfaces. For atomically flat crystals the AFM has been used to determine atomic positions to <1 A resolution. In the case of samples with topographic features larger than atoms, the probe tip of the AFM 'convolutes' with the size and shape of surface features. This has hindered the use of AFM for molecular level structural determination of proteins at the liquid/solid interface. The work presented in this dissertation covers the development of the surface oriented molecular sizing (SOMS) technique which makes use of the angstrom height resolution of the AFM and a physically based mathematical framework for the analysis of the height distribution of adsorbed protein molecules. The surface adsorption and orientation (SAO) model is developed using statistical thermodynamics to model the expected height distributions for molecules adsorbed on a surface. The SOMS technique will be shown to be viable through studies of ferritin and concanavalin A (Con A) at the water/mica interface. Using this technique we are able to determine both the three-dimensional size and the oligomerization state of the adsorbed molecules. This technique will then be utilized for the examination of denaturation of Con A at the interface, by a number of mechanisms. Further, the structural and orientational changes in Con A as a function of pH will also be presented. The final chapter of this dissertation will present an extension of these studies to the deposition and structure of Con A thin films on mica.

Charged groups at binding interfaces of the PsbO subunit of photosystem II: A combined bioinformatics and simulation study.

PubMed

Del Val, Coral; Bondar, Ana-Nicoleta

2017-06-01

PsbO is an extrinsic subunit of photosystem II engaged in complex binding interactions within photosystem II. At the interface between PsbO, D1 and D2 subunits of photosystem II, a cluster of charged and polar groups of PsbO is part of an extended hydrogen-bond network thought to participate in proton transfer. The precise role of specific amino acid residues at this complex binding interface remains a key open question. Here, we address this question by carrying out extensive bioinformatics analyses and molecular dynamics simulations of PsbO proteins with mutations at the binding interface. We find that PsbO proteins from cyanobacteria vs. plants have specific preferences for the number and composition of charged amino acid residues that may ensure that PsbO proteins avoid aggregation and expose long unstructured loops for binding to photosystem II. A cluster of conserved charged groups with dynamic hydrogen bonds provides PsbO with structural plasticity at the binding interface with photosystem II. Copyright © 2017. Published by Elsevier B.V.

A Thermodynamic Description of the Adsorption of Simple Water-Soluble Peptoids to Silica.

PubMed

Calkins, Anna L; Yin, Jennifer; Rangel, Jacenda L; Landry, Madeleine R; Fuller, Amelia A; Stokes, Grace Y

2016-11-08

The first report of a water-soluble peptoid adsorbed to silica monitored by second harmonic generation (SHG) at the liquid/solid interface is presented here. The molecular insights gained from these studies will inform the design and preparation of novel peptoid coatings. Simple 6- and 15-residue peptoids were dissolved in phosphate buffered saline and adsorbed to bare silica surfaces. Equilibrium binding constants and relative surface concentrations of adsorbed peptoids were determined from fits to the Langmuir model. Complementary fluorescence spectroscopy studies were used to quantify the maximum surface excess. Binding constants, determined here by SHG, were comparable to those previously reported for cationic proteins and small molecules. Enthalpies and free energies of adsorption were determined to elucidate thermodynamic driving forces. Circular dichroism spectra confirm that minimal conformational changes occur when peptoids are adsorbed to silica while pH studies indicate that electrostatic interactions impact adsorption.

Binding of small molecules at interface of protein-protein complex - A newer approach to rational drug design.

PubMed

Gurung, A B; Bhattacharjee, A; Ajmal Ali, M; Al-Hemaid, F; Lee, Joongku

2017-02-01

Protein-protein interaction is a vital process which drives many important physiological processes in the cell and has also been implicated in several diseases. Though the protein-protein interaction network is quite complex but understanding its interacting partners using both in silico as well as molecular biology techniques can provide better insights for targeting such interactions. Targeting protein-protein interaction with small molecules is a challenging task because of druggability issues. Nevertheless, several studies on the kinetics as well as thermodynamic properties of protein-protein interactions have immensely contributed toward better understanding of the affinity of these complexes. But, more recent studies on hot spots and interface residues have opened up new avenues in the drug discovery process. This approach has been used in the design of hot spot based modulators targeting protein-protein interaction with the objective of normalizing such interactions.

pH-dependence of single-protein adsorption and diffusion at a liquid chromatographic interface.

PubMed

Kisley, Lydia; Poongavanam, Mohan-Vivekanandan; Kourentzi, Katerina; Willson, Richard C; Landes, Christy F

2016-02-01

pH is a common mobile phase variable used to control protein separations due to the tunable nature of amino acid and adsorbent charge. Like other column variables such as column density and ligand loading density, pH is usually optimized empirically. Single-molecule spectroscopy extracts molecular-scale data to provide a framework for mechanistic optimization of pH. The adsorption and diffusion of a model globular protein, α-lactalbumin, was studied by single-molecule microscopy at a silica-aqueous interface analogous to aqueous normal phase and hydrophilic interaction chromatography and capillary electrophoresis interfaces at varied pH. Electrostatic repulsion resulting in free diffusion was observed at pH above the isoelectric point of the protein. In contrast, at low pH strong adsorption and surface diffusion with either no (D ∼ 0.01 μm(2) /s) or translational (D ∼ 0.3 μm(2) /s) motion was observed where the protein likely interacted with the surface through electrostatic, hydrophobic, and hydrogen bonding forces. The fraction of proteins immobilized could be increased by lowering the pH. These results show that retention of proteins at the silica interface cannot be viewed solely as an adsorption/desorption process and that the type of surface diffusion, which ultimately leads to ensemble chromatographic separations, can be controlled by tuning long-range electrostatic and short-range hydrophobic and hydrogen bonding forces with pH. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A discriminatory function for prediction of protein-DNA interactions based on alpha shape modeling.

PubMed

Zhou, Weiqiang; Yan, Hong

2010-10-15

Protein-DNA interaction has significant importance in many biological processes. However, the underlying principle of the molecular recognition process is still largely unknown. As more high-resolution 3D structures of protein-DNA complex are becoming available, the surface characteristics of the complex become an important research topic. In our work, we apply an alpha shape model to represent the surface structure of the protein-DNA complex and developed an interface-atom curvature-dependent conditional probability discriminatory function for the prediction of protein-DNA interaction. The interface-atom curvature-dependent formalism captures atomic interaction details better than the atomic distance-based method. The proposed method provides good performance in discriminating the native structures from the docking decoy sets, and outperforms the distance-dependent formalism in terms of the z-score. Computer experiment results show that the curvature-dependent formalism with the optimal parameters can achieve a native z-score of -8.17 in discriminating the native structure from the highest surface-complementarity scored decoy set and a native z-score of -7.38 in discriminating the native structure from the lowest RMSD decoy set. The interface-atom curvature-dependent formalism can also be used to predict apo version of DNA-binding proteins. These results suggest that the interface-atom curvature-dependent formalism has a good prediction capability for protein-DNA interactions. The code and data sets are available for download on http://www.hy8.com/bioinformatics.htm kenandzhou@hotmail.com.

Relating protein conformational changes to packing efficiency and disorder

PubMed Central

Bhardwaj, Nitin; Gerstein, Mark

2009-01-01

Changes in protein conformation play key roles in facilitating various biochemical processes, ranging from signaling and phosphorylation to transport and catalysis. While various factors that drive these motions such as environmental changes and binding of small molecules are well understood, specific causative effects on the structural features of the protein due to these conformational changes have not been studied on a large scale. Here, we study protein conformational changes in relation to two key structural metrics: packing efficiency and disorder. Packing has been shown to be crucial for protein stability and function by many protein design and engineering studies. We study changes in packing efficiency during conformational changes, thus extending the analysis from a static context to a dynamic perspective and report some interesting observations. First, we study various proteins that adopt alternate conformations and find that tendencies to show motion and change in packing efficiency are correlated: residues that change their packing efficiency show larger motions. Second, our results suggest that residues that show higher changes in packing during motion are located on the changing interfaces which are formed during these conformational changes. These changing interfaces are slightly different from shear or static interfaces that have been analyzed in previous studies. Third, analysis of packing efficiency changes in the context of secondary structure shows that, as expected, residues buried in helices show the least change in packing efficiency, whereas those embedded in bends are most likely to change packing. Finally, by relating protein disorder to motions, we show that marginally disordered residues which are ordered enough to be crystallized but have sequence patterns indicative of disorder show higher dislocation and a higher change in packing than ordered ones and are located mostly on the changing interfaces. Overall, our results demonstrate that between the two conformations, the cores of the proteins remain mostly intact, whereas the interfaces display the most elasticity, both in terms of disorder and change in packing efficiency. By doing a variety of tests, we also show that our observations are robust to the solvation state of the proteins. PMID:19472340

Breakdown of the Debye polarization ansatz at protein-water interfaces

NASA Astrophysics Data System (ADS)

Fernández Stigliano, Ariel

2013-06-01

The topographical and physico-chemical complexity of protein-water interfaces scales down to the sub-nanoscale range. At this level of confinement, we demonstrate that the dielectric structure of interfacial water entails a breakdown of the Debye ansatz that postulates the alignment of polarization with the protein electrostatic field. The tendencies to promote anomalous polarization are determined for each residue type and a particular kind of structural defect is shown to provide the predominant causal context.

Local functional descriptors for surface comparison based binding prediction

PubMed Central

2012-01-01

Background Molecular recognition in proteins occurs due to appropriate arrangements of physical, chemical, and geometric properties of an atomic surface. Similar surface regions should create similar binding interfaces. Effective methods for comparing surface regions can be used in identifying similar regions, and to predict interactions without regard to the underlying structural scaffold that creates the surface. Results We present a new descriptor for protein functional surfaces and algorithms for using these descriptors to compare protein surface regions to identify ligand binding interfaces. Our approach uses descriptors of local regions of the surface, and assembles collections of matches to compare larger regions. Our approach uses a variety of physical, chemical, and geometric properties, adaptively weighting these properties as appropriate for different regions of the interface. Our approach builds a classifier based on a training corpus of examples of binding sites of the target ligand. The constructed classifiers can be applied to a query protein providing a probability for each position on the protein that the position is part of a binding interface. We demonstrate the effectiveness of the approach on a number of benchmarks, demonstrating performance that is comparable to the state-of-the-art, with an approach with more generality than these prior methods. Conclusions Local functional descriptors offer a new method for protein surface comparison that is sufficiently flexible to serve in a variety of applications. PMID:23176080

Molecular interactions within the halophilic, thermophilic, and mesophilic prokaryotic ribosomal complexes: clues to environmental adaptation.

PubMed

Mallik, Saurav; Kundu, Sudip

2015-01-01

Using the available crystal structures of 50S ribosomal subunits from three prokaryotic species: Escherichia coli (mesophilic), Thermus thermophilus (thermophilic), and Haloarcula marismortui (halophilic), we have analyzed different structural features of ribosomal RNAs (rRNAs), proteins, and of their interfaces. We have correlated these structural features with the environmental adaptation strategies of the corresponding species. While dense intra-rRNA packing is observed in thermophilic, loose intra-rRNA packing is observed in halophilic (both compared to mesophilic). Interestingly, protein-rRNA interfaces of both the extremophiles are densely packed compared to that of the mesophilic. The intersubunit bridge regions are almost devoid of cavities, probably ensuring the proper formation of each bridge (by not allowing any loosely packed region nearby). During rRNA binding, the ribosomal proteins experience some structural transitions. Here, we have analyzed the intrinsically disordered and ordered regions of the ribosomal proteins, which are subjected to such transitions. The intrinsically disordered and disorder-to-order transition sites of the thermophilic and mesophilic ribosomal proteins are simultaneously (i) highly conserved and (ii) slowly evolving compared to rest of the protein structure. Although high conservation is observed at such sites of halophilic ribosomal proteins, but slow rate of evolution is absent. Such differences between thermophilic, mesophilic, and halophilic can be explained from their environmental adaptation strategy. Interestingly, a universal biophysical principle evident by a linear relationship between the free energy of interface formation, interface area, and structural changes of r-proteins during assembly is always maintained, irrespective of the environmental conditions.

Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions.

PubMed

Wachsmuth, Leah M; Johnson, Meredith G; Gavenonis, Jason

2017-06-01

Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface*

PubMed Central

Chin, Stephanie; Yang, Donghe; Miles, Andrew J.; Eckford, Paul D. W.; Molinski, Steven; Wallace, B. A.; Bear, Christine E.

2017-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation. PMID:28003367

Modeling Ionization Events iduced by Protein Protein Binding

NASA Astrophysics Data System (ADS)

Mitra, Rooplekha; Shyam, Radhey; Alexov, Emil

2009-11-01

The association of two or more biological macromolecules dramatically change the environment of the amino acids situated at binding interface and could change ionization states of titratable groups. The change of ionization due to the binding results in proton uptake/release and causes pH-dependence of the binding free energy. We apply computational method, as implemented in Multi Conformation Continuum Electrostatics (MCCE) algorithm, to study protonation evens on a large set of protein-protein complexes. Our results indicate that proton uptake/release is a common phenomena in protein binding since in vast majority of the cases (70%) the binding caused at least 0.5 units proton change. The proton uptake/release was further investigated with respect to interfacial area and charges of the monomers and it was found that macroscopic characteristics are not important determinants. Instead, charge complementarity across the interface and the number of unpaired ionizable groups at the interface are the primary source of proton uptake/release.

Computational Design of Self-Assembling Cyclic Protein Homo-oligomers

PubMed Central

Fallas, Jorge A.; Ueda, George; Sheffler, William; Nguyen, Vanessa; McNamara, Dan E.; Sankaran, Banumathi; Pereira, Jose Henrique; Parmeggiani, Fabio; Brunette, TJ; Cascio, Duilio; Yeates, Todd R.; Zwart, Peter; Baker, David

2016-01-01

Self-assembling cyclic protein homo-oligomers play important roles in biology and the ability to generate custom homo-oligomeric structures could enable new approaches to probe biological function. Here we report a general approach to design cyclic homo-oligomers that employs a new residue pair transform method for assessing the design ability of a protein-protein interface. This method is sufficiently rapid to enable systematic enumeration of cyclically docked arrangements of a monomer followed by sequence design of the newly formed interfaces. We use this method to design interfaces onto idealized repeat proteins that direct their assembly into complexes that possess cyclic symmetry. Of 96 designs that were experimentally characterized, 21 were found to form stable monodisperse homo-oligomers in solution, and 15 (4 homodimers, 6 homotrimers, 6 homotetramers and 1 homopentamer) had solution small angle X-ray scattering data consistent with the design models. X-ray crystal structures were obtained for five of the designs and each of these were shown to be very close to their design model. PMID:28338692

S-Layer Protein-Based Biosensors.

PubMed

Schuster, Bernhard

2018-04-11

The present paper highlights the application of bacterial surface (S-) layer proteins as versatile components for the fabrication of biosensors. One technologically relevant feature of S-layer proteins is their ability to self-assemble on many surfaces and interfaces to form a crystalline two-dimensional (2D) protein lattice. The S-layer lattice on the surface of a biosensor becomes part of the interface architecture linking the bioreceptor to the transducer interface, which may cause signal amplification. The S-layer lattice as ultrathin, highly porous structure with functional groups in a well-defined special distribution and orientation and an overall anti-fouling characteristics can significantly raise the limit in terms of variety and the ease of bioreceptor immobilization, compactness of bioreceptor molecule arrangement, sensitivity, specificity, and detection limit for many types of biosensors. The present paper discusses and summarizes examples for the successful implementation of S-layer lattices on biosensor surfaces in order to give a comprehensive overview on the application potential of these bioinspired S-layer protein-based biosensors.

Probing alpha-helical and beta-sheet structures of peptides at solid/liquid interfaces with SFG.

PubMed

Chen, Xiaoyun; Wang, Jie; Sniadecki, Jason J; Even, Mark A; Chen, Zhan

2005-03-29

We demonstrated that sum frequency generation (SFG) vibrational spectroscopy can distinguish different secondary structures of proteins or peptides adsorbed at solid/liquid interfaces. The SFG spectrum for tachyplesin I at the polystyrene (PS)/solution interface has a fingerprint peak corresponding to the B1/B3 mode of the antiparallel beta-sheet. This peak disappeared upon the addition of dithiothreitol, which can disrupt the beta-sheet structure. The SFG spectrum indicative of the MSI594 alpha-helical structure was observed at the PS/MSI594 solution interface. This research validates SFG as a powerful technique for revealing detailed secondary structures of interfacial proteins and peptides.

Acid-base chemistry of frustrated water at protein interfaces.

PubMed

Fernández, Ariel

2016-01-01

Water molecules at a protein interface are often frustrated in hydrogen-bonding opportunities due to subnanoscale confinement. As shown, this condition makes them behave as a general base that may titrate side-chain ammonium and guanidinium cations. Frustration-based chemistry is captured by a quantum mechanical treatment of proton transference and shown to remove same-charge uncompensated anticontacts at the interface found in the crystallographic record and in other spectroscopic information on the aqueous interface. Such observations are untenable within classical arguments, as hydronium is a stronger acid than ammonium or guanidinium. Frustration enables a directed Grotthuss mechanism for proton transference stabilizing same-charge anticontacts. © 2015 Federation of European Biochemical Societies.

Growth inhibition at the ice prismatic plane induced by a spruce budworm antifreeze protein: a molecular dynamics simulation study.

PubMed

Nada, H; Furukawa, Y

2011-11-28

A molecular dynamics simulation was conducted to investigate the growth kinetics at the ice prismatic interface to which a spruce budworm antifreeze protein was bound. Two initial binding conformations of the protein at the interface--one energetically stable and the other energetically unstable--were examined. For both binding conformations, the growth of ice was observed around the protein. A sharp decrease in the rate of ice growth was observed around the protein that initially had the energetically stable binding conformation. Simulation results suggest that the observed decrease in the ice growth rate was attributable to melting point depression caused by the Gibbs-Thomson effect. The protein that initially had the energetically unstable binding conformation markedly relaxed so as to stably bind to the prismatic plane interface of the grown ice; thereafter, a decrease in the ice growth rate was observed as well. However, the binding conformation that the protein approached during the relaxation was different from that of the protein that initially had the energetically stable binding conformation. Thus, the simulation indicates the existence of two binding conformations for inducing a decrease in the ice growth rate. The results are possibly related to the hyperactivity of a spruce budworm antifreeze protein in real systems.

Fragment-based protein-protein interaction antagonists of a viral dimeric protease

PubMed Central

Gable, Jonathan E.; Lee, Gregory M.; Acker, Timothy M.; Hulce, Kaitlin R.; Gonzalez, Eric R.; Schweigler, Patrick; Melkko, Samu; Farady, Christopher J.; Craik, Charles S.

2016-01-01

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose response determination was performed as a confirmation screen and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed via NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80% of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogs. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces. PMID:26822284

Interaction between dimer interface residues of native and mutated SOD1 protein: a theoretical study.

PubMed

Keerthana, S P; Kolandaivel, P

2015-04-01

Cu-Zn superoxide dismutase 1 (SOD1) is a highly conserved bimetallic protein enzyme, used for the scavenging the superoxide radicals (O2 (-)) produced due to aerobic metabolism in the mitochondrial respiratory chain. Over 100 mutations have been identified and found to be in the homodimeric structure of SOD1. The enzyme has to be maintained in its dimeric state for the structural stability and enzymatic activity. From our investigation, we found that the mutations apart from the dimer interface residues are found to affect the dimer stability of protein and hence enhancing the aggregation and misfolding tendency of mutated protein. The homodimeric state of SOD1 is found to be held together by the non-covalent interactions. The molecular dynamics simulation has been used to study the hydrogen bond interactions between the dimer interface residues of the monomers in native and mutated forms of SOD1 in apo- and holo-states. The results obtained by this analysis reveal the fact that the loss of hydrogen bond interactions between the monomers of the dimer is responsible for the reduced stability of the apo- and holo-mutant forms of SOD1. The conformers with dimer interface residues in native and mutated protein obtained by the molecular dynamics simulation is subjected to quantum mechanical study using M052X/6-31G(d) level of theory. The charge transfer between N-H···O interactions in the dimer interface residues were studied. The weak interaction between the monomers of the dimer accounts for the reduced dimerization and enhanced deformation energy in the mutated SOD1 protein.

Characterizing Electrolyte and Platinum Interface in PEM Fuel Cells Using CO Displacement

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

Garrick, Taylor R.; Moylan, Thomas E.; Yarlagadda, Venkata

Relatively large O 2 transport resistance at the ionomer and Pt interface has been thought to be responsible for the large performance loss at high power for a low Pt loading proton-exchange-membrane fuel cell. A facile method to characterize the interface in the fuel cell electrode is needed. In this study, the CO displacement method was explored on polycrystalline Pt and carbon-supported Pt nanoparticles. The displacement charge coverages were used to quantify the adsorption of perchlorate, sulfate, and perfluorosulfonic acid ionomer. The application of this method in a fuel cell electrode was demonstrated.

Characterizing Electrolyte and Platinum Interface in PEM Fuel Cells Using CO Displacement

DOE PAGES

Garrick, Taylor R.; Moylan, Thomas E.; Yarlagadda, Venkata; ...

2016-12-13

Relatively large O 2 transport resistance at the ionomer and Pt interface has been thought to be responsible for the large performance loss at high power for a low Pt loading proton-exchange-membrane fuel cell. A facile method to characterize the interface in the fuel cell electrode is needed. In this study, the CO displacement method was explored on polycrystalline Pt and carbon-supported Pt nanoparticles. The displacement charge coverages were used to quantify the adsorption of perchlorate, sulfate, and perfluorosulfonic acid ionomer. The application of this method in a fuel cell electrode was demonstrated.

Vitronectin as a Micromanager of Cell Response in Material-Driven Fibronectin Nanonetworks.

PubMed

Cantini, Marco; Gomide, Karina; Moulisova, Vladimira; González-García, Cristina; Salmerón-Sánchez, Manuel

2017-09-01

Surface functionalization strategies of synthetic materials for regenerative medicine applications comprise the development of microenvironments that recapitulate the physical and biochemical cues of physiological extracellular matrices. In this context, material-driven fibronectin (FN) nanonetworks obtained from the adsorption of the protein on poly(ethyl acrylate) provide a robust system to control cell behavior, particularly to enhance differentiation. This study aims at augmenting the complexity of these fibrillar matrices by introducing vitronectin, a lower-molecular-weight multifunctional glycoprotein and main adhesive component of serum. A cooperative effect during co-adsorption of the proteins is observed, as the addition of vitronectin leads to increased fibronectin adsorption, improved fibril formation, and enhanced vitronectin exposure. The mobility of the protein at the material interface increases, and this, in turn, facilitates the reorganization of the adsorbed FN by cells. Furthermore, the interplay between interface mobility and engagement of vitronectin receptors controls the level of cell fusion and the degree of cell differentiation. Ultimately, this work reveals that substrate-induced protein interfaces resulting from the cooperative adsorption of fibronectin and vitronectin fine-tune cell behavior, as vitronectin micromanages the local properties of the microenvironment and consequently short-term cell response to the protein interface and higher order cellular functions such as differentiation.

OpenVirtualToxLab--a platform for generating and exchanging in silico toxicity data.

PubMed

Vedani, Angelo; Dobler, Max; Hu, Zhenquan; Smieško, Martin

2015-01-22

The VirtualToxLab is an in silico technology for estimating the toxic potential--endocrine and metabolic disruption, some aspects of carcinogenicity and cardiotoxicity--of drugs, chemicals and natural products. The technology is based on an automated protocol that simulates and quantifies the binding of small molecules towards a series of currently 16 proteins, known or suspected to trigger adverse effects: 10 nuclear receptors (androgen, estrogen α, estrogen β, glucocorticoid, liver X, mineralocorticoid, peroxisome proliferator-activated receptor γ, progesterone, thyroid α, thyroid β), four members of the cytochrome P450 enzyme family (1A2, 2C9, 2D6, 3A4), a cytosolic transcription factor (aryl hydrocarbon receptor) and a potassium ion channel (hERG). The toxic potential of a compound--its ability to trigger adverse effects--is derived from its computed binding affinities toward these very proteins: the computationally demanding simulations are executed in client-server model on a Linux cluster of the University of Basel. The graphical-user interface supports all computer platforms, allows building and uploading molecular structures, inspecting and downloading the results and, most important, rationalizing any prediction at the atomic level by interactively analyzing the binding mode of a compound with its target protein(s) in real-time 3D. Access to the VirtualToxLab is available free of charge for universities, governmental agencies, regulatory bodies and non-profit organizations. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

A feature-based approach to modeling protein-protein interaction hot spots.

