Effect of resveratrol or ascorbic acid on the stability of α-tocopherol in O/W emulsions stabilized by whey protein isolate: Simultaneous encapsulation of the vitamin and the protective antioxidant.

PubMed

Wang, Lei; Gao, Yahui; Li, Juan; Subirade, Muriel; Song, Yuanda; Liang, Li

2016-04-01

Food proteins have been widely used as carrier materials due to their multiple functional properties. Hydrophobic bioactives are generally dissolved in the oil phase of O/W emulsions. Ligand-binding properties provide the possibility of binding bioactives to the protein membrane of oil droplets. In this study, the influence of whey protein isolate (WPI) concentration and amphiphilic resveratrol or hydrophilic ascorbic acid on the decomposition of α-tocopherol in the oil phase of WPI emulsions is considered. Impact of ascorbic acid, in the continuous phase, on the decomposition depended on the vitamin concentration. Resveratrol partitioned into the oil-water interface and the cis-isomer contributed most of the protective effect of this polyphenol. About 94% of α-tocopherol and 50% of resveratrol were found in the oil droplets stabilized by 0.01% WPI. These results suggest the feasibility of using the emulsifying and ligand-binding properties of WPI to produce carriers for simultaneous encapsulation of bioactives with different physicochemical properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Modeling and Experimental Investigation of the Effects of Antigen Density, Binding Affinity, and Antigen Expression Ratio on Bispecific Antibody Binding to Cell Surface Targets.

PubMed

Rhoden, John J; Dyas, Gregory L; Wroblewski, Victor J

2016-05-20

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Acid-base and copper-binding properties of three organic matter fractions isolated from a forest floor soil solution

NASA Astrophysics Data System (ADS)

van Schaik, Joris W. J.; Kleja, Dan B.; Gustafsson, Jon Petter

2010-02-01

Vast amounts of knowledge about the proton- and metal-binding properties of dissolved organic matter (DOM) in natural waters have been obtained in studies on isolated humic and fulvic (hydrophobic) acids. Although macromolecular hydrophilic acids normally make up about one-third of DOM, their proton- and metal-binding properties are poorly known. Here, we investigated the acid-base and Cu-binding properties of the hydrophobic (fulvic) acid fraction and two hydrophilic fractions isolated from a soil solution. Proton titrations revealed a higher total charge for the hydrophilic acid fractions than for the hydrophobic acid fraction. The most hydrophilic fraction appeared to be dominated by weak acid sites, as evidenced by increased slope of the curve of surface charge versus pH at pH values above 6. The titration curves were poorly predicted by both Stockholm Humic Model (SHM) and NICA-Donnan model calculations using generic parameter values, but could be modelled accurately after optimisation of the proton-binding parameters (pH ⩽ 9). Cu-binding isotherms for the three fractions were determined at pH values of 4, 6 and 9. With the optimised proton-binding parameters, the SHM model predictions for Cu binding improved, whereas the NICA-Donnan predictions deteriorated. After optimisation of Cu-binding parameters, both models described the experimental data satisfactorily. Iron(III) and aluminium competed strongly with Cu for binding sites at both pH 4 and pH 6. The SHM model predicted this competition reasonably well, but the NICA-Donnan model underestimated the effects significantly at pH 6. Overall, the Cu-binding behaviour of the two hydrophilic acid fractions was very similar to that of the hydrophobic acid fraction, despite the differences observed in proton-binding characteristics. These results show that for modelling purposes, it is essential to include the hydrophilic acid fraction in the pool of 'active' humic substances.

Electronic and optical properties of exciton, trions and biexciton in II-VI parabolic quantum dot

NASA Astrophysics Data System (ADS)

Sujanah, P.; John Peter, A.; Woo Lee, Chang

2015-08-01

Binding energies of exciton, trions and biexciton and their interband optical transition energies are studied in a CdTe/ZnTe quantum dot nanostructure taking into consideration the geometrical confinement effect. The radial spread of the wavefunctions, binding energies, optical transition energies, oscillator strength, radiative life time and the absorption coefficients of exciton, positively and negatively charged excitons and biexciton are carried out. It is found that the ratio of the radiative life time of exciton with the trions and biexciton enhances with the reduction of geometrical confinement. The results show that (i) the binding energies of exciton, positive and negative trions and the biexciton have strong influence on the reduction of geometrical confinement effect, (ii) the binding energy is found to decrease from the binding energies of exciton to positive trion through biexciton and negative trion binding energies, (iii) the oscillator strength of trions is found to be lesser than exciton and the biexciton and (iv) the electronic and optical properties of exciton, trions and the biexciton are considerably dependent on the spatial confinement, incident photon energy and the radiative life time. The obtained results are in good agreement with the other existing literature.

Binding of ring-substituted indole-3-acetic acids to human serum albumin.

PubMed

Soskić, Milan; Magnus, Volker

2007-07-01

The plant hormone, indole-3-acetic acid (IAA), and its ring-substituted derivatives have recently attracted attention as promising pro-drugs in cancer therapy. Here we present relative binding constants to human serum albumin for IAA and 34 of its derivatives, as obtained using the immobilized protein bound to a support suitable for high-performance liquid chromatography. We also report their octanol-water partition coefficients (logK(ow)) computed from retention data on a C(18) coated silica gel column. A four-parameter QSPR (quantitative structure-property relationships) model, based on physico-chemical properties, is put forward, which accounts for more than 96% of the variations in the binding affinities of these compounds. The model confirms the importance of lipophilicity as a global parameter governing interaction with serum albumin, but also assigns significant roles to parameters specifically related to the molecular topology of ring-substituted IAAs. Bulky substituents at ring-position 6 increase affinity, those at position 2 obstruct binding, while no steric effects were noted at other ring-positions. Electron-withdrawing substituents at position 5 enhance binding, but have no obvious effect at other ring positions.

Does TATA matter? A structural exploration of the selectivity determinants in its complexes with TATA box-binding protein.

PubMed Central

Pastor, N; Pardo, L; Weinstein, H

1997-01-01

The binding of the TATA box-binding protein (TBP) to a TATA sequence in DNA is essential for eukaryotic basal transcription. TBP binds in the minor groove of DNA, causing a large distortion of the DNA helix. Given the apparent stereochemical equivalence of AT and TA basepairs in the minor groove, DNA deformability must play a significant role in binding site selection, because not all AT-rich sequences are bound effectively by TBP. To gain insight into the precise role that the properties of the TATA sequence have in determining the specificity of the DNA substrates of TBP, the solution structure and dynamics of seven DNA dodecamers have been studied by using molecular dynamics simulations. The analysis of the structural properties of basepair steps in these TATA sequences suggests a reason for the preference for alternating pyrimidine-purine (YR) sequences, but indicates that these properties cannot be the sole determinant of the sequence specificity of TBP. Rather, recognition depends on the interplay between the inherent deformability of the DNA and steric complementarity at the molecular interface. Images FIGURE 2 PMID:9251783

Characteristics of low-and high-fat beef patties: effect of high hydrostatic pressure.

PubMed

Carballo, J; Fernandez, P; Carrascosa, A V; Solas, M T; Colmenero, F J

1997-01-01

The purpose of this study was to analyze the consequences of applying high pressures (100 and 300 MPa for 5 or 20 min) on characteristics such as water- and fat-binding properties, texture, color, microstructure, and microbiology of low-fat (9.2%) and high-fat (20.3%) beef patties. In nonpressurized patties, the low-fat product exhibited significantly poorer (P < 0.05) binding properties and higher (P < 0.05) Kramer shear force and Kramer energy than did high-fat patties. Although high pressure did not clearly influence the binding properties of low- and high-fat beef patties, it did produce a rise in the Kramer shear force and energy which were more pronounced at 300 MPa. High pressures altered patty color, the extent of alteration depending on fat content, pressure, and pressurizing time. Pressurizing high- and low-fat beef patties at 300 MPa not only produced a lethal effect (P < 0.05) on microorganisms, but caused sublethal damage as well.

Effect of altering local protein fluctuations using artificial intelligence

NASA Astrophysics Data System (ADS)

Nishiyama, Katsuhiko

2017-03-01

The fluctuations in Arg111, a significantly fluctuating residue in cathepsin K, were locally regulated by modifying Arg111 to Gly111. The binding properties of 15 dipeptides in the modified protein were analyzed by molecular simulations, and modeled as decision trees using artificial intelligence. The decision tree of the modified protein significantly differed from that of unmodified cathepsin K, and the Arg-to-Gly modification exerted a remarkable effect on the peptide binding properties. By locally regulating the fluctuations of a protein, we may greatly alter the original functions of the protein, enabling novel applications in several fields.

Mapping multivalency and differential affinities within large intrinsically disordered protein complexes with segmental motion analysis.

PubMed

Milles, Sigrid; Lemke, Edward A

2014-07-07

Intrinsically disordered proteins (IDPs) can bind to multiple interaction partners. Numerous binding regions in the IDP that act in concert through complex cooperative effects facilitate such interactions, but complicate studying IDP complexes. To address this challenge we developed a combined fluorescence correlation and time-resolved polarization spectroscopy approach to study the binding properties of the IDP nucleoporin153 (Nup153) to nuclear transport receptors (NTRs). The detection of segmental backbone mobility of Nup153 within the unperturbed complex provided a readout of local, region-specific binding properties that are usually masked in measurements of the whole IDP. The binding affinities of functionally and structurally diverse NTRs to distinct regions of Nup153 can differ by orders of magnitudes-a result with implications for the diversity of transport routes in nucleocytoplasmic transport. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of ionic compositions of the medium on monosodium glutamate binding to taste epithelial cells.

PubMed

Hayashi, Y; Tsunenari, T; Mori, T

1999-03-01

Monosodium glutamate and nucleotides are umami taste substances in animals and have a synergistic effect on each other. We studied the ligand-binding properties of the glutamate receptors in taste epithelial cells isolated from bovine tongue. Specific glutamate binding was observed in an enriched suspension of taste receptor cells in Hanks' balanced salt solution, while no specific glutamate binding was apparent in the absence of divalent ions or when the cells had been depolarized by a high content of potassium in Hanks' balanced salt solution. There was no significant difference between the release of glutamate under depolarized or divalent ion-free conditions and under normal conditions. However, glutamate was easily released from the depolarized cells in the absence of divalent ions. These data suggest that the binding of glutamate to receptors depends on divalent ions, which also have an effect on maintaining binding between glutamate and receptors.

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

Carrageenans as a new source of drugs with metal binding properties.

PubMed

Khotimchenko, Yuri S; Khozhaenko, Elena V; Khotimchenko, Maxim Y; Kolenchenko, Elena A; Kovalev, Valeri V

2010-04-01

Carrageenans are abundant and safe non-starch polysaccharides exerting their biological effects in living organisms. Apart from their known pro-inflammation properties and some pharmacological activity, carrageenans can also strongly bind and hold metal ions. This property can be used for creation of the new drugs for elimination of metals from the body or targeted delivery of these metal ions for healing purposes. Metal binding activity of different carrageenans in aqueous solutions containing Y(3+) or Pb(2+) ions was studied in a batch sorption system. The metal uptake by carrageenans is not affected by the change of the pH within the range from 2.0 to 6.0. The rates and binding capacities of carrageenans regarding metal ions were evaluated. The Langmuir, Freundlich and BET sorption models were applied to describe the isotherms and constants, and the sorption isothermal data could be explained well by the Langmuir equation. The results obtained through the study suggest that kappa-, iota-, and lambda-carrageenans are favorable sorbents. The largest amount of Y(3+) and Pb(2+) ions are bound by iota-carrageenan. Therefore, it can be concluded that this type of polysaccharide is the more appropriate substance for elaboration of the drugs with high selective metal binding properties.

Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor.

PubMed

Yin, Donghua; He, Yali; Perera, Minoli A; Hong, Seoung Soo; Marhefka, Craig; Stourman, Nina; Kirkovsky, Leonid; Miller, Duane D; Dalton, James T

2003-01-01

The purposes of the present studies were to examine the androgen receptor (AR) binding ability and in vitro functional activity of multiple series of nonsteroidal compounds derived from known antiandrogen pharmacophores and to investigate the structure-activity relationships (SARs) of these nonsteroidal compounds. The AR binding properties of sixty-five nonsteroidal compounds were assessed by a radioligand competitive binding assay with the use of cytosolic AR prepared from rat prostates. The AR agonist and antagonist activities of high-affinity ligands were determined by the ability of the ligand to regulate AR-mediated transcriptional activation in cultured CV-1 cells, using a cotransfection assay. Nonsteroidal compounds with diverse structural features demonstrated a wide range of binding affinity for the AR. Ten compounds, mainly from the bicalutamide-related series, showed a binding affinity superior to the structural pharmacophore from which they were derived. Several SARs regarding nonsteroidal AR binding were revealed from the binding data, including stereoisomeric conformation, steric effect, and electronic effect. The functional activity of high-affinity ligands ranged from antagonist to full agonist for the AR. Several structural features were found to be determinative of agonist and antagonist activities. The nonsteroidal AR agonists identified from the present studies provided a pool of candidates for further development of selective androgen receptor modulators (SARMs) for androgen therapy. Also, these studies uncovered or confirmed numerous important SARs governing AR binding and functional properties by nonsteroidal molecules, which would be valuable in the future structural optimization of SARMs.

The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex.

PubMed

Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

2013-07-01

A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy=2,2-bipyridine, Hbcpip=2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5±1.4)×10(5) M(-1) in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of Various Material Properties on the Adhesive Stage of Fretting

NASA Technical Reports Server (NTRS)

Buckley, D. H.

1974-01-01

Various properties of metals and alloys were studied with respect to their effect on the initial stage of the fretting process, namely adhesion. Crystallographic orientation, crystal structure, interfacial binding energies of dissimiliar metal, segregation of alloy constituents and the nature and structure of surface films were found to influence adhesion. High atomic density, low surface energy grain orientations exhibited lower adhesion than other orientations. Knowledge of interfacial surface binding energies assists in predicting adhesive transfer and wear. Selective surface segregation of alloy constituents accomplishes both a reduction in adhesion and improved surface oxidation characteristics. Equivalent surface coverages of various adsorbed species indicate that some are markedly more effective in inhibiting adhesion than others.

Cotton and Protein Interactions

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

Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.

The adsorbent properties of important wound fluid proteins and cotton cellulose are reviewed. This review focuses on the adsorption of albumin to cotton-based wound dressings and some chemically modified derivatives targeted for chronic wounds. Adsorption of elastase in the presence of albumin was examined as a model to understand the interactive properties of these wound fluid components with cotton fibers. In the chronic non-healing wound, elastase appears to be over-expressed, and it digests tissue and growth factors, interfering with the normal healing process. Albumin is the most prevalent protein in wound fluid, and in highly to moderately exudative wounds, itmore » may bind significantly to the fibers of wound dressings. Thus, the relative binding properties of both elastase and albumin to wound dressing fibers are of interest in the design of more effective wound dressings. The present work examines the binding of albumin to two different derivatives of cotton, and quantifies the elastase binding to the same derivatives following exposure of albumin to the fiber surface. An HPLC adsorption technique was employed coupled with a colorimetric enzyme assay to quantify the relative binding properties of albumin and elastase to cotton. The results of wound protein binding are discussed in relation to the porosity and surface chemistry interactions of cotton and wound proteins. Studies are directed to understanding the implications of protein adsorption phenomena in terms of fiber-protein models that have implications for rationally designing dressings for chronic wounds.« less

Lead-binding capacity of calcium pectates with different molecular weight.

PubMed

Khotimchenko, Maksim; Makarova, Ksenia; Khozhaenko, Elena; Kovalev, Valeri

2017-04-01

Nowadays, heavy metal contamination of environment is considered as a serious threat to public health because of toxicity of these pollutants and the lack of effective materials with metal-binding properties. Some biopolymers such as pectins were proposed for removal of metal ions from industrial water disposals. Chemical structure of pectins is quite variable and substantially affects their metal binding properties. In this work, relationship between molecular weight and Pb(II)-binding capacity of calcium pectates was investigated in a batch sorption system. The results showed that all pectate samples are able to form complexes with Pb(II) ions. The effects of contact time, pH of the media and equilibrium metal concentration on metal-binding process were tested in experiments. The equilibrium time min required for uptake of Pb(II) by pectate compounds was found to be 60min. Langmuir and Freundlich models were applied for description of interactions between pectates and metal ions. Binding capacity of low molecular pectate was highest among all the samples tested. Langmuir model was figured out to be the best fit within the whole range of pH values. These results demonstrate that calcium pectate with low molecular weight is more promising agent for elimination of Pb(II) ions from contaminated wastewaters. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of salts on protein-surface interactions: applications for column chromatography.

PubMed

Tsumoto, Kouhei; Ejima, Daisuke; Senczuk, Anna M; Kita, Yoshiko; Arakawa, Tsutomu

2007-07-01

Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein-protein or protein-surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein-protein or protein-surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers. Copyright 2007 Wiley-Liss, Inc.

Structural, thermal, functional, antioxidant & antimicrobial properties of β-d-glucan extracted from baker's yeast (Saccharomyces cereviseae)-Effect of γ-irradiation.

PubMed

Khan, Asma Ashraf; Gani, Adil; Masoodi, F A; Amin, Furheen; Wani, Idrees Ahmed; Khanday, Firdous Ahmad; Gani, Asir

2016-04-20

This study was carried out to evaluate the effect of γ-irradiation (0, 5, 10, 20, 30 & 50kGy) on the structural, functional, antioxidant and antimicrobial properties of yeast β-d-glucan. The samples were characterized by ATR-FTIR, gel permeation chromatography (GPC) and the thermal properties were studied using DSC. There was a decrease in the average molecular weight of β-d-glucan as the irradiation dose increased. The functional properties of irradiated yeast β-d-glucan were largely influenced by the action of gamma radiation like swelling power and viscosity decreases with increase in the irradiation dose while as fat binding capacity, emulsifying properties, foaming properties and bile acid binding capacity shows an increasing trend. All the antioxidant properties carried out using six different assays increased significantly (p≤0.05) in a dose dependent manner. The antibacterial activity of yeast β-d-glucan also showed an increasing trend with increase in the irradiation dose from 5 to 50kDa. Copyright © 2016 Elsevier Ltd. All rights reserved.

Anti-tumor Study of Chondroitin Sulfate-Methotrexate Nanogels

NASA Astrophysics Data System (ADS)

Wang, Jinyu; Zhao, Weibo; Chen, Haixiao; Qin, An; Zhu, Peizhi

2017-10-01

Self-assembly nanogels (NGs) were formed by bioconjugating methotrexate (MTX) with chondroitin sulfate (CS). MTX-CS NGs can greatly enhance the solubility and improve the delivery efficacy of MTX due to the CD44 binding property of CS. Vivo experiments revealed that MTX-CS NGs showed less toxicity than MTX. MTX-CS NGs can improve the anti-tumor effect while reducing the side effects of MTX. Due to their CD44 binding property, chondroitin sulfate-drug conjugates could be a promising and efficient platform for improving the solubility of sparingly soluble drug molecules as well as targeted delivery to cancer cells and tumor tissues.

E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion.

PubMed

Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu; Funahashi, Tohru; Shimomura, Iichiro; Kihara, Shinji

2016-02-05

Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of new composite biosorbents from olive pomace wastes

NASA Astrophysics Data System (ADS)

Pagnanelli, Francesca; Viggi, Carolina Cruz; Toro, Luigi

2010-06-01

In this study olive pomace was used as a source of binding substances for the development of composite biosorbents to be used in heavy metal removal from aqueous solutions. The aim was to obtain biosorbent material with an increased concentration of binding sites. The effects of two different extraction procedures (one using only methanol and the other one hexane followed by methanol) on the binding properties of olive pomace were tested by potentiometric titrations and batch biosorption tests for copper and cadmium removal. Titration modelling evidenced that both kinds of extractions generated a solid with a reduced amount of protonatable sites. Biosorption tests were organized according to full factorial designs. Analysis of variance denoted that both kinds of extractions determined a statistically significant negative effect on metal biosorption. In the case of cadmium extractions also determined a significant decrease of selectivity with respect to olive pomace. When the acid-base and binding properties of the substances extracted were determined, they were adsorbed onto a synthetic resin (octadecylsilane) and calcium alginate beads. In this way two kinds of composite biosorbents have been obtained both having an increased concentration of binding substances with respect to native olive pomace, also working more efficiently in metal removal.

Assessment of amsacrine binding with DNA using UV-visible, circular dichroism and Raman spectroscopic techniques.

PubMed

Jangir, Deepak Kumar; Dey, Sanjay Kumar; Kundu, Suman; Mehrotra, Ranjana

2012-09-03

Proper understanding of the mechanism of binding of drugs to their targets in cell is a fundamental requirement to develop new drug therapy regimen. Amsacrine is a rationally designed anticancer drug, used to treat leukemia and lymphoma. Binding with cellular DNA is a crucial step in its mechanism of cytotoxicity. Despite numerous studies, DNA binding properties of amsacrine are poorly understood. Its reversible binding with DNA does not permit X-ray crystallography or NMR spectroscopic evaluation of amsacrine-DNA complexes. In the present work, interaction of amsacrine with calf thymus DNA is investigated at physiological conditions. UV-visible, FT-Raman and circular dichroism spectroscopic techniques were employed to determine the binding mode, binding constant, sequence specificity and conformational effects of amsacrine binding to native calf thymus DNA. Our results illustrate that amsacrine interacts with DNA by and large through intercalation between base pairs. Binding constant of the amsacrine-DNA complex was found to be K=1.2±0.1×10(4) M(-1) which is indicative of moderate type of binding of amsacrine to DNA. Raman spectroscopic results suggest that amsacrine has a binding preference of intercalation between AT base pairs of DNA. Minor groove binding is also observed in amsacrine-DNA complexes. These results are in good agreement with in silico investigation of amsacrine binding to DNA and thus provide detailed insight into DNA binding properties of amsacrine, which could ultimately, renders its cytotoxic efficacy. Copyright © 2012 Elsevier B.V. All rights reserved.

The effect of macromolecular crowding on the electrostatic component of barnase-barstar binding: a computational, implicit solvent-based study.

PubMed

Qi, Helena W; Nakka, Priyanka; Chen, Connie; Radhakrishnan, Mala L

2014-01-01

Macromolecular crowding within the cell can impact both protein folding and binding. Earlier models of cellular crowding focused on the excluded volume, entropic effect of crowding agents, which generally favors compact protein states. Recently, other effects of crowding have been explored, including enthalpically-related crowder-protein interactions and changes in solvation properties. In this work, we explore the effects of macromolecular crowding on the electrostatic desolvation and solvent-screened interaction components of protein-protein binding. Our simple model enables us to focus exclusively on the electrostatic effects of water depletion on protein binding due to crowding, providing us with the ability to systematically analyze and quantify these potentially intuitive effects. We use the barnase-barstar complex as a model system and randomly placed, uncharged spheres within implicit solvent to model crowding in an aqueous environment. On average, we find that the desolvation free energy penalties incurred by partners upon binding are lowered in a crowded environment and solvent-screened interactions are amplified. At a constant crowder density (fraction of total available volume occupied by crowders), this effect generally increases as the radius of model crowders decreases, but the strength and nature of this trend can depend on the water probe radius used to generate the molecular surface in the continuum model. In general, there is huge variation in desolvation penalties as a function of the random crowder positions. Results with explicit model crowders can be qualitatively similar to those using a lowered "effective" solvent dielectric to account for crowding, although the "best" effective dielectric constant will likely depend on multiple system properties. Taken together, this work systematically demonstrates, quantifies, and analyzes qualitative intuition-based insights into the effects of water depletion due to crowding on the electrostatic component of protein binding, and it provides an initial framework for future analyses.

The effect of feeding high corn oil on fatty-acid-binding-protein isolated from rat liver.

PubMed

Catalá, A

1987-12-01

Fatty-acid-binding-protein isolated from liver of rats receiving normal or high fat diet was studied by three different methods. The effect of high fat diet on the thermal stability of the protein was determined employing differential scanning calorimetry. Fatty acids have a stabilizing effect on the thermal stability of the protein. In order to determine the relative binding affinity of native and delipidated protein a Sephadex G-50 assay was employed using [1-14C] oleate as ligand. The delipidated protein exhibited greater binding of oleate than did the native material. Increases in the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes in vitro were also observed when protein obtained from both sources were delipidated. The results suggest that high corn oil diet would modify the properties of fatty-acid-binding-protein in the uptake and cytosolic transport of long-chain fatty acids.

Effects of egg-adaptation on receptor-binding and antigenic properties of recent influenza A (H3N2) vaccine viruses.

PubMed

Parker, Lauren; Wharton, Stephen A; Martin, Stephen R; Cross, Karen; Lin, Yipu; Liu, Yan; Feizi, Ten; Daniels, Rodney S; McCauley, John W

2016-06-01

Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012-2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.

The Effect of Macromolecular Crowding on the Electrostatic Component of Barnase–Barstar Binding: A Computational, Implicit Solvent-Based Study

PubMed Central

Qi, Helena W.; Nakka, Priyanka; Chen, Connie; Radhakrishnan, Mala L.

2014-01-01

Macromolecular crowding within the cell can impact both protein folding and binding. Earlier models of cellular crowding focused on the excluded volume, entropic effect of crowding agents, which generally favors compact protein states. Recently, other effects of crowding have been explored, including enthalpically-related crowder–protein interactions and changes in solvation properties. In this work, we explore the effects of macromolecular crowding on the electrostatic desolvation and solvent-screened interaction components of protein–protein binding. Our simple model enables us to focus exclusively on the electrostatic effects of water depletion on protein binding due to crowding, providing us with the ability to systematically analyze and quantify these potentially intuitive effects. We use the barnase–barstar complex as a model system and randomly placed, uncharged spheres within implicit solvent to model crowding in an aqueous environment. On average, we find that the desolvation free energy penalties incurred by partners upon binding are lowered in a crowded environment and solvent-screened interactions are amplified. At a constant crowder density (fraction of total available volume occupied by crowders), this effect generally increases as the radius of model crowders decreases, but the strength and nature of this trend can depend on the water probe radius used to generate the molecular surface in the continuum model. In general, there is huge variation in desolvation penalties as a function of the random crowder positions. Results with explicit model crowders can be qualitatively similar to those using a lowered “effective” solvent dielectric to account for crowding, although the “best” effective dielectric constant will likely depend on multiple system properties. Taken together, this work systematically demonstrates, quantifies, and analyzes qualitative intuition-based insights into the effects of water depletion due to crowding on the electrostatic component of protein binding, and it provides an initial framework for future analyses. PMID:24915485

Fabrication of NdCeCuO and effects of binding agents on the growth, micro-structural and electrical properties

NASA Astrophysics Data System (ADS)

Altin, S.; Aksan, M. A.; Turkoglu, S.; Yakinci, M. E.

2011-12-01

NdCeCuO superconducting samples were fabricated using ethyl alcohol, acetone and ethylenediaminetetraacetic acid (EDTA) as binding agents. For evaporation of binding agents, the samples were heat treated at 1050 °C for 24 h and then at 950 °C for 6-48 h under argon atmosphere to obtain the superconducting phase. The best superconducting performance was found in the sample heat treated at 1050 °C for 24 h and then 950 °C for 12 h which was fabricated by using acetone as binding agent. The T c and T 0 value was found to be ∼25 K and 23.4 K, respectively. Grain size in the samples fabricated was calculated using Scherer equation and SEM data. It was found that grain size strongly depends on the binding agents and heat treatment conditions. Some cracks and voids on the surface of the samples were observed, which influences the superconducting and electrical transport properties of the samples.

Preclinical Evaluation of [(18)F]THK-5105 Enantiomers: Effects of Chirality on Its Effectiveness as a Tau Imaging Radiotracer.

PubMed

Tago, Tetsuro; Furumoto, Shozo; Okamura, Nobuyuki; Harada, Ryuichi; Adachi, Hajime; Ishikawa, Yoichi; Yanai, Kazuhiko; Iwata, Ren; Kudo, Yukitsuka

2016-04-01

Noninvasive imaging of tau and amyloid-β pathologies would facilitate diagnosis of Alzheimer's disease (AD). Recently, we have developed [(18)F]THK-5105 for selective detection of tau pathology by positron emission tomography (PET). The purpose of this study was to clarify biological properties of optically pure [(18)F]THK-5105 enantiomers. Binding for tau aggregates in AD brain section was evaluated by autoradiography (ARG). In vitro binding assays were performed to evaluate the binding properties of enantiomers for AD brain homogenates. The pharmacokinetics in the normal mouse brains was assessed by ex vivo biodistribution assay The ARG of enantiomers showed the high accumulation of radioactivity corresponding to the distribution of tau deposits. In vitro binding assays revealed that (S)-[(18)F]THK-5105 has slower dissociation from tau than (R)-[(18)F]THK-5105. Biodistribution assays indicated that (S)-[(18)F]THK-5105 eliminated faster from the mouse brains and blood compared with (R)-[(18)F]THK-5105. (S)-[(18)F]THK-5105 could be more suitable than (R)-enantiomer for a tau imaging agent.

Large-scale binding ligand prediction by improved patch-based method Patch-Surfer2.0

PubMed Central

Zhu, Xiaolei; Xiong, Yi; Kihara, Daisuke

2015-01-01

Motivation: Ligand binding is a key aspect of the function of many proteins. Thus, binding ligand prediction provides important insight in understanding the biological function of proteins. Binding ligand prediction is also useful for drug design and examining potential drug side effects. Results: We present a computational method named Patch-Surfer2.0, which predicts binding ligands for a protein pocket. By representing and comparing pockets at the level of small local surface patches that characterize physicochemical properties of the local regions, the method can identify binding pockets of the same ligand even if they do not share globally similar shapes. Properties of local patches are represented by an efficient mathematical representation, 3D Zernike Descriptor. Patch-Surfer2.0 has significant technical improvements over our previous prototype, which includes a new feature that captures approximate patch position with a geodesic distance histogram. Moreover, we constructed a large comprehensive database of ligand binding pockets that will be searched against by a query. The benchmark shows better performance of Patch-Surfer2.0 over existing methods. Availability and implementation: http://kiharalab.org/patchsurfer2.0/ Contact: dkihara@purdue.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25359888

Effects of mutations at amino acid 61 in the arm of TF1 on its DNA-binding properties.

PubMed

Sayre, M H; Geiduschek, E P

1990-12-20

Transcription factor 1 (TF1) is the Bacillus subtilis phage SPO1-encoded member of the family of bacterial DNA-binding proteins that includes Escherichia coli HU and integration host factor (IHF). We have initiated a mutational analysis of the TF1 molecule to understand better its unique DNA-binding properties and to investigate its physiological function. We report here the consequences of mutating the putative DNA-binding "arms" of TF1. At position 61 in its primary sequence, TF1 contains a Phe residue in place of the Arg residue found in all other known members of the HU family. Substituting polar, uncharged residues for Phe61 substantially reduced the DNA-binding affinity and site-selectivity of TF1 in vitro, whereas the substitution of Tyr had no effect. Substituting Trp or Arg for Phe61 had little effect on the affinity of TF1 for SPO1 DNA, but altered the electrophoretic mobilities of protein-DNA complexes in non-denaturing gels. The Arg61 substitution increased the affinity of the protein for non-specific sites on thymine-containing DNA, thus reducing the natural preference of TF1 for (5-hydroxymethyluracil)-containing DNA. The Phe61-to-Arg mutation was also correlated with decreased phage yield and aberrant regulation of viral protein synthesis in vivo.