PubMed

Cho, Kyu-il; Kim, Dongsup; Lee, Doheon

2009-05-01

Identifying features that effectively represent the energetic contribution of an individual interface residue to the interactions between proteins remains problematic. Here, we present several new features and show that they are more effective than conventional features. By combining the proposed features with conventional features, we develop a predictive model for interaction hot spots. Initially, 54 multifaceted features, composed of different levels of information including structure, sequence and molecular interaction information, are quantified. Then, to identify the best subset of features for predicting hot spots, feature selection is performed using a decision tree. Based on the selected features, a predictive model for hot spots is created using support vector machine (SVM) and tested on an independent test set. Our model shows better overall predictive accuracy than previous methods such as the alanine scanning methods Robetta and FOLDEF, and the knowledge-based method KFC. Subsequent analysis yields several findings about hot spots. As expected, hot spots have a larger relative surface area burial and are more hydrophobic than other residues. Unexpectedly, however, residue conservation displays a rather complicated tendency depending on the types of protein complexes, indicating that this feature is not good for identifying hot spots. Of the selected features, the weighted atomic packing density, relative surface area burial and weighted hydrophobicity are the top 3, with the weighted atomic packing density proving to be the most effective feature for predicting hot spots. Notably, we find that hot spots are closely related to pi-related interactions, especially pi . . . pi interactions.

Adsorption kinetics of c-Fos and c-Jun to air-water interfaces.

PubMed

Del Boca, Maximiliano; Nobre, Thatyane Morimoto; Zaniquelli, Maria Elisabete Darbello; Maggio, Bruno; Borioli, Graciela A

2007-11-01

The kinetics of adsorption to air-water interfaces of the biomembrane active transcription factors c-Fos, c-Jun and their mixtures is investigated. The adsorption process shows three distinct stages: a lag time, a fast pseudo zero-order stage, and a halting stage. The initial stage determines the course of the process, which is concentration dependent until the end of the fast stage. We show that c-Fos has faster adsorption kinetics than c-Jun over all three stages and that the interaction between both proteins is apparent in the adsorption profiles of the mixtures. Protein molecular reorganization at the interface determines the transition to the final adsorption stage of the pure proteins as well as that of the mixtures.

Study of Fluorinated Quantum Dots-Protein Interactions at the Oil/Water Interface by Interfacial Surface Tension Changes.

PubMed

Carrillo-Carrión, Carolina; Gallego, Marta; Parak, Wolfgang J; Carril, Mónica

2018-05-08

Understanding the interaction of nanoparticles with proteins and how this interaction modifies the nanoparticles’ surface is crucial before their use for biomedical applications. Since fluorinated materials are emerging as potential imaging probes and delivery vehicles, their interaction with proteins of biological interest must be studied in order to be able to predict their performance in real scenarios. It is known that fluorinated planar surfaces may repel the unspecific adsorption of proteins but little is known regarding the same process on fluorinated nanoparticles due to the scarce examples in the literature. In this context, the aim of this work is to propose a simple and fast methodology to study fluorinated nanoparticle-protein interactions based on interfacial surface tension (IFT) measurements. This technique is particularly interesting for fluorinated nanoparticles due to their increased hydrophobicity. Our study is based on the determination of IFT variations due to the interaction of quantum dots of ca. 5 nm inorganic core/shell diameter coated with fluorinated ligands (QD_F) with several proteins at the oil/water interface. Based on the results, we conclude that the presence of QD_F do not disrupt protein spontaneous film formation at the oil/water interface. Even if at very low concentrations of proteins the film formation in the presence of QD_F shows a slower rate, the final interfacial tension reached is similar to that obtained in the absence of QD_F. The differential behaviour of the studied proteins (bovine serum albumin, fibrinogen and apotransferrin) has been discussed on the basis of the adsorption affinity of each protein towards DCM/water interface and their different sizes. Additionally, it has been clearly demonstrated that the proposed methodology can serve as a complementary technique to other reported direct and indirect methods for the evaluation of nanoparticle-protein interactions at low protein concentrations.

A view of the E2-CD81 interface at the binding site of a neutralizing antibody against hepatitis C virus.

PubMed

Harman, Christine; Zhong, Lilin; Ma, Li; Liu, Peter; Deng, Lu; Zhao, Zhong; Yan, Hailing; Struble, Evi; Virata-Theimer, Maria Luisa; Zhang, Pei

2015-01-01

Hepatitis C virus (HCV) glycoprotein E2 is considered a major target for generating neutralizing antibodies against HCV, primarily due to its role of engaging host entry factors, such as CD81, a key cell surface protein associated with HCV entry. Based on a series of biochemical analyses in combination with molecular docking, we present a description of a potential binding interface formed between the E2 protein and CD81. The virus side of this interface includes a hydrophobic helix motif comprised of residues W(437)LAGLF(442), which encompasses the binding site of a neutralizing monoclonal antibody, mAb41. The helical conformation of this motif provides a structural framework for the positioning of residues F442 and Y443, serving as contact points for the interaction with CD81. The cell side of this interface likewise involves a surface-exposed hydrophobic helix, namely, the D-helix of CD81, which coincides with the binding site of 1D6, a monoclonal anti-CD81 antibody known to block HCV entry. Our illustration of this virus-host interface suggests an important role played by the W(437)LAGLF(442) helix of the E2 protein in the hydrophobic interaction with the D-helix of CD81, thereby facilitating our understanding of the mechanism for antibody-mediated neutralization of HCV. Characterization of the interface established between a virus and host cells can provide important information that may be used for the control of virus infections. The interface that enables hepatitis C virus (HCV) to infect human liver cells has not been well understood because of the number of cell surface proteins, factors, and conditions found to be associated with the infection process. Based on a series of biochemical analyses in combination with molecular docking, we present such an interface, consisting of two hydrophobic helical structures, from the HCV E2 surface glycoprotein and the CD81 protein, a major host cell receptor recognized by all HCV strains. Our study reveals the critical role played by hydrophobic interactions in the formation of this virus-host interface, thereby contributing to our understanding of the mechanism for antibody-mediated neutralization of HCV. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Effect of mutation at the interface of Trp-repressor dimeric protein: a steered molecular dynamics simulation.

PubMed

Miño, German; Baez, Mauricio; Gutierrez, Gonzalo

2013-09-01

The strength of key interfacial contacts that stabilize protein-protein interactions have been studied by computer simulation. Experimentally, changes in the interface are evaluated by generating specific mutations at one or more points of the protein structure. Here, such an evaluation is performed by means of steered molecular dynamics and use of a dimeric model of tryptophan repressor and in-silico mutants as a test case. Analysis of four particular cases shows that, in principle, it is possible to distinguish between wild-type and mutant forms by examination of the total energy and force-extension profiles. In particular, detailed atomic level structural analysis indicates that specific mutations at the interface of the dimeric model (positions 19 and 39) alter interactions that appear in the wild-type form of tryptophan repressor, reducing the energy and force required to separate both subunits.

Structure-based energetics of protein interfaces guide Foot-and-Mouth Disease virus vaccine design

PubMed Central

Scott, Katherine; Burman, Alison; Loureiro, Silvia; Ren, Jingshan; Porta, Claudine; Ginn, Helen M.; Jackson, Terry; Perez-Martin, Eva; Siebert, C. Alistair; Paul, Guntram; Huiskonen, Juha T.; Jones, Ian M.; Esnouf, Robert M.; Fry, Elizabeth E.; Maree, Francois F.; Charleston, Bryan; Stuart, David I.

2018-01-01

Summary Virus capsids are primed for disassembly yet capsid integrity is key to generating a protective immune response. Here we devise a computational method to assess relative stability of protein-protein interfaces and use it to design improved candidate vaccines for two of the least stable, but globally important, serotypes of Foot-and-Mouth Disease virus (FMDV), O and SAT2. FMDV capsids comprise identical pentameric protein subunits held together by tenuous non-covalent interactions, and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. We use a novel restrained molecular dynamics strategy, to rank mutations predicted to strengthen the pentamer interfaces to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralising antibody responses to stabilised particles over parental viruses and wild-type capsids. PMID:26389739

A functional protein retention and release multilayer with high stability

NASA Astrophysics Data System (ADS)

Nie, Kun; An, Qi; Zhang, Yihe

2016-04-01

Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by varying the number of capping layers. Furthermore, we demonstrate that the protein-loaded interfacial layers could not only be used to construct catalytic-active interfaces, but also be integrated as the power-generating unit to propel a macroscopic floating device.Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by varying the number of capping layers. Furthermore, we demonstrate that the protein-loaded interfacial layers could not only be used to construct catalytic-active interfaces, but also be integrated as the power-generating unit to propel a macroscopic floating device. Electronic supplementary information (ESI) available: UV-vis spectra of the substrate covered by only polymeric (PAH/PAA)5/(PAH/DAS)5 multilayers, UV-vis spectra of the covalently cross-linked (PAH/SiO2 NP)5/(PAH/DAS)5 hybrid multilayers and (CAT/PAH)5/(PAH/DAS)5 films. TEM of the mesoporous silica. See DOI: 10.1039/c6nr01671e

Quantifying Density Fluctuations in Volumes of All Shapes and Sizes Using Indirect Umbrella Sampling

NASA Astrophysics Data System (ADS)

Patel, Amish J.; Varilly, Patrick; Chandler, David; Garde, Shekhar

2011-10-01

Water density fluctuations are an important statistical mechanical observable and are related to many-body correlations, as well as hydrophobic hydration and interactions. Local water density fluctuations at a solid-water surface have also been proposed as a measure of its hydrophobicity. These fluctuations can be quantified by calculating the probability, P v ( N), of observing N waters in a probe volume of interest v. When v is large, calculating P v ( N) using molecular dynamics simulations is challenging, as the probability of observing very few waters is exponentially small, and the standard procedure for overcoming this problem (umbrella sampling in N) leads to undesirable impulsive forces. Patel et al. (J. Phys. Chem. B 114:1632, 2010) have recently developed an indirect umbrella sampling (INDUS) method, that samples a coarse-grained particle number to obtain P v ( N) in cuboidal volumes. Here, we present and demonstrate an extension of that approach to volumes of other basic shapes, like spheres and cylinders, as well as to collections of such volumes. We further describe the implementation of INDUS in the NPT ensemble and calculate P v ( N) distributions over a broad range of pressures. Our method may be of particular interest in characterizing the hydrophobicity of interfaces of proteins, nanotubes and related systems.

Effects of short-time preheating on ice growth in antifreeze polypeptides solutions in a narrow space

NASA Astrophysics Data System (ADS)

Miyamoto, T.; Nishi, N.; Waku, T.; Tanaka, N.; Hagiwara, Y.

2018-03-01

We conducted experiments on the unidirectional freezing of solutions of winter flounder antifreeze protein or of a polypeptide which was based on twelve amino-acid residues of this protein. The temperature in the solutions and ice was measured with a small thermocouple. The interface temperature was defined as the temperature at the tip of the serrated or pectinate interface. The interface temperature of these solutions was lower than that of pure water. To vary this supercooling activity of these solutes, we preheated the solutions and cooled them before conducting identical experiments. It was found that short-time preheating caused further decreases in the interface temperature and interface velocities. Furthermore, the inclined interfaces and the narrow liquid regions inside the ice area became wider. To investigate the reasons for these changes, we measured aggregates of the solutes in the solutions. These aggregates were found to become larger as a result of preheating. Thus, it can be concluded that these large aggregates attenuated the ice growth by their interaction with ice. Finally, we carried out similar measurements by using pH-adjusted solutions of the protein to produce aggregates without preheating, and obtained similar supercooling enhancement by the aggregates. Thus, the effects of thermal denaturation on the supercooling were not significant in the preheating.

Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM.

PubMed

Tuncbag, Nurcan; Gursoy, Attila; Nussinov, Ruth; Keskin, Ozlem

2011-08-11

Prediction of protein-protein interactions at the structural level on the proteome scale is important because it allows prediction of protein function, helps drug discovery and takes steps toward genome-wide structural systems biology. We provide a protocol (termed PRISM, protein interactions by structural matching) for large-scale prediction of protein-protein interactions and assembly of protein complex structures. The method consists of two components: rigid-body structural comparisons of target proteins to known template protein-protein interfaces and flexible refinement using a docking energy function. The PRISM rationale follows our observation that globally different protein structures can interact via similar architectural motifs. PRISM predicts binding residues by using structural similarity and evolutionary conservation of putative binding residue 'hot spots'. Ultimately, PRISM could help to construct cellular pathways and functional, proteome-scale annotation. PRISM is implemented in Python and runs in a UNIX environment. The program accepts Protein Data Bank-formatted protein structures and is available at http://prism.ccbb.ku.edu.tr/prism_protocol/.

Equilibrium of adsorption of mixed milk protein/surfactant solutions at the water/air interface.

PubMed

Kotsmar, C; Grigoriev, D O; Xu, F; Aksenenko, E V; Fainerman, V B; Leser, M E; Miller, R

2008-12-16

Ellipsometry and surface profile analysis tensiometry were used to study and compare the adsorption behavior of beta-lactoglobulin (BLG)/C10DMPO, beta-casein (BCS)/C10DMPO and BCS/C12DMPO mixtures at the air/solution interface. The adsorption from protein/surfactant mixed solutions is of competitive nature. The obtained adsorption isotherms suggest a gradual replacement of the protein molecules at the interface with increasing surfactant concentration for all studied mixed systems. The thickness, refractive index, and the adsorbed amount of the respective adsorption layers, determined by ellipsometry, decrease monotonically and reach values close to those for a surface covered only by surfactant molecules, indicating the absence of proteins from a certain surfactant concentration on. These results correlate with the surface tension data. A continuous increase of adsorption layer thickness was observed up to this concentration, caused by the desorption of segments of the protein and transforming the thin surface layer into a rather diffuse and thick one. Replacement and structural changes of the protein molecules are discussed in terms of protein structure and surface activity of surfactant molecules. Theoretical models derived recently were used for the quantitative description of the equilibrium state of the mixed surface layers.

A Computational Investigation of Small-Molecule Engagement of Hot Spots at Protein-Protein Interaction Interfaces.

PubMed

Xu, David; Si, Yubing; Meroueh, Samy O

2017-09-25

The binding affinity of a protein-protein interaction is concentrated at amino acids known as hot spots. It has been suggested that small molecules disrupt protein-protein interactions by either (i) engaging receptor protein hot spots or (ii) mimicking hot spots of the protein ligand. Yet, no systematic studies have been done to explore how effectively existing small-molecule protein-protein interaction inhibitors mimic or engage hot spots at protein interfaces. Here, we employ explicit-solvent molecular dynamics simulations and end-point MM-GBSA free energy calculations to explore this question. We select 36 compounds for which high-quality binding affinity and cocrystal structures are available. Five complexes that belong to three classes of protein-protein interactions (primary, secondary, and tertiary) were considered, namely, BRD4•H4, XIAP•Smac, MDM2•p53, Bcl-xL•Bak, and IL-2•IL-2Rα. Computational alanine scanning using MM-GBSA identified hot-spot residues at the interface of these protein interactions. Decomposition energies compared the interaction of small molecules with individual receptor hot spots to those of the native protein ligand. Pharmacophore analysis was used to investigate how effectively small molecules mimic the position of hot spots of the protein ligand. Finally, we study whether small molecules mimic the effects of the native protein ligand on the receptor dynamics. Our results show that, in general, existing small-molecule inhibitors of protein-protein interactions do not optimally mimic protein-ligand hot spots, nor do they effectively engage protein receptor hot spots. The more effective use of hot spots in future drug design efforts may result in smaller compounds with higher ligand efficiencies that may lead to greater success in clinical trials.

Organization of the ER–Golgi interface for membrane traffic control

PubMed Central

Brandizzi, Federica; Barlowe, Charles

2014-01-01

Coat protein complex I (COPI) and COPII are required for bidirectional membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. While these core coat machineries and other transport factors are highly conserved across species, high-resolution imaging studies indicate that the organization of the ER–Golgi interface is varied in eukaryotic cells. Regulation of COPII assembly, in some cases to manage distinct cellular cargo, is emerging as one important component in determining this structure. Comparison of the ER–Golgi interface across different systems, particularly mammalian and plant cells, reveals fundamental elements and distinct organization of this interface. A better understanding of how these interfaces are regulated to meet varying cellular secretory demands should provide key insights into the mechanisms that control efficient trafficking of proteins and lipids through the secretory pathway. PMID:23698585

Xanthan/chitosan gold chip for metal enhanced protein biomarker detection.

PubMed

Domnanich, Patrick; Peña, Dacimoneida Brito; Preininger, Claudia

2011-01-15

Protein microarrays for disease diagnostics are required to accurately quantify analytes in the low pg/mL range. This task is hampered by weak signal strengths and too low detector sensitivity. Herein we present reflective gold chips coated with polyelectrolyte multilayers (PEMs) for signal enhancement in immunoassays for melanoma-relevant biomarkers. Among tested (semi)natural polysaccharides (xanthan, chitosan, carboxymethylcellulose, hyaluronic acid) PEMs composed of xanthan and chitosan performed best in terms of detection of low analyte concentrations (ED10), spot morphology, fluorescence background and variability (<10%). Fluorescence signals on gold slides with a 75 nm coating of seven crosslinked polyelectrolyte double layers were up to 50 times higher than on bare glass slides. In comparison to commercial substrates the signal to noise ratio is enhanced by up to factor 11. Furthermore sandwich assays for interleukins 6, 8, 10, tumour necrosis factor alpha (TNFα), vascular endothelial growth factor A (VEGF-A) and S100B show working ranges which cover significantly lower concentrations (up to 38-fold). Not limited to above assays the presented substrates, which combine a biocompatible interface with metal-based signal amplification, are a valuable tool in a variety of biosensor applications. Copyright © 2010 Elsevier B.V. All rights reserved.

SURFACE INACTIVATION OF BACTERIAL VIRUSES AND OF PROTEINS

PubMed Central

Adams, Mark H.

1948-01-01

1. The seven bacterial viruses of the T group active against E. coli, are rapidly inactivated at gas-liquid interfaces. 2. The kinetics of this inactivation whether brought about by shaking or by bubbling with nitrogen are those of a first order reaction. 3. This inactivation may be prevented by the addition of enough protein to maintain the gas-liquid interface in a saturated condition. 4. The analogy between this phenomenon and the surface denaturation of proteins is pointed out and discussed. PMID:18917025

RosettaRemodel: A Generalized Framework for Flexible Backbone Protein Design

PubMed Central

Huang, Po-Ssu; Ban, Yih-En Andrew; Richter, Florian; Andre, Ingemar; Vernon, Robert; Schief, William R.; Baker, David

2011-01-01

We describe RosettaRemodel, a generalized framework for flexible protein design that provides a versatile and convenient interface to the Rosetta modeling suite. RosettaRemodel employs a unified interface, called a blueprint, which allows detailed control over many aspects of flexible backbone protein design calculations. RosettaRemodel allows the construction and elaboration of customized protocols for a wide range of design problems ranging from loop insertion and deletion, disulfide engineering, domain assembly, loop remodeling, motif grafting, symmetrical units, to de novo structure modeling. PMID:21909381

Evaluation of the adhesion on the nano-scaled polymeric film systems.

PubMed

Park, Tae Sung; Park, Ik Keun; Yoshida, Sanichiro

2017-04-01

We applied scanning acoustic microscopy known as the V(z) curve technique to photoresist thin-film systems for the evaluation of the adhesive strength at the film-substrate interface. Through the measurement of the SAW (Surface Acoustic Wave) velocity, the V(z) curve analysis allows us to quantify the stiffness of the film-substrate interface. In addition, we conducted a nano-scratch test to quantify the ultimate strength of the adhesion through the evaluation of the critical load. To vary the adhesive conditions, we prepared thin-film specimens with three different types of pre-coating surface treatments, i.e., oxygen-plasma bombardment, HMDS (Hexametyldisilazane) treatment and untreated. The magnitudes of the quantified stiffness and ultimate strength are found consistent with each other for all the specimens tested, indicating that the pre-coating surface treatment can strengthen both the stiffness and ultimate strength of the adhesion. The results of this study demonstrate the usefulness of the V(Z) analysis as a nondestructive method to evaluate the adhesion strength of nano-structured thin-film systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of Genes Involved in Biofilm Formation and Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens▿ †

PubMed Central

Rollefson, Janet B.; Levar, Caleb E.; Bond, Daniel R.

2009-01-01

Electron transfer from cells to metals and electrodes by the Fe(III)-reducing anaerobe Geobacter sulfurreducens requires proper expression of redox proteins and attachment mechanisms to interface bacteria with surfaces and neighboring cells. We hypothesized that transposon mutagenesis would complement targeted knockout studies in Geobacter spp. and identify novel genes involved in this process. Escherichia coli mating strains and plasmids were used to develop a conjugation protocol and deliver mini-Himar transposons, creating a library of over 8,000 mutants that was anaerobically arrayed and screened for a range of phenotypes, including auxotrophy for amino acids, inability to reduce Fe(III) citrate, and attachment to surfaces. Following protocol validation, mutants with strong phenotypes were further characterized in a three-electrode system to simultaneously quantify attachment, biofilm development, and respiratory parameters, revealing mutants defective in Fe(III) reduction but unaffected in electron transfer to electrodes (such as an insertion in GSU1330, a putative metal export protein) or defective in electrode reduction but demonstrating wild-type biofilm formation (due to an insertion upstream of the NHL domain protein GSU2505). An insertion in a putative ATP-dependent transporter (GSU1501) eliminated electrode colonization but not Fe(III) citrate reduction. A more complex phenotype was demonstrated by a mutant containing an insertion in a transglutaminase domain protein (GSU3361), which suddenly ceased to respire when biofilms reached approximately 50% of the wild-type levels. As most insertions were not in cytochromes but rather in transporters, two-component signaling proteins, and proteins of unknown function, this collection illustrates how biofilm formation and electron transfer are separate but complementary phenotypes, controlled by multiple loci not commonly studied in Geobacter spp. PMID:19395486

Ferroelectric hydration shells around proteins: electrostatics of the protein-water interface.

PubMed

LeBard, David N; Matyushov, Dmitry V

2010-07-22

Numerical simulations of hydrated proteins show that protein hydration shells are polarized into a ferroelectric layer with large values of the average dipole moment magnitude and the dipole moment variance. The emergence of the new polarized mesophase dramatically alters the statistics of electrostatic fluctuations at the protein-water interface. The linear response relation between the average electrostatic potential and its variance breaks down, with the breadth of the electrostatic fluctuations far exceeding the expectations of the linear response theories. The dynamics of these non-Gaussian electrostatic fluctuations are dominated by a slow (approximately = 1 ns) component that freezes in at the temperature of the dynamical transition of proteins. The ferroelectric shell propagates 3-5 water diameters into the bulk.

Database of amino acid-nucleotide contacts in contacts in DNA-homeodomain protein

NASA Astrophysics Data System (ADS)

Grokhlina, T. I.; Zrelov, P. V.; Ivanov, V. V.; Polozov, R. V.; Chirgadze, Yu. N.; Sivozhelezov, V. S.

2013-09-01

The analysis of amino acid-nucleotide contacts in interfaces of the protein-DNA complexes, intended to find consistencies in the protein-DNA recognition, is a complex problem that requires an analysis of the physicochemical characteristics of these contacts and the positions of the participating amino acids and nucleotides in the chains of the protein and the DNA, respectively, as well as conservatism of these contacts. Thus, those heterogeneous data should be systematized. For this purpose we have developed a database of amino acid-nucleotide contacts ANTPC (Amino acid Nucleotide Type Position Conservation) following the archetypal example of the proteins in the homeodomain family. We show that it can be used to compare and classify the interfaces of the protein-DNA complexes.

Role of anisotropy in determining stability of electrodeposition at solid-solid interfaces

DOE PAGES

Ahmad, Zeeshan; Viswanathan, Venkatasubramanian

2017-10-24

Here, we investigate the stability of electrodeposition at solid-solid interfaces for materials exhibiting an anisotropic mechanical response. The stability of electrodeposition or resistance to the formation of dendrites is studied within a linear stability analysis. The deformation and stress equations are solved using the Stroh formalism and faithfully recover the boundary conditions at the interface. The stability parameter is used to quantify the stability of different solid-solid interfaces incorporating the full anisotropy of the elastic tensor of the two materials. Our results show a high degree of variability in the stability parameter depending on the crystallographic orientation of the solidsmore » in contact, and point to opportunities for exploiting this effect in developing Li metal anodes.« less

Biotribological properties at the stem-cement interface lubricated with different media.

PubMed

Zhang, H Y; Luo, J B; Zhou, M; Zhang, Y; Huang, Y L

2013-04-01

Debonding of the stem-cement interface occurs inevitably in-vivo under physiological loading, and pseudo-synovial fluid is subsequently pumped into this interface, serving as the lubricant. However, the influence of protein adsorption onto the femoral stem surface has not been well taken into consideration in previous in vitro studies. The biotribological properties at the stem-cement interface were investigated through a series of fretting frictional tests using polished stainless steel 316L stem and smooth bone cement, lubricated by three different media at body temperature, i.e. 100% calf serum, 25% calf serum, and 0.9% saline solution. The surface characterization of the femoral stem was evaluated sequentially using optical microscope, optical interferometer, scanning electron microscope, and Raman spectroscopy. The friction coefficient generally kept stable during the test, and the minimum value (0.254) was obtained when 100% calf serum was used as the lubricant. Slight scratches were detected within the contact area for the stainless steel 316L stems lubricated by 100% calf serum and 25% calf serum, which was further surrounded by the adsorbed protein film with alveolate feature. Additionally, a wear scar was present within the contact area when 0.9% saline solution was used as the lubricant. Protein adsorption onto the stainless steel 316L stem surface affected the biotribological properties at the stem-cement interface under oscillatory fretting mechanism. Generation of wear debris at the stem-cement interface may be postponed by modification of physicochemical properties of the femoral stem to promote protein adsorption. Copyright © 2013 Elsevier Ltd. All rights reserved.

Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding

PubMed Central

Liko, Idlir; Degiacomi, Matteo T.; Mohammed, Shabaz; Yoshikawa, Shinya; Schmidt, Carla; Robinson, Carol V.

2016-01-01

Bovine cytochrome c oxidase is an integral membrane protein complex comprising 13 protein subunits and associated lipids. Dimerization of the complex has been proposed; however, definitive evidence for the dimer is lacking. We used advanced mass spectrometry methods to investigate the oligomeric state of cytochrome c oxidase and the potential role of lipids and posttranslational modifications in its subunit interfaces. Mass spectrometry of the intact protein complex revealed that both the monomer and the dimer are stabilized by large lipid entities. We identified these lipid species from the purified protein complex, thus implying that they interact specifically with the enzyme. We further identified phosphorylation and acetylation sites of cytochrome c oxidase, located in the peripheral subunits and in the dimer interface, respectively. Comparing our phosphorylation and acetylation sites with those found in previous studies of bovine, mouse, rat, and human cytochrome c oxidase, we found that whereas some acetylation sites within the dimer interface are conserved, suggesting a role for regulation and stabilization of the dimer, phosphorylation sites were less conserved and more transient. Our results therefore provide insights into the locations and interactions of lipids with acetylated residues within the dimer interface of this enzyme, and thereby contribute to a better understanding of its structure in the natural membrane. Moreover dimeric cytochrome c oxidase, comprising 20 transmembrane, six extramembrane subunits, and associated lipids, represents the largest integral membrane protein complex that has been transferred via electrospray intact into the gas phase of a mass spectrometer, representing a significant technological advance. PMID:27364008

The effect of protein–precipitant interfaces and applied shear on the nucleation and growth of lysozyme crystals

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

Reis, Nuno M.; Chirgadze, Dimitri Y.; Blundell, Tom L.

The nucleation of lysozyme in microbatch experiments was linked to the formation of protein–precipitant interfaces. The use of oscillatory shear allowed decreasing the nucleation rate and extending the growth period for lysozyme crystals, presumably through the control of the number of interfaces and removal of impurities or defects. This paper is concerned with the effect of protein–precipitant interfaces and externally applied shear on the nucleation and growth kinetics of hen egg-white lysozyme crystals. The early stages of microbatch crystallization of lysozyme were explored using both optical and confocal fluorescence microscopy imaging. Initially, an antisolvent (precipitant) was added to a proteinmore » drop and the optical development of the protein–precipitant interface was followed with time. In the presence of the water-soluble polymer poly(ethylene glycol) (PEG) a sharp interface was observed to form immediately within the drop, giving an initial clear separation between the lighter protein solution and the heavier precipitant. This interface subsequently became unstable and quickly developed within a few seconds into several unstable ‘fingers’ that represented regions of high concentration-gradient interfaces. Confocal microscopy demonstrated that the subsequent nucleation of protein crystals occurred preferentially in the region of these interfaces. Additional experiments using an optical shearing system demonstrated that oscillatory shear significantly decreased nucleation rates whilst extending the growth period of the lysozyme crystals. The experimental observations relating to both nucleation and growth have relevance in developing efficient and reliable protocols for general crystallization procedures and the controlled crystallization of single large high-quality protein crystals for use in X-ray crystallography.« less

Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins.

PubMed

Pradhan, Mohan R; Pal, Arumay; Hu, Zhongqiao; Kannan, Srinivasaraghavan; Chee Keong, Kwoh; Lane, David P; Verma, Chandra S

2016-02-01

Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using "Dehydrons." "Dehydrons" are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein-protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non-conserved dehydron-residues) from other interaction interfaces (conserved dehydron-residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation. © 2015 Wiley Periodicals, Inc.

Modulating surface rheology by electrostatic protein/polysaccharide interactions.

PubMed

Ganzevles, Renate A; Zinoviadou, Kyriaki; van Vliet, Ton; Cohen, Martien A; de Jongh, Harmen H

2006-11-21

There is a large interest in mixed protein/polysaccharide layers at air-water and oil-water interfaces because of their ability to stabilize foams and emulsions. Mixed protein/polysaccharide adsorbed layers at air-water interfaces can be prepared either by adsorption of soluble protein/polysaccharide complexes or by sequential adsorption of complexes or polysaccharides to a previously formed protein layer. Even though the final protein and polysaccharide bulk concentrations are the same, the behavior of the adsorbed layers can be very different, depending on the method of preparation. The surface shear modulus of a sequentially formed beta-lactoglobulin/pectin layer can be up to a factor of 6 higher than that of a layer made by simultaneous adsorption. Furthermore, the surface dilatational modulus and surface shear modulus strongly (up to factors of 2 and 7, respectively) depend on the bulk -lactoglobulin/pectin mixing ratio. On the basis of the surface rheological behavior, a mechanistic understanding of how the structure of the adsorbed layers depends on the protein/polysaccharide interaction in bulk solution, mixing ratio, ionic strength, and order of adsorption to the interface (simultaneous or sequential) is derived. Insight into the effect of protein/polysaccharide interactions on the properties of adsorbed layers provides a solid basis to modulate surface rheological behavior.

Effects of surfaces and leachables on the stability of biopharmaceuticals.

PubMed

Bee, Jared S; Randolph, Theodore W; Carpenter, John F; Bishop, Steven M; Dimitrova, Mariana N

2011-10-01

Therapeutic proteins are exposed to various potential contact surfaces, particles, and leachables during manufacturing, shipping, storage, and delivery. In this review, we present published examples of interfacial- or leachable-induced aggregation or particle formation, and discuss the mitigation strategies that were successfully utilized. Adsorption to interfaces or interactions with leachables and/or particles in some cases has been reported to cause protein aggregation or particle formation. Identification of the cause(s) of particle formation involving minute amounts of protein over extended periods of time can be challenging. Various formulation strategies such as addition of a nonionic surfactant (e.g., polysorbate) have been demonstrated to effectively mitigate adsorption-induced protein aggregation. However, not all stability problems associated with interfaces or leachables are best resolved by formulation optimization. Detectable leachables do not necessarily have any adverse impact on the protein but control of the leachable source is preferred when there is a concern. In other cases, preventing protein aggregation and particle formation may require manufacturing process and/or equipment changes, use of compatible materials at contact interfaces, and so on. This review summarizes approaches that have been used to minimize protein aggregation and particle formation during manufacturing and fill-finish operations, product storage and transportation, and delivery of protein therapeutics. Copyright © 2011 Wiley-Liss, Inc.

Accurate prediction of interfacial residues in two-domain proteins using evolutionary information: implications for three-dimensional modeling.

PubMed

Bhaskara, Ramachandra M; Padhi, Amrita; Srinivasan, Narayanaswamy

2014-07-01

With the preponderance of multidomain proteins in eukaryotic genomes, it is essential to recognize the constituent domains and their functions. Often function involves communications across the domain interfaces, and the knowledge of the interacting sites is essential to our understanding of the structure-function relationship. Using evolutionary information extracted from homologous domains in at least two diverse domain architectures (single and multidomain), we predict the interface residues corresponding to domains from the two-domain proteins. We also use information from the three-dimensional structures of individual domains of two-domain proteins to train naïve Bayes classifier model to predict the interfacial residues. Our predictions are highly accurate (∼85%) and specific (∼95%) to the domain-domain interfaces. This method is specific to multidomain proteins which contain domains in at least more than one protein architectural context. Using predicted residues to constrain domain-domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. We believe that these results can be of considerable interest toward rational protein and interaction design, apart from providing us with valuable information on the nature of interactions. © 2013 Wiley Periodicals, Inc.

CD4-gp120 interaction interface - a gateway for HIV-1 infection in human: molecular network, modeling and docking studies.

PubMed

Pandey, Deeksha; Podder, Avijit; Pandit, Mansi; Latha, Narayanan

2017-09-01

The major causative agent for Acquired Immune Deficiency Syndrome (AIDS) is Human Immunodeficiency Virus-1 (HIV-1). HIV-1 is a predominant subtype of HIV which counts on human cellular mechanism virtually in every aspect of its life cycle. Binding of viral envelope glycoprotein-gp120 with human cell surface CD4 receptor triggers the early infection stage of HIV-1. This study focuses on the interaction interface between these two proteins that play a crucial role for viral infectivity. The CD4-gp120 interaction interface has been studied through a comprehensive protein-protein interaction network (PPIN) analysis and highlighted as a useful step towards identifying potential therapeutic drug targets against HIV-1 infection. We prioritized gp41, Nef and Tat proteins of HIV-1 as valuable drug targets at early stage of viral infection. Lack of crystal structure has made it difficult to understand the biological implication of these proteins during disease progression. Here, computational protein modeling techniques and molecular dynamics simulations were performed to generate three-dimensional models of these targets. Besides, molecular docking was initiated to determine the desirability of these target proteins for already available HIV-1 specific drugs which indicates the usefulness of these protein structures to identify an effective drug combination therapy against AIDS.

3dRPC: a web server for 3D RNA-protein structure prediction.

PubMed

Huang, Yangyu; Li, Haotian; Xiao, Yi

2018-04-01

RNA-protein interactions occur in many biological processes. To understand the mechanism of these interactions one needs to know three-dimensional (3D) structures of RNA-protein complexes. 3dRPC is an algorithm for prediction of 3D RNA-protein complex structures and consists of a docking algorithm RPDOCK and a scoring function 3dRPC-Score. RPDOCK is used to sample possible complex conformations of an RNA and a protein by calculating the geometric and electrostatic complementarities and stacking interactions at the RNA-protein interface according to the features of atom packing of the interface. 3dRPC-Score is a knowledge-based potential that uses the conformations of nucleotide-amino-acid pairs as statistical variables and that is used to choose the near-native complex-conformations obtained from the docking method above. Recently, we built a web server for 3dRPC. The users can easily use 3dRPC without installing it locally. RNA and protein structures in PDB (Protein Data Bank) format are the only needed input files. It can also incorporate the information of interface residues or residue-pairs obtained from experiments or theoretical predictions to improve the prediction. The address of 3dRPC web server is http://biophy.hust.edu.cn/3dRPC. yxiao@hust.edu.cn.

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel.

PubMed

Li-Smerin, Y; Hackos, D H; Swartz, K J

2000-02-01

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.

The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex.

PubMed

Brzovic, Peter S; Heikaus, Clemens C; Kisselev, Leonid; Vernon, Robert; Herbig, Eric; Pacheco, Derek; Warfield, Linda; Littlefield, Peter; Baker, David; Klevit, Rachel E; Hahn, Steven

2011-12-23

The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation, allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a "fuzzy" complex, where the Gcn4-Gal11 interface cannot be described by a single conformation. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets. Copyright © 2011 Elsevier Inc. All rights reserved.

Conformational dynamics of amyloid proteins at the aqueous interface

NASA Astrophysics Data System (ADS)

Armbruster, Matthew; Horst, Nathan; Aoki, Brendy; Malik, Saad; Soto, Patricia

2013-03-01

Amyloid proteins is a class of proteins that exhibit distinct monomeric and oligomeric conformational states hallmark of deleterious neurological diseases for which there are not yet cures. Our goal is to examine the extent of which the aqueous/membrane interface modulates the folding energy landscape of amyloid proteins. To this end, we probe the dynamic conformational ensemble of amyloids (monomer prion protein and Alzheimer's Ab protofilaments) interacting with model bilayers. We will present the results of our coarse grain molecular modeling study in terms of the existence of preferential binding spots of the amyloid to the bilayer and the response of the bilayer to the interaction with the amyloid. NSF Nebraska EPSCoR First Award

Comparison of Pancreas Juice Proteins from Cancer Versus Pancreatitis Using Quantitative Proteomic Analysis

PubMed Central

Chen, Ru; Pan, Sheng; Cooke, Kelly; Moyes, Kara White; Bronner, Mary P.; Goodlett, David R.; Aebersold, Ruedi; Brentnall, Teresa A.

2008-01-01

Objectives Pancreatitis is an inflammatory condition of the pancreas. However, it often shares many molecular features with pancreatic cancer. Biomarkers present in pancreatic cancer frequently occur in the setting of pancreatitis. The efforts to develop diagnostic biomarkers for pancreatic cancer have thus been complicated by the false-positive involvement of pancreatitis. Methods In an attempt to develop protein biomarkers for pancreatic cancer, we previously use quantitative proteomics to identify and quantify the proteins from pancreatic cancer juice. Pancreatic juice is a rich source of proteins that are shed by the pancreatic ductal cells. In this study, we used a similar approach to identify and quantify proteins from pancreatitis juice. Results In total, 72 proteins were identified and quantified in the comparison of pancreatic juice from pancreatitis patients versus pooled normal control juice. Nineteen of the juice proteins were overexpressed, and 8 were underexpressed in pancreatitis juice by at least 2-fold compared with normal pancreatic juice. Of these 27 differentially expressed proteins in pancreatitis, 9 proteins were also differentially expressed in the pancreatic juice from pancreatic cancer patient. Conclusions Identification of these differentially expressed proteins from pancreatitis juice provides useful information for future study of specific pancreatitis-associated proteins and to eliminate potential false-positive biomarkers for pancreatic cancer. PMID:17198186

Comparison of pancreas juice proteins from cancer versus pancreatitis using quantitative proteomic analysis.

PubMed

Chen, Ru; Pan, Sheng; Cooke, Kelly; Moyes, Kara White; Bronner, Mary P; Goodlett, David R; Aebersold, Ruedi; Brentnall, Teresa A

2007-01-01

Pancreatitis is an inflammatory condition of the pancreas. However, it often shares many molecular features with pancreatic cancer. Biomarkers present in pancreatic cancer frequently occur in the setting of pancreatitis. The efforts to develop diagnostic biomarkers for pancreatic cancer have thus been complicated by the false-positive involvement of pancreatitis. In an attempt to develop protein biomarkers for pancreatic cancer, we previously use quantitative proteomics to identify and quantify the proteins from pancreatic cancer juice. Pancreatic juice is a rich source of proteins that are shed by the pancreatic ductal cells. In this study, we used a similar approach to identify and quantify proteins from pancreatitis juice. In total, 72 proteins were identified and quantified in the comparison of pancreatic juice from pancreatitis patients versus pooled normal control juice. Nineteen of the juice proteins were overexpressed, and 8 were underexpressed in pancreatitis juice by at least 2-fold compared with normal pancreatic juice. Of these 27 differentially expressed proteins in pancreatitis, 9 proteins were also differentially expressed in the pancreatic juice from pancreatic cancer patient. Identification of these differentially expressed proteins from pancreatitis juice provides useful information for future study of specific pancreatitis-associated proteins and to eliminate potential false-positive biomarkers for pancreatic cancer.

Shock Interaction with a Finite Thickness Two-Gas Interface

NASA Astrophysics Data System (ADS)

Labenski, John; Kim, Yong

2006-03-01

A dual-driver shock tube was used to investigate the growth rate of a finite thickness two-gas interface after shock forcing. One driver was used to create an argon-refrigerant interface as the contact surface behind a weak shock wave. The other driver, at the opposite end of the driven section, generates a stronger shock of Mach 1.1 to 1.3 to force the interface back in front of the detector station. Two schlieren systems record the density fluctuations while light scattering detectors record the density of the refrigerant as a function of position over the interface during both it's initial passage and return. A pair of digital cameras take stereo images of the interface, as mapped out by the tracer particles under illumination by a Q-switched ruby laser. The amount of time that the interface is allowed to travel up the driven section determines the interaction time as a control. Comparisons made between the schlieren signals, light scattering detector outputs, and the images quantify the fingered characteristics of the interface and its growth due to shock forcing. The results show that the interface has a distribution of thicknesses and that the interaction with a shock further broadens the interface.

Deciphering the shape and deformation of secondary structures through local conformation analysis

PubMed Central

2011-01-01

Background Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation. Results Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface) reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations. Conclusion The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons. PMID:21284872

The role of charged surface residues in the binding ability of small hubs in protein-protein interaction networks

PubMed Central

Patil, Ashwini; Nakamura, Haruki

2007-01-01

Hubs are highly connected proteins in a protein-protein interaction network. Previous work has implicated disordered domains and high surface charge as the properties significant in the ability of hubs to bind multiple proteins. While conformational flexibility of disordered domains plays an important role in the binding ability of large hubs, high surface charge is the dominant property in small hubs. In this study, we further investigate the role of the high surface charge in the binding ability of small hubs in the absence of disordered domains. Using multipole expansion, we find that the charges are highly distributed over the hub surfaces. Residue enrichment studies show that the charged residues in hubs are more prevalent on the exposed surface, with the exception of Arg, which is predominantly found at the interface, as compared to non-hubs. This suggests that the charged residues act primarily from the exposed surface rather than the interface to affect the binding ability of small hubs. They do this through (i) enhanced intra-molecular electrostatic interactions to lower the desolvation penalty, (ii) indirect long – range intermolecular interactions with charged residues on the partner proteins for better complementarity and electrostatic steering, and (iii) increased solubility for enhanced diffusion-controlled rate of binding. Along with Arg, we also find a high prevalence of polar residues Tyr, Gln and His and the hydrophobic residue Met at the interfaces of hubs, all of which have the ability to form multiple types of interactions, indicating that the interfaces of hubs are optimized to participate in multiple interactions. PMID:27857564

The role of charged surface residues in the binding ability of small hubs in protein-protein interaction networks.

PubMed

Patil, Ashwini; Nakamura, Haruki

2007-01-01

Hubs are highly connected proteins in a protein-protein interaction network. Previous work has implicated disordered domains and high surface charge as the properties significant in the ability of hubs to bind multiple proteins. While conformational flexibility of disordered domains plays an important role in the binding ability of large hubs, high surface charge is the dominant property in small hubs. In this study, we further investigate the role of the high surface charge in the binding ability of small hubs in the absence of disordered domains. Using multipole expansion, we find that the charges are highly distributed over the hub surfaces. Residue enrichment studies show that the charged residues in hubs are more prevalent on the exposed surface, with the exception of Arg, which is predominantly found at the interface, as compared to non-hubs. This suggests that the charged residues act primarily from the exposed surface rather than the interface to affect the binding ability of small hubs. They do this through (i) enhanced intra-molecular electrostatic interactions to lower the desolvation penalty, (ii) indirect long - range intermolecular interactions with charged residues on the partner proteins for better complementarity and electrostatic steering, and (iii) increased solubility for enhanced diffusion-controlled rate of binding. Along with Arg, we also find a high prevalence of polar residues Tyr, Gln and His and the hydrophobic residue Met at the interfaces of hubs, all of which have the ability to form multiple types of interactions, indicating that the interfaces of hubs are optimized to participate in multiple interactions.

Deciphering the shape and deformation of secondary structures through local conformation analysis.

PubMed

Baussand, Julie; Camproux, Anne-Claude

2011-02-01

Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation. Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface) reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations. The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons.

Orientation determination of interfacial beta-sheet structures in situ.

PubMed

Nguyen, Khoi Tan; King, John Thomas; Chen, Zhan

2010-07-01

Structural information such as orientations of interfacial proteins and peptides is important for understanding properties and functions of such biological molecules, which play crucial roles in biological applications and processes such as antimicrobial selectivity, membrane protein activity, biocompatibility, and biosensing performance. The alpha-helical and beta-sheet structures are the most widely encountered secondary structures in peptides and proteins. In this paper, for the first time, a method to quantify the orientation of the interfacial beta-sheet structure using a combined attenuated total reflectance Fourier transformation infrared spectroscopic (ATR-FTIR) and sum frequency generation (SFG) vibrational spectroscopic study was developed. As an illustration of the methodology, the orientation of tachyplesin I, a 17 amino acid peptide with an antiparallel beta-sheet, adsorbed to polymer surfaces as well as associated with a lipid bilayer was determined using the regular and chiral SFG spectra, together with polarized ATR-FTIR amide I signals. Both the tilt angle (theta) and the twist angle (psi) of the beta-sheet at interfaces are determined. The developed method in this paper can be used to obtain in situ structural information of beta-sheet components in complex molecules. The combination of this method and the existing methodology that is currently used to investigate alpha-helical structures will greatly broaden the application of optical spectroscopy in physical chemistry, biochemistry, biophysics, and structural biology.

CNA web server: rigidity theory-based thermal unfolding simulations of proteins for linking structure, (thermo-)stability, and function

PubMed Central

Krüger, Dennis M.; Rathi, Prakash Chandra; Pfleger, Christopher; Gohlke, Holger

2013-01-01

The Constraint Network Analysis (CNA) web server provides a user-friendly interface to the CNA approach developed in our laboratory for linking results from rigidity analyses to biologically relevant characteristics of a biomolecular structure. The CNA web server provides a refined modeling of thermal unfolding simulations that considers the temperature dependence of hydrophobic tethers and computes a set of global and local indices for quantifying biomacromolecular stability. From the global indices, phase transition points are identified where the structure switches from a rigid to a floppy state; these phase transition points can be related to a protein’s (thermo-)stability. Structural weak spots (unfolding nuclei) are automatically identified, too; this knowledge can be exploited in data-driven protein engineering. The local indices are useful in linking flexibility and function and to understand the impact of ligand binding on protein flexibility. The CNA web server robustly handles small-molecule ligands in general. To overcome issues of sensitivity with respect to the input structure, the CNA web server allows performing two ensemble-based variants of thermal unfolding simulations. The web server output is provided as raw data, plots and/or Jmol representations. The CNA web server, accessible at http://cpclab.uni-duesseldorf.de/cna or http://www.cnanalysis.de, is free and open to all users with no login requirement. PMID:23609541

Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase.