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

Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi

Background: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. Methods and Results: In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partiallymore » abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Conclusion: Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. - Highlights: • E-selectin ligand (ESL)-1 was identified as an adiponectin (APN)-binding protein. • ESL-1 bound to APN at its N-terminal 6th-10th amino acids. • shESL-1 reduced the suppressive effect of APN on adhesion of THP-1 cells to HUVECs. • Interaction with ESL may be involved in the anti-atherogenic effects of APN.« less

Oxygen binding properties of hemoglobin from the white rhinoceros (beta 2-GLU) and the tapir.

PubMed

Baumann, R; Mazur, G; Braunitzer, G

1984-04-01

The beta-chain of rhinoceros hemoglobin contains glutamic acid at position beta 2, and important site for the binding of organic phosphates. We have investigated the oxygen binding properties of this hemoglobin and its interaction with ATP, 2,3-diphosphoglycerate, CO2 and chloride. The results show that the presence of GLU at position beta 2 nearly abolishes the effect of organic phosphates and CO2, whereas the oxygen-linked binding of chloride is not affected. Thus rhinoceros hemoglobin has only protons and chloride anions as major allosteric effectors for the control of its oxygen affinity. From the results obtained with hemoglobin solutions it can be calculated that the blood oxygen affinity of the rhinoceros must be rather high with a P50 of about 20 torr at pH 7.4 and 37 degrees C, which conforms with observations obtained for other large mammals.

DNA binding of supramolecular mixed-metal complexes

NASA Astrophysics Data System (ADS)

Swavey, Shawn; Williams, Rodd L.; Fang, Zhenglai; Milkevitch, Matthew; Brewer, Karen J.

2001-10-01

The high binding affinity of cisplatin toward DNA has led to its popularity as an anticancer agent. Due to cumulative drug resistance and toxic side effects, researchers are exploring related metallodrugs. Our approach involves the use of supramolecular complexes. These mixed-metal complexes incorporate a reactive platinum moiety bridged by a polyazine ligand to a light absorbing metal-based chromophore. The presence of the light absorber allows excitation of these systems, opening up the possibility of photoactivation. The use of a supramolecular design allows components of the assembly to be varied to enhance device function and light absorbing properties. Aspects of our molecular design process and results on the DNA binding properties for a number of these mixed-metal complexes will be discussed.

Theoretical and experimental study of organic nano-material for acetate anion based on 1, 10-phenanthroline.

PubMed

Shang, Xuefang; Zhao, Yuan; Wei, Xiaofang; Feng, Yaqian; Li, Xin; Gao, Shuyan; Xu, Xiufang

2015-01-01

New phenanthroline derivatives (1, 2, 3, 4) containing phenol groups have been synthesized and optimized. The nano-material of compound 2 was also developed. Their binding properties were evaluated for various biological anions (F(-), Cl(-), Br(-), I(-), AcO(-) and H(2)PO(4)(-)) by theoretical investigation, UV-vis, fluorescence, (1)HNMR titration experiments and these compounds all showed strong binding ability for AcO(-) without the interference of other anions tested. The anion binding ability could be regularized by electron push-pull properties of the ortho- or para- substituent on benzene. Theoretical investigation analysis revealed the effect of intramolecular hydrogen bond existed between -OH and other atoms in the structure of these compounds.

In Silico Docking and Electrophysiological Characterization of Lacosamide Binding Sites on Collapsin Response Mediator Protein-2 Identifies a Pocket Important in Modulating Sodium Channel Slow Inactivation*

PubMed Central

Wang, Yuying; Brittain, Joel M.; Jarecki, Brian W.; Park, Ki Duk; Wilson, Sarah M.; Wang, Bo; Hale, Rachel; Meroueh, Samy O.; Cummins, Theodore R.; Khanna, Rajesh

2010-01-01

The anti-epileptic drug (R)-lacosamide ((2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide (LCM)) modulates voltage-gated sodium channels (VGSCs) by preferentially interacting with slow inactivated sodium channels, but the observation that LCM binds to collapsin response mediator protein 2 (CRMP-2) suggests additional mechanisms of action for LCM. We postulated that CRMP-2 levels affects the actions of LCM on VGSCs. CRMP-2 labeling by LCM analogs was competitively displaced by excess LCM in rat brain lysates. Manipulation of CRMP-2 levels in the neuronal model system CAD cells affected slow inactivation of VGSCs without any effects on other voltage-dependent properties. In silico docking was performed to identify putative binding sites in CRMP-2 that may modulate the effects of LCM on VGSCs. These studies identified five cavities in CRMP-2 that can accommodate LCM. CRMP-2 alanine mutants of key residues within these cavities were functionally similar to wild-type CRMP-2 as assessed by similar levels of enhancement in dendritic complexity of cortical neurons. Next, we examined the effects of expression of wild-type and mutant CRMP-2 constructs on voltage-sensitive properties of VGSCs in CAD cells: 1) steady-state voltage-dependent activation and fast-inactivation properties were not affected by LCM, 2) CRMP-2 single alanine mutants reduced the LCM-mediated effects on the ability of endogenous Na+ channels to transition to a slow inactivated state, and 3) a quintuplicate CRMP-2 alanine mutant further decreased this slow inactivated fraction. Collectively, these results identify key CRMP-2 residues that can coordinate LCM binding thus making it more effective on its primary clinical target. PMID:20538611

Zampanolide Binding to Tubulin Indicates Cross-Talk of Taxane Site with Colchicine and Nucleotide Sites.

PubMed

Field, Jessica J; Pera, Benet; Gallego, Juan Estévez; Calvo, Enrique; Rodríguez-Salarichs, Javier; Sáez-Calvo, Gonzalo; Zuwerra, Didier; Jordi, Michel; Andreu, José M; Prota, Andrea E; Ménchon, Grégory; Miller, John H; Altmann, Karl-Heinz; Díaz, J Fernando

2018-03-23

The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to β-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 10 5 M -1 in the case of unmodified tubulin to 1.4 ± 0.3 × 10 5 M -1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin.

Binding of anti-apoptotic Bcl-2 with different BH3 peptides: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Zhang, Dawei; Liu, Huihui; Cui, Jinglan

2018-01-01

In this work, molecular dynamics simulation and free energy calculations are utilized to study how different BH3 peptides originating from Bax, Bim, Bik and Noxa interact with Bcl-2, one of the main members of anti-apoptotic proteins. The effects of peptide length, sequence and helical content on the binding affinity are discussed, on which a novel BH3-like peptide is designed in silico with an improved binding property.

Quantum transport in alkane molecular wires: Effects of binding modes and anchoring groups

NASA Astrophysics Data System (ADS)

Sheng, W.; Li, Z. Y.; Ning, Z. Y.; Zhang, Z. H.; Yang, Z. Q.; Guo, H.

2009-12-01

Effects of binding modes and anchoring groups on nonequilibrium electronic transport properties of alkane molecular wires are investigated from atomic first-principles based on density functional theory and nonequilibrium Green's function formalism. Four typical binding modes, top, bridge, hcp-hollow, and fcc-hollow, are considered at one of the two contacts. For wires with three different anchoring groups, dithiol, diamine, or dicarboxylic acid, the low bias conductances resulting from the four binding modes are all found to have either a high or a low value, well consistent with recent experimental observations. The trend can be rationalized by the behavior of electrode-induced gap states at small bias. When bias increases to higher values, states from the anchoring groups enter into the bias window and contribute significantly to the tunneling process so that transport properties become more complicated for the four binding modes. Other low bias behaviors including the values of the inverse length scale for tunneling characteristic, contact resistance, and the ratios of the high/low conductance values are also calculated and compared to experimental results. The conducting capabilities of the three anchoring groups are found to decrease from dithiol, diamine to dicarboxylic-acid, largely owing to a decrease in binding strength to the electrodes. Our results give a clear microscopic picture to the transport physics and provide reasonable qualitative explanations for the corresponding experimental data.

Effects of membrane properties on the binding activities of the HN and HC heavy-chain domains of botulinum neurotoxin A.

PubMed

Ayyar, B Vijayalakshmi; Atassi, M Zouhair

2016-12-01

Binding behaviors of the H N and the H C domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A H N and H C regions were prepared (H N 519-845 and H C 967-1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of H N 519-845 to Neuro 2a cells without affecting H C 967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both H C 967-1296 and H N 519-845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both H C 967-1296 and H N 519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K + ) depletion buffer lowered the binding of both H C 967-1296 and H N 519-845 to the cells, but seemed to exert a more pronounced effect on the binding of H C 967-1296 than on the binding of H N 519-845. Results indicate that while both the H N and H C domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology.

PubMed

List, K; Høyer-Hansen, G; Rønne, E; Danø, K; Behrendt, N

1999-01-01

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interference with conformational properties of the receptor critical for ligand binding. This distinction is central when employing the antibodies as tools in the elucidation of the structure-function relationship of the protein in question. We have studied the effect of monoclonal antibodies against the urokinase plasminogen activator receptor (uPAR), a protein located on the surface of various types of malignant and normal cells which is involved in the direction of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (BIA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former antibody efficiently blocked the receptor against subsequent ligand binding but was unable to promote the dissociation of a preformed receptor-ligand complex. The latter antibody was capable of binding the preformed complex, forming a transient trimolecular assembly, and promoting the dissociation of the uPA/uPAR complex. The continuous recording of binding and dissociation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this receptor reveals the presence of a determinant which influences the binding properties of a remote site in the molecular structure and which could be an important target for a putative synthetic antagonist.

Kinetics and equilibrium of solute diffusion into human hair.

PubMed

Wang, Liming; Chen, Longjian; Han, Lujia; Lian, Guoping

2012-12-01

The uptake kinetics of five molecules by hair has been measured and the effects of pH and physical chemical properties of molecules were investigated. A theoretical model is proposed to analyze the experimental data. The results indicate that the binding affinity of solute to hair, as characterized by hair-water partition coefficient, scales to the hydrophobicity of the solute and decreases dramatically as the pH increases to the dissociation constant. The effective diffusion coefficient of solute depended not only on the molecular size as most previous studies suggested, but also on the binding affinity as well as solute dissociation. It appears that the uptake of molecules by hair is due to both hydrophobic interaction and ionic charge interaction. Based on theoretical considerations of the cellular structure, composition and physical chemical properties of hair, quantitative-structure-property-relationships (QSPR) have been proposed to predict the hair-water partition coefficient (PC) and the effective diffusion coefficient (D (e)) of solute. The proposed QSPR models fit well with the experimental data. This paper could be taken as a reference for investigating the adsorption properties for polymeric materials, fibres, and biomaterials.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes.

PubMed

Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying

2015-01-01

Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

PubMed Central

2015-01-01

Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes. PMID:26681483

Effects of metal on the biochemical properties of Helicobacter pylori HypB, a maturation factor of [NiFe]-hydrogenase and urease.

PubMed

Sydor, Andrew M; Liu, Jenny; Zamble, Deborah B

2011-03-01

The biosyntheses of the [NiFe]-hydrogenase and urease enzymes in Helicobacter pylori require several accessory proteins for proper construction of the nickel-containing metallocenters. The hydrogenase accessory proteins HypA and HypB, a GTPase, have been implicated in the nickel delivery steps of both enzymes. In this study, the metal-binding properties of H. pylori HypB were characterized, and the effects of metal binding on the biochemical behavior of the protein were examined. The protein can bind stoichiometric amounts of Zn(II) or Ni(II), each with nanomolar affinity. Mutation of Cys106 and His107, which are located between two major GTPase motifs, results in undetectable Ni(II) binding, and the Zn(II) affinity is weakened by 2 orders of magnitude. These two residues are also required for the metal-dependent dimerization observed in the presence of Ni(II) but not Zn(II). The addition of metals to the protein has distinct impacts on GTPase activity, with zinc significantly reducing GTP hydrolysis to below detectable levels and nickel only slightly altering the k(cat) and K(m) of the reaction. The regulation of HypB activities by metal binding may contribute to the maturation of the nickel-containing enzymes.

Impact of the alkyl chain length on binding of imidazolium-based ionic liquids to bovine serum albumin

NASA Astrophysics Data System (ADS)

Zhang, Mengyue; Wang, Ying; Zhang, Hongmei; Cao, Jian; Fei, Zhenghao; Wang, Yanqing

2018-05-01

The effects of six imidazolium-based ionic liquids (ILs) with different alkyl chain length ([CnMim]Cl, n = 2, 4, 6, 8, 10, 12) on the structure and functions of bovine serum albumin (BSA) were studied by multi-spectral methods and molecular docking. ILs with the longer alkyl chain length have the stronger binding interaction with BSA and the greater conformational damage to protein. The effects of ILs on the functional properties of BSA were further studied by the determination of non-enzyme esterase activity, β-fibrosis and other properties of BSA. The thermal stability of BSA was reduced, the rate of the formation of beta sheet structures of BSA was lowered, and the esterase-like activity of BSA were decreased with the increase of ILs concentration. Simultaneous molecular modeling technique revealed the favorable binding sites of ILs on protein. The hydrophobic force and polar interactions were the mainly binding forces of them. The calculated results are in a good agreement with the spectroscopic experiments. These studies on the impact of the alkyl chain length on binding of imidazolium-based ionic liquids to BSA are of great significance for understanding and developing the application of ionic liquid in life and physiological system.

Predicting and analyzing DNA-binding domains using a systematic approach to identifying a set of informative physicochemical and biochemical properties

PubMed Central

2011-01-01

Background Existing methods of predicting DNA-binding proteins used valuable features of physicochemical properties to design support vector machine (SVM) based classifiers. Generally, selection of physicochemical properties and determination of their corresponding feature vectors rely mainly on known properties of binding mechanism and experience of designers. However, there exists a troublesome problem for designers that some different physicochemical properties have similar vectors of representing 20 amino acids and some closely related physicochemical properties have dissimilar vectors. Results This study proposes a systematic approach (named Auto-IDPCPs) to automatically identify a set of physicochemical and biochemical properties in the AAindex database to design SVM-based classifiers for predicting and analyzing DNA-binding domains/proteins. Auto-IDPCPs consists of 1) clustering 531 amino acid indices in AAindex into 20 clusters using a fuzzy c-means algorithm, 2) utilizing an efficient genetic algorithm based optimization method IBCGA to select an informative feature set of size m to represent sequences, and 3) analyzing the selected features to identify related physicochemical properties which may affect the binding mechanism of DNA-binding domains/proteins. The proposed Auto-IDPCPs identified m=22 features of properties belonging to five clusters for predicting DNA-binding domains with a five-fold cross-validation accuracy of 87.12%, which is promising compared with the accuracy of 86.62% of the existing method PSSM-400. For predicting DNA-binding sequences, the accuracy of 75.50% was obtained using m=28 features, where PSSM-400 has an accuracy of 74.22%. Auto-IDPCPs and PSSM-400 have accuracies of 80.73% and 82.81%, respectively, applied to an independent test data set of DNA-binding domains. Some typical physicochemical properties discovered are hydrophobicity, secondary structure, charge, solvent accessibility, polarity, flexibility, normalized Van Der Waals volume, pK (pK-C, pK-N, pK-COOH and pK-a(RCOOH)), etc. Conclusions The proposed approach Auto-IDPCPs would help designers to investigate informative physicochemical and biochemical properties by considering both prediction accuracy and analysis of binding mechanism simultaneously. The approach Auto-IDPCPs can be also applicable to predict and analyze other protein functions from sequences. PMID:21342579

Plant-derived micronutrients suppress monocyte adhesion to cultured human aortic endothelial cell layer by modulating its extracellular matrix composition.