PubMed

Mehrotra, Sonali; B Ningappa, Mylarappa; Raman, Jayalakshmi; Anand, Ranjith P; Balaram, Hemalatha

2012-04-01

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion. Copyright © 2012 Elsevier B.V. All rights reserved.

Cryo-EM Data Are Superior to Contact and Interface Information in Integrative Modeling.

PubMed

de Vries, Sjoerd J; Chauvot de Beauchêne, Isaure; Schindler, Christina E M; Zacharias, Martin

2016-02-23

Protein-protein interactions carry out a large variety of essential cellular processes. Cryo-electron microscopy (cryo-EM) is a powerful technique for the modeling of protein-protein interactions at a wide range of resolutions, and recent developments have caused a revolution in the field. At low resolution, cryo-EM maps can drive integrative modeling of the interaction, assembling existing structures into the map. Other experimental techniques can provide information on the interface or on the contacts between the monomers in the complex. This inevitably raises the question regarding which type of data is best suited to drive integrative modeling approaches. Systematic comparison of the prediction accuracy and specificity of the different integrative modeling paradigms is unavailable to date. Here, we compare EM-driven, interface-driven, and contact-driven integrative modeling paradigms. Models were generated for the protein docking benchmark using the ATTRACT docking engine and evaluated using the CAPRI two-star criterion. At 20 Å resolution, EM-driven modeling achieved a success rate of 100%, outperforming the other paradigms even with perfect interface and contact information. Therefore, even very low resolution cryo-EM data is superior in predicting heterodimeric and heterotrimeric protein assemblies. Our study demonstrates that a force field is not necessary, cryo-EM data alone is sufficient to accurately guide the monomers into place. The resulting rigid models successfully identify regions of conformational change, opening up perspectives for targeted flexible remodeling. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Cryo-EM Data Are Superior to Contact and Interface Information in Integrative Modeling

PubMed Central

de Vries, Sjoerd J.; Chauvot de Beauchêne, Isaure; Schindler, Christina E.M.; Zacharias, Martin

2016-01-01

Protein-protein interactions carry out a large variety of essential cellular processes. Cryo-electron microscopy (cryo-EM) is a powerful technique for the modeling of protein-protein interactions at a wide range of resolutions, and recent developments have caused a revolution in the field. At low resolution, cryo-EM maps can drive integrative modeling of the interaction, assembling existing structures into the map. Other experimental techniques can provide information on the interface or on the contacts between the monomers in the complex. This inevitably raises the question regarding which type of data is best suited to drive integrative modeling approaches. Systematic comparison of the prediction accuracy and specificity of the different integrative modeling paradigms is unavailable to date. Here, we compare EM-driven, interface-driven, and contact-driven integrative modeling paradigms. Models were generated for the protein docking benchmark using the ATTRACT docking engine and evaluated using the CAPRI two-star criterion. At 20 Å resolution, EM-driven modeling achieved a success rate of 100%, outperforming the other paradigms even with perfect interface and contact information. Therefore, even very low resolution cryo-EM data is superior in predicting heterodimeric and heterotrimeric protein assemblies. Our study demonstrates that a force field is not necessary, cryo-EM data alone is sufficient to accurately guide the monomers into place. The resulting rigid models successfully identify regions of conformational change, opening up perspectives for targeted flexible remodeling. PMID:26846888

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.

The De Novo Design of Protein-Protein Interfaces

DTIC Science & Technology

it was our intention to add to this body by engineering de novo (from scratch) protein/protein complexes. Using this inverse approach we have furthered...key physical features needed to drive specific protein/protein interactions. It is considered inverse because, instead of studying natural complexes

Recurring sequence-structure motifs in (βα)8-barrel proteins and experimental optimization of a chimeric protein designed based on such motifs.

PubMed

Wang, Jichao; Zhang, Tongchuan; Liu, Ruicun; Song, Meilin; Wang, Juncheng; Hong, Jiong; Chen, Quan; Liu, Haiyan

2017-02-01

An interesting way of generating novel artificial proteins is to combine sequence motifs from natural proteins, mimicking the evolutionary path suggested by natural proteins comprising recurring motifs. We analyzed the βα and αβ modules of TIM barrel proteins by structure alignment-based sequence clustering. A number of preferred motifs were identified. A chimeric TIM was designed by using recurring elements as mutually compatible interfaces. The foldability of the designed TIM protein was then significantly improved by six rounds of directed evolution. The melting temperature has been improved by more than 20°C. A variety of characteristics suggested that the resulting protein is well-folded. Our analysis provided a library of peptide motifs that is potentially useful for different protein engineering studies. The protein engineering strategy of using recurring motifs as interfaces to connect partial natural proteins may be applied to other protein folds. Copyright © 2016 Elsevier B.V. All rights reserved.

ATAQS: A computational software tool for high throughput transition optimization and validation for selected reaction monitoring mass spectrometry

PubMed Central

2011-01-01

Background Since its inception, proteomics has essentially operated in a discovery mode with the goal of identifying and quantifying the maximal number of proteins in a sample. Increasingly, proteomic measurements are also supporting hypothesis-driven studies, in which a predetermined set of proteins is consistently detected and quantified in multiple samples. Selected reaction monitoring (SRM) is a targeted mass spectrometric technique that supports the detection and quantification of specific proteins in complex samples at high sensitivity and reproducibility. Here, we describe ATAQS, an integrated software platform that supports all stages of targeted, SRM-based proteomics experiments including target selection, transition optimization and post acquisition data analysis. This software will significantly facilitate the use of targeted proteomic techniques and contribute to the generation of highly sensitive, reproducible and complete datasets that are particularly critical for the discovery and validation of targets in hypothesis-driven studies in systems biology. Result We introduce a new open source software pipeline, ATAQS (Automated and Targeted Analysis with Quantitative SRM), which consists of a number of modules that collectively support the SRM assay development workflow for targeted proteomic experiments (project management and generation of protein, peptide and transitions and the validation of peptide detection by SRM). ATAQS provides a flexible pipeline for end-users by allowing the workflow to start or end at any point of the pipeline, and for computational biologists, by enabling the easy extension of java algorithm classes for their own algorithm plug-in or connection via an external web site. This integrated system supports all steps in a SRM-based experiment and provides a user-friendly GUI that can be run by any operating system that allows the installation of the Mozilla Firefox web browser. Conclusions Targeted proteomics via SRM is a powerful new technique that enables the reproducible and accurate identification and quantification of sets of proteins of interest. ATAQS is the first open-source software that supports all steps of the targeted proteomics workflow. ATAQS also provides software API (Application Program Interface) documentation that enables the addition of new algorithms to each of the workflow steps. The software, installation guide and sample dataset can be found in http://tools.proteomecenter.org/ATAQS/ATAQS.html PMID:21414234

Communication between filamentous pathogens and plants at the biotrophic interface.

PubMed

Yi, Mihwa; Valent, Barbara

2013-01-01

Fungi and oomycetes that colonize living plant tissue form extensive interfaces with plant cells in which the cytoplasm of the microorganism is closely aligned with the host cytoplasm for an extended distance. In all cases, specialized biotrophic hyphae function to hijack host cellular processes across an interfacial zone consisting of a hyphal plasma membrane, a specialized interfacial matrix, and a plant-derived membrane. The interface is the site of active secretion by both players. This cross talk at the interface determines the winner in adversarial relationships and establishes the partnership in mutualistic relationships. Fungi and oomycetes secrete many specialized effector proteins for controlling the host, and they can stimulate remarkable cellular reorganization even in distant plant cells. Breakthroughs in live-cell imaging of fungal and oomycete encounter sites, including live-cell imaging of pathogens secreting fluorescently labeled effector proteins, have led to recent progress in understanding communication across the interface.

Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex.

PubMed

Toogood, Helen S; van Thiel, Adam; Basran, Jaswir; Sutcliffe, Mike J; Scrutton, Nigel S; Leys, David

2004-07-30

The crystal structure of the human electron transferring flavoprotein (ETF).medium chain acyl-CoA dehydrogenase (MCAD) complex reveals a dual mode of protein-protein interaction, imparting both specificity and promiscuity in the interaction of ETF with a range of structurally distinct primary dehydrogenases. ETF partitions the functions of partner binding and electron transfer between (i) the recognition loop, which acts as a static anchor at the ETF.MCAD interface, and (ii) the highly mobile redox active FAD domain. Together, these enable the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. Disorders in amino acid or fatty acid catabolism can be attributed to mutations at the protein-protein interface. Crucially, complex formation triggers mobility of the FAD domain, an induced disorder that contrasts with general models of protein-protein interaction by induced fit mechanisms. The subsequent interfacial motion in the MCAD.ETF complex is the basis for the interaction of ETF with structurally diverse protein partners. Solution studies using ETF and MCAD with mutations at the protein-protein interface support this dynamic model and indicate ionic interactions between MCAD Glu(212) and ETF Arg alpha(249) are likely to transiently stabilize productive conformations of the FAD domain leading to enhanced electron transfer rates between both partners.

Single-molecule resolution of protein dynamics on polymeric membrane surfaces: the roles of spatial and population heterogeneity.

PubMed

Langdon, Blake B; Mirhossaini, Roya B; Mabry, Joshua N; Sriram, Indira; Lajmi, Ajay; Zhang, Yanxia; Rojas, Orlando J; Schwartz, Daniel K

2015-02-18

Although polymeric membranes are widely used in the purification of protein pharmaceuticals, interactions between biomolecules and membrane surfaces can lead to reduced membrane performance and damage to the product. In this study, single-molecule fluorescence microscopy provided direct observation of bovine serum albumin (BSA) and human monoclonal antibody (IgG) dynamics at the interface between aqueous buffer and polymeric membrane materials including regenerated cellulose and unmodified poly(ether sulfone) (PES) blended with either polyvinylpyrrolidone (PVP), polyvinyl acetate-co-polyvinylpyrrolidone (PVAc-PVP), or polyethylene glycol methacrylate (PEGM) before casting. These polymer surfaces were compared with model surfaces composed of hydrophilic bare fused silica and hydrophobic trimethylsilane-coated fused silica. At extremely dilute protein concentrations (10(-3)-10(-7) mg/mL), protein surface exchange was highly dynamic with protein monomers desorbing from the surface within ∼1 s after adsorption. Protein oligomers (e.g., nonspecific dimers, trimers, or larger aggregates), although less common, remained on the surface for 5 times longer than monomers. Using newly developed super-resolution methods, we could localize adsorption sites with ∼50 nm resolution and quantify the spatial heterogeneity of the various surfaces. On a small anomalous subset of the adsorption sites, proteins adsorbed preferentially and tended to reside for significantly longer times (i.e., on "strong" sites). Proteins resided for shorter times overall on surfaces that were more homogeneous and exhibited fewer strong sites (e.g., PVAc-PVP/PES). We propose that strong surface sites may nucleate protein aggregation, initiated preferentially by protein oligomers, and accelerate ultrafiltration membrane fouling. At high protein concentrations (0.3-1.0 mg/mL), fewer strong adsorption sites were observed, and surface residence times were reduced. This suggests that at high concentrations, adsorbed proteins block strong sites from further protein adsorption. Importantly, this demonstrates that strong binding sites can be modified by changing solution conditions. Membrane surfaces are intrinsically heterogeneous; by employing single-molecule techniques, we have provided a new framework for understanding protein interactions with such surfaces.

Three-dimensional stress field around a membrane protein: atomistic and coarse-grained simulation analysis of gramicidin A.

PubMed

Yoo, Jejoong; Cui, Qiang

2013-01-08

Using both atomistic and coarse-grained (CG) models, we compute the three-dimensional stress field around a gramicidin A (gA) dimer in lipid bilayers that feature different degrees of negative hydrophobic mismatch. The general trends in the computed stress field are similar at the atomistic and CG levels, supporting the use of the CG model for analyzing the mechanical features of protein/lipid/water interfaces. The calculations reveal that the stress field near the protein-lipid interface exhibits a layered structure with both significant repulsive and attractive regions, with the magnitude of the stress reaching 1000 bar in certain regions. Analysis of density profiles and stress field distributions helps highlight the Trp residues at the protein/membrane/water interface as mechanical anchors, suggesting that similar analysis is useful for identifying tension sensors in other membrane proteins, especially membrane proteins involved in mechanosensation. This work fosters a connection between microscopic and continuum mechanics models for proteins in complex environments and makes it possible to test the validity of assumptions commonly made in continuum mechanics models for membrane mediated processes. For example, using the calculated stress field, we estimate the free energy of membrane deformation induced by the hydrophobic mismatch, and the results for regions beyond the annular lipids are in general consistent with relevant experimental data and previous theoretical estimates using elasticity theory. On the other hand, the assumptions of homogeneous material properties for the membrane and a bilayer thickness at the protein/lipid interface being independent of lipid type (e.g., tail length) appear to be oversimplified, highlighting the importance of annular lipids of membrane proteins. Finally, the stress field analysis makes it clear that the effect of even rather severe hydrophobic mismatch propagates to only about two to three lipid layers, thus putting a limit on the range of cooperativity between membrane proteins in crowded cellular membranes. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Vibrational Stark effect spectroscopy at the interface of Ras and Rap1A bound to the Ras binding domain of RalGDS reveals an electrostatic mechanism for protein-protein interaction.

PubMed

Stafford, Amy J; Ensign, Daniel L; Webb, Lauren J

2010-11-25

Electrostatic fields at the interface of the Ras binding domain of the protein Ral guanine nucleotide dissociation stimulator (RalGDS) with the structurally analogous GTPases Ras and Rap1A were measured with vibrational Stark effect (VSE) spectroscopy. Eleven residues on the surface of RalGDS that participate in this protein-protein interaction were systematically mutated to cysteine and subsequently converted to cyanocysteine in order to introduce a nitrile VSE probe in the form of the thiocyanate (SCN) functional group. The measured SCN absorption energy on the monomeric protein was compared with solvent-accessible surface area (SASA) calculations and solutions to the Poisson-Boltzmann equation using Boltzmann-weighted structural snapshots from molecular dynamics simulations. We found a weak negative correlation between SASA and measured absorption energy, indicating that water exposure of protein surface amino acids can be estimated from experimental measurement of the magnitude of the thiocyanate absorption energy. We found no correlation between calculated field and measured absorption energy. These results highlight the complex structural and electrostatic nature of the protein-water interface. The SCN-labeled RalGDS was incubated with either wild-type Ras or wild-type Rap1A, and the formation of the docked complex was confirmed by measurement of the dissociation constant of the interaction. The change in absorption energy of the thiocyanate functional group due to complex formation was related to the change in electrostatic field experienced by the nitrile functional group when the protein-protein interface forms. At some locations, the nitrile experiences the same shift in field when bound to Ras and Rap1A, but at others, the change in field is dramatically different. These differences identify residues on the surface of RalGDS that direct the specificity of RalGDS binding to its in vivo binding partner, Rap1A, through an electrostatic mechanism.

Hot-spot analysis to dissect the functional protein-protein interface of a tRNA-modifying enzyme.

PubMed

Jakobi, Stephan; Nguyen, Tran Xuan Phong; Debaene, François; Metz, Alexander; Sanglier-Cianférani, Sarah; Reuter, Klaus; Klebe, Gerhard

2014-10-01

Interference with protein-protein interactions of interfaces larger than 1500 Ų by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization. © 2014 Wiley Periodicals, Inc.

Benchmarking protein-protein interface predictions: why you should care about protein size.

PubMed

Martin, Juliette

2014-07-01

A number of predictive methods have been developed to predict protein-protein binding sites. Each new method is traditionally benchmarked using sets of protein structures of various sizes, and global statistics are used to assess the quality of the prediction. Little attention has been paid to the potential bias due to protein size on these statistics. Indeed, small proteins involve proportionally more residues at interfaces than large ones. If a predictive method is biased toward small proteins, this can lead to an over-estimation of its performance. Here, we investigate the bias due to the size effect when benchmarking protein-protein interface prediction on the widely used docking benchmark 4.0. First, we simulate random scores that favor small proteins over large ones. Instead of the 0.5 AUC (Area Under the Curve) value expected by chance, these biased scores result in an AUC equal to 0.6 using hypergeometric distributions, and up to 0.65 using constant scores. We then use real prediction results to illustrate how to detect the size bias by shuffling, and subsequently correct it using a simple conversion of the scores into normalized ranks. In addition, we investigate the scores produced by eight published methods and show that they are all affected by the size effect, which can change their relative ranking. The size effect also has an impact on linear combination scores by modifying the relative contributions of each method. In the future, systematic corrections should be applied when benchmarking predictive methods using data sets with mixed protein sizes. © 2014 Wiley Periodicals, Inc.

Fragment-Based Protein-Protein Interaction Antagonists of a Viral Dimeric Protease.

PubMed

Gable, Jonathan E; Lee, Gregory M; Acker, Timothy M; Hulce, Kaitlin R; Gonzalez, Eric R; Schweigler, Patrick; Melkko, Samu; Farady, Christopher J; Craik, Charles S

2016-04-19

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

SnapDock—template-based docking by Geometric Hashing

PubMed Central

Estrin, Michael; Wolfson, Haim J.

2017-01-01

Abstract Motivation: A highly efficient template-based protein–protein docking algorithm, nicknamed SnapDock, is presented. It employs a Geometric Hashing-based structural alignment scheme to align the target proteins to the interfaces of non-redundant protein–protein interface libraries. Docking of a pair of proteins utilizing the 22 600 interface PIFACE library is performed in < 2 min on the average. A flexible version of the algorithm allowing hinge motion in one of the proteins is presented as well. Results: To evaluate the performance of the algorithm a blind re-modelling of 3547 PDB complexes, which have been uploaded after the PIFACE publication has been performed with success ratio of about 35%. Interestingly, a similar experiment with the template free PatchDock docking algorithm yielded a success rate of about 23% with roughly 1/3 of the solutions different from those of SnapDock. Consequently, the combination of the two methods gave a 42% success ratio. Availability and implementation: A web server of the application is under development. Contact: michaelestrin@gmail.com or wolfson@tau.ac.il PMID:28881968

Multistep building of a soft plant protein film at the air-water interface.

PubMed

Poirier, Alexandre; Banc, Amélie; Stocco, Antonio; In, Martin; Ramos, Laurence

2018-09-15

Gliadins are edible wheat storage proteins well known for their surface active properties. In this paper, we present experimental results on the interfacial properties of acidic solutions of gliadin studied over 5 decades of concentrations, from 0.001 to 110 g/L. Dynamic pendant drop tensiometry reveals that the surface pressure Π of gliadin solutions builds up in a multistep process. The series of curves of the time evolution of Π collected at different bulk protein concentrations C can be merged onto a single master curve when Π is plotted as a function of αt where t is the time elapsed since the formation of the air/water interface and α is a shift parameter that varies with C as a power law with an exponent 2. The existence of such time-concentration superposition, which we evidence for the first time, indicates that the same mechanisms govern the surface tension evolution at all concentrations and are accelerated by an increase of the bulk concentration. The scaling of α with C is consistent with a kinetic of adsorption controlled by the diffusion of the proteins in the bulk. Moreover, we show that the proteins adsorption at the air/water interface is kinetically irreversible. Correlated evolutions of the optical and elastic properties of the interfaces, as probed by ellipsometry and surface dilatational rheology respectively, provide a consistent physical picture of the building up of the protein interfacial layer. A progressive coverage of the interface by the proteins occurs at low Π. This stage is followed, at higher Π, by conformational rearrangements of the protein film, which are identified by a strong increase of the dissipative viscoelastic properties of the film concomitantly with a peculiar evolution of its optical profile that we have rationalized. In the last stage, at even higher surface pressure, the adsorption is arrested; the optical profile is not modified while the elasticity of the interfacial layer dramatically increases with the surface pressure, presumably due to the film ageing. Copyright © 2018 Elsevier Inc. All rights reserved.

A script to highlight hydrophobicity and charge on protein surfaces

PubMed Central

Hagemans, Dominique; van Belzen, Ianthe A. E. M.; Morán Luengo, Tania; Rüdiger, Stefan G. D.

2015-01-01

The composition of protein surfaces determines both affinity and specificity of protein-protein interactions. Matching of hydrophobic contacts and charged groups on both sites of the interface are crucial to ensure specificity. Here, we propose a highlighting scheme, YRB, which highlights both hydrophobicity and charge in protein structures. YRB highlighting visualizes hydrophobicity by highlighting all carbon atoms that are not bound to nitrogen and oxygen atoms. The charged oxygens of glutamate and aspartate are highlighted red and the charged nitrogens of arginine and lysine are highlighted blue. For a set of representative examples, we demonstrate that YRB highlighting intuitively visualizes segments on protein surfaces that contribute to specificity in protein-protein interfaces, including Hsp90/co-chaperone complexes, the SNARE complex and a transmembrane domain. We provide YRB highlighting in form of a script that runs using the software PyMOL. PMID:26528483

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for the Design of Functional Monomeric Proteins

PubMed Central

Joseph, Prem Raj B.; Poluri, Krishna Mohan; Gangavarapu, Pavani; Rajagopalan, Lavanya; Raghuwanshi, Sandeep; Richardson, Ricardo M.; Garofalo, Roberto P.; Rajarathnam, Krishna

2013-01-01

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins. PMID:24048001

In situ characterization of the brain-microdevice interface using Device Capture Histology

PubMed Central

Woolley, Andrew J.; Desai, Himanshi A.; Steckbeck, Mitchell A.; Patel, Neil K.; Otto, Kevin J.

2011-01-01

Accurate assessment of brain-implantable microdevice bio-integration remains a formidable challenge. Prevailing histological methods require device extraction prior to tissue processing, often disrupting and removing the tissue of interest which had been surrounding the device. The Device-Capture Histology method, presented here, overcomes many limitations of the conventional Device-Explant Histology method, by collecting the device and surrounding tissue intact for subsequent labeling. With the implant remaining in situ, accurate and precise imaging of the morphologically preserved tissue at the brain/microdevice interface can then be collected and quantified. First, this article presents the Device-Capture Histology method for obtaining and processing the intact, undisturbed microdevice-tissue interface, and images using fluorescent labeling and confocal microscopy. Second, this article gives examples of how to quantify features found in the captured peridevice tissue. We also share histological data capturing 1) the impact of microdevice implantation on tissue, 2) the effects of an experimental anti-inflammatory coating, 3) a dense grouping of cell nuclei encapsulating a long-term implant, and 4) atypical oligodendrocyte organization neighboring a longterm implant. Data sets collected using the Device-Capture Histology method are presented to demonstrate the significant advantages of processing the intact microdevice-tissue interface, and to underscore the utility of the method in understanding the effects of the brain-implantable microdevices on nearby tissue. PMID:21802446

Frictional interactions in forming processes: New studies with transparent sapphire strip-drawing dies

NASA Astrophysics Data System (ADS)

Rao, R. S.; Lu, C. Y.; Wright, P. K.; Devenpeck, M. L.; Richmond, O.; Appleby, E. J.

1982-05-01

This research is concerned with the frictional interactions at the toolwork interfaces in the machining and strip-drawing processes. A novel feature is that transparent sapphire (single crystal Al2O3) is being used as the tool and die material. This allows the tribological features of the interface to be directly observed and recorded on movie-film. These qualitative studies provide information on the role of lubricants. In addition, techniques are being developed to quantify the velocity gradient along the interface. For example, in the drawing work it has been found that tracer markings (e.g. dye-spots), applied to the undrawn strip, remain intact during drawing and can be tracked along the sapphire/strip interface. Such data will be used as input to a finite-element, elasto-plastic-workhardening model of the deformation process. The latter can compute strip deformation characteristics, drawing forces and local coefficients of friction at the interface. Introductory results will be presented in this paper, obtained from drawing tin-plated mild steel with sapphire and cemented carbide dies. Drawing loads and die-separating forces will be presented and movie-films of the action of tracer markings at the interface shown. In order to demonstrate how this data can be used in an analysis of a large strain deformation process with friction, initial results from running the FIPDEF elasto-plastic code will be discussed. From a commercial viewpoint research on strip-drawing is of special interest to the can-making industry. From a physical viewpoint stripdrawing is of particular interest because it is a symmetrical, plane strain deformation and, in comparison with other metal processing operations, it is more readily modeled. However, until now the elasto-plastic codes that have been developed to predictively model drawing have had limitations: the most notable being that of quantifying the friction conditions at the die-work interface. Hence the specification of the latter is the ultimate goal of this research program.