PubMed

Ivanov, Vadim; Ivanova, Svetlana; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

2008-07-01

Monocyte adhesion to endothelium plays an important role in atherosclerosis. We investigated the effects of micronutrients on monocyte-binding properties of extracellular matrix (ECM) produced by human aortic endothelial cells (AoEC). Confluent cultures of AoEC were exposed to ascorbic acid, quercetin, gotu kola extract (10% asiatic acid), green tea extract (40% epigallocatechin gallate), or a mixture of these micronutrients for 48 hours. AoEC-produced ECM was exposed by differential treatment. U937 monocyte adhesion was assayed by fluorescence. ECM composition was assayed immunochemically and with radiolabeled metabolic precursors. AoEC exposure to micronutrients reduced ECM capacity to bind monocytes in a dose-dependent manner. This effect was accompanied by profound changes in the ECM composition. Correlation analysis revealed that changes in monocyte adhesion to ECM had the strongest positive correlation with ECM content for laminin (CC = 0.9681, P < 0.01), followed by fibronectin, collagens type III, I, and IV, biglycan, heparan sulfate, and elastin. The strongest negative correlation was with chondroitin sulfate (CC = -0.9623, P < 0.01), followed by perlecan and versican. Individual micronutrients had diverse effects on ECM composition and binding properties, and their mixture was the most effective treatment. In conclusion, micronutrient-dependent reduction of monocyte adhesion to endothelium is partly mediated through specific modulation of ECM composition and properties.

Effective Mass Theory of 2D Excitons Revisited

NASA Astrophysics Data System (ADS)

Gonzalez, Joseph; Oleynik, Ivan

Two-dimensional (2D) semiconducting materials possess an exceptionally unique set of electronic and excitonic properties due to the combined effects of quantum and dielectric confinement. Reliable determination of exciton binding energies from both first-principles many-body perturbation theory (GW/BSE) and experiment is very challenging due to the enormous computational expense as well as the tremendous technical difficulties in experiment.. Very recently, effective mass theories of 2D excitons have been developed as an attractive alternative for inexpensive and accurate evaluation of the exciton binding energies. In this presentation, we evaluate two effective mass theory approaches by Velizhanin et al and Olsen et al in predicting exciton binding energies across a wide range of 2D materials. We specifically analyze the trends related to the varying screening lengths and exciton effective masses. We also extended the effective mass theory of 2D excitons to include effects of electron and hole mass anisotropies (mx ≠ my) , the latter showing a substantial influence on exciton binding energies. The recent predictions of exciton binding energies being independent of the exciton effective mass and a linear correlation with the band gap of a specific material are also critically reexamined.

Excitons in boron nitride single layer

NASA Astrophysics Data System (ADS)

Galvani, Thomas; Paleari, Fulvio; Miranda, Henrique P. C.; Molina-Sánchez, Alejandro; Wirtz, Ludger; Latil, Sylvain; Amara, Hakim; Ducastelle, François

2016-09-01

Boron nitride single layer belongs to the family of two-dimensional materials whose optical properties are currently receiving considerable attention. Strong excitonic effects have already been observed in the bulk and still stronger effects are predicted for single layers. We present here a detailed study of these properties by combining ab initio calculations and a tight-binding Wannier analysis in both real and reciprocal space. Due to the simplicity of the band structure with single valence (π ) and conduction (π*) bands the tight-binding analysis becomes quasiquantitative with only two adjustable parameters and provides tools for a detailed analysis of the exciton properties. Strong deviations from the usual hydrogenic model are evidenced. The ground-state exciton is not a genuine Frenkel exciton, but a very localized tightly bound one. The other ones are similar to those found in transition-metal dichalcogenides and, although more localized, can be described within a Wannier-Mott scheme.

Exopolysaccharide from Ganoderma applanatum as a Promising Bioactive Compound with Cytostatic and Antibacterial Properties

PubMed Central

Osińska-Jaroszuk, Monika; Błachowicz, Adriana; Wydrych, Jerzy; Polak, Jolanta; Jarosz-Wilkołazka, Anna; Kandefer-Szerszeń, Martyna

2014-01-01

A new exopolysaccharide preparation isolated from stationary cultures of the white rot fungus Ganoderma applanatum (GpEPS) was tested in terms of its bioactive properties including its cytotoxic and immunostimulatory effect. The results indicate that the tested GpEPS (at concentrations above 22.85 µg/mL and 228.5 µg/mL) may exhibit selective activity against tumor cells (cell lines SiHa) and stimulate production of TNF-α THP-1-derived macrophages at the level of 752.17 pg/mL. The GpEPS showed antibacterial properties against Staphyloccoccus aureus and a toxic effect against Vibrio fischeri cells (82.8% cell damage). High cholesterol-binding capacity and triglycerides-binding capacity (57.9% and 41.6% after 24 h of incubation with the tested substances, resp.) were also detected for the investigated samples of GpEPS. PMID:25114920

Binding mechanisms of DNA/RNA nucleobases adsorbed on graphene under charging: first-principles van der Waals study

NASA Astrophysics Data System (ADS)

Gürel, Hikmet Hakan; Salmankurt, Bahadır

2017-06-01

Graphene is a 2D material that has attracted much attention due to its outstanding properties. Because of its high surface area and unique chemical and physical properties, graphene is a good candidate for biological applications. For this reason, a deep understanding of the mechanism of interaction of graphene with biomolecules is required. In this study, theoretical investigation of van der Waals effects has been conducted using density functional theory. Here we show that the order of the binding energies of five nucleobases with graphene is G  >  A  >  T  >  C  >   U. This trend is in good agreement with most of the theoretical and experimental data. Also, the effects of charging on the electronic and structural properties of the graphene-nucleubase systems are studied for the first time. We show that the binding energy can be changed by adding or removing an electron from the system. The results presented in this work provide fundamental insights into the quantum interactions of DNA with carbon-based nanostructures and will be useful for developments in biotechnology and nanotechnology.

Excitons, trions, and biexcitons in transition-metal dichalcogenides: Magnetic-field dependence

NASA Astrophysics Data System (ADS)

Van der Donck, M.; Zarenia, M.; Peeters, F. M.

2018-05-01

The influence of a perpendicular magnetic field on the binding energy and structural properties of excitons, trions, and biexcitons in monolayers of semiconducting transition metal dichalcogenides (TMDs) is investigated. The stochastic variational method (SVM) with a correlated Gaussian basis is used to calculate the different properties of these few-particle systems. In addition, we present a simplified variational approach which supports the SVM results for excitons as a function of magnetic field. The exciton diamagnetic shift is compared with recent experimental results, and we extend this concept to trions and biexcitons. The effect of a local potential fluctuation, which we model by a circular potential well, on the binding energy of trions and biexcitons is investigated and found to significantly increase the binding of those excitonic complexes.

Effect of amino acid mutations on intra-dimer tubulin-tubulin binding strength of microtubules.

PubMed

Liu, Ning; Pidaparti, Ramana; Wang, Xianqiao

2017-12-11

Energetic interactions inside αβ-tubulin dimers of a microtubule (MT) with atomic resolutions are of importance in determining the mechanical properties and structural stability of the MT as well as designing self-assembled functional structures from it. Here, we carry out several comprehensive atomistic simulations to investigate the interaction properties within αβ-tubulin dimers and effect of residue mutations on the intra-dimer tubulin-tubulin (IDTT) binding strength. Results indicate that the force-displacement responses of the dimer could be roughly divided into three stages involving increasing, decreasing, and fluctuating forces. Energetic analysis shows that electrostatic interactions dominate the IDTT binding strength. Further per-residue energetic analysis shows that the major part of the interface interaction energy (approximately 72% for α-tubulin and 62% for β-tubulin) comes from amino acid residues with net charges, namely arginine (ARG), lysine (LYS), glutamic acid (GLU), aspartic acid (ASP). Residue mutations are completed for ARG105 on α-tubulin and ASP251 on β-tubulin to study the effect of mutations on the IDTT binding strength. Results indicate that stiffness, rupture force, and interface interaction energy of αβ-tubulin dimer can be improved by up to 28%, 13% and 28%, respectively. Overall, our results provide a thorough atomistic understanding of the IDTT binding strength within αβ-tubulin heterodimers and help pave the way for eventually designing and controlling the self-assembled functional structures from MTs.

Large-scale binding ligand prediction by improved patch-based method Patch-Surfer2.0.

PubMed

Zhu, Xiaolei; Xiong, Yi; Kihara, Daisuke

2015-03-01

Ligand binding is a key aspect of the function of many proteins. Thus, binding ligand prediction provides important insight in understanding the biological function of proteins. Binding ligand prediction is also useful for drug design and examining potential drug side effects. We present a computational method named Patch-Surfer2.0, which predicts binding ligands for a protein pocket. By representing and comparing pockets at the level of small local surface patches that characterize physicochemical properties of the local regions, the method can identify binding pockets of the same ligand even if they do not share globally similar shapes. Properties of local patches are represented by an efficient mathematical representation, 3D Zernike Descriptor. Patch-Surfer2.0 has significant technical improvements over our previous prototype, which includes a new feature that captures approximate patch position with a geodesic distance histogram. Moreover, we constructed a large comprehensive database of ligand binding pockets that will be searched against by a query. The benchmark shows better performance of Patch-Surfer2.0 over existing methods. http://kiharalab.org/patchsurfer2.0/ CONTACT: dkihara@purdue.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Studies on the contributions of steric and polarity effects to the H2S-binding properties of Vitreoscilla hemoglobin

NASA Astrophysics Data System (ADS)

Wang, Dandan; Wang, Hui; Li, Haichao; Liu, Li; Li, Zhengqiang

2017-01-01

We have reported recently that Vitreoscilla hemoglobin (VHb) is a potential H2S receptor and storage molecule in bacterial metabolism. In this study, molecular cloning and site-directed mutagenesis were employed to investigate the structural basis for H2S binding. Association and dissociation rate constants (kon and koff) were determined using stopped-flow rapid-scanning spectrophotometry and compared with those for wild type VHb. Several unanticipated factors were found to govern H2S binding properties, due to the distinct structure of VHb. The results presented in this paper show that: i) bulkier residues at positions E7 and E11 decrease H2S binding accessibility, while the residue located at position B10 blocks bound H2S from escaping. ii) hydroxyl sidechains within the distal heme pocket reduce H2S reactivity to VHb; iii) Pro(E8) is involved in moving the E7-E10 loop region to trigger opening of the distal heme pocket to facilitate H2S binding.

Binding energies of benzene on coinage metal surfaces: Equal stability on different metals

NASA Astrophysics Data System (ADS)

Maaß, Friedrich; Jiang, Yingda; Liu, Wei; Tkatchenko, Alexandre; Tegeder, Petra

2018-06-01

Interfaces between organic molecules and inorganic solids adapt a prominent role in fundamental science, catalysis, molecular sensors, and molecular electronics. The molecular adsorption geometry, which is dictated by the strength of lateral and vertical interactions, determines the electronic structure of the molecule/substrate system. In this study, we investigate the binding properties of benzene on the noble metal surfaces Au(111), Ag(111), and Cu(111), respectively, using temperature-programmed desorption and first-principles calculations that account for non-locality of both electronic exchange and correlation effects. In the monolayer regime, we observed for all three systems a decrease of the binding energy with increasing coverage due to repulsive adsorbate/adsorbate interactions. Although the electronic properties of the noble metal surfaces are rather different, the binding strength of benzene on these surfaces is equal within the experimental error (accuracy of 0.05 eV), in excellent agreement with our calculations. This points toward the existence of a universal trend for the binding energy of aromatic molecules resulting from a subtle balance between Pauli repulsion and many-body van der Waals attraction.

Bioinspired assemblies of plant cell wall polymers unravel the affinity properties of carbohydrate-binding modules.

PubMed

Paës, Gabriel; von Schantz, Laura; Ohlin, Mats

2015-09-07

Lignocellulose-acting enzymes play a central role in the biorefinery of plant biomass to make fuels, chemicals and materials. These enzymes are often appended to carbohydrate binding modules (CBMs) that promote substrate targeting. When used in plant materials, which are complex assemblies of polymers, the binding properties of CBMs can be difficult to understand and predict, thus limiting the efficiency of enzymes. In order to gain more information on the binding properties of CBMs, some bioinspired model assemblies that contain some of the polymers and covalent interactions found in the plant cell walls have been designed. The mobility of three engineered CBMs has been investigated by FRAP in these assemblies, while varying the parameters related to the polymer concentration, the physical state of assemblies and the oligomerization state of CBMs. The features controlling the mobility of the CBMs in the assemblies have been quantified and hierarchized. We demonstrate that the parameters can have additional or opposite effects on mobility, depending on the CBM tested. We also find evidence of a relationship between the mobility of CBMs and their binding strength. Overall, bioinspired assemblies are able to reveal the unique features of affinity of CBMs. In particular, the results show that oligomerization of CBMs and the presence of ferulic acid motifs in the assemblies play an important role in the binding affinity of CBMs. Thus we propose that these features should be finely tuned when CBMs are used in plant cell walls to optimise bioprocesses.

The Staphylococcus aureus group II biotin protein ligase BirA is an effective regulator of biotin operon transcription and requires the DNA binding domain for full enzymatic activity.

PubMed

Henke, Sarah K; Cronan, John E

2016-11-01

Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins. However, the S. aureus BirA was reported to dimerize and bind DNA in the absence of biotin or biotinoyl-AMP (Soares da Costa et al. (2014) Mol Microbiol 91: 110-120). These in vitro results argued that the protein would be unable to respond to the levels of biotin or acceptor proteins and thus would lack the regulatory properties of the other characterized BirA proteins. We tested the regulatory function of the protein using an in vivo model system and examined its DNA binding properties in vitro using electrophoretic mobility shift and fluorescence anisotropy analyses. We report that the S. aureus BirA is an effective regulator of biotin operon transcription and that the prior data can be attributed to artifacts of mobility shift analyses. We also report that deletion of the DNA binding domain of the S. aureus BirA results in loss of virtually all of its ligation activity. © 2016 John Wiley & Sons Ltd.

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

PubMed

Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

2017-05-01

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

Quasiparticle and optical properties of strained stanene and stanane.

PubMed

Lu, Pengfei; Wu, Liyuan; Yang, Chuanghua; Liang, Dan; Quhe, Ruge; Guan, Pengfei; Wang, Shumin

2017-06-20

Quasiparticle band structures and optical properties of two dimensional stanene and stanane (fully hydrogenated stanene) are studied by the GW and GW plus Bethe-Salpeter equation (GW-BSE) approaches, with inclusion of the spin-orbit coupling (SOC). The SOC effect is significant for the electronic and optical properties in both stanene and stanane, compared with their group IV-enes and IV-anes counterparts. Stanene is a semiconductor with a quasiparticle band gap of 0.10 eV. Stanane has a sizable band gap of 1.63 eV and strongly binding exciton with binding energy of 0.10 eV. Under strain, the quasiparticle band gap and optical spectrum of both stanene and stanane are tunable.

Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

PubMed

Shemon, Anne N; Heil, Gary L; Granovsky, Alexey E; Clark, Mathew M; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R; Koide, Shohei

2010-05-05

Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

In Silico and In Vitro Analysis of Bacoside A Aglycones and Its Derivatives as the Constituents Responsible for the Cognitive Effects of Bacopa monnieri

PubMed Central

Ramasamy, Seetha; Chin, Sek Peng; Sukumaran, Sri Devi; Buckle, Michael James Christopher; Kiew, Lik Voon; Chung, Lip Yong

2015-01-01

Bacopa monnieri has been used in Ayurvedic medicine to improve memory and cognition. The active constituent responsible for its pharmacological effects is bacoside A, a mixture of dammarane-type triterpenoid saponins containing sugar chains linked to a steroid aglycone skeleton. Triterpenoid saponins have been reported to be transformed in vivo to metabolites that give better biological activity and pharmacokinetic characteristics. Thus, the activities of the parent compounds (bacosides), aglycones (jujubogenin and pseudojujubogenin) and their derivatives (ebelin lactone and bacogenin A1) were compared using a combination of in silico and in vitro screening methods. The compounds were docked into 5-HT1A, 5-HT2A, D1, D2, M1 receptors and acetylcholinesterase (AChE) using AutoDock and their central nervous system (CNS) drug-like properties were determined using Discovery Studio molecular properties and ADMET descriptors. The compounds were screened in vitro using radioligand receptor binding and AChE inhibition assays. In silico studies showed that the parent bacosides were not able to dock into the chosen CNS targets and had poor molecular properties as a CNS drug. In contrast, the aglycones and their derivatives showed better binding affinity and good CNS drug-like properties, were well absorbed through the intestines and had good blood brain barrier (BBB) penetration. Among the compounds tested in vitro, ebelin lactone showed binding affinity towards M1 (Ki = 0.45 μM) and 5-HT2A (4.21 μM) receptors. Bacoside A and bacopaside X (9.06 μM) showed binding affinity towards the D1 receptor. None of the compounds showed any inhibitory activity against AChE. Since the stimulation of M1 and 5-HT2A receptors has been implicated in memory and cognition and ebelin lactone was shown to have the strongest binding energy, highest BBB penetration and binding affinity towards M1 and 5-HT2A receptors, we suggest that B. monnieri constituents may be transformed in vivo to the active form before exerting their pharmacological activity. PMID:25965066

In Silico and In Vitro Analysis of Bacoside A Aglycones and Its Derivatives as the Constituents Responsible for the Cognitive Effects of Bacopa monnieri.

PubMed

Ramasamy, Seetha; Chin, Sek Peng; Sukumaran, Sri Devi; Buckle, Michael James Christopher; Kiew, Lik Voon; Chung, Lip Yong

2015-01-01

Bacopa monnieri has been used in Ayurvedic medicine to improve memory and cognition. The active constituent responsible for its pharmacological effects is bacoside A, a mixture of dammarane-type triterpenoid saponins containing sugar chains linked to a steroid aglycone skeleton. Triterpenoid saponins have been reported to be transformed in vivo to metabolites that give better biological activity and pharmacokinetic characteristics. Thus, the activities of the parent compounds (bacosides), aglycones (jujubogenin and pseudojujubogenin) and their derivatives (ebelin lactone and bacogenin A1) were compared using a combination of in silico and in vitro screening methods. The compounds were docked into 5-HT1A, 5-HT2A, D1, D2, M1 receptors and acetylcholinesterase (AChE) using AutoDock and their central nervous system (CNS) drug-like properties were determined using Discovery Studio molecular properties and ADMET descriptors. The compounds were screened in vitro using radioligand receptor binding and AChE inhibition assays. In silico studies showed that the parent bacosides were not able to dock into the chosen CNS targets and had poor molecular properties as a CNS drug. In contrast, the aglycones and their derivatives showed better binding affinity and good CNS drug-like properties, were well absorbed through the intestines and had good blood brain barrier (BBB) penetration. Among the compounds tested in vitro, ebelin lactone showed binding affinity towards M1 (Ki = 0.45 μM) and 5-HT2A (4.21 μM) receptors. Bacoside A and bacopaside X (9.06 μM) showed binding affinity towards the D1 receptor. None of the compounds showed any inhibitory activity against AChE. Since the stimulation of M1 and 5-HT2A receptors has been implicated in memory and cognition and ebelin lactone was shown to have the strongest binding energy, highest BBB penetration and binding affinity towards M1 and 5-HT2A receptors, we suggest that B. monnieri constituents may be transformed in vivo to the active form before exerting their pharmacological activity.

Distinctive receptor binding properties of the surface glycoprotein of a natural feline leukemia virus isolate with unusual disease spectrum.

PubMed

Bolin, Lisa L; Chandhasin, Chandtip; Lobelle-Rich, Patricia A; Albritton, Lorraine M; Levy, Laura S

2011-05-13

Feline leukemia virus (FeLV)-945, a member of the FeLV-A subgroup, was previously isolated from a cohort of naturally infected cats. An unusual multicentric lymphoma of non-T-cell origin was observed in natural and experimental infection with FeLV-945. Previous studies implicated the FeLV-945 surface glycoprotein (SU) as a determinant of disease outcome by an as yet unknown mechanism. The present studies demonstrate that FeLV-945 SU confers distinctive properties of binding to the cell surface receptor. Virions bearing the FeLV-945 Env protein were observed to bind the cell surface receptor with significantly increased efficiency, as was soluble FeLV-945 SU protein, as compared to the corresponding virions or soluble protein from a prototype FeLV-A isolate. SU proteins cloned from other cohort isolates exhibited increased binding efficiency comparable to or greater than FeLV-945 SU. Mutational analysis implicated a domain containing variable region B (VRB) to be the major determinant of increased receptor binding, and identified a single residue, valine 186, to be responsible for the effect. The FeLV-945 SU protein binds its cell surface receptor, feTHTR1, with significantly greater efficiency than does that of prototype FeLV-A (FeLV-A/61E) when present on the surface of virus particles or in soluble form, demonstrating a 2-fold difference in the relative dissociation constant. The results implicate a single residue, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with the receptor-binding domain through residue glutamine 110 to effect increased binding affinity. Through its increased receptor binding affinity, FeLV-945 SU might function in pathogenesis by increasing the rate of virus entry and spread in vivo, or by facilitating entry into a novel target cell with a low receptor density.

Modulating inhibitors of transthyretin fibrillogenesis via sulfation: polychlorinated biphenyl sulfates as models.

PubMed

Grimm, Fabian A; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W; Duffel, Michael W

2015-02-25

Small molecules that bind with high affinity to thyroxine (T4) binding sites on transthyretin (TTR) kinetically stabilize the protein's tetrameric structure, thereby efficiently decreasing the rate of tetramer dissociation in TTR related amyloidoses. Current research efforts aim to optimize the amyloid inhibiting properties of known inhibitors, such as derivatives of biphenyls, dibenzofurans and benzooxazoles, by chemical modification. In order to test the hypothesis that sulfate group substituents can improve the efficiencies of such inhibitors, we evaluated the potential of six polychlorinated biphenyl sulfates to inhibit TTR amyloid fibril formation in vitro. In addition, we determined their binding orientations and molecular interactions within the T4 binding site by molecular docking simulations. Utilizing this combined experimental and computational approach, we demonstrated that sulfation significantly improves the amyloid inhibiting properties as compared to both parent and hydroxylated PCBs. Importantly, several PCB sulfates were of equal or higher potency than some of the most effective previously described inhibitors. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Modulating Inhibitors of Transthyretin Fibrillogenesis via Sulfation: Polychlorinated Biphenyl Sulfates as Models1

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.; Duffel, Michael W.

2015-01-01

Small molecules that bind with high affinity to thyroxine (T4) binding sites on transthyretin (TTR) kinetically stabilize the protein’s tetrameric structure, thereby efficiently decreasing the rate of tetramer dissociation in TTR related amyloidoses. Current research efforts aim to optimize the amyloid inhibiting properties of known inhibitors, such as derivatives of biphenyls, dibenzofurans and benzooxazoles, by chemical modification. In order to test the hypothesis that sulfate group substituents can improve the efficiencies of such inhibitors, we evaluated the potential of six polychlorinated biphenyl sulfates to inhibit TTR amyloid fibril formation in vitro. In addition, we determined their binding orientations and molecular interactions within the T4 binding site by molecular docking simulations. Utilizing this combined experimental and computational approach, we demonstrated that sulfation significantly improves the amyloid inhibiting properties as compared to both parent and hydroxylated PCBs. Importantly, several PCB sulfates were of equal or higher potency than some of the most effective previously described inhibitors. PMID:25595224

Rapid comparison of protein binding site surfaces with Property Encoded Shape Distributions (PESD)

PubMed Central

Das, Sourav; Kokardekar, Arshad

2009-01-01

Patterns in shape and property distributions on the surface of binding sites are often conserved across functional proteins without significant conservation of the underlying amino-acid residues. To explore similarities of these sites from the viewpoint of a ligand, a sequence and fold-independent method was created to rapidly and accurately compare binding sites of proteins represented by property-mapped triangulated Gauss-Connolly surfaces. Within this paradigm, signatures for each binding site surface are produced by calculating their property-encoded shape distributions (PESD), a measure of the probability that a particular property will be at a specific distance to another on the molecular surface. Similarity between the signatures can then be treated as a measure of similarity between binding sites. As postulated, the PESD method rapidly detected high levels of similarity in binding site surface characteristics even in cases where there was very low similarity at the sequence level. In a screening experiment involving each member of the PDBBind 2005 dataset as a query against the rest of the set, PESD was able to retrieve a binding site with identical E.C. (Enzyme Commission) numbers as the top match in 79.5% of cases. The ability of the method in detecting similarity in binding sites with low sequence conservations were compared with state-of-the-art binding site comparison methods. PMID:19919089

How the extra methylene group affects the ligation properties of Glu vs. Asp and Gln vs. Asn amino acids: a DFT/PCM study.

PubMed

Dudev, Todor; Doudeva, Lyudmila

2017-02-01

The effect of the extra methylene group on the ligation properties of glutamic (Glu) vs. aspartic (Asp) acid, and glutamine (Gln) vs. asparagine (Asn) amino acids-two pairs of protein building blocks differing by the length of their side chains-has been studied by employing DFT calculations combined with polarizable continuum model (PCM) computations. Complexes of the nominal species with partner ligands of various structures, charge states, and degree of solvent exposure have been examined. The results obtained reveal that the difference in the alkyl chain length of these amino acid residues does not affect the mode of their binding. This, however, influences the thermodynamics of the ligand-ligand and ligand-metal recognition thus bestowing unique ligation characteristics on the competing entities. The calculations reveal that the competition between the longer-chain and shorter-chain analogs is entropy driven and that the differential electronic effects are of minor importance for the process. Thus, the outcome of the rivalry between Asp and Glu, and Asn and Gln is almost unaffected by the nature of the partner ligand, its charge state and, in most cases, the dielectric properties of the binding site. The longer-chain Glu, as opposed to its shorter-chain Asp counterpart, is the preferred partner ligand in various protein binding sites. Contrariwise, the shorter-chain Asn binds more favorably to the respective binding sites than its longer-chain Gln analog. The results obtained shed additional light on the intimate mechanism of the ligand-ligand and ligand-metal recognition in proteins and could be employed as guidelines in protein engineering and design.

Effects of pH on transport properties of articular cartilages.

PubMed

Loret, Benjamin; Simões, Fernando M F

2010-02-01

Articular cartilages swell and shrink depending on the ionic strength of the electrolyte they are in contact with. This electro-chemo-mechanical coupling is due to the presence of fixed electrical charges on proteoglycans (PGs). In addition, at nonphysiological pH, collagen fibers become charged. Therefore, variation of the pH of the electrolyte has strong implications on the electrical charge of cartilages and, by the same token, on their transport and mechanical properties. Articular cartilages are viewed as three-phase multi-species porous media. The constitutive framework is phrased in the theory of thermodynamics of porous media. Acid-base reactions, as well as calcium binding, are embedded in this framework. Although macroscopic in nature, the model accounts for a number of biochemical details defining collagen and PGs. The change of the electrical charge is due to the binding of hydrogen ions on specific sites of PGs and collagen. Simulations are performed mimicking laboratory experiments where either the ionic strength or the pH of the bath, the cartilage piece is in contact with, is varied. They provide the evolutions of the chemical compositions of mobile ions, of the sites of acid-base reactions and calcium binding, and of the charges of collagen and glycosaminoglycans, at constant volume fraction of water. Emphasis is laid on the effects of pH, ionic strength and calcium binding on the transport properties of cartilages, and, in particular, on the electrical conductivity and electro-osmotic coefficient.

Saw palmetto extracts potently and noncompetitively inhibit human alpha1-adrenoceptors in vitro.

PubMed

Goepel, M; Hecker, U; Krege, S; Rübben, H; Michel, M C

1999-02-15

We wanted to test whether phytotherapeutic agents used in the treatment of lower urinary tract symptoms have alpha1-adrenoceptor antagonistic properties in vitro. Preparations of beta-sitosterol and extracts of stinging nettle, medicinal pumpkin, and saw palmetto were obtained from several pharmaceutical companies. They were tested for their ability to inhibit [3H]tamsulosin binding to human prostatic alpha1-adrenoceptors and [3H]prazosin binding to cloned human alpha1A- and alpha1B-adrenoceptors. Inhibition of phenylephrine-stimulated [3H]inositol phosphate formation by cloned receptors was also investigated. Up to the highest concentration which could be tested, preparations of beta-sitosterol, stinging nettle, and medicinal pumpkin were without consistent inhibitory effect in all assays. In contrast, all tested saw palmetto extracts inhibited radioligand binding to human alpha1-adrenoceptors and agonist-induced [3H]inositol phosphate formation. Saturation binding experiments in the presence of a single saw palmetto extract concentration indicated a noncompetitive antagonism. The relationship between active concentrations in vitro and recommended therapeutic doses for the saw palmetto extracts was slightly lower than that for several chemically defined alpha1-adrenoceptor antagonists. Saw palmetto extracts have alpha1-adrenoceptor-inhibitory properties. If bioavailability and other pharmacokinetic properties of these ingredients are similar to those of the chemically defined alpha1-adrenoceptor antagonists, alpha1-adrenoceptor antagonism might be involved in the therapeutic effects of these extracts in patients with lower urinary tract symptoms suggestive of benign prostatic obstruction.

Pharmaceutical-grade albumin: impaired drug-binding capacity in vitro

PubMed Central

Olsen, Harald; Andersen, Anders; Nordbø, Arve; Kongsgaard, Ulf E; Børmer, Ole P

2004-01-01

Background Albumin is the most abundant protein in blood plasma, and due to its ligand binding properties, serves as a circulating depot for endogenous and exogenous (e.g. drugs) compounds. Hence, the unbound drug is the pharmacologically active drug. Commercial human albumin preparations are frequently used during surgery and in critically ill patients. Recent studies have indicated that the use of pharmaceutical-grade albumin is controversial in critically ill patients. In this in vitro study we investigated the drug binding properties of pharmaceutical-grade albumins (Baxter/Immuno, Octapharma, and Pharmacia & Upjohn), native human serum, and commercially available human serum albumin from Sigma Chemical Company. Methods The binding properties of the various albumin solutions were tested in vitro by means of ultrafiltration. Naproxen, warfarin, and digitoxin were used as ligands. HPLC was used to quantitate the total and free drug concentrations. The data were fitted to a model of two classes of binding sites for naproxen and warfarin and one class for digitoxin, using Microsoft Excel and Graphpad Prism. Results The drugs were highly bound to albumin (95–99.5%). The highest affinity (lowest K1) was found with naproxen. Pharmaceutical-grade albumin solutions displayed significantly lower drug-binding capacity compared to native human serum and Sigma albumin. Thus, the free fraction was considerably higher, approximately 40 times for naproxen and 5 and 2 times for warfarin and digitoxin, respectively. The stabilisers caprylic acid and N-acetyl-DL-tryptophan used in the manufacturing procedure seem to be of importance. Adding the stabilisers to human serum and Sigma albumin reduced the binding affinity whereas charcoal treatment of the pharmaceutical-grade albumin from Octapharma almost restored the specific binding capacity. Conclusion This in vitro study demonstrates that the specific binding for warfarin and digitoxin is significantly reduced and for naproxen no longer detectable in pharmaceutical-grade albumin. It further shows that the addition of stabilisers may be of major importance for this effect. PMID:15046641

dsRNA binding properties of RDE-4 and TRBP reflect their distinct roles in RNAi.

PubMed

Parker, Greg S; Maity, Tuhin Subhra; Bass, Brenda L

2008-12-26

Double-stranded RNA (dsRNA)-binding proteins facilitate Dicer functions in RNA interference. Caenorhabditis elegans RDE-4 facilitates cleavage of long dsRNA to small interfering RNA (siRNA), while human trans-activation response RNA-binding protein (TRBP) functions downstream to pass siRNA to the RNA-induced silencing complex. We show that these distinct in vivo roles are reflected in in vitro binding properties. RDE-4 preferentially binds long dsRNA, while TRBP binds siRNA with an affinity that is independent of dsRNA length. These properties are mechanistically based on the fact that RDE-4 binds cooperatively, via contributions from multiple domains, while TRBP binds noncooperatively. Our studies offer a paradigm for how dsRNA-binding proteins, which are not sequence specific, discern dsRNA length. Additionally, analyses of the ability of RDE-4 deletion constructs and RDE-4/TRBP chimeras to reconstitute Dicer activity suggest RDE-4 promotes activity using its dsRNA-binding motif 2 to bind dsRNA, its linker region to interact with Dicer, and its C-terminus for Dicer activation.

Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

PubMed Central

Li, Guangwei; Chen, Xiulin; Li, Boliao; Zhang, Guohui; Li, Yiping; Wu, Junxiang

2016-01-01

Background The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications. Methodology/Principal Finding Two antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition. Conclusion Two rGmolGOBPs exhibit different binding characteristics for tested ligands. rGmolGOBP1 has dual functions in recognition of host plant volatiles and sex pheromone components, while rGmolGOBP2 is mainly involved in minor sex pheromone component dodecanol perception. This study also provides empirical evidence for the predicted functions of key amino acids in recombinant protein ligand-binding characteristics. PMID:27152703

High-affinity binding of laurate to naturally occurring mutants of human serum albumin and proalbumin.

PubMed

Kragh-Hansen, U; Pedersen, A O; Galliano, M; Minchiotti, L; Brennan, S O; Tárnoky, A L; Franco, M H; Salzano, F M

1996-12-15

Binding of laurate (n-dodecanoate) to genetic variants of albumin or its proprotein and to normal albumin isolated from the same heterozygous carriers was studied by a kinetic dialysis technique at physiological pH. The first stoichiometric association constant for binding to proalbumin Lille (Arg-2-->His) and albumin (Alb) Roma (Glu321-->Lys) was increased to 126% and 136% respectively compared with that for binding to normal albumin, whereas the constant for Alb Maku (Lys541-->Glu) was decreased to 80%. In contrast, normal laurate-binding properties were found for as many as nine other albumin variants with single amino acid substitutions. Because the net charges of all these mutants were different from that of normal albumin, the results suggest that the examples of modified laurate binding are not caused by long-range electrostatic effects. Rather, the three positions mentioned are located close to different binding sites for the fatty acid anion. The most pronounced effect was observed for the glycosylated Alb Casebrook, the binding constant of which was decreased to 20%. Binding to the glycosylated Alb Redhill was also decreased, but to a smaller extent (68%). These decreases in binding are caused by partial or total blocking of the high-affinity site by the oligosaccharides, by the negative charges of the oligosaccharides, and/or by conformational changes induced by these bulky moieties. Laurate binding to two chain-termination mutants (Alb Catania and Alb Venezia) was normal, indicating that the C-terminus of albumin is not important for binding. By using different preparations of normal albumin as controls in the binding experiments, it was also possible to compare the effect of various methods for isolation and defatting on laurate binding.

Ionic Strength, Surface Charge, and Packing Density Effects on the Properties of Peptide Self-Assembled Monolayers.

PubMed

Leo, Norman; Liu, Juan; Archbold, Ian; Tang, Yongan; Zeng, Xiangqun

2017-02-28

The various environmental parameters of packing density, ionic strength, and solution charge were examined for their effects on the properties of the immobilized peptide mimotope CH19 (CGSGSGSQLGPYELWELSH) that binds with the therapeutic antibody Trastuzumab (Herceptin) on a gold substrate. The immobilization of CH19 onto gold was examined with a quartz crystal microbalance (QCM). The QCM data showed the presence of intermolecular interactions resulting in the increase of viscoelastic properties of the peptide self-assembled monolayer (SAM). The CH19 SAM was diluted with CS7 (CGSGSGS) to decrease the packing density as CH19/CS7. The packing density and ionic strength parameters were evaluated by atomic force microscopy (AFM), ellipsometry, and QCM. AFM and ellipsometry showed a distinct conformational difference between CH19 and CH19/CS7, indicating a relationship between packing density and conformational state of the immobilized peptide. The CH19 SAM thickness was 40 Å with a rough topology, while the CH19/CS7 SAM thickness was 20 Å with a smooth topology. The affinity studies showed that the affinity of CH19 and CH19/CS7 to Trastuzumab were both on the order of 10 7 M -1 in undiluted PBS buffer, while the dilution of the buffer by 1000× increased both SAMs affinities to Trastuzumab to the order of 10 15 M -2 and changed the binding behavior from noncooperative to cooperative binding. This indicated that ionic strength had a more pronounced effect on binding properties of the CH19 SAM than packing density. Electrochemical impedance spectroscopy (EIS) was conducted on the CH19/CS7 SAM, which showed an increase in impedance after each EIS measurement cycle. Cyclic voltammetry on the CH19/CS7 SAM decreased impedance to near initial values. The impact of the packing density, buffer ionic strength, and local charge perturbation of the peptide SAM properties was interpreted based on the titratable sites in CH19 that could participate in the proton transfer and water equilibrium.

Aqueous Synthesis of PEGylated Quantum Dots with Increased Colloidal Stability and Reduced Cytotoxicity.

PubMed

Ulusoy, Mehriban; Jonczyk, Rebecca; Walter, Johanna-Gabriela; Springer, Sergej; Lavrentieva, Antonina; Stahl, Frank; Green, Mark; Scheper, Thomas

2016-02-17

Ligands used on the surface of colloidal nanoparticles (NPs) have a significant impact on physiochemical properties of NPs and their interaction in biological environments. In this study, we report a one-pot aqueous synthesis of 3-mercaptopropionic acid (MPA)-functionalized CdTe/CdS/ZnS quantum dots (Qdots) in the presence of thiol-terminated methoxy polyethylene glycol (mPEG) molecules as a surface coordinating ligand. The resulting mPEG-Qdots were characterized by using ζ potential, FTIR, thermogravimetric (TG) analysis, and microscale thermophoresis (MST) studies. We investigated the effect of mPEG molecules and their grafting density on the Qdots photophysical properties, colloidal stability, protein binding affinity, and in vitro cellular toxicity. Moreover, cellular binding features of the resulting Qdots were examined by using three-dimensional (3D) tumor-like spheroids, and the results were discussed in detail. Promisingly, mPEG ligands were found to increase colloidal stability of Qdots, reduce adsorption of proteins to the Qdot surface, and mitigate Qdot-induced side effects to a great extent. Flow cytometry and confocal microscopy studies revealed that PEGylated Qdots exhibited distinctive cellular interactions with respect to their mPEG grafting density. As a result, mPEG molecules demonstrated a minimal effect on the ZnS shell deposition and the Qdot fluorescence efficiency at a low mPEG density, whereas they showed pronounced effect on Qdot colloidal stability, protein binding affinity, cytotoxicity, and nonspecific binding at a higher mPEG grafting amount.

Contrasting effects of photochemical and microbial degradation on Cu(II) binding with fluorescent DOM from different origins.

PubMed

Xu, Huacheng; Guan, Dong-Xing; Zou, Li; Lin, Hui; Guo, Laodong

2018-08-01

Effects of photochemical and microbial degradation on variations in composition and molecular-size of dissolved organic matter (DOM) from different sources (algal and soil) and the subsequent influence on Cu(II) binding were investigated using UV-Vis, fluorescence excitation-emission matrices coupled with parallel factor analysis, flow field-flow fractionation (FlFFF), and metal titration. The degradation processes resulted in an initial rapid decline in the bulk dissolved organic carbon and chromophoric and fluorescent DOM components, followed by a small or little decrease. Specifically, photochemical reaction decreased the aromaticity, humification and apparent molecular weights of all DOM samples, whereas a reverse trend was observed during microbial degradation. The FlFFF fractograms revealed that coagulation of both protein- and humic-like DOM induced an increase in molecular weights for algal-DOM, while the molecular weight enhancement for allochthonous soil samples was mainly attributed to the self-assembly of humic-like components. The Cu(II) binding capacity of algal-derived humic-like and fulvic-like DOM consistently increased during photo- and bio-degradation, while the soil-derived DOM exhibited a slight decline in Cu(II) binding capacity during photo-degradation but a substantial increase during microbial degradation, indicating source- and degradation-dependent metal binding heterogeneities. Pearson correlation analysis demonstrated that the Cu(II) binding potential was mostly related with aromaticity and molecular size for allochthonous soil-derived DOM, but was regulated by both DOM properties and specific degradation processes for autochthonous algal-derived DOM. This study highlighted the coupling role of inherent DOM properties and external environmental processes in regulating metal binding, and provided new insights into metal-DOM interactions and the behavior and fate of DOM-bound metals in aquatic environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Fluorescence analysis of competition of phenylbutazone and methotrexate in binding to serum albumin in combination treatment in rheumatology

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

2009-04-01

Combination of several drugs is often necessary especially during long-them therapy. The competition between drugs can cause a decrease of the amount of a drug bound to albumin. This results in an increase of the free, biological active fraction of the drug. The aim of the presented study was to describe the competition between phenylbutazone (Phe) and methotrexate (MTX), two drugs recommended for the treatment of rheumatology in binding to bovine (BSA) and human (HSA) serum albumin in the high affinity binding site. Fluorescence analysis was used to estimate the effect of drugs on the protein fluorescence and to define the binding and quenching properties of drugs-serum albumin complexes. The effect of the displacement of one drug from the complex of the other with serum albumin has been described on the basis of the comparison of the quenching curves and binding constants for the binary and ternary systems. The conclusion that both Phe and MTX form a binding site in the same subdomain (IIA) points to the necessity of using a monitoring therapy owning to the possible increase of the uncontrolled toxic effects.

Preparation and DNA-binding properties of substituted triostin antibiotics.

PubMed

Cornish, A; Fox, K R; Waring, M J

1983-02-01

Novel derivatives of the triostin group of antibiotics were prepared by supplementing cultures of the producing organism Streptomyces triostinicus with a variety of aromatic carboxylic acids. Five new antibiotics, each having both the natural quinoxaline chromophores replaced by a substituted ring system, were purified to homogeneity and characterized by high-pressure liquid chromatography and nuclear magnetic resonance. Their antibacterial activities and DNA-binding properties were investigated. Addition of a halogen atom at position 6 of the quinoxaline ring or an amino group at position 3 had little effect on either the biological activity or the DNA-binding characteristics. The bis-3-amino derivative is fluorescent, and its fluorescence is strongly quenched by calf thymus DNA and polydeoxyguanylate-polydeoxycytidylate but not by polydeoxyadenylate-polydeoxythymidylate, suggesting that it binds preferentially to guanosine-cytosine-rich sequences in natural DNA. Binding constants for the bis-6-chloro and bis-3-amino derivatives do not differ greatly from those of unsubstituted triostin A. The analogs having two quinoline chromophores or a chlorine atom in position 7 of the quinoxaline ring display little or no detectable antibacterial activity, in marked contrast to the other congeners. Bis-7-chloro-triostin A binds conspicuously more tightly to polydeoxyadenylate-polydeoxythymidylate than to any other polynucleotide tested.

Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

PubMed Central

de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; Gomes da Silva, Lúcia Patrícia Bezerra; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Gois Ruiz, Ana Lucia Tasca; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

2015-01-01

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

Effect of Escherichia coli DNA binding protein on the transcription of single-stranded phage M13 DNA by Escherichia coli RNA polymerase

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

Niyogi, S.K.; Ratrie, H. III; Datta, A.K.

E. coli DNA binding protein strongly inhibits the transcription of single-stranded rather than double-stranded phage M13 DNA by E. coli RNA polymerase. This inhibition cannot be significantly overcome by increasing the concentration of RNA polymerase. Nor does the order of addition of binding protein affect its inhibitory property: inhibition is evident whether binding protein is added before or after the formation of the RNA polymerase--DNA complex. Inhibition is also observed if binding protein is added at various times after initiation of RNA synthesis. Maximal inhibition occurs at a binding protein-to-DNA ratio (w/w) of about 8:1. This corresponds to one bindingmore » protein molecule covering about 30 nucleotides, in good agreement with values obtained by physical measurements.« less

Experimental and computational studies on the effects of valganciclovir as an antiviral drug on calf thymus DNA.

PubMed

Shahabadi, Nahid; Pourfoulad, Mehdi; Moghadam, Neda Hosseinpour

2017-01-02

DNA-binding properties of an antiviral drug, valganciclovir (valcyte) was studied by using emission, absorption, circular dichroism, viscosity, differential pulse voltammetry, fluorescence techniques, and computational studies. The drug bound to calf thymus DNA (ct-DNA) in a groove-binding mode. The calculated binding constant of UV-vis, K a , is comparable to groove-binding drugs. Competitive fluorimetric studies with Hoechst 33258 showed that valcyte could displace the DNA-bound Hoechst 33258. The drug could not displace intercalated methylene blue from DNA double helix. Furthermore, the induced detectable changes in the CD spectrum of ct-DNA as well as changes in its viscosity confirm the groove-binding mode. In addition, an integrated molecular docking was employed to further investigate the binding interactions between valcyte and calf thymus DNA.

Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands

PubMed Central

Granovsky, Alexey E.; Clark, Mathew M.; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R.; Koide, Shohei

2010-01-01

Background Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. Methods/Findings In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. Conclusions/Significance This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential. PMID:20463977

Effects of flow changes on radiotracer binding: Simultaneous measurement of neuroreceptor binding and cerebral blood flow modulation.

PubMed

Sander, Christin Y; Mandeville, Joseph B; Wey, Hsiao-Ying; Catana, Ciprian; Hooker, Jacob M; Rosen, Bruce R

2017-01-01

The potential effects of changes in blood flow on the delivery and washout of radiotracers has been an ongoing question in PET bolus injection studies. This study provides practical insight into this topic by experimentally measuring cerebral blood flow (CBF) and neuroreceptor binding using simultaneous PET/MRI. Hypercapnic challenges (7% CO 2 ) were administered to non-human primates in order to induce controlled increases in CBF, measured with pseudo-continuous arterial spin labeling. Simultaneously, dopamine D 2 /D 3 receptor binding of [ 11 C]raclopride or [ 18 F]fallypride was monitored with dynamic PET. Experiments showed that neither time activity curves nor quantification of binding through binding potentials ( BP ND ) were measurably affected by CBF increases, which were larger than two-fold. Simulations of experimental procedures showed that even large changes in CBF should have little effect on the time activity curves of radiotracers, given a set of realistic assumptions. The proposed method can be applied to experimentally assess the flow sensitivity of other radiotracers. Results demonstrate that CBF changes, which often occur due to behavioral tasks or pharmacological challenges, do not affect PET [ 11 C]raclopride or [ 18 F]fallypride binding studies and their quantification. The results from this study suggest flow effects may have limited impact on many PET neuroreceptor tracers with similar properties.

Effects of Low-Energy Excitations on Spectral Properties at Higher Binding Energy: The Metal-Insulator Transition of VO2

NASA Astrophysics Data System (ADS)

Gatti, Matteo; Panaccione, Giancarlo; Reining, Lucia

2015-03-01

The effects of electron interaction on spectral properties can be understood in terms of coupling between excitations. In transition-metal oxides, the spectral function close to the Fermi level and low-energy excitations between d states have attracted particular attention. In this work we focus on photoemission spectra of vanadium dioxide over a wide (10 eV) range of binding energies. We show that there are clear signatures of the metal-insulator transition over the whole range due to a cross coupling of the delocalized s and p states with low-energy excitations between the localized d states. This coupling can be understood by advanced calculations based on many-body perturbation theory in the G W approximation. We also advocate the fact that tuning the photon energy up to the hard-x-ray range can help to distinguish fingerprints of correlation from pure band-structure effects.

Interrelations of secondary structure stability and DNA-binding affinity in the bacteriophage SPO1-encoded type II DNA-binding protein TF1.