Tuning the Two-Dimensional Electron Liquid at Oxide Interfaces by Buffer-Layer-Engineered Redox Reactions.

PubMed

Chen, Yunzhong; Green, Robert J; Sutarto, Ronny; He, Feizhou; Linderoth, Søren; Sawatzky, George A; Pryds, Nini

2017-11-08

Polar discontinuities and redox reactions provide alternative paths to create two-dimensional electron liquids (2DELs) at oxide interfaces. Herein, we report high mobility 2DELs at interfaces involving SrTiO 3 (STO) achieved using polar La 7/8 Sr 1/8 MnO 3 (LSMO) buffer layers to manipulate both polarities and redox reactions from disordered overlayers grown at room temperature. Using resonant X-ray reflectometry experiments, we quantify redox reactions from oxide overlayers on STO as well as polarity induced electronic reconstruction at epitaxial LSMO/STO interfaces. The analysis reveals how these effects can be combined in a STO/LSMO/disordered film trilayer system to yield high mobility modulation doped 2DELs, where the buffer layer undergoes a partial transformation from perovskite to brownmillerite structure. This uncovered interplay between polar discontinuities and redox reactions via buffer layers provides a new approach for the design of functional oxide interfaces.

PIA: An Intuitive Protein Inference Engine with a Web-Based User Interface.

PubMed

Uszkoreit, Julian; Maerkens, Alexandra; Perez-Riverol, Yasset; Meyer, Helmut E; Marcus, Katrin; Stephan, Christian; Kohlbacher, Oliver; Eisenacher, Martin

2015-07-02

Protein inference connects the peptide spectrum matches (PSMs) obtained from database search engines back to proteins, which are typically at the heart of most proteomics studies. Different search engines yield different PSMs and thus different protein lists. Analysis of results from one or multiple search engines is often hampered by different data exchange formats and lack of convenient and intuitive user interfaces. We present PIA, a flexible software suite for combining PSMs from different search engine runs and turning these into consistent results. PIA can be integrated into proteomics data analysis workflows in several ways. A user-friendly graphical user interface can be run either locally or (e.g., for larger core facilities) from a central server. For automated data processing, stand-alone tools are available. PIA implements several established protein inference algorithms and can combine results from different search engines seamlessly. On several benchmark data sets, we show that PIA can identify a larger number of proteins at the same protein FDR when compared to that using inference based on a single search engine. PIA supports the majority of established search engines and data in the mzIdentML standard format. It is implemented in Java and freely available at https://github.com/mpc-bioinformatics/pia.

A multiprotein binding interface in an intrinsically disordered region of the tumor suppressor protein interferon regulatory factor-1.

PubMed

Narayan, Vikram; Halada, Petr; Hernychová, Lenka; Chong, Yuh Ping; Žáková, Jitka; Hupp, Ted R; Vojtesek, Borivoj; Ball, Kathryn L

2011-04-22

The interferon-regulated transcription factor and tumor suppressor protein IRF-1 is predicted to be largely disordered outside of the DNA-binding domain. One of the advantages of intrinsically disordered protein domains is thought to be their ability to take part in multiple, specific but low affinity protein interactions; however, relatively few IRF-1-interacting proteins have been described. The recent identification of a functional binding interface for the E3-ubiquitin ligase CHIP within the major disordered domain of IRF-1 led us to ask whether this region might be employed more widely by regulators of IRF-1 function. Here we describe the use of peptide aptamer-based affinity chromatography coupled with mass spectrometry to define a multiprotein binding interface on IRF-1 (Mf2 domain; amino acids 106-140) and to identify Mf2-binding proteins from A375 cells. Based on their function as known transcriptional regulators, a selection of the Mf2 domain-binding proteins (NPM1, TRIM28, and YB-1) have been validated using in vitro and cell-based assays. Interestingly, although NPM1, TRIM28, and YB-1 all bind to the Mf2 domain, they have differing amino acid specificities, demonstrating the degree of combinatorial diversity and specificity available through linear interaction motifs.

Spatial Competition: Roughening of an Experimental Interface.

PubMed

Allstadt, Andrew J; Newman, Jonathan A; Walter, Jonathan A; Korniss, G; Caraco, Thomas

2016-07-28

Limited dispersal distance generates spatial aggregation. Intraspecific interactions are then concentrated within clusters, and between-species interactions occur near cluster boundaries. Spread of a locally dispersing invader can become motion of an interface between the invading and resident species, and spatial competition will produce variation in the extent of invasive advance along the interface. Kinetic roughening theory offers a framework for quantifying the development of these fluctuations, which may structure the interface as a self-affine fractal, and so induce a series of temporal and spatial scaling relationships. For most clonal plants, advance should become spatially correlated along the interface, and width of the interface (where invader and resident compete directly) should increase as a power function of time. Once roughening equilibrates, interface width and the relative location of the most advanced invader should each scale with interface length. We tested these predictions by letting white clover (Trifolium repens) invade ryegrass (Lolium perenne). The spatial correlation of clover growth developed as anticipated by kinetic roughening theory, and both interface width and the most advanced invader's lead scaled with front length. However, the scaling exponents differed from those predicted by recent simulation studies, likely due to clover's growth morphology.

Spatial Competition: Roughening of an Experimental Interface

PubMed Central

Allstadt, Andrew J.; Newman, Jonathan A.; Walter, Jonathan A.; Korniss, G.; Caraco, Thomas

2016-01-01

Limited dispersal distance generates spatial aggregation. Intraspecific interactions are then concentrated within clusters, and between-species interactions occur near cluster boundaries. Spread of a locally dispersing invader can become motion of an interface between the invading and resident species, and spatial competition will produce variation in the extent of invasive advance along the interface. Kinetic roughening theory offers a framework for quantifying the development of these fluctuations, which may structure the interface as a self-affine fractal, and so induce a series of temporal and spatial scaling relationships. For most clonal plants, advance should become spatially correlated along the interface, and width of the interface (where invader and resident compete directly) should increase as a power function of time. Once roughening equilibrates, interface width and the relative location of the most advanced invader should each scale with interface length. We tested these predictions by letting white clover (Trifolium repens) invade ryegrass (Lolium perenne). The spatial correlation of clover growth developed as anticipated by kinetic roughening theory, and both interface width and the most advanced invader’s lead scaled with front length. However, the scaling exponents differed from those predicted by recent simulation studies, likely due to clover’s growth morphology. PMID:27465518

Electrochemical impedance spectroscopy for quantitative interface state characterization of planar and nanostructured semiconductor-dielectric interfaces

NASA Astrophysics Data System (ADS)

Meng, Andrew C.; Tang, Kechao; Braun, Michael R.; Zhang, Liangliang; McIntyre, Paul C.

2017-10-01

The performance of nanostructured semiconductors is frequently limited by interface defects that trap electronic carriers. In particular, high aspect ratio geometries dramatically increase the difficulty of using typical solid-state electrical measurements (multifrequency capacitance- and conductance-voltage testing) to quantify interface trap densities (D it). We report on electrochemical impedance spectroscopy (EIS) to characterize the energy distribution of interface traps at metal oxide/semiconductor interfaces. This method takes advantage of liquid electrolytes, which provide conformal electrical contacts. Planar Al2O3/p-Si and Al2O3/p-Si0.55Ge0.45 interfaces are used to benchmark the EIS data against results obtained from standard electrical testing methods. We find that the solid state and EIS data agree very well, leading to the extraction of consistent D it energy distributions. Measurements carried out on pyramid-nanostructured p-Si obtained by KOH etching followed by deposition of a 10 nm ALD-Al2O3 demonstrate the application of EIS to trap characterization of a nanostructured dielectric/semiconductor interface. These results show the promise of this methodology to measure interface state densities for a broad range of semiconductor nanostructures such as nanowires, nanofins, and porous structures.

Rate theory of ion pairing at the water liquid-vapor interface: A case of sodium iodide.

PubMed

Dang, Liem X; Schenter, Gregory K

2018-06-14

Studies on ion pairing at interfaces have been intensified recently because of their importance in many chemical reactive phenomena, such as ion-ion interactions that are affected by interfaces and their influence on kinetic processes. In this study, we performed simulations to examine the thermodynamics and kinetics of small polarizable sodium iodide ions in the bulk and near the water liquid-vapor interface. Using classical transition state theory, we calculated the dissociation rates and corrected them with transmission coefficients obtained from the reactive flux formalism and Grote-Hynes theory. Our results show that in addition to affecting the free energy of ions in solution, the interfacial environments significantly influence the kinetics of ion pairing. The results on the relaxation time obtained using the reactive flux formalism and Grote-Hynes theory present an unequivocal picture that the interface suppresses ion dissociation. The effects of the use of molecular models on the ion interactions as well as the ion-pair configurations at the interface are also quantified and discussed.

Rate theory of ion pairing at the water liquid-vapor interface: A case of sodium iodide

NASA Astrophysics Data System (ADS)

Dang, Liem X.; Schenter, Gregory K.

2018-06-01

Studies on ion pairing at interfaces have been intensified recently because of their importance in many chemical reactive phenomena, such as ion-ion interactions that are affected by interfaces and their influence on kinetic processes. In this study, we performed simulations to examine the thermodynamics and kinetics of small polarizable sodium iodide ions in the bulk and near the water liquid-vapor interface. Using classical transition state theory, we calculated the dissociation rates and corrected them with transmission coefficients obtained from the reactive flux formalism and Grote-Hynes theory. Our results show that in addition to affecting the free energy of ions in solution, the interfacial environments significantly influence the kinetics of ion pairing. The results on the relaxation time obtained using the reactive flux formalism and Grote-Hynes theory present an unequivocal picture that the interface suppresses ion dissociation. The effects of the use of molecular models on the ion interactions as well as the ion-pair configurations at the interface are also quantified and discussed.

The Excess Chemical Potential of Water at the Interface with a Protein from End Point Simulations.

PubMed

Zhang, Bin W; Cui, Di; Matubayasi, Nobuyuki; Levy, Ronald M

2018-05-03

We use end point simulations to estimate the excess chemical potential of water in the homogeneous liquid and at the interface with a protein in solution. When the pure liquid is taken as the reference, the excess chemical potential of interfacial water is the difference between the solvation free energy of a water molecule at the interface and in the bulk. Using the homogeneous liquid as an example, we show that the solvation free energy for growing a water molecule can be estimated by applying UWHAM to the simulation data generated from the initial and final states (i.e., "the end points") instead of multistate free energy perturbation simulations because of the possible overlaps of the configurations sampled at the end points. Then end point simulations are used to estimate the solvation free energy of water at the interface with a protein in solution. The estimate of the solvation free energy at the interface from two simulations at the end points agrees with the benchmark using 32 states within a 95% confidence interval for most interfacial locations. The ability to accurately estimate the excess chemical potential of water from end point simulations facilitates the statistical thermodynamic analysis of diverse interfacial phenomena. Our focus is on analyzing the excess chemical potential of water at protein receptor binding sites with the goal of using this information to assist in the design of tight binding ligands.

Vibrational energy flow through the green fluorescent protein-water interface: communication maps and thermal boundary conductance.

PubMed

Xu, Yao; Leitner, David M

2014-07-17

We calculate communication maps for green fluorescent protein (GFP) to elucidate energy transfer pathways between the chromophore and other parts of the protein in the ground and excited state. The approach locates energy transport channels from the chromophore to remote regions of the protein via residues and water molecules that hydrogen bond to the chromophore. We calculate the thermal boundary conductance between GFP and water over a wide range of temperature and find that the interface between the protein and the cluster of water molecules in the β-barrel poses negligible resistance to thermal flow, consistent with facile vibrational energy transfer from the chromophore to the β-barrel waters observed in the communication maps.

Interleukin-4 and interleukin-13 compromise the sinonasal epithelial barrier and perturb intercellular junction protein expression.

PubMed

Wise, Sarah K; Laury, Adrienne M; Katz, Elizabeth H; Den Beste, Kyle A; Parkos, Charles A; Nusrat, Asma

2014-05-01

Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a "leaky" epithelial barrier phenotype. We hypothesize that T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13 modulate epithelial junction proteins, thereby contributing to the leaky epithelial barrier. Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n = 6) and 68.6% (n = 8) of baseline, respectively. Tight junction protein junctional adhesion molecule-A (JAM-A) expression decreased 42.2% with IL-4 exposure (n = 9) and 37.5% with IL-13 exposure (n = 9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n = 9) and 32.9% with IL-13 exposure (n = 9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or zonula occludens-1 (ZO-1) with IL-4 or IL-13 exposure. Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity. © 2014 ARS-AAOA, LLC.

System-Level and Granger Network Analysis of Integrated Proteomic and Metabolomic Dynamics Identifies Key Points of Grape Berry Development at the Interface of Primary and Secondary Metabolism.

PubMed

Wang, Lei; Sun, Xiaoliang; Weiszmann, Jakob; Weckwerth, Wolfram

2017-01-01

Grapevine is a fruit crop with worldwide economic importance. The grape berry undergoes complex biochemical changes from fruit set until ripening. This ripening process and production processes define the wine quality. Thus, a thorough understanding of berry ripening is crucial for the prediction of wine quality. For a systemic analysis of grape berry development we applied mass spectrometry based platforms to analyse the metabolome and proteome of Early Campbell at 12 stages covering major developmental phases. Primary metabolites involved in central carbon metabolism, such as sugars, organic acids and amino acids together with various bioactive secondary metabolites like flavonols, flavan-3-ols and anthocyanins were annotated and quantified. At the same time, the proteomic analysis revealed the protein dynamics of the developing grape berries. Multivariate statistical analysis of the integrated metabolomic and proteomic dataset revealed the growth trajectory and corresponding metabolites and proteins contributing most to the specific developmental process. K-means clustering analysis revealed 12 highly specific clusters of co-regulated metabolites and proteins. Granger causality network analysis allowed for the identification of time-shift correlations between metabolite-metabolite, protein- protein and protein-metabolite pairs which is especially interesting for the understanding of developmental processes. The integration of metabolite and protein dynamics with their corresponding biochemical pathways revealed an energy-linked metabolism before veraison with high abundances of amino acids and accumulation of organic acids, followed by protein and secondary metabolite synthesis. Anthocyanins were strongly accumulated after veraison whereas other flavonoids were in higher abundance at early developmental stages and decreased during the grape berry developmental processes. A comparison of the anthocyanin profile of Early Campbell to other cultivars revealed similarities to Concord grape and indicates the strong effect of genetic background on metabolic partitioning in primary and secondary metabolism.

System-Level and Granger Network Analysis of Integrated Proteomic and Metabolomic Dynamics Identifies Key Points of Grape Berry Development at the Interface of Primary and Secondary Metabolism

PubMed Central

Wang, Lei; Sun, Xiaoliang; Weiszmann, Jakob; Weckwerth, Wolfram

2017-01-01

Grapevine is a fruit crop with worldwide economic importance. The grape berry undergoes complex biochemical changes from fruit set until ripening. This ripening process and production processes define the wine quality. Thus, a thorough understanding of berry ripening is crucial for the prediction of wine quality. For a systemic analysis of grape berry development we applied mass spectrometry based platforms to analyse the metabolome and proteome of Early Campbell at 12 stages covering major developmental phases. Primary metabolites involved in central carbon metabolism, such as sugars, organic acids and amino acids together with various bioactive secondary metabolites like flavonols, flavan-3-ols and anthocyanins were annotated and quantified. At the same time, the proteomic analysis revealed the protein dynamics of the developing grape berries. Multivariate statistical analysis of the integrated metabolomic and proteomic dataset revealed the growth trajectory and corresponding metabolites and proteins contributing most to the specific developmental process. K-means clustering analysis revealed 12 highly specific clusters of co-regulated metabolites and proteins. Granger causality network analysis allowed for the identification of time-shift correlations between metabolite-metabolite, protein- protein and protein-metabolite pairs which is especially interesting for the understanding of developmental processes. The integration of metabolite and protein dynamics with their corresponding biochemical pathways revealed an energy-linked metabolism before veraison with high abundances of amino acids and accumulation of organic acids, followed by protein and secondary metabolite synthesis. Anthocyanins were strongly accumulated after veraison whereas other flavonoids were in higher abundance at early developmental stages and decreased during the grape berry developmental processes. A comparison of the anthocyanin profile of Early Campbell to other cultivars revealed similarities to Concord grape and indicates the strong effect of genetic background on metabolic partitioning in primary and secondary metabolism. PMID:28713396

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.

Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoring.

PubMed

Lange, Vinzenz; Malmström, Johan A; Didion, John; King, Nichole L; Johansson, Björn P; Schäfer, Juliane; Rameseder, Jonathan; Wong, Chee-Hong; Deutsch, Eric W; Brusniak, Mi-Youn; Bühlmann, Peter; Björck, Lars; Domon, Bruno; Aebersold, Ruedi

2008-08-01

In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors*

PubMed Central

Aurass, Philipp; Gerlach, Thomas; Becher, Dörte; Voigt, Birgit; Karste, Susanne; Bernhardt, Jörg; Riedel, Katharina; Hecker, Michael; Flieger, Antje

2016-01-01

Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phase-specific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests their phase specific function. The distinct temporal or spatial presence of such proteins might have important implications for functional assignments in the future or for use as life-stage specific markers for pathogen analysis. PMID:26545400

Deletion of internal structured repeats increases the stability of a leucine-rich repeat protein, YopM

PubMed Central

Barrick, Doug

2011-01-01

Mapping the stability distributions of proteins in their native folded states provides a critical link between structure, thermodynamics, and function. Linear repeat proteins have proven more amenable to this kind of mapping than globular proteins. C-terminal deletion studies of YopM, a large, linear leucine-rich repeat (LRR) protein, show that stability is distributed quite heterogeneously, yet a high level of cooperativity is maintained [1]. Key components of this distribution are three interfaces that strongly stabilize adjacent sequences, thereby maintaining structural integrity and promoting cooperativity. To better understand the distribution of interaction energy around these critical interfaces, we studied internal (rather than terminal) deletions of three LRRs in this region, including one of these stabilizing interfaces. Contrary to our expectation that deletion of structured repeats should be destabilizing, we find that internal deletion of folded repeats can actually stabilize the native state, suggesting that these repeats are destabilizing, although paradoxically, they are folded in the native state. We identified two residues within this destabilizing segment that deviate from the consensus sequence at a position that normally forms a stacked leucine ladder in the hydrophobic core. Replacement of these nonconsensus residues with leucine is stabilizing. This stability enhancement can be reproduced in the context of nonnative interfaces, but it requires an extended hydrophobic core. Our results demonstrate that different LRRs vary widely in their contribution to stability, and that this variation is context-dependent. These two factors are likely to determine the types of rearrangements that lead to folded, functional proteins, and in turn, are likely to restrict the pathways available for the evolution of linear repeat proteins. PMID:21764506

Probing binding hot spots at protein-RNA recognition sites.

PubMed

Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

2016-01-29

We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Amino Acid Interaction (INTAA) web server.

PubMed

Galgonek, Jakub; Vymetal, Jirí; Jakubec, David; Vondrášek, Jirí

2017-07-03

Large biomolecules-proteins and nucleic acids-are composed of building blocks which define their identity, properties and binding capabilities. In order to shed light on the energetic side of interactions of amino acids between themselves and with deoxyribonucleotides, we present the Amino Acid Interaction web server (http://bioinfo.uochb.cas.cz/INTAA/). INTAA offers the calculation of the residue Interaction Energy Matrix for any protein structure (deposited in Protein Data Bank or submitted by the user) and a comprehensive analysis of the interfaces in protein-DNA complexes. The Interaction Energy Matrix web application aims to identify key residues within protein structures which contribute significantly to the stability of the protein. The application provides an interactive user interface enhanced by 3D structure viewer for efficient visualization of pairwise and net interaction energies of individual amino acids, side chains and backbones. The protein-DNA interaction analysis part of the web server allows the user to view the relative abundance of various configurations of amino acid-deoxyribonucleotide pairs found at the protein-DNA interface and the interaction energies corresponding to these configurations calculated using a molecular mechanical force field. The effects of the sugar-phosphate moiety and of the dielectric properties of the solvent on the interaction energies can be studied for the various configurations. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A decision framework for identifying models to estimate forest ecosystem services gains from restoration

USGS Publications Warehouse

Christin, Zachary; Bagstad, Kenneth J.; Verdone, Michael

2016-01-01

Restoring degraded forests and agricultural lands has become a global conservation priority. A growing number of tools can quantify ecosystem service tradeoffs associated with forest restoration. This evolving “tools landscape” presents a dilemma: more tools are available, but selecting appropriate tools has become more challenging. We present a Restoration Ecosystem Service Tool Selector (RESTS) framework that describes key characteristics of 13 ecosystem service assessment tools. Analysts enter information about their decision context, services to be analyzed, and desired outputs. Tools are filtered and presented based on five evaluative criteria: scalability, cost, time requirements, handling of uncertainty, and applicability to benefit-cost analysis. RESTS uses a spreadsheet interface but a web-based interface is planned. Given the rapid evolution of ecosystem services science, RESTS provides an adaptable framework to guide forest restoration decision makers toward tools that can help quantify ecosystem services in support of restoration.

Parametric Modeling of the Safety Effects of NextGen Terminal Maneuvering Area Conflict Scenarios

NASA Technical Reports Server (NTRS)

Rogers, William H.; Waldron, Timothy P.; Stroiney, Steven R.

2011-01-01

The goal of this work was to analytically identify and quantify the issues, challenges, technical hurdles, and pilot-vehicle interface issues associated with conflict detection and resolution (CD&R)in emerging operational concepts for a NextGen terminal aneuvering area, including surface operations. To this end, the work entailed analytical and trade studies focused on modeling the achievable safety benefits of different CD&R strategies and concepts in the current and future airport environment. In addition, crew-vehicle interface and pilot performance enhancements and potential issues were analyzed based on review of envisioned NextGen operations, expected equipage advances, and human factors expertise. The results of perturbation analysis, which quantify the high-level performance impact of changes to key parameters such as median response time and surveillance position error, show that the analytical model developed could be useful in making technology investment decisions.

Symmetry and lattice mismatch induced strain accommodation near and away from correlated perovskite interfaces

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

Vailionis, A.; Boschker, H.; Max Planck Institute for Solid State Research, 70569 Stuttgart

2014-09-29

Distinct MnO{sub 6} octahedral distortions near and away from the La{sub 0.67}Sr{sub 0.33}MnO{sub 3}/SrTiO{sub 3}(001) (LSMO/STO) interface are quantified using synchrotron x-ray diffraction and dynamical x-ray diffraction simulations. Three structural regions of stress accommodation throughout the film thickness were resolved: near the LSMO/STO interface, intermediate region farther from the interface, and the main layer away from the interface. The results show that within the first two unit cells stress is accommodated by the suppression of octahedral rotations in the film, leading to the expansion of the c-axis lattice parameter. Farther from the interface film structure acquires octahedral tilts similar tomore » thicker perovskite films under tensile stress, leading to a reduced c-axis parameter. We demonstrate that these regions are related to two different strain coupling mechanisms: symmetry mismatch at the interface and lattice mismatch in the rest of the film. The findings suggest new routes for strain engineering in correlated perovskite heterostructures.« less

Critical Comparison of Biomembrane Force Fields: Protein-Lipid Interactions at the Membrane Interface.