PubMed

Andera, L; Spangler, C J; Galeone, A; Mayol, L; Geiduschek, E P

1994-02-11

TF1, a homodimeric DNA-binding and -bending protein with a preference for hydroxymethyluracil-containing DNA is the Bacillus subtilis-encoded homolog of the bacterial HU proteins and of the E. coli integration host factor. A temperature-sensitive mutation at amino acid 25 of TF1 (L25-->A) and two intragenic second site revertants at amino acids 15 (E15-->G) and 32 (L32-->I) were previously identified and their effects on virus development were examined. The DNA-binding properties of these proteins and the thermal stability of their secondary structures have now been analyzed. Amino acids 15 and 32 are far removed from the putative DNA-binding domains of TF1 but changes there exert striking effects on DNA affinity that correlate with effects on structure. The double mutant protein TF1-G15I32 binds to a preferred site in hydroxymethyluracil-containing DNA 40 times more tightly, denatures at higher temperature (delta tm = 21 degrees C), and also exchanges subunits much more slowly than does the wild-type protein. The L25-->A mutation makes TF1 secondary structure and DNA-binding highly salt concentration-dependent. The E15-->G mutation partly suppresses this effect: secondary structure of TF1-A25G15 is restored at 21 degrees C by 1 M NaCl or, at low NaCl concentration, by binding to DNA.

Development of multifunctional, heterodimeric isoindoline-1,3-dione derivatives as cholinesterase and β-amyloid aggregation inhibitors with neuroprotective properties.

PubMed

Guzior, Natalia; Bajda, Marek; Skrok, Mirosław; Kurpiewska, Katarzyna; Lewiński, Krzysztof; Brus, Boris; Pišlar, Anja; Kos, Janko; Gobec, Stanislav; Malawska, Barbara

2015-03-06

The presented study describes the synthesis, pharmacological evaluation (AChE and BuChE inhibition, beta amyloid anti-aggregation effect and neuroprotective effect), molecular modeling and crystallographic studies of a novel series of isoindoline-1,3-dione derivatives. The target compounds were designed as dual binding site acetylcholinesterase inhibitors with an arylalkylamine moiety binding at the catalytic site of the enzyme and connected via an alkyl chain to a heterocyclic fragment, capable of binding at the peripheral anionic site of AChE. Among these molecules, compound 15b was found to be the most potent and selective AChE inhibitor (IC50EeAChE = 0.034 μM). Moreover, compound 13b in addition to AChE inhibition (IC50 EeAChE = 0.219 μM) possesses additional properties, such as the ability to inhibit Aβ aggregation (65.96% at 10 μM) and a neuroprotective effect against Aβ toxicity at 1 and 3 μM. Compound 13b emerges as a promising multi-target ligand for the further development of the therapy for age-related neurodegenerative disorders. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Methyl group reorientation under ligand binding probed by pseudocontact shifts.

PubMed

Lescanne, Mathilde; Ahuja, Puneet; Blok, Anneloes; Timmer, Monika; Akerud, Tomas; Ubbink, Marcellus

2018-06-02

Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1-3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.

Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor

PubMed Central

Kuban-Jankowska, Alicja; Sahu, Kamlesh K.; Gorska, Magdalena; Tuszynski, Jack A.; Wozniak, Michal

2016-01-01

Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding. PMID:26735581

Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor.

PubMed

Kuban-Jankowska, Alicja; Sahu, Kamlesh K; Gorska, Magdalena; Tuszynski, Jack A; Wozniak, Michal

2016-01-19

Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding.

Probing the General Time Scale Question of Boronic Acid Binding with Sugars in Aqueous Solution at Physiological pH

PubMed Central

Ni, Nanting; Laughlin, Sarah; Wang, Yingji; Feng, You; Zheng, Yujun

2012-01-01

The boronic acid group is widely used in chemosensor design due to its ability to reversibly bind diol-containing compounds. The thermodynamic properties of the boronic acid-diol binding process have been investigated extensively. However, there are few studies of the kinetic properties of such binding processes. In this report, stopped-flow method was used for the first time to study the kinetic properties of the binding between three model arylboronic acids, 4-, 5-, and 8-isoquinolinylboronic acids, and various sugars. With all the boronic acid-diol pair sexamined, reactions were complete within seconds. The kon values with various sugars follow the order of D-fructose >D-tagatose>D-mannose >D-glucose. This trend tracks the thermodynamic binding affinities for these sugars and demonstrates that the “on” rate is the key factor determining the binding constant. PMID:22464680

Modular cell biology: retroactivity and insulation

PubMed Central

Del Vecchio, Domitilla; Ninfa, Alexander J; Sontag, Eduardo D

2008-01-01

Modularity plays a fundamental role in the prediction of the behavior of a system from the behavior of its components, guaranteeing that the properties of individual components do not change upon interconnection. Just as electrical, hydraulic, and other physical systems often do not display modularity, nor do many biochemical systems, and specifically, genetic networks. Here, we study the effect of interconnections on the input–output dynamic characteristics of transcriptional components, focusing on a property, which we call ‘retroactivity', that plays a role analogous to non-zero output impedance in electrical systems. In transcriptional networks, retroactivity is large when the amount of transcription factor is comparable to, or smaller than, the amount of promoter-binding sites, or when the affinity of such binding sites is high. To attenuate the effect of retroactivity, we propose a feedback mechanism inspired by the design of amplifiers in electronics. We introduce, in particular, a mechanism based on a phosphorylation–dephosphorylation cycle. This mechanism enjoys a remarkable insulation property, due to the fast timescales of the phosphorylation and dephosphorylation reactions. PMID:18277378

Influence of enzymatic hydrolysis on the allergenic reactivity of processed cashew and pistachio.

PubMed

Cuadrado, Carmen; Cheng, Hsiaopo; Sanchiz, Africa; Ballesteros, Isabel; Easson, Michael; Grimm, Casey C; Dieguez, M Carmen; Linacero, Rosario; Burbano, Carmen; Maleki, Soheila J

2018-02-15

Cashew and pistachio allergies are considered a serious health problem. Previous studies have shown that thermal processing, pressurization and enzymatic hydrolysis may reduce the allergenic properties of food by changing the protein structure. This study assesses the allergenic properties of cashew and pistachio after thermal treatment (boiling and autoclaving), with or without pressure (autoclaving), and multiple enzymatic treatments under sonication, by SDS-PAGE, western blot and ELISA, with serum IgE of allergic individuals, and mass spectroscopy. Autoclaving and enzymatic hydrolysis under sonication separately induced a measurable reduction in the IgE binding properties of pastes made from treated cashew and pistachio nuts. These treatments were more effective with pistachio allergens. However, heat combined with enzymatic digestion was necessary to markedly lower IgE binding to cashew allergens. The findings identify highly effective simultaneous processing conditions to reduce or even abolish the allergenic potency of cashew and pistachio. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of phloretin on the binding of 1-anilino-8-naphtalene sulfonate (ANS) to 1,2-Dimyristoyl-sn-glycero-3-phosphocoline (DMPC) vesicles in the gel and liquid-crystalline state.

PubMed

Cutró, Andrea C; Montich, Guillermo; Roveri, Oscar A

2015-02-01

Phloretin is a known modifier of the internal dipole potential of lipid membranes. We studied the interaction of phloretin with model lipid membranes and how it influences the membrane dipole organization using ANS as fluorescent probe. The fluorescence increase observed when ANS binds to DMPC liposomes in gel phase (13 °C) was 2.5 times larger in the presence of phloretin. This effect was due to an increase in ANS affinity, which can be related to the known capability of phloretin in decreasing the dipole potential. Conversely, when the experiments were carried out at 33 °C (liquid crystalline phase), phloretin completely inhibited the increase in ANS fluorescence. In addition, phloretin only affected the electrical properties of the membrane in the gel phase, whereas it modifies structural ones in the liquid-crystalline state. We postulate that phloretin was bound only to the DMPC interface in the gel phase decreasing the surface negative charge density without modifying the structural properties of the ANS binding sites. In the liquid-crystalline phase instead, it increased the accessibility of water to the ANS binding sites decreasing the intrinsic affinity and the fluorescence quantum yield of ANS.

dsRNA binding properties of RDE-4 and TRBP reflect their distinct roles in RNAi

PubMed Central

Parker, Greg S.; Maity, Tuhin Subhra; Bass, Brenda L.

2008-01-01

SUMMARY dsRNA binding proteins (dsRBPs) facilitate Dicer functions in RNAi. C. elegans RDE-4 facilitates cleavage of long dsRNA to siRNA, while human TRBP functions downstream to pass siRNA to RISC. We show that these distinct in vivo roles are reflected in in vitro binding properties. RDE-4 preferentially binds long dsRNA, while TRBP binds siRNA with an affinity that is independent of dsRNA length. These properties are mechanistically based in the fact that RDE-4 binds cooperatively, via contributions from multiple domains, while TRBP binds non-cooperatively. Our studies offer a paradigm for how dsRBPs, which are not sequence-specific, discern dsRNA length. Additionally, analyses of the ability of RDE-4 deletion constructs and RDE-4/TRBP chimeras to reconstitute Dicer activity suggest RDE-4 promotes activity using its dsRBM2 to bind dsRNA, its linker region to interact with Dicer, and its C-terminus for Dicer activation. PMID:18948111

Revealing kinetics and state-dependent binding properties of IKur-targeting drugs that maximize atrial fibrillation selectivity

NASA Astrophysics Data System (ADS)

Ellinwood, Nicholas; Dobrev, Dobromir; Morotti, Stefano; Grandi, Eleonora

2017-09-01

The KV1.5 potassium channel, which underlies the ultra-rapid delayed-rectifier current (IKur) and is predominantly expressed in atria vs. ventricles, has emerged as a promising target to treat atrial fibrillation (AF). However, while numerous KV1.5-selective compounds have been screened, characterized, and tested in various animal models of AF, evidence of antiarrhythmic efficacy in humans is still lacking. Moreover, current guidelines for pre-clinical assessment of candidate drugs heavily rely on steady-state concentration-response curves or IC50 values, which can overlook adverse cardiotoxic effects. We sought to investigate the effects of kinetics and state-dependent binding of IKur-targeting drugs on atrial electrophysiology in silico and reveal the ideal properties of IKur blockers that maximize anti-AF efficacy and minimize pro-arrhythmic risk. To this aim, we developed a new Markov model of IKur that describes KV1.5 gating based on experimental voltage-clamp data in atrial myocytes from patient right-atrial samples in normal sinus rhythm. We extended the IKur formulation to account for state-specificity and kinetics of KV1.5-drug interactions and incorporated it into our human atrial cell model. We simulated 1- and 3-Hz pacing protocols in drug-free conditions and with a [drug] equal to the IC50 value. The effects of binding and unbinding kinetics were determined by examining permutations of the forward (kon) and reverse (koff) binding rates to the closed, open, and inactivated states of the KV1.5 channel. We identified a subset of ideal drugs exhibiting anti-AF electrophysiological parameter changes at fast pacing rates (effective refractory period prolongation), while having little effect on normal sinus rhythm (limited action potential prolongation). Our results highlight that accurately accounting for channel interactions with drugs, including kinetics and state-dependent binding, is critical for developing safer and more effective pharmacological anti-AF options.

Fungal chitinases: diversity, mechanistic properties and biotechnological potential.

PubMed

Hartl, Lukas; Zach, Simone; Seidl-Seiboth, Verena

2012-01-01

Chitin derivatives, chitosan and substituted chito-oligosaccharides have a wide spectrum of applications ranging from medicine to cosmetics and dietary supplements. With advancing knowledge about the substrate-binding properties of chitinases, enzyme-based production of these biotechnologically relevant sugars from biological resources is becoming increasingly interesting. Fungi have high numbers of glycoside hydrolase family 18 chitinases with different substrate-binding site architectures. As presented in this review, the large diversity of fungal chitinases is an interesting starting point for protein engineering. In this review, recent data about the architecture of the substrate-binding clefts of fungal chitinases, in connection with their hydrolytic and transglycolytic abilities, and the development of chitinase inhibitors are summarized. Furthermore, the biological functions of chitinases, chitin and chitosan utilization by fungi, and the effects of these aspects on biotechnological applications, including protein overexpression and autolysis during industrial processes, are discussed in this review.

Calculating Water Thermodynamics in the Binding Site of Proteins - Applications of WaterMap to Drug Discovery.

PubMed

Cappel, Daniel; Sherman, Woody; Beuming, Thijs

2017-01-01

The ability to accurately characterize the solvation properties (water locations and thermodynamics) of biomolecules is of great importance to drug discovery. While crystallography, NMR, and other experimental techniques can assist in determining the structure of water networks in proteins and protein-ligand complexes, most water molecules are not fully resolved and accurately placed. Furthermore, understanding the energetic effects of solvation and desolvation on binding requires an analysis of the thermodynamic properties of solvent involved in the interaction between ligands and proteins. WaterMap is a molecular dynamics-based computational method that uses statistical mechanics to describe the thermodynamic properties (entropy, enthalpy, and free energy) of water molecules at the surface of proteins. This method can be used to assess the solvent contributions to ligand binding affinity and to guide lead optimization. In this review, we provide a comprehensive summary of published uses of WaterMap, including applications to lead optimization, virtual screening, selectivity analysis, ligand pose prediction, and druggability assessment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Monoclonal antibody binding to the macrophage-specific receptor sialoadhesin alters the phagocytic properties of human and mouse macrophages.

PubMed

De Schryver, Marjorie; Cappoen, Davie; Elewaut, Dirk; Nauwynck, Hans J; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L

2017-02-01

Sialoadhesin (Sn) is a surface receptor expressed on macrophages in steady state conditions, but during inflammation, Sn can be upregulated both on macrophages and on circulating monocytes. It was shown for different species that Sn becomes internalized after binding with monoclonal antibodies. These features suggest that Sn is a potential target for immunotherapies. In this study, human and mouse macrophages were treated with anti-Sn monoclonal antibodies or F(ab') 2 fragments and the effect of their binding to Sn on phagocytosis was analyzed. Binding of antibodies to Sn resulted in delayed and reduced phagocytosis of fluorescent beads. No effect was observed on Fc-mediated phagocytosis or phagocytosis of bacteria by human macrophages. In contrast, an enhanced phagocytosis of bacteria by mouse macrophages was detected. These results showed that stimulation of Sn could have different effects on macrophage phagocytosis, depending both on the type of phagocytosis and cellular background. Copyright © 2016 Elsevier Inc. All rights reserved.

Biochemical and pharmacological characterization of the thyrotropin releasing hormone (TRH) receptor from clonal GH sub 4 C sub 1 pituitary cells

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

Phillips, W.J.

1987-01-01

The effect of drugs with anesthetic properties on the activity of the pituitary thyrotropin-releasing hormone (TRH) receptor was determined in the clonal GH{sub 4}C{sub 1} somatomammotropic cell line. Classic local anesthetics and other drugs with anesthetic activity inhibited binding of ({sup 3}H)methyl-TRH to cell receptors at concentrations known to produce anesthetic effects on the membrane. The inhibition of TRH receptor binding by tetracaine was competitive and temperature and pH dependent. Verapamil and tetracaine inhibited TRH-stimulated prolactin secretion at concentrations that inhibited peptide binding. TRH-stimulated prolactin secretion was equivalent with or without Ca{sup 2+} channel activity. Verapamil and tetracaine also inhibitedmore » basal prolactin and secretion stimulated by drugs that bypass membrane receptors, db-cAMP and TPA. These results indicate that inhibition of TRH binding and responses by diverse drugs results from an anesthetic effect on the cell membrane.« less

Theoretical Studies for Dendrimer-Based Drug Delivery.