PubMed

Sandoval-Perez, Angelica; Pluhackova, Kristyna; Böckmann, Rainer A

2017-05-09

Molecular dynamics (MD) simulations offer the possibility to study biological processes at high spatial and temporal resolution often not reachable by experiments. Corresponding biomolecular force field parameters have been developed for a wide variety of molecules ranging from inorganic ligands and small organic molecules over proteins and lipids to nucleic acids. Force fields have typically been parametrized and validated on thermodynamic observables and structural characteristics of individual compounds, e.g. of soluble proteins or lipid bilayers. Less strictly, due to the added complexity and missing experimental data to compare to, force fields have hardly been tested on the properties of mixed systems, e.g. on protein-lipid systems. Their selection and combination for mixed systems is further complicated by the partially differing parametrization strategies. Additionally, the presence of other compounds in the system may shift the subtle balance of force field parameters. Here, we assessed the protein-lipid interactions as described in the four atomistic force fields GROMOS54a7, CHARMM36 and the two force field combinations Amber14sb/Slipids and Amber14sb/Lipid14. Four observables were compared, focusing on the membrane-water interface: the conservation of the secondary structure of transmembrane proteins, the positioning of transmembrane peptides relative to the lipid bilayer, the insertion depth of side chains of unfolded peptides absorbed at the membrane interface, and the ability to reproduce experimental insertion energies of Wimley-White peptides at the membrane interface. Significant differences between the force fields were observed that affect e.g. membrane insertion depths and tilting of transmembrane peptides.

Characterizing hydrophobicity at the nanoscale: a molecular dynamics simulation study.

PubMed

Bandyopadhyay, Dibyendu; Choudhury, Niharendu

2012-06-14

We use molecular dynamics (MD) simulations of water near nanoscopic surfaces to characterize hydrophobic solute-water interfaces. By using nanoscopic paraffin like plates as model solutes, MD simulations in isothermal-isobaric ensemble have been employed to identify characteristic features of such an interface. Enhanced water correlation, density fluctuations, and position dependent compressibility apart from surface specific hydrogen bond distribution and molecular orientations have been identified as characteristic features of such interfaces. Tetrahedral order parameter that quantifies the degree of tetrahedrality in the water structure and an orientational order parameter, which quantifies the orientational preferences of the second solvation shell water around a central water molecule, have also been calculated as a function of distance from the plate surface. In the vicinity of the surface these two order parameters too show considerable sensitivity to the surface hydrophobicity. The potential of mean force (PMF) between water and the surface as a function of the distance from the surface has also been analyzed in terms of direct interaction and induced contribution, which shows unusual effect of plate hydrophobicity on the solvent induced PMF. In order to investigate hydrophobic nature of these plates, we have also investigated interplate dewetting when two such plates are immersed in water.

Pattern formation study of dissolution-driven convection

NASA Astrophysics Data System (ADS)

Aljahdaly, Noufe; Hadji, Layachi

2017-11-01

A three-dimensional pattern formation analysis is performed to investigate the dissolution-driven convection induced by the sequestration of carbon dioxide. We model this situation by considering a Rayleigh-Taylor like base state consisting of carbon-rich heavy brine overlying a carbon-free layer and seek, through a linear stability analysis, the instability threshold conditions as function of the thickness of the CO2-rich brine layer. Our model accounts for carbon diffusion anisotropy, permeability dependence on depth and the presence of a first order chemical reaction between the carbon-rich brine and host mineralogy. A small amplitude nonlinear stability analysis is performed to isolate the preferred regular pattern and solute flux conditions at the interface. The latter are used to derive equations for the time and space evolution of the interface as it migrates upward. We quantify the terminal time when the interface reaches the top boundary as function of the type of solute boundary conditions at the top boundary thereby also quantifying the beginning of the shutdown regime. The analysis will also shed light on the development of the three-dimensional fingering pattern that is observed when the constant flux regime is attained.

Enzymatic biofuel cell-based self-powered biosensing of protein kinase activity and inhibition via thiophosphorylation-mediated interface engineering.

PubMed

Gu, Chengcheng; Gai, Panpan; Han, Lei; Yu, Wen; Liu, Qingyun; Li, Feng

2018-05-24

We developed a facile and ultrasensitive enzymatic biofuel cell (EBFC)-based self-powered biosensor of protein kinase A (PKA) activity and inhibition via thiophosphorylation-mediated interface engineering. The detection limit was down to 0.00022 U mL-1 (S/N = 3). In addition, the PKA activities from MCF-7 and A549 cell lysates were analyzed and achieved reliable results.

Evaluation of a commercial electro-kinetically pumped sheath-flow nanospray interface coupled to an automated capillary zone electrophoresis system.

PubMed

Peuchen, Elizabeth H; Zhu, Guije; Sun, Liangliang; Dovichi, Norman J

2017-03-01

Capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) is attracting renewed attention for proteomic and metabolomic analysis. An important reason for this interest is the maturation and commercialization of interfaces for coupling CZE with ESI-MS. One of these interfaces is an electro-kinetically pumped sheath flow nanospray interface developed by the Dovichi group, in which a very low sheath flow is generated based on electroosmosis within a glass emitter. CMP Scientific has commercialized this interface as the EMASS-II ion source. In this work, we compared the performance of the EMASS-II ion source with our in-house system. The performance of the systems is equivalent. We also coupled the EMASS-II ion source with a PrinCE Next|480 capillary electrophoresis autosampler and an Orbitrap mass spectrometer, and analyzed this system's performance in terms of sensitivity, reproducibility, and separation performance for separation of tryptic digests, intact proteins, and amino acids. The system produced reproducible analysis of BSA digest; the RSDs of peptide intensity and migration time across 24 runs were less than 20 and 6%, respectively. The system produced a linear calibration curve of intensity across a 30-fold range of tryptic digest concentration. The combination of a commercial autosampler and electrospray interface efficiently separated amino acids, peptides, and intact proteins, and only required 5 μL of sample for analysis. Graphical Abstract The commercial and locally constructed versions of the interface provide similar numbers of protein identifications from a Xenopus laevis fertilized egg digest.

The dopamine D2 receptor dimer and its interaction with homobivalent antagonists: homology modeling, docking and molecular dynamics.

PubMed

Kaczor, Agnieszka A; Jörg, Manuela; Capuano, Ben

2016-09-01

In order to apply structure-based drug design techniques to G protein-coupled receptor complexes, it is essential to model their 3D structure and to identify regions that are suitable for selective drug binding. For this purpose, we have developed and tested a multi-component protocol to model the inactive conformation of the dopamine D2 receptor dimer, suitable for interaction with homobivalent antagonists. Our approach was based on protein-protein docking, applying the Rosetta software to obtain populations of dimers as present in membranes with all the main possible interfaces. Consensus scoring based on the values and frequencies of best interfaces regarding four scoring parameters, Rosetta interface score, interface area, free energy of binding and energy of hydrogen bond interactions indicated that the best scored dimer model possesses a TM4-TM5-TM7-TM1 interface, which is in agreement with experimental data. This model was used to study interactions of the previously published dopamine D2 receptor homobivalent antagonists based on clozapine,1,4-disubstituted aromatic piperidines/piperazines and arylamidoalkyl substituted phenylpiperazine pharmacophores. It was found that the homobivalent antagonists stabilize the receptor-inactive conformation by maintaining the ionic lock interaction, and change the dimer interface by disrupting a set of hydrogen bonds and maintaining water- and ligand-mediated hydrogen bonds in the extracellular and intracellular part of the interface. Graphical Abstract Structure of the final model of the dopamine D2 receptor homodimer, indicating the distancebetween Tyr37 and Tyr 5.42 in the apo form (left) and in the complex with the ligand (right).

AlphaSpace: Fragment-Centric Topographical Mapping To Target Protein–Protein Interaction Interfaces

PubMed Central

2016-01-01

Inhibition of protein–protein interactions (PPIs) is emerging as a promising therapeutic strategy despite the difficulty in targeting such interfaces with drug-like small molecules. PPIs generally feature large and flat binding surfaces as compared to typical drug targets. These features pose a challenge for structural characterization of the surface using geometry-based pocket-detection methods. An attractive mapping strategy—that builds on the principles of fragment-based drug discovery (FBDD)—is to detect the fragment-centric modularity at the protein surface and then characterize the large PPI interface as a set of localized, fragment-targetable interaction regions. Here, we introduce AlphaSpace, a computational analysis tool designed for fragment-centric topographical mapping (FCTM) of PPI interfaces. Our approach uses the alpha sphere construct, a geometric feature of a protein’s Voronoi diagram, to map out concave interaction space at the protein surface. We introduce two new features—alpha-atom and alpha-space—and the concept of the alpha-atom/alpha-space pair to rank pockets for fragment-targetability and to facilitate the evaluation of pocket/fragment complementarity. The resulting high-resolution interfacial map of targetable pocket space can be used to guide the rational design and optimization of small molecule or biomimetic PPI inhibitors. PMID:26225450

Manufacture and characterisation of EmDerm-novel hierarchically structured bio-active scaffolds for tissue regeneration.

PubMed

Lim, Xuxin; Potter, Matthew; Cui, Zhanfeng; Dye, Julian F

2018-06-05

There are significant challenges for using emulsion templating as a method of manufacturing macro-porous protein scaffolds. Issues include protein denaturation by adsorption at hydrophobic interfaces, emulsion instability, oil droplet and surfactant removal after protein gelation, and compatible cross-linking methods. We investigated an oil-in-water macro-emulsion stabilised with a surfactant blend, as a template for manufacturing protein-based nano-structured bio-intelligent scaffolds (EmDerm) with tuneable micro-scale porosity for tissue regeneration. Prototype EmDerm scaffolds were made using either collagen, through thermal gelation, fibrin, through enzymatic coagulation or collagen-fibrin composite. Pore size was controlled via surfactant-to-oil phase ratio. Scaffolds were crosslink-stabilised with EDC/NHS for varying durations. Scaffold micro-architecture and porosity were characterised with SEM, and mechanical properties by tensiometry. Hydrolytic and proteolytic degradation profiles were quantified by mass decrease over time. Human dermal fibroblasts, endothelial cells and bone marrow derived mesenchymal stem cells were used to investigate cytotoxicity and cell proliferation within each scaffold. EmDerm scaffolds showed nano-scale based hierarchical structures, with mean pore diameters ranging from 40-100 microns. The Young's modulus range was 1.1-2.9 MPa, and ultimate tensile strength was 4-16 MPa. Degradation rate was related to cross-linking duration. Each EmDerm scaffold supported excellent cell ingress and proliferation compared to the reference materials Integra™ and Matriderm™. Emulsion templating is a novel rapid method of fabricating nano-structured fibrous protein scaffolds with micro-scale pore dimensions. These scaffolds hold promising clinical potential for regeneration of the dermis and other soft tissues, e.g., for burns or chronic wound therapies.

MetaPro-IQ: a universal metaproteomic approach to studying human and mouse gut microbiota.

PubMed

Zhang, Xu; Ning, Zhibin; Mayne, Janice; Moore, Jasmine I; Li, Jennifer; Butcher, James; Deeke, Shelley Ann; Chen, Rui; Chiang, Cheng-Kang; Wen, Ming; Mack, David; Stintzi, Alain; Figeys, Daniel

2016-06-24

The gut microbiota has been shown to be closely associated with human health and disease. While next-generation sequencing can be readily used to profile the microbiota taxonomy and metabolic potential, metaproteomics is better suited for deciphering microbial biological activities. However, the application of gut metaproteomics has largely been limited due to the low efficiency of protein identification. Thus, a high-performance and easy-to-implement gut metaproteomic approach is required. In this study, we developed a high-performance and universal workflow for gut metaproteome identification and quantification (named MetaPro-IQ) by using the close-to-complete human or mouse gut microbial gene catalog as database and an iterative database search strategy. An average of 38 and 33 % of the acquired tandem mass spectrometry (MS) spectra was confidently identified for the studied mouse stool and human mucosal-luminal interface samples, respectively. In total, we accurately quantified 30,749 protein groups for the mouse metaproteome and 19,011 protein groups for the human metaproteome. Moreover, the MetaPro-IQ approach enabled comparable identifications with the matched metagenome database search strategy that is widely used but needs prior metagenomic sequencing. The response of gut microbiota to high-fat diet in mice was then assessed, which showed distinct metaproteome patterns for high-fat-fed mice and identified 849 proteins as significant responders to high-fat feeding in comparison to low-fat feeding. We present MetaPro-IQ, a metaproteomic approach for highly efficient intestinal microbial protein identification and quantification, which functions as a universal workflow for metaproteomic studies, and will thus facilitate the application of metaproteomics for better understanding the functions of gut microbiota in health and disease.

Ischemia-reperfusion impairs blood-brain barrier function and alters tight junction protein expression in the ovine fetus

PubMed Central

Chen, Xiaodi; Threlkeld, Steven W.; Cummings, Erin E.; Juan, Ilona; Makeyev, Oleksandr; Besio, Walter G.; Gaitanis, John; Banks, William A.; Sadowska, Grazyna B.; Stonestreet, Barbara S.

2012-01-01

The blood-brain barrier is a restrictive interface between the brain parenchyma and the intravascular compartment. Tight junctions contribute to the integrity of the blood-brain barrier. Hypoxic-ischemic damage to the blood-brain barrier could be an important component of fetal brain injury. We hypothesized that increases in blood-brain barrier permeability after ischemia depend upon the duration of reperfusion and that decreases in tight junction proteins are associated with the ischemia-related impairment in blood-brain barrier function in the fetus. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (Ki) and tight junction proteins by Western immunoblot in fetal sheep at 127 days-of-gestation without ischemia, and 4-, 24-, or 48-h after ischemia. The largest increase in Ki (P<0.05) was 4-h after ischemia. Occludin and claudin-5 expressions decreased at 4-h, but returned toward control levels 24- and 48-h after ischemia. Zonula occludens-1 and -2 decreased after ischemia. Inverse correlations between Ki and tight junction proteins suggest that the decreases in tight junction proteins contribute to impaired blood-brain barrier function after ischemia. We conclude that impaired blood-brain barrier function is an important component of hypoxic-ischemic brain injury in the fetus, and that increases in quantitatively measured barrier permeability (Ki) change as a function of the duration of reperfusion after ischemia. The largest increase in permeability occurs 4-h after ischemia and blood-brain barrier function improves early after injury because the blood-brain barrier is less permeable 24- and 48- than 4-h after ischemia. Changes in the tight junction molecular composition are associated with increases in blood-brain barrier permeability after ischemia. PMID:22986172

Protein resistance efficacy of PEO-silane amphiphiles: Dependence on PEO-segment length and concentration

PubMed Central

Rufin, Marc A.; Barry, Mikayla E.; Adair, Paige A.; Hawkins, Melissa L.; Raymond, Jeffery E.; Grunlan, Melissa A.

2016-01-01

In contrast to modification with conventional PEO-silanes (i.e. no siloxane tether), silicones with dramatically enhanced protein resistance have been previously achieved via bulk-modification with poly (ethylene oxide) (PEO)-silane amphiphiles α-(EtO)3Si(CH2)2-oligodimethylsiloxane13-block-PEOn-OCH3 when n = 8 and 16 but not when n = 3. In this work, their efficacy was evaluated in terms of optimal PEO-segment length and minimum concentration required in silicone. For each PEO-silane amphiphile (n = 3, 8, and 16), five concentrations (5, 10, 25, 50, and 100 μmol per 1 g silicone) were evaluated. Efficacy was quantified in terms of the modified silicones’ abilities to undergo rapid, water-driven surface restructuring to form hydrophilic surfaces as well as resistance to fibrinogen adsorption. Only n = 8 and 16 were effective, with a lower minimum concentration in silicone required for n = 8 (10 μmol per 1 g silicone) versus n = 16 (25 μmol per 1 g silicone). Statement of Significance Silicone is commonly used for implantable medical devices, but its hydrophobic surface promotes protein adsorption which leads to thrombosis and infection. Typical methods to incorporate poly(ethylene oxide) (PEO) into silicones have not been effective due to the poor migration of PEO to the surface-biological interface. In this work, PEO-silane amphiphiles – comprised of a siloxane tether (m = 13) and variable PEO segment lengths (n = 3, 8, 16) – were blended into silicone to improve its protein resistance. The efficacy of the amphiphiles was determined to be dependent on PEO length. With the intermediate PEO length (n = 8), water-driven surface restructuring and resulting protein resistance was achieved with a concentration of only 1.7 wt%. PMID:27090588

Quantitative Mass Spectrometry by Isotope Dilution and Multiple Reaction Monitoring (MRM).

PubMed

Russo, Paul; Hood, Brian L; Bateman, Nicholas W; Conrads, Thomas P

2017-01-01

Selected reaction monitoring (SRM) is used in molecular profiling to detect and quantify specific known proteins in complex mixtures. Using isotope dilution (Barnidge et al., Anal Chem 75(3):445-451, 2003) methodologies, peptides can be quantified without the need for an antibody-based method. Selected reaction monitoring assays employ electrospray ionization mass spectrometry (ESI-MS) followed by two stages of mass selection: a first stage where the mass of the peptide ion is selected and, after fragmentation by collision-induced dissociation (CID), a second stage (tandem MS) where either a single (e.g., SRM) or multiple (multiple reaction monitoring, MRM) specific peptide fragment ions are transmitted for detection. The MRM experiment is accomplished by specifying the parent masses of the selected endogenous and isotope-labeled peptides for MS/MS fragmentation and then monitoring fragment ions of interest, using their intensities/abundances and relative ratios to quantify the parent protein of interest. In this example protocol, we will utilize isotope dilution MRM-MS to quantify in absolute terms the total levels of the protein of interest, ataxia telangiectasia mutated (ATM) serine/threonine protein kinase. Ataxia telangiectasia mutated (ATM) phosphorylates several key proteins that initiate activation of the DNA damage checkpoint leading to cell cycle arrest.

Effect of quantifying peptide release on ruminal protein degradation determined using the inhibitor in vitro system

USDA-ARS?s Scientific Manuscript database

The aim of this work was to compare use of an o-phthaldialdehyde (OPA) colorimetric assay (OPA-C), which responds to both free AA and peptides, with an OPA fluorimetric assay (OPA-F), which is insensitive to peptides, to quantify rates of ruminal protein degradation in the inhibitor in vitro system ...

Anomalous Dynamics of Water Confined in Protein-Protein and Protein-DNA Interfaces.

PubMed

Chong, Song-Ho; Ham, Sihyun

2016-10-06

Confined water often exhibits anomalous properties not observable in the bulk phase. Although water in hydrophobic confinement has been the focus of intense investigation, the behavior of water confined between hydrophilic surfaces, which are more frequently found in biological systems, has not been fully explored. Here, we investigate using molecular dynamics simulations dynamical properties of the water confined in hydrophilic protein-protein and protein-DNA interfaces. We find that the interfacial water exhibits glassy slow relaxations even at 300 K. In particular, the rotational dynamics show a logarithmic decay that was observed in glass-forming liquids at deeply supercooled states. We argue that such slow water dynamics are indeed induced by the hydrophilic binding surfaces, which is in opposition to the picture that the hydration water slaves protein motions. Our results will significantly impact the view on the role of water in biomolecular interactions.

Potential Interference of Protein-Protein Interactions by Graphyne.

PubMed

Luan, Binquan; Huynh, Tien; Zhou, Ruhong

2016-03-10

Graphyne has attracted tremendous attention recently due to its many potentially superior properties relative to those of graphene. Although extensive efforts have been devoted to explore the applicability of graphyne as an alternative nanomaterial for state-of-the-art nanotechnology (including biomedical applications), knowledge regarding its possible adverse effects to biological cells is still lacking. Here, using large-scale all-atom molecular dynamics simulations, we investigate the potential toxicity of graphyne by interfering a protein-protein interaction (ppI). We found that graphyne could indeed disrupt the ppIs by cutting through the protein-protein interface and separating the protein complex into noncontacting ones, due to graphyne's dispersive and hydrophobic interaction with the hydrophobic residues residing at the dimer interface. Our results help to elucidate the mechanism of interaction between graphyne and ppI networks within a biological cell and provide insights for its hazard reduction.

Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.

PubMed

Varadi, Mihaly; Zsolyomi, Fruzsina; Guharoy, Mainak; Tompa, Peter

2015-01-01

Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches

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

Murtaugh, Megan L.; Fanning, Sean W.; Sharma, Tressa M.

2012-09-05

There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein-protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton-linkage dictate that the presence of multiple ionizable groups, which undergo a pK{sub a} change on protein binding, are necessary to result in highly pH-dependent binding. To test this hypothesis, a novelmore » combinatorial histidine library was developed where every possible combination of histidine and wild-type residue is sampled throughout the interface of a model anti-RNase A single domain VHH antibody. Antibodies were coselected for high-affinity binding and pH-sensitivity using an in vitro, dual-function selection strategy. The resulting antibodies retained near wild-type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine 'hot-spots,' which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH-sensitive protein affinity reagents for a number of different applications.« less

Sequential protein unfolding through a carbon nanotube pore

NASA Astrophysics Data System (ADS)

Xu, Zhonghe; Zhang, Shuang; Weber, Jeffrey K.; Luan, Binquan; Zhou, Ruhong; Li, Jingyuan

2016-06-01

An assortment of biological processes, like protein degradation and the transport of proteins across membranes, depend on protein unfolding events mediated by nanopore interfaces. In this work, we exploit fully atomistic simulations of an artificial, CNT-based nanopore to investigate the nature of ubiquitin unfolding. With one end of the protein subjected to an external force, we observe non-canonical unfolding behaviour as ubiquitin is pulled through the pore opening. Secondary structural elements are sequentially detached from the protein and threaded into the nanotube, interestingly, the remaining part maintains native-like characteristics. The constraints of the nanopore interface thus facilitate the formation of stable ``unfoldon'' motifs above the nanotube aperture that can exist in the absence of specific native contacts with the other secondary structure. Destruction of these unfoldons gives rise to distinct force peaks in our simulations, providing us with a sensitive probe for studying the kinetics of serial unfolding events. Our detailed analysis of nanopore-mediated protein unfolding events not only provides insight into how related processes might proceed in the cell, but also serves to deepen our understanding of structural arrangements which form the basis for protein conformational stability.An assortment of biological processes, like protein degradation and the transport of proteins across membranes, depend on protein unfolding events mediated by nanopore interfaces. In this work, we exploit fully atomistic simulations of an artificial, CNT-based nanopore to investigate the nature of ubiquitin unfolding. With one end of the protein subjected to an external force, we observe non-canonical unfolding behaviour as ubiquitin is pulled through the pore opening. Secondary structural elements are sequentially detached from the protein and threaded into the nanotube, interestingly, the remaining part maintains native-like characteristics. The constraints of the nanopore interface thus facilitate the formation of stable ``unfoldon'' motifs above the nanotube aperture that can exist in the absence of specific native contacts with the other secondary structure. Destruction of these unfoldons gives rise to distinct force peaks in our simulations, providing us with a sensitive probe for studying the kinetics of serial unfolding events. Our detailed analysis of nanopore-mediated protein unfolding events not only provides insight into how related processes might proceed in the cell, but also serves to deepen our understanding of structural arrangements which form the basis for protein conformational stability. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr00410e

Amyloid-beta-sheet formation at the air-water interface.

PubMed Central

Schladitz, C; Vieira, E P; Hermel, H; Möhwald, H

1999-01-01

An amyloid(1-40) solution rich in coil, turn, and alpha-helix, but poor in beta-sheet, develops monolayers with a high beta-sheet content when spread at the air-water interface. These monolayers are resistant to repeated compression-dilatation cycles and interaction with trifluoroethanol. The secondary structure motifs were detected by circular dichroism (CD) in solution and with infrared reflection-absorption spectroscopy (IRRAS) at the interface. Hydrophobic influences are discussed for the structure conversion in an effort to understand the completely unknown reason for the natural change of the normal prion protein cellular (PrP(C)) into the abnormal prion protein scrapie (PrP(Sc)). PMID:10585952

Characterization of mechanical unfolding intermediates of membrane proteins by coarse grained molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Yamada, Tatsuya; Mitaku, Shigeki; Yamato, Takahisa

2018-01-01

Single-molecule force spectroscopy by atomic force microscopy allows us to get insight into the mechanical unfolding of membrane proteins, and a typical experiment exhibits characteristic patterns on the force distance curves. The origin of these patterns, however, has not been fully understood yet. We performed coarse-grained simulation of the forced unfolding of halorodopsin, reproduced the characteristic features of the experimental force distance curves. A further examination near the membrane-water interface indicated the existence of a motif for the force peak formation, i.e., the occurrence of hydrophobic residues in the upper interface region and hydrophilic residues below the lower interface region.