PubMed

Bello, Martiniano; Fragoso-Vázquez, Jonathan; Correa-Basurto, José

2017-01-01

Numerous theoretical studies have been performed to iteratively optimize the physicochemical properties such as dendrimer size and surface constituents in solution, as well as their molecular recognition properties for drugs, lipid membranes, nucleic acids and proteins, etc. Molecular modeling approaches such as docking and molecular dynamic (MD) simulations have supported experimental efforts by providing important insights into the structural properties of dendrimers in solution and possible binding properties of drugs at the atomic level. We review the utilization of molecular modelling tools to obtain insight into the study and design of dendrimers, with particular importance placed on the improvement of binding properties of dendrimers for their use as drug nanocarriers and to increase the water solubility properties and drug delivery. The modeling studies discussed in this review have provided substantial insight into the physicochemical properties of dendrimers in solution, including solvent pH and counterion distribution, at the atomic level, as well as the elucidation of some of the key interactions in solution of unmodified and modified dendrimers with some drugs of pharmaceutics interest and biological systems such as nucleic acids, proteins and lipid membranes. the described studies illustrate that whether simulations will be run at the all-atom or coarse-grained level, physicochemical conditions such as the type of force field, the treatment of electrostatics effects, counterion distribution, protonation state of dendrimers, and dendrimer concentrations which have been probed to play a crucial role in the structural behavior and binding properties must be prudently incorporated in the simulations. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

26 CFR 1.197-1T - Certain elections for intangible property (temporary).

Code of Federal Regulations, 2010 CFR

2010-04-01

...) to apply prior law to property acquired pursuant to a written binding contract in effect on August 10... section 13261 of OBRA '93 (e.g., the amendment of section 1253 in the case of a franchise, trademark, or... section 197 intangible with an unadjusted basis of $180. Under prior law, no amortization or depreciation...

Binding site stoichiometry and the effects of phosphorylation on human α1 homomeric glycine receptors

PubMed Central

Gentet, Luc J; Clements, John D

2002-01-01

The kinetic properties of the human α1 homomeric glycine receptor were investigated. Receptors were expressed in HEK 293 cells, and glycine was applied to outside-out membrane patches with sub-millisecond solution exchange. The activation time course of the glycine response was used to investigate receptor stoichiometry. The unbinding of three strychnine molecules and the cooperative binding of two glycine molecules were required to activate the channel. The effects of phosphorylation on glycine receptor kinetics were investigated by pretreating cells with phosphorylators or with phosphatases. Phosphorylation accelerated desensitisation, but slowed deactivation and recovery from desensitisation. A chemical-kinetic model was developed that reproduced the experimental observations. The model suggests that only three binding sites on the glycine channel are functional, while the remaining two binding sites are ‘silent’, possibly due to strong negative cooperativity. PMID:12356883

Action of novel antipsychotics at human dopamine D3 receptors coupled to G protein and ERK1/2 activation.

PubMed

Bruins Slot, Liesbeth A; Palmier, Christiane; Tardif, Stéphanie; Cussac, Didier

2007-08-01

The effects of new generation antipsychotic drugs (APDs) targeting dopamine D(2) and serotonin 5-HT(1A) receptors were compared with typical and atypical APDs on phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and measures of G protein activation in CHO cell lines stably expressing the human dopamine D(3) receptor. The preferential dopamine D(3) agonists (+)-7-OH-DPAT and PD128907, like dopamine and quinelorane, efficaciously stimulated ERK 1/2 phosphorylation at dopamine D(3) receptors. In contrast, in [(35)S]GTPgammaS binding experiments, (+)-7-OH-DPAT exhibited partial agonist properties, while PD128907 and quinelorane maintained full agonist properties. The preferential dopamine D(3) ligand BP 897 and the antidyskinetic sarizotan partially activated ERK 1/2 phosphorylation while exerting no agonist activity on GTPgammaS binding, suggesting signal amplification at the MAP kinase level. Antipsychotics differed in their ability to inhibit both agonist-stimulated GTPgammaS binding and ERK 1/2 phosphorylation, but all typical and atypical compounds tested acted as dopamine D(3) receptor antagonists with the exception of n-desmethylclozapine, the active metabolite of clozapine, which partially activated dopamine D(3) receptor-mediated ERK 1/2 phosphorylation. Among the new generation dopamine D(2)/serotonin 5-HT(1A) antipsychotics, only F 15063 and SLV313 acted as pure dopamine D(3) receptor antagonists, bifeprunox was highly efficacious whereas SSR181507 and aripiprazole showed marked partial agonist properties for ERK 1/2 phosphorylation. In contrast, in the GTPgammaS binding study, aripiprazole was devoid of agonist properties and bifeprunox, and to an even lesser extent SSR181507, only weakly stimulated GTPgammaS binding. In summary, these findings underline the differences of dopamine D(3) properties of new generation antipsychotics which may need to be considered in understanding their diverse therapeutic actions.

Biological properties of prolactin-releasing peptide analogs with a modified aromatic ring of a C-terminal phenylalanine amide.

PubMed

Maletínská, Lenka; Spolcová, Andrea; Maixnerová, Jana; Blechová, Miroslava; Zelezná, Blanka

2011-09-01

Prolactin-releasing peptide (PrRP)-induced secretion of prolactin is not currently considered a primary function of PrRP, but the development of late-onset obesity in both PrRP and PrRP receptor knock-out mice indicates the unique anorexigenic properties of PrRP. In our recent study, we showed comparable potencies of peptides PrRP31 and PrRP20 in binding, intracellular signaling and prolactin release in pituitary RC-4B/C cells, and anorexigenic effect after central administration in fasted mice. In the present study, eight analogs of PrRP20 with C-terminal Phe amide modified with a bulky side chain or a halogenated aromatic ring revealed high binding potency, activation of mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) and prolactin release in RC-4B/C cells. In particular, [PheNO(2)(31)]PrRP20, [1-Nal(31)]PrRP20, [2-Nal(31)]PrRP20 and [Tyr(31)]PrRP20 showed not only in vitro effects comparable or higher than those of PrRP20, but also a very significant and long-lasting anorexigenic effect after central administration in fasted mice. The design of potent and long-lasting PrRP analogs with selective anorexigenic properties promises to contribute to the study of food intake disorders. Copyright © 2011 Elsevier Inc. All rights reserved.

Probing the binding interaction of a phenazinium dye with serum transport proteins: a combined fluorometric and circular dichroism study.

PubMed

Bose, Debosreeta; Sarkar, Deboleena; Chattopadhyay, Nitin

2010-01-01

In the present investigation, an attempt has been made to study the interaction of phenosafranin (PSF), a cationic phenazinium dye with the transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), employing steady-state and time-resolved fluorometric and circular dichroism (CD) techniques. The photophysical properties of the dye are altered on binding with the serum proteins. An explicit study with respect to the modification of the fluorescence and fluorescence anisotropy upon binding, effect of denaturant, fluorescence lifetime and CD measurements reveal that the dye binds to both BSA and HSA with almost the same affinity. Far-UV CD spectra indicate a decrease in the percentage of alpha-helicity only for BSA upon binding with the probe. Near-UV CD responses indicate an alteration in the tertiary structure of both the transport proteins because of binding.

Non-intercalative, deoxyribose binding of boric acid to calf thymus DNA.

PubMed

Ozdemir, Ayse; Gursaclı, Refiye Tekiner; Tekinay, Turgay

2014-05-01

The present study characterizes the effects of the boric acid binding on calf thymus DNA (ct-DNA) by spectroscopic and calorimetric methods. UV-Vis absorbance spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), isothermal titration calorimetry (ITC), and Fourier transform infrared (FT-IR) spectroscopy were employed to characterize binding properties. Changes in the secondary structure of ct-DNA were determined by CD spectroscopy. Sizes and morphologies of boric acid-DNA complexes were determined by transmission electron microscopy (TEM). The kinetics of boric acid binding to calf thymus DNA (ct-DNA) was investigated by isothermal titration calorimetry (ITC). ITC results revealed that boric acid exhibits a moderate affinity to ct-DNA with a binding constant (K a) of 9.54 × 10(4) M(-1). FT-IR results revealed that boric acid binds to the deoxyribose sugar of DNA without disrupting the B-conformation at tested concentrations.

Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

PubMed

Sugitani, Norie; Voehler, Markus W; Roh, Michelle S; Topolska-Woś, Agnieszka M; Chazin, Walter J

2017-10-13

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The effect of fibre loading and graphene on the mechanical properties of goat hair fibre epoxy composite

NASA Astrophysics Data System (ADS)

Jayaseelan, J.; Vijayakumar, K. R.; Ethiraj, N.; Sivabalan, T.; nallayan, W. Andrew

2017-12-01

Composite materials are heterogenous materials containing one or more solid phases. In recent years cost-effective composite making is an ideal task. Hence we have come out with a natural fibre composite, which contains goat hair and epoxy as a binding element, with the combination of Graphene as a main source of enhanced mechanical property. Fabrication of natural composite consists of five layers of goat hair sandwiched in epoxy matrix. These composites made are tested for mechanical properties including Tensile strength, Flexural strength, Inter laminar shear and Impact strength. The mechanical properties of the six composite sets are analyzed and reported.

Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

PubMed

He, Mingyue; Hamon, Maureen; Liu, Hong; Corper, Adam L; Taussig, Michael J

2006-09-01

The crystal structures of the Fab' fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 10(4)-fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position.

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

PubMed

Verrier, C S; Roodi, N; Yee, C J; Bailey, L R; Jensen, R A; Bustin, M; Parl, F F

1997-07-01

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

Properties of inhibitors of methane hydrate formation via molecular dynamics simulations.

PubMed

Anderson, Brian J; Tester, Jefferson W; Borghi, Gian Paolo; Trout, Bernhardt L

2005-12-21

Within the framework of a proposed two-step mechanism for hydrate inhibition, the energy of binding of four inhibitor molecules (PEO, PVP, PVCap, and VIMA) to a hydrate surface is estimated with molecular dynamic simulations. One key feature of this proposed mechanism is that the binding of an inhibitor molecule to the surface of an ensuing hydrate crystal disrupts growth and therein crystallization. It is found through the molecular dynamic simulations that inhibitor molecules that experimentally exhibit better inhibition strength also have higher free energies of binding, an indirect confirmation of our proposed mechanism. Inhibitors increasing in effectiveness, PEO < PVP < PVCap < VIMA, have increasingly negative (exothermic) binding energies of -0.2 < -20.6 < -37.5 < -45.8 kcal/mol and binding free energies of increasing favorability (+0.4 approximately = +0.5 < -9.4 < -15.1 kcal/mol). Furthermore, the effect of an inhibitor molecule on the local liquid water structure under hydrate-forming conditions was examined and correlated to the experimental effectiveness of the inhibitors. Two molecular characteristics that lead to strongly binding inhibitors were found: (1) a charge distribution on the edge of the inhibitor that mimics the charge separation in the water molecules on the surface of the hydrate and (2) the congruence of the size of the inhibitor with respect to the available space at the hydrate-surface binding site. Equipped with this molecular-level understanding of the process of hydrate inhibition via low-dosage kinetic hydrate inhibitors we can design new, more effective inhibitor molecules.

Pharmacological profile of essential oils derived from Lavandula angustifolia and Melissa officinalis with anti-agitation properties: focus on ligand-gated channels.

PubMed

Huang, Liping; Abuhamdah, Sawsan; Howes, Melanie-Jayne R; Dixon, Christine L; Elliot, Mark S J; Ballard, Clive; Holmes, Clive; Burns, Alistair; Perry, Elaine K; Francis, Paul T; Lees, George; Chazot, Paul L

2008-11-01

Both Melissa officinalis (Mo) and Lavandula angustifolia (La) essential oils have putative anti-agitation properties in humans, indicating common components with a depressant action in the central nervous system. A dual radioligand binding and electrophysiological study, focusing on a range of ligand-gated ion channels, was performed with a chemically validated essential oil derived from La, which has shown clinical benefit in treating agitation. La inhibited [35S] TBPS binding to the rat forebrain gamma aminobutyric acid (GABA)(A) receptor channel (apparent IC50 = 0.040 +/- 0.001 mg mL(-1)), but had no effect on N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or nicotinic acetylcholine receptors. A 50:50 mixture of Mo and La essential oils inhibited [3H] flunitrazepam binding, whereas the individual oils had no significant effect. Electrophysiological analyses with rat cortical primary cultures demonstrated that La reversibly inhibited GABA-induced currents in a concentration-dependent manner (0.01-1 mg mL(-1)), whereas no inhibition of NMDA- or AMPA-induced currents was noted. La elicited a significant dose-dependent reduction in both inhibitory and excitatory transmission, with a net depressant effect on neurotransmission (in contrast to the classic GABA(A) antagonist picrotoxin which evoked profound epileptiform burst firing in these cells). These properties are similar to those recently reported for Mo. The anti-agitation effects in patients and the depressant effects of La we report in neural membranes in-vitro are unlikely to reflect a sedative interaction with any of the ionotropic receptors examined here. These data suggest that components common to the two oils are worthy of focus to identify the actives underlying the neuronal depressant and anti-agitation activities reported.

Characterization of ( sup 3 H)alprazolam binding to central benzodiazepine receptors

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

McCabe, R.T.; Mahan, D.R.; Smith, R.B.

1990-10-01

The binding of the triazolobenzodiazepine ({sup 3}H)alprazolam was studied to characterize the in vitro interactions with benzodiazepine receptors in membrane preparations of rat brain. Studies using nonequilibrium and equilibrium binding conditions for ({sup 3}H)alprazolam resulted in high specific to nonspecific (signal to noise) binding ratios. The binding of ({sup 3}H)alprazolam was saturable and specific with a low nanomolar affinity for benzodiazepine receptors in the rat brain. The Kd was 4.6 nM and the Bmax was 2.6 pmol/mg protein. GABA enhanced ({sup 3}H)alprazolam binding while several benzodiazepine receptor ligands were competitive inhibitors of this drug. Compounds that bind to other receptormore » sites had a very weak or negligible effect on ({sup 3}H)alprazolam binding. Alprazolam, an agent used as an anxiolytic and in the treatment of depression, acts in vitro as a selective and specific ligand for benzodiazepine receptors in the rat brain. The biochemical binding profile does not appear to account for the unique therapeutic properties which distinguish this compound from the other benzodiazepines in its class.« less

Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

PubMed

Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

2009-10-01

Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

Biophysical Fitness Landscapes for Transcription Factor Binding Sites

PubMed Central

Haldane, Allan; Manhart, Michael; Morozov, Alexandre V.

2014-01-01

Phenotypic states and evolutionary trajectories available to cell populations are ultimately dictated by complex interactions among DNA, RNA, proteins, and other molecular species. Here we study how evolution of gene regulation in a single-cell eukaryote S. cerevisiae is affected by interactions between transcription factors (TFs) and their cognate DNA sites. Our study is informed by a comprehensive collection of genomic binding sites and high-throughput in vitro measurements of TF-DNA binding interactions. Using an evolutionary model for monomorphic populations evolving on a fitness landscape, we infer fitness as a function of TF-DNA binding to show that the shape of the inferred fitness functions is in broad agreement with a simple functional form inspired by a thermodynamic model of two-state TF-DNA binding. However, the effective parameters of the model are not always consistent with physical values, indicating selection pressures beyond the biophysical constraints imposed by TF-DNA interactions. We find little statistical support for the fitness landscape in which each position in the binding site evolves independently, indicating that epistasis is common in the evolution of gene regulation. Finally, by correlating TF-DNA binding energies with biological properties of the sites or the genes they regulate, we are able to rule out several scenarios of site-specific selection, under which binding sites of the same TF would experience different selection pressures depending on their position in the genome. These findings support the existence of universal fitness landscapes which shape evolution of all sites for a given TF, and whose properties are determined in part by the physics of protein-DNA interactions. PMID:25010228

Glycosaminoglycans are interactants of Langerin: comparison with gp120 highlights an unexpected calcium-independent binding mode.

PubMed

Chabrol, Eric; Nurisso, Alessandra; Daina, Antoine; Vassal-Stermann, Emilie; Thepaut, Michel; Girard, Eric; Vivès, Romain R; Fieschi, Franck

2012-01-01

Langerin is a C-type lectin specifically expressed in Langerhans cells. As recently shown for HIV, Langerin is thought to capture pathogens and mediate their internalisation into Birbeck Granules for elimination. However, the precise functions of Langerin remain elusive, mostly because of the lack of information on its binding properties and physiological ligands. Based on recent reports that Langerin binds to sulfated sugars, we conducted