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.

Interplay Between Hydrophobic Effect and Dipole Interactions in Peptide Aggregation

NASA Astrophysics Data System (ADS)

Ganesan, Sai; Matysiak, Silvina

In the past decade, the development of various coarse-grained models for proteins have provided key insights into the driving forces in folding and aggregation.We recently developed a low resolution Water Explicit Polarizable PROtein coarse-grained Model by adding oppositely charged dummy particles inside protein backbone beads.With this model,we were able to achieve significant α/ β secondary structure content,without any added bias.We now extend the model to study peptide aggregation at hydrophobic-hydrophilic interface using elastin-like octapeptides (GV)4 as a model system.A condensation-ordering mechanism of aggregation is observed in water.Our results suggest that backbone interpeptide dipolar interactions,not hydrophobicity,plays a more significant role in fibril-like peptide aggregation.We observe a cooperative effect in hydrogen bonding or dipolar interactions, with increase in aggregate size in water and interface.Based on this cooperative effect, we provide a potential explanation for the observed nucleus size in peptide aggregation pathways.Without dipolar particles,peptide aggregation is not observed at the hydrophilic-hydrophobic interface.Thus,the presence of dipoles,not hydrophobicity plays a key role in aggregation observed at hydrophobic interfaces.

A Procedure for Measuring Latencies in Brain-Computer Interfaces

PubMed Central

Wilson, J. Adam; Mellinger, Jürgen; Schalk, Gerwin; Williams, Justin

2011-01-01

Brain-computer interface (BCI) systems must process neural signals with consistent timing in order to support adequate system performance. Thus, it is important to have the capability to determine whether a particular BCI configuration (i.e., hardware, software) provides adequate timing performance for a particular experiment. This report presents a method of measuring and quantifying different aspects of system timing in several typical BCI experiments across a range of settings, and presents comprehensive measures of expected overall system latency for each experimental configuration. PMID:20403781

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

VanderLinden, Ryan T.; Hemmis, Casey W.; Yao, Tingting

This work presents that the 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to themore » C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 with independent energetics. In conclusion, these findings provide a detailed characterization of interactions that are important for proteasome function, indicate ubiquitin affinities that are consistent with the role of RPN13 as a proteasomal ubiquitin receptor, and have major implications for the development of novel anticancer therapeutics.« less

Microbial response to environmental gradients in a ceramic-based diffusion system.

PubMed

Wolfaardt, G M; Hendry, M J; Birkham, T; Bressel, A; Gardner, M N; Sousa, A J; Korber, D R; Pilaski, M

2008-05-01

A solid, porous matrix was used to establish steady-state concentration profiles upon which microbial responses to concentration gradients of nutrients or antimicrobial agents could be quantified. This technique relies on the development of spatially defined concentration gradients across a ceramic plate resulting from the diffusion of solutes through the porous ceramic matrix. A two-dimensional, finite-element numerical transport model was used to predict the establishment of concentration profiles, after which concentration profiles of conservative tracers were quantified fluorometrically and chemically at the solid-liquid interface to verify the simulated profiles. Microbial growth responses to nutrient, hypochloride, and antimicrobial concentration gradients were then quantified using epifluorescent or scanning confocal laser microscopy. The observed microbial response verified the establishment and maintenance of stable concentration gradients along the solid-liquid interface. These results indicate the ceramic diffusion system has potential for the isolation of heterogeneous microbial communities as well as for testing the efficacy of antimicrobial agents. In addition, the durability of the solid matrix allowed long-term investigations, making this approach preferable to conventional gel-stabilized systems that are impeded by erosion as well as expansion or shrinkage of the gel. Copyright 2008 Wiley Periodicals, Inc.

Dissolved oxygen transfer to sediments by sweep and eject motions in aquatic environments

USGS Publications Warehouse

O'Connor, B.L.; Hondzo, Miki

2008-01-01

Dissolved oxygen (DO) concentrations were quantified near the sediment-water interface to evaluate DO transfer to sediments in a laboratory recirculating flume and open channel under varying fluid-flow conditions. DO concentration fluctuations were observed within the diffusive sublayer, as defined by the time-averaged DO concentration gradient near the sediment-water interface. Evaluation of the DO concentration fluctuations along with detailed fluid-flow characterizations were used to quantify quasi-periodic sweep and eject motions (bursting events) near the sediments. Bursting events dominated the Reynolds shear stresses responsible for momentum and mass fluctuations near the sediment bed. Two independent methods for detecting bursting events using DO concentration and velocity data produced consistent results. The average time between bursting events was scaled with wall variables and was incorporated into a similarity model to describe the dimensionless mass transfer coefficient (Sherwood number, Sh) in terms of the Reynolds number, Re, and Schmidt number, Sc, which described transport in the flow. The scaling of bursting events was employed with the similarity model to quantify DO transfer to sediments and results showed a high degree of agreement with experimental data. ?? 2008, by the American Society of Limnology and Oceanography, Inc.

Sequence composition and environment effects on residue fluctuations in protein structures

NASA Astrophysics Data System (ADS)

Ruvinsky, Anatoly M.; Vakser, Ilya A.

2010-10-01

Structure fluctuations in proteins affect a broad range of cell phenomena, including stability of proteins and their fragments, allosteric transitions, and energy transfer. This study presents a statistical-thermodynamic analysis of relationship between the sequence composition and the distribution of residue fluctuations in protein-protein complexes. A one-node-per-residue elastic network model accounting for the nonhomogeneous protein mass distribution and the interatomic interactions through the renormalized inter-residue potential is developed. Two factors, a protein mass distribution and a residue environment, were found to determine the scale of residue fluctuations. Surface residues undergo larger fluctuations than core residues in agreement with experimental observations. Ranking residues over the normalized scale of fluctuations yields a distinct classification of amino acids into three groups: (i) highly fluctuating-Gly, Ala, Ser, Pro, and Asp, (ii) moderately fluctuating-Thr, Asn, Gln, Lys, Glu, Arg, Val, and Cys, and (iii) weakly fluctuating-Ile, Leu, Met, Phe, Tyr, Trp, and His. The structural instability in proteins possibly relates to the high content of the highly fluctuating residues and a deficiency of the weakly fluctuating residues in irregular secondary structure elements (loops), chameleon sequences, and disordered proteins. Strong correlation between residue fluctuations and the sequence composition of protein loops supports this hypothesis. Comparing fluctuations of binding site residues (interface residues) with other surface residues shows that, on average, the interface is more rigid than the rest of the protein surface and Gly, Ala, Ser, Cys, Leu, and Trp have a propensity to form more stable docking patches on the interface. The findings have broad implications for understanding mechanisms of protein association and stability of protein structures.

Modulation of dimerization by residues distant from the interface in bovine neurophysin-II.

PubMed

Zheng, C; Peyton, D; Breslow, E

1997-09-01

The crystal structure of bovine neurophysin-II in its liganded state (Chen et al. [1991] Proc. Natl. Acad. Sci. USA 88, 4240-4244) indicates that the 1-6 sequence has a disordered conformation, lacks noncovalent contacts to other regions of the protein and is distant from the monomer-monomer interface. Cleavage of the 1-6 sequence by Staphylococcus protease V8 yielded a protein that, for the first time, crystallized in both liganded and unliganded states. Insights into the role of the 1-6 sequence in the unliganded state were obtained by NMR and related biophysical comparisons of the native and des-1-6 proteins. NMR spectra demonstrated that the environment and/or conformation of residues in the 1-6 sequence differed in liganded and unliganded states. Additionally, the unliganded des-1-6 protein exhibited a dimerization constant four to five times that of the native protein, potentially accounting for the observation that its peptide affinity was also increased. NMR studies further indicated that the increased dimerization constant of the des-1-6 protein correlated with the presence in the native protein of two isoenergetic forms of the monomer, in contrast to only a single form in the des-1-6 protein, as evidenced by signals from an internal dimerization-sensitive alpha-proton. Thus, the 1-6 sequence reduces the dimerization constant by stabilization of an alternative monomer conformation. A second product of Staphylococcus protease V8 digestion of the native protein was identified as the des-1-6 protein with an internal clip after binding site residue Glu-47, the clip presumably breaking the short 3,10 helix that most directly connects the interface to the interface to the binding site. This product, although unable to bind peptide, retained the dimerization constant of the des-1-6 protein, suggesting a lack of importance of the helix in dimerization and contrasting with the effects of the 1-6 sequence. A model is proposed in which the 1-6 sequence stabilizes the second conformation of the unliganded monomer via interactions affecting the loop region that separates the two neurophysin domains and which has been shown to influence neurophysin self-association.

Structural elucidation of the interaction between neurodegenerative disease-related tau protein with model lipid membranes

NASA Astrophysics Data System (ADS)

Jones, Emmalee M.

A protein's sequence of amino acids determines how it folds. That folded structure is linked to protein function, and misfolding to dysfunction. Protein misfolding and aggregation into beta-sheet rich fibrillar aggregates is connected with over 20 neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized in part by misfolding, aggregation and deposition of the microtubule associated tau protein into neurofibrillary tangles (NFTs). However, two questions remain: What is tau's fibrillization mechanism, and what is tau's cytotoxicity mechanism? Tau is prone to heterogeneous interactions, including with lipid membranes. Lipids have been found in NFTs, anionic lipid vesicles induced aggregation of the microtubule binding domain of tau, and other protein aggregates induced ion permeability in cells. This evidence prompted our investigation of tau's interaction with model lipid membranes to elucidate the structural perturbations those interactions induced in tau protein and in the membrane. We show that although tau is highly charged and soluble, it is highly surface active and preferentially interacts with anionic membranes. To resolve molecular-scale structural details of tau and model membranes, we utilized X-ray and neutron scattering techniques. X-ray reflectivity indicated tau aggregated at air/water and anionic lipid membrane interfaces and penetrated into membranes. More significantly, membrane interfaces induced tau protein to partially adopt a more compact conformation with density similar to folded protein and ordered structure characteristic of beta-sheet formation. This suggests possible membrane-based mechanisms of tau aggregation. Membrane morphological changes were seen using fluorescence microscopy, and X-ray scattering techniques showed tau completely disrupts anionic membranes, suggesting an aggregate-based cytotoxicity mechanism. Further investigation of protein constructs and a "hyperphosphorylation" disease mimic helped clarify the role of the microtubule binding domain in anionic lipid affinity and demonstrated even "hyperphosphorylation" did not prevent interaction with anionic membranes. Additional studies investigated more complex membrane models to increase physiological relevance. These insights revealed structural changes in tau protein and lipid membranes after interaction. We observed tau's affinity for interfaces, and aggregation and compaction once tau partitions to interfaces. We observed the beginnings of beta-sheet formation in tau at anionic lipid membranes. We also examined disruption to the membrane on a molecular scale.

Comparison of the orogenic displacement of sodium caseinate with the caseins from the air-water interface by nonionic surfactants.

PubMed

Woodward, N C; Gunning, A P; Mackie, A R; Wilde, P J; Morris, V J

2009-06-16

Displacement of sodium caseinate from the air-water interface by nonionic surfactants Tween 20 and Tween 60 was observed by atomic force microscopy (AFM). The interfacial structure was sampled by Langmuir-Blodgett deposition onto freshly cleaved mica substrates. Protein displacement occurred through an orogenic mechanism: it involved the nucleation and growth of surfactant domains within the protein network, followed by failure of the protein network. The surface pressure at which failure of the protein network occurred was essentially independent of the type of surfactant. The major component of sodium caseinate is beta-casein, and previous studies at the air-water interface have shown that beta-casein networks are weak, failing at surface pressures below that observed for sodium caseinate. The other components of sodium caseinate are alpha(s)- and kappa-caseins. Studies of the displacement of alpha(s)-caseins from air-water interfaces show that these proteins also form weak networks that fail at surface pressures below that observed for sodium caseinate. However, kappa-casein was found to form strong networks that resisted displacement and failed at surface pressures comparable to those observed for sodium caseinate. The AFM images of the displacement suggest that, despite kappa-casein being a minor component, it dominates the failure of sodium caseinate networks: alpha(s)-casein and beta-casein are preferentially desorbed at lower surface pressures, allowing the residual kappa-casein to control the breakdown of the sodium caseinate network at higher surface pressures.

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

PubMed Central

Park, Jin Hyoung; Jin, Jong Hwa; Lim, Myung Sin; An, Hyun Joo; Kim, Jong Won; Lee, Gyun Min

2017-01-01

Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality. PMID:28281648

Complex of a Protective Antibody with its Ebola Virus GP Peptide Epitope: Unusual Features of a Vlambdalx Light Chain

DTIC Science & Technology

2008-01-01

Bioinformatics, 19, ii246–ii255. 52. Lawrence, M. C. & Colman, P. M. (1993). Shape complementarity at protein / protein interfaces . J. Mol. Biol. 234, 946...envelope spike, which is the sole protein expressed on the surface of the Ebola virus and is involved in receptor binding, tropism, and viral entry.6–9 It...variable light chain/heavy chain (VL/VH) interface of 13F6-1-2, ∼1025 Å2 surface area is buried on VL Fig. 1. Nucleotide and translated amino acid

Complex of a Protective Antibody with its Ebola Virus GP Peptide Epitope: Unusual Features of a V lambda x Light Chain

DTIC Science & Technology

2007-10-01

twists. Bioinformatics, 19, ii246–ii255. 52. Lawrence, M. C. & Colman, P. M. (1993). Shape complementarity at protein / protein interfaces . J. Mol. Biol...envelope spike, which is the sole protein expressed on the surface of the Ebola virus and is involved in receptor binding, tropism, and viral entry.6–9 It...26 At the variable light chain/heavy chain (VL/VH) interface of 13F6-1-2, ∼1025 Å2 surface area is buried on VL Fig. 1. Nucleotide and translated amino

Cathodic protection of coastal prestressed concrete piles : prevention of hydrogen embrittlement.

DOT National Transportation Integrated Search

1998-01-01

Assessing the effect of cathodic protection (CP) on a chloride-contaminated bridge pile involved defining the hydrogen embrittlement behavior of the pearlitic reinforcement and quantifying the local (i.e., at the steel/concrete interface) chemical an...

Unraveling Interfaces between Energy Metabolism and Cell Cycle in Plants.

PubMed

Siqueira, João Antonio; Hardoim, Pablo; Ferreira, Paulo C G; Nunes-Nesi, Adriano; Hemerly, Adriana S

2018-06-19

Oscillation in energy levels is widely variable in dividing and differentiated cells. To synchronize cell proliferation and energy fluctuations, cell cycle-related proteins have been implicated in the regulation of mitochondrial energy-generating pathways in yeasts and animals. Plants have chloroplasts and mitochondria, coordinating the cell energy flow. Recent findings suggest an integrated regulation of these organelles and the nuclear cell cycle. Furthermore, reports indicate a set of interactions between the cell cycle and energy metabolism, coordinating the turnover of proteins in plants. Here, we discuss how cell cycle-related proteins directly interact with energy metabolism-related proteins to modulate energy homeostasis and cell cycle progression. We provide interfaces between cell cycle and energy metabolism-related proteins that could be explored to maximize plant yield. Copyright © 2018 Elsevier Ltd. All rights reserved.

Protein-Precipitant-Specific Criteria for the Impact of Reduced Gravity on Crystal Perfection

NASA Technical Reports Server (NTRS)

Vekilov, Peter G.; Witherow, W. (Technical Monitor)

2003-01-01

The objective of this research is to provide quantitative criteria for the impact of reduced or enhanced convective transport on protein crystal perfection. Our earlier work strongly suggests that the magnitude of (lattice defect-inducing) fluctuations in the crystallization rate of proteins arise from the coupling of bulk transport and nonlinear interface kinetics. Hence, we surmised that, depending on the relative weight of bulk transport and interface kinetics in the control of the crystallization process on Earth, these fluctuations can either increase or decrease under reduced gravity conditions. The sign and magnitude of these changes depend on the specific protein-precipitant system. As a consequence, space environments can be either beneficial or detrimental for achieving structural perfection in protein crystals. The task objectives consist in systematic investigations of this hypothesis.

Automated crystallographic system for high-throughput protein structure determination.

PubMed

Brunzelle, Joseph S; Shafaee, Padram; Yang, Xiaojing; Weigand, Steve; Ren, Zhong; Anderson, Wayne F

2003-07-01

High-throughput structural genomic efforts require software that is highly automated, distributive and requires minimal user intervention to determine protein structures. Preliminary experiments were set up to test whether automated scripts could utilize a minimum set of input parameters and produce a set of initial protein coordinates. From this starting point, a highly distributive system was developed that could determine macromolecular structures at a high throughput rate, warehouse and harvest the associated data. The system uses a web interface to obtain input data and display results. It utilizes a relational database to store the initial data needed to start the structure-determination process as well as generated data. A distributive program interface administers the crystallographic programs which determine protein structures. Using a test set of 19 protein targets, 79% were determined automatically.

Driving force behind adsorption-induced protein unfolding: a time-resolved X-ray reflectivity study on lysozyme adsorbed at an air/water interface.

PubMed

Yano, Yohko F; Uruga, Tomoya; Tanida, Hajime; Toyokawa, Hidenori; Terada, Yasuko; Takagaki, Masafumi; Yamada, Hironari

2009-01-06

Time-resolved X-ray reflectivity measurements for lysozyme (LSZ) adsorbed at an air/water interface were performed to study the mechanism of adsorption-induced protein unfolding. The time dependence of the density profile at the air/water interface revealed that the molecular conformation changed significantly during adsorption. Taking into account previous work using Fourier transform infrared (FTIR) spectroscopy, we propose that the LSZ molecules initially adsorbed on the air/water interface have a flat unfolded structure, forming antiparallel beta-sheets as a result of hydrophobic interactions with the gas phase. In contrast, as adsorption continues, a second layer forms in which the molecules have a very loose structure having random coils as a result of hydrophilic interactions with the hydrophilic groups that protrude from the first layer.

Proline substitution of dimer interface β-strand residues as a strategy for the design of functional monomeric proteins.

PubMed

Joseph, Prem Raj B; Poluri, Krishna Mohan; Gangavarapu, Pavani; Rajagopalan, Lavanya; Raghuwanshi, Sandeep; Richardson, Ricardo M; Garofalo, Roberto P; Rajarathnam, Krishna

2013-09-17

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline's unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

On the Hofmeister effect: fluctuations at the protein-water interface and the surface tension.

PubMed

Bogár, Ferenc; Bartha, Ferenc; Násztor, Zoltán; Fábián, László; Leitgeb, Balázs; Dér, András

2014-07-24

We performed molecular dynamics simulations on the tryptophane-cage miniprotein using a nonpolarizable force field, in order to model the effect of concentrated water solutions of neutral salts on protein conformation, which is a manifestation of Hofmeister effects. From the equilibrium values and the fluctuations of the solvent accessible surface area of the miniprotein, the salt-induced changes of the mean value of protein-water interfacial tension were determined. At 300 K, the chaotropic ClO4(-) and NO3(-) decreased the interfacial tension according to their position in the Hofmeister series (by approximately 5 and 2.7 mN/m, respectively), while the kosmotropic F(-) increased it (by 1 mN/m). These values were compared to those obtained from the Gibbs equation using the excess surface adsorption calculated from the probability distribution of the water molecules and ions around the miniprotein, and the two sets were found to be very close to each other. Our results present a direct evidence for the central role of interfacial tension and fluctuations at the protein-water interface in Hofmeister phenomena, and provide a computational method for the determination of the protein-water interfacial tension, establishing a link between the phenomenological and microscopic description of protein-water interfaces.

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

Ahmad, Zeeshan; Viswanathan, Venkatasubramanian

Here, we investigate the stability of electrodeposition at solid-solid interfaces for materials exhibiting an anisotropic mechanical response. The stability of electrodeposition or resistance to the formation of dendrites is studied within a linear stability analysis. The deformation and stress equations are solved using the Stroh formalism and faithfully recover the boundary conditions at the interface. The stability parameter is used to quantify the stability of different solid-solid interfaces incorporating the full anisotropy of the elastic tensor of the two materials. Our results show a high degree of variability in the stability parameter depending on the crystallographic orientation of the solidsmore » in contact, and point to opportunities for exploiting this effect in developing Li metal anodes.« less

Docking glycosaminoglycans to proteins: analysis of solvent inclusion

NASA Astrophysics Data System (ADS)

Samsonov, Sergey A.; Teyra, Joan; Pisabarro, M. Teresa

2011-05-01

Glycosaminoglycans (GAGs) are anionic polysaccharides, which participate in key processes in the extracellular matrix by interactions with protein targets. Due to their charged nature, accurate consideration of electrostatic and water-mediated interactions is indispensable for understanding GAGs binding properties. However, solvent is often overlooked in molecular recognition studies. Here we analyze the abundance of solvent in GAG-protein interfaces and investigate the challenges of adding explicit solvent in GAG-protein docking experiments. We observe PDB GAG-protein interfaces being significantly more hydrated than protein-protein interfaces. Furthermore, by applying molecular dynamics approaches we estimate that about half of GAG-protein interactions are water-mediated. With a dataset of eleven GAG-protein complexes we analyze how solvent inclusion affects Autodock 3, eHiTs, MOE and FlexX docking. We develop an approach to de novo place explicit solvent into the binding site prior to docking, which uses the GRID program to predict positions of waters and to locate possible areas of solvent displacement upon ligand binding. To investigate how solvent placement affects docking performance, we compare these results with those obtained by taking into account information about the solvent position in the crystal structure. In general, we observe that inclusion of solvent improves the results obtained with these methods. Our data show that Autodock 3 performs best, though it experiences difficulties to quantitatively reproduce experimental data on specificity of heparin/heparan sulfate disaccharides binding to IL-8. Our work highlights the current challenges of introducing solvent in protein-GAGs recognition studies, which is crucial for exploiting the full potential of these molecules for rational engineering.

Protein-Protein Interactions in Clathrin Vesicular Assembly: Radial Distribution of Evolutionary Constraints in Interfaces

PubMed Central

Gadkari, Rupali A.; Srinivasan, Narayanaswamy

2012-01-01

In eukaryotic organisms clathrin-coated vesicles are instrumental in the processes of endocytosis as well as intracellular protein trafficking. Hence, it is important to understand how these vesicles have evolved across eukaryotes, to carry cargo molecules of varied shapes and sizes. The intricate nature and functional diversity of the vesicles are maintained by numerous interacting protein partners of the vesicle system. However, to delineate functionally important residues participating in protein-protein interactions of the assembly is a daunting task as there are no high-resolution structures of the intact assembly available. The two cryoEM structures closely representing intact assembly were determined at very low resolution and provide positions of Cα atoms alone. In the present study, using the method developed by us earlier, we predict the protein-protein interface residues in clathrin assembly, taking guidance from the available low-resolution structures. The conservation status of these interfaces when investigated across eukaryotes, revealed a radial distribution of evolutionary constraints, i.e., if the members of the clathrin vesicular assembly can be imagined to be arranged in spherical manner, the cargo being at the center and clathrins being at the periphery, the detailed phylogenetic analysis of these members of the assembly indicated high-residue variation in the members of the assembly closer to the cargo while high conservation was noted in clathrins and in other proteins at the periphery of the vesicle. This points to the strategy adopted by the nature to package diverse proteins but transport them through a highly conserved mechanism. PMID:22384024

EasyModeller: A graphical interface to MODELLER

PubMed Central

2010-01-01

Background MODELLER is a program for automated protein Homology Modeling. It is one of the most widely used tool for homology or comparative modeling of protein three-dimensional structures, but most users find it a bit difficult to start with MODELLER as it is command line based and requires knowledge of basic Python scripting to use it efficiently. Findings The study was designed with an aim to develop of "EasyModeller" tool as a frontend graphical interface to MODELLER using Perl/Tk, which can be used as a standalone tool in windows platform with MODELLER and Python preinstalled. It helps inexperienced users to perform modeling, assessment, visualization, and optimization of protein models in a simple and straightforward way. Conclusion EasyModeller provides a graphical straight forward interface and functions as a stand-alone tool which can be used in a standard personal computer with Microsoft Windows as the operating system. PMID:20712861

Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome

NASA Astrophysics Data System (ADS)

Poirot, Olivier; Timsit, Youri

2016-05-01

From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through “molecular synapses”, ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the “sensory-proteins” innervate the functional ribosomal sites, while the “inter-proteins” interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing.

Quantifying protein microstructure and electrostatic effects on the change in Gibbs free energy of binding in immobilized metal affinity chromatography.

PubMed

Pathange, Lakshmi P; Bevan, David R; Zhang, Chenming

2008-03-01

Electrostatic forces play a major role in maintaining both structural and functional properties of proteins. A major component of protein electrostatics is the interactions between the charged or titratable amino acid residues (e.g., Glu, Lys, and His), whose pK(a) (or the change of the pK(a)) value could be used to study protein electrostatics. Here, we report the study of electrostatic forces through experiments using a well-controlled model protein (T4 lysozyme) and its variants. We generated 10 T4 lysozyme variants, in which the electrostatic environment of the histidine residue was perturbed by altering charged and neutral amino acid residues at various distances from the histidine (probe) residue. The electrostatic perturbations were theoretically quantified by calculating the change in free energy (DeltaDeltaG(E)) using Coulomb's law. On the other hand, immobilized metal affinity chromatography (IMAC) was used to quantify these perturbations in terms of protein binding strength or change in free energy of binding (DeltaDeltaG(B)), which varies from -0.53 to 0.99 kcal/mol. For most of the variants, there is a good correlation (R(2) = 0.97) between the theoretical DeltaDeltaG(E) and experimental DeltaDeltaG(B) values. However, there are three deviant variants, whose histidine residue was found to be involved in site-specific interactions (e.g., ion pair and steric hindrance), which were further investigated by molecular dynamics simulation. This report demonstrates that the electrostatic (DeltaDeltaG(Elec)) and microstructural effects (DeltaDeltaG(Micro)) in a protein can be quantified by IMAC through surface histidine mediated protein-metal ion interaction and that the unique microstructure around a histidine residue can be identified by identifying the abnormal binding behaviors during IMAC.

Self-consistent-field calculations of proteinlike incorporations in polyelectrolyte complex micelles

NASA Astrophysics Data System (ADS)

Lindhoud, Saskia; Stuart, Martien A. Cohen; Norde, Willem; Leermakers, Frans A. M.

2009-11-01

Self-consistent field theory is applied to model the structure and stability of polyelectrolyte complex micelles with incorporated protein (molten globule) molecules in the core. The electrostatic interactions that drive the micelle formation are mimicked by nearest-neighbor interactions using Flory-Huggins χ parameters. The strong qualitative comparison with experimental data proves that the Flory-Huggins approach is reasonable. The free energy of insertion of a proteinlike molecule into the micelle is nonmonotonic: there is (i) a small repulsion when the protein is inside the corona; the height of the insertion barrier is determined by the local osmotic pressure and the elastic deformation of the core, (ii) a local minimum occurs when the protein molecule is at the core-corona interface; the depth (a few kBT ’s) is related to the interfacial tension at the core-corona interface and (iii) a steep repulsion (several kBT ) when part of the protein molecule is dragged into the core. Hence, the protein molecules reside preferentially at the core-corona interface and the absorption as well as the release of the protein molecules has annealed rather than quenched characteristics. Upon an increase of the ionic strength it is possible to reach a critical micellization ionic (CMI) strength. With increasing ionic strength the aggregation numbers decrease strongly and only few proteins remain associated with the micelles near the CMI.

A Multiprotein Binding Interface in an Intrinsically Disordered Region of the Tumor Suppressor Protein Interferon Regulatory Factor-1*

PubMed Central

Narayan, Vikram; Halada, Petr; Hernychová, Lenka; Chong, Yuh Ping; Žáková, Jitka; Hupp, Ted R.; Vojtesek, Borivoj; Ball, Kathryn L.

2011-01-01

The interferon-regulated transcription factor and tumor suppressor protein IRF-1 is predicted to be largely disordered outside of the DNA-binding domain. One of the advantages of intrinsically disordered protein domains is thought to be their ability to take part in multiple, specific but low affinity protein interactions; however, relatively few IRF-1-interacting proteins have been described. The recent identification of a functional binding interface for the E3-ubiquitin ligase CHIP within the major disordered domain of IRF-1 led us to ask whether this region might be employed more widely by regulators of IRF-1 function. Here we describe the use of peptide aptamer-based affinity chromatography coupled with mass spectrometry to define a multiprotein binding interface on IRF-1 (Mf2 domain; amino acids 106–140) and to identify Mf2-binding proteins from A375 cells. Based on their function as known transcriptional regulators, a selection of the Mf2 domain-binding proteins (NPM1, TRIM28, and YB-1) have been validated using in vitro and cell-based assays. Interestingly, although NPM1, TRIM28, and YB-1 all bind to the Mf2 domain, they have differing amino acid specificities, demonstrating the degree of combinatorial diversity and specificity available through linear interaction motifs. PMID:21245151

Interplay between binding affinity and kinetics in protein-protein interactions.

PubMed

Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

2016-07-01

To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

What induces pocket openings on protein surface patches involved in protein-protein interactions?

PubMed

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-X(L), IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

What induces pocket openings on protein surface patches involved in protein-protein interactions?

NASA Astrophysics Data System (ADS)

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-XL, IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

Protein modeling and molecular dynamics simulation of the two novel surfactant proteins SP-G and SP-H.

PubMed

Rausch, Felix; Schicht, Martin; Bräuer, Lars; Paulsen, Friedrich; Brandt, Wolfgang

2014-11-01

Surfactant proteins are well known from the human lung where they are responsible for the stability and flexibility of the pulmonary surfactant system. They are able to influence the surface tension of the gas-liquid interface specifically by directly interacting with single lipids. This work describes the generation of reliable protein structure models to support the experimental characterization of two novel putative surfactant proteins called SP-G and SP-H. The obtained protein models were complemented by predicted posttranslational modifications and placed in a lipid model system mimicking the pulmonary surface. Molecular dynamics simulations of these protein-lipid systems showed the stability of the protein models and the formation of interactions between protein surface and lipid head groups on an atomic scale. Thereby, interaction interface and strength seem to be dependent on orientation and posttranslational modification of the protein. The here presented modeling was fundamental for experimental localization studies and the simulations showed that SP-G and SP-H are theoretically able to interact with lipid systems and thus are members of the surfactant protein family.

Quantifying Protein Concentrations Using Smartphone Colorimetry: A New Method for an Established Test

ERIC Educational Resources Information Center

Gee, Clifford T.; Kehoe, Eric; Pomerantz, William C. K.; Penn, R. Lee

2017-01-01

Proteins are involved in nearly every biological process, which makes them of interest to a range of scientists. Previous work has shown that hand-held cameras can be used to determine the concentration of colored analytes in solution, and this paper extends the approach to reactions involving a color change in order to quantify protein…

Stokesian swimming of a helical swimmer across an interface

NASA Astrophysics Data System (ADS)

Godinez, Francisco; Ramos, Armando; Zenit, Roberto

2016-11-01

Microorganisms swim in flows dominated by viscous effects but in many instances the motion occurs across heterogeneous environments where the fluid properties may vary. To our knowledge, the effect of such in-homogeneity has not been addressed in depth. We conduct experiments in which a magnetic self-propelled helical swimmer displaces across the interface between two immiscible density stratified fluids. As the swimmer crosses the interface, at a fixed rotation rate, its speed is reduced and a certain volume of the lower fluid is dragged across. We quantify the drift volume and the change of swimming speed for different swimming speeds and different fluid combinations. We relate the reduction of the swimming speed with the interfacial tension of the interface. We also compare the measurements of the drift volume with some recent calculations found in the literature.

Molecular modeling of lipase binding to a substrate-water interface.

PubMed

Gruber, Christian C; Pleiss, Jürgen

2012-01-01

Interactions of lipases with hydrophobic substrate-water interfaces are of great interest to design improved lipase variants and engineer reaction conditions. This chapter describes the necessary steps to carry out molecular dynamics simulations of Candida antarctica lipase B at tributyrin-water interface using the GROMACS simulation software. Special attention is drawn to the preparation of the protein and the substrate-water interface and to the analysis of the obtained trajectory.

The Structural Interface between HIV-1 Vif and Human APOBEC3H.

PubMed

Ooms, Marcel; Letko, Michael; Simon, Viviana

2017-03-01

Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H α-helixes α3 and α4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes α3 and α4 interact with the Vif β-sheet (β2-β5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif β-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection. IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV-1 Vif and A3H and successfully generated a Vif-A3H interaction model. Importantly, we find that the HIV-1 Vif-A3H interface is distinct from the Vif-A3G and Vif-A3F interfaces, with a small Vif region being important for recognition of both A3G and A3H. Our Vif-A3H structure model informs on how both proteins interact and could guide toward approaches to block the Vif-A3H interface to target HIV replication. Copyright © 2017 American Society for Microbiology.

A Diffuse Interface Model with Immiscibility Preservation

PubMed Central

Tiwari, Arpit; Freund, Jonathan B.; Pantano, Carlos

2013-01-01

A new, simple, and computationally efficient interface capturing scheme based on a diffuse interface approach is presented for simulation of compressible multiphase flows. Multi-fluid interfaces are represented using field variables (interface functions) with associated transport equations that are augmented, with respect to an established formulation, to enforce a selected interface thickness. The resulting interface region can be set just thick enough to be resolved by the underlying mesh and numerical method, yet thin enough to provide an efficient model for dynamics of well-resolved scales. A key advance in the present method is that the interface regularization is asymptotically compatible with the thermodynamic mixture laws of the mixture model upon which it is constructed. It incorporates first-order pressure and velocity non-equilibrium effects while preserving interface conditions for equilibrium flows, even within the thin diffused mixture region. We first quantify the improved convergence of this formulation in some widely used one-dimensional configurations, then show that it enables fundamentally better simulations of bubble dynamics. Demonstrations include both a spherical bubble collapse, which is shown to maintain excellent symmetry despite the Cartesian mesh, and a jetting bubble collapse adjacent a wall. Comparisons show that without the new formulation the jet is suppressed by numerical diffusion leading to qualitatively incorrect results. PMID:24058207

Footprint Contact Area and Interface Pressure Comparison Between the Knotless and Knot-Tying Transosseous-Equivalent Technique for Rotator Cuff Repair.

PubMed

Kim, Sung-Jae; Kim, Sung-Hwan; Moon, Hyun-Soo; Chun, Yong-Min

2016-01-01

To quantify and compare the footprint contact area and interface pressure on the greater tuberosity between knotless and knot-tying transosseous-equivalent (TOE) repair using pressure-sensitive film. We used 11 pairs of fresh-frozen cadaveric shoulders (22 specimens), in which rotator cuff tears were created before repair. Each pair was randomized to either conventional medial knot-tying TOE repair (group A) or medial knotless TOE repair using the modified Mason-Allen technique (group B). Pressure-sensitive film was used to quantify the pressurized contact area and interface pressure between the greater tuberosity and supraspinatus tendon. The mean pressurized contact area was 33.2 ± 2.5 mm(2) for group A and 28.4 ± 2.4 mm(2) for group B. There was a significant difference between groups (P = .005). Although the overall contact configuration of both groups was similar and showed an M shape, group A showed a greater pressurized configuration around the medial row. The mean interface pressure was 0.20 ± 0.02 MPa for group A and 0.17 ± 0.02 MPa for group B. There was a significant difference between groups (P = .001). Contrary to our hypothesis, in this time-zero study, medial knotless TOE repair using a modified Mason-Allen suture produced a significantly inferior footprint contact area and interface pressure compared with conventional medial knot-tying TOE repair. Even though we found a statistically significant difference between the 2 repair methods, it is still unknown if this statistical difference seen in our study has any clinical and radiologic significance. Copyright © 2016 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Effects of bulk and free surface shear flows on amyloid fibril formation

NASA Astrophysics Data System (ADS)

Posada, David; Sorci, Mirco; Belfort, Georges; Hirsa, Amir

2008-11-01

Amyloid diseases such as Alzheimer's and Huntington's, among others, are characterized by the conversion of monomers to oligomers (precursors) and then to amyloid fibrils. Besides factors such as concentration, pH, and ionic strength, evidence exists that shearing flow strongly influences amyloid formation in vitro. Also, during fibrillation in the presence of either gas or solid surfaces, both the polarity and roughness of the surfaces play a significant role in the kinetics of the fibrillation process. By studying the nucleation and growth of a model system (insulin fibrils) in a well-defined flow field, we can identify the flow and interfacial conditions that impact protein aggregation kinetics. The present flow system consists of an annular region, bounded by stationary inner and outer cylinders and driven by rotation of the floor, with either a hydrophobic (air) or hydrophilic (solid) interface. We show both the combined and separated effects of shear and interfacial hydrophobicity on the fibrillation process, and the use of interfacial shear viscosity as a parameter for quantifying the oligomerization process.

Docking-based modeling of protein-protein interfaces for extensive structural and functional characterization of missense mutations.

PubMed

Barradas-Bautista, Didier; Fernández-Recio, Juan

2017-01-01

Next-generation sequencing (NGS) technologies are providing genomic information for an increasing number of healthy individuals and patient populations. In the context of the large amount of generated genomic data that is being generated, understanding the effect of disease-related mutations at molecular level can contribute to close the gap between genotype and phenotype and thus improve prevention, diagnosis or treatment of a pathological condition. In order to fully characterize the effect of a pathological mutation and have useful information for prediction purposes, it is important first to identify whether the mutation is located at a protein-binding interface, and second to understand the effect on the binding affinity of the affected interaction/s. Computational methods, such as protein docking are currently used to complement experimental efforts and could help to build the human structural interactome. Here we have extended the original pyDockNIP method to predict the location of disease-associated nsSNPs at protein-protein interfaces, when there is no available structure for the protein-protein complex. We have applied this approach to the pathological interaction networks of six diseases with low structural data on PPIs. This approach can almost double the number of nsSNPs that can be characterized and identify edgetic effects in many nsSNPs that were previously unknown. This can help to annotate and interpret genomic data from large-scale population studies, and to achieve a better understanding of disease at molecular level.

Docking-based modeling of protein-protein interfaces for extensive structural and functional characterization of missense mutations

PubMed Central

2017-01-01

Next-generation sequencing (NGS) technologies are providing genomic information for an increasing number of healthy individuals and patient populations. In the context of the large amount of generated genomic data that is being generated, understanding the effect of disease-related mutations at molecular level can contribute to close the gap between genotype and phenotype and thus improve prevention, diagnosis or treatment of a pathological condition. In order to fully characterize the effect of a pathological mutation and have useful information for prediction purposes, it is important first to identify whether the mutation is located at a protein-binding interface, and second to understand the effect on the binding affinity of the affected interaction/s. Computational methods, such as protein docking are currently used to complement experimental efforts and could help to build the human structural interactome. Here we have extended the original pyDockNIP method to predict the location of disease-associated nsSNPs at protein-protein interfaces, when there is no available structure for the protein-protein complex. We have applied this approach to the pathological interaction networks of six diseases with low structural data on PPIs. This approach can almost double the number of nsSNPs that can be characterized and identify edgetic effects in many nsSNPs that were previously unknown. This can help to annotate and interpret genomic data from large-scale population studies, and to achieve a better understanding of disease at molecular level. PMID:28841721

The interface of protein structure, protein biophysics, and molecular evolution

PubMed Central

Liberles, David A; Teichmann, Sarah A; Bahar, Ivet; Bastolla, Ugo; Bloom, Jesse; Bornberg-Bauer, Erich; Colwell, Lucy J; de Koning, A P Jason; Dokholyan, Nikolay V; Echave, Julian; Elofsson, Arne; Gerloff, Dietlind L; Goldstein, Richard A; Grahnen, Johan A; Holder, Mark T; Lakner, Clemens; Lartillot, Nicholas; Lovell, Simon C; Naylor, Gavin; Perica, Tina; Pollock, David D; Pupko, Tal; Regan, Lynne; Roger, Andrew; Rubinstein, Nimrod; Shakhnovich, Eugene; Sjölander, Kimmen; Sunyaev, Shamil; Teufel, Ashley I; Thorne, Jeffrey L; Thornton, Joseph W; Weinreich, Daniel M; Whelan, Simon

2012-01-01

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction. PMID:22528593

Biomechanical evaluation of a transtibial pull-out meniscal root repair: challenging the bungee effect.

PubMed

Cerminara, Anthony J; LaPrade, Christopher M; Smith, Sean D; Ellman, Michael B; Wijdicks, Coen A; LaPrade, Robert F

2014-12-01

A common treatment for posterior meniscal root tears is transtibial pull-out repair, which has been biomechanically reported to restore tibiofemoral contact mechanics to those of the intact knee. Biomechanical data suggest that there is significant displacement of the repaired meniscal root with cyclic loading, which may be responsible for the poor healing and meniscal extrusion demonstrated in some clinical studies. The purpose of this study was to quantify the time-zero displacement of the posterior meniscal root in response to cyclic loading after transtibial pull-out repair and to quantify the individual contributions to displacement of the following: (1) suture elongation, (2) button-bone interface, and (3) meniscus-suture interface. The meniscus-suture interface was hypothesized to result in significantly more displacement than the button-bone interface or suture elongation. Descriptive laboratory study. Transtibial pull-out repair of the posterior medial meniscal root was performed in 6 porcine knees, and cyclic displacement was measured using a loading protocol representative of postoperative rehabilitation. Displacement from (1) suture elongation, (2) the button-bone interface, and (3) the meniscus-suture interface was determined by cyclically loading 6 specimens for each construct using the same loading protocol to determine the contribution of each component to the overall displacement of the repair construct. After 1000 cycles, the repair construct displaced by a mean of 3.28 mm (95% CI, 2.07-4.49). The meniscus-suture component (mean, 2.52 mm; 95% CI, 2.21-2.83) displaced significantly more than the button-bone component (mean, 0.90 mm; 95% CI, 0.64-1.15; P = .006) and suture elongation component (mean, 0.71 mm; 95% CI, 0.36-1.06; P = .006) after 1000 cycles. Displacement of the button-bone and suture elongation components was not significantly different after 1000 cycles (P = .720). There was substantial displacement of the posterior medial meniscal root repaired with the transtibial pull-out technique under a cyclic loading protocol simulating postoperative rehabilitation. The meniscus-suture interface contributed to significantly more displacement than the button-bone interface and suture elongation in the transtibial pull-out repair construct. The results provide a framework for optimizing the transtibial pull-out repair technique. Future studies should focus on improving suture fixation strength within the meniscus-suture interface. © 2014 The Author(s).

Tack coat optimization for HMA overlays : accelerated pavement test report.

DOT National Transportation Integrated Search

2009-02-01

Interface bonding between hot-mix asphalt (HMA) overlays and Portland cement concrete (PCC) pavements is one : of the most significant factors affecting overlay service life. This study was performed to quantify the effects of HMA type, : tack coat t...

ProBiS-CHARMMing: Web Interface for Prediction and Optimization of Ligands in Protein Binding Sites.

PubMed

Konc, Janez; Miller, Benjamin T; Štular, Tanja; Lešnik, Samo; Woodcock, H Lee; Brooks, Bernard R; Janežič, Dušanka

2015-11-23

Proteins often exist only as apo structures (unligated) in the Protein Data Bank, with their corresponding holo structures (with ligands) unavailable. However, apoproteins may not represent the amino-acid residue arrangement upon ligand binding well, which is especially problematic for molecular docking. We developed the ProBiS-CHARMMing web interface by connecting the ProBiS ( http://probis.cmm.ki.si ) and CHARMMing ( http://www.charmming.org ) web servers into one functional unit that enables prediction of protein-ligand complexes and allows for their geometry optimization and interaction energy calculation. The ProBiS web server predicts ligands (small compounds, proteins, nucleic acids, and single-atom ligands) that may bind to a query protein. This is achieved by comparing its surface structure against a nonredundant database of protein structures and finding those that have binding sites similar to that of the query protein. Existing ligands found in the similar binding sites are then transposed to the query according to predictions from ProBiS. The CHARMMing web server enables, among other things, minimization and potential energy calculation for a wide variety of biomolecular systems, and it is used here to optimize the geometry of the predicted protein-ligand complex structures using the CHARMM force field and to calculate their interaction energies with the corresponding query proteins. We show how ProBiS-CHARMMing can be used to predict ligands and their poses for a particular binding site, and minimize the predicted protein-ligand complexes to obtain representations of holoproteins. The ProBiS-CHARMMing web interface is freely available for academic users at http://probis.nih.gov.

Luciferase Protein Complementation Assays for Bioluminescence Imaging of Cells and Mice

PubMed Central

Luker, Gary D.; Luker, Kathryn E.

2015-01-01

Summary Protein fragment complementation assays (PCAs) with luciferase reporters currently are the preferred method for detecting and quantifying protein-protein interactions in living animals. At the most basic level, PCAs involve fusion of two proteins of interest to enzymatically inactive fragments of luciferase. Upon association of the proteins of interest, the luciferase fragments are capable of reconstituting enzymatic activity to generate luminescence in vivo. In addition to bi-molecular luciferase PCAs, unimolecular biosensors for hormones, kinases, and proteases also have been developed using target peptides inserted between inactive luciferase fragments. Luciferase PCAs offer unprecedented opportunities to quantify dynamics of protein-protein interactions in intact cells and living animals, but successful use of luciferase PCAs in cells and mice involves careful consideration of many technical factors. This chapter discusses the design of luciferase PCAs appropriate for animal imaging, including construction of reporters, incorporation of reporters into cells and mice, imaging techniques, and data analysis. PMID:21153371

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

Baden, Elizabeth M.; Owen, Barbara A.L.; Peterson, Francis C.

Amyloidoses are devastating and currently incurable diseases in which the process of amyloid formation causes fatal cellular and organ damage. The molecular mechanisms underlying amyloidoses are not well known. In this study, we address the structural basis of immunoglobulin light chain amyloidosis, which results from deposition of light chains produced by clonal plasma cells. We compare light chain amyloidosis protein AL-09 to its wild-type counterpart, the kl O18/O8 light chain germline. Crystallographic studies indicate that both proteins form dimers. However, AL-09 has an altered dimer interface that is rotated 90 degrees from the kl O18/O8 dimer interface. The three non-conservativemore » mutations in AL-09 are located within the dimer interface, consistent with their role in the decreased stability of this amyloidogenic protein. Moreover, AL-09 forms amyloid fibrils more quickly than kl O18/O8 in vitro. These results support the notion that the increased stability of the monomer and delayed fibril formation, together with a properly formed dimer, may be protective against amyloidogenesis. This could open a new direction into rational drug design for amyloidogenic proteins.« less

Adsorption of hydrophobin/β-casein mixtures at the solid-liquid interface.

PubMed

Tucker, I M; Petkov, J T; Penfold, J; Thomas, R K; Cox, A R; Hedges, N

2016-09-15

The adsorption behaviour of mixtures of the proteins β-casein and hydrophobin at the hydrophilic solid-liquid surface have been studied by neutron reflectivity. The results of measurements from sequential adsorption and co-adsorption from solution are contrasted. The adsorption properties of protein mixtures are important for a wide range of applications. Because of competing factors the adsorption behaviour of protein mixtures at interfaces is often difficult to predict. This is particularly true for mixtures containing hydrophobin as hydrophobin possesses some unusual surface properties. At β-casein concentrations ⩾0.1wt% β-casein largely displaces a pre-adsorbed layer of hydrophobin at the interface, similar to that observed in hydrophobin-surfactant mixtures. In the composition and concentration range studied here for the co-adsorption of β-casein-hydrophobin mixtures the adsorption is dominated by the β-casein adsorption. The results provide an important insight into how the competitive adsorption in protein mixtures of hydrophobin and β-casein can impact upon the modification of solid surface properties and the potential for a wide range of colloid stabilisation applications. Copyright © 2016 Elsevier Inc. All rights reserved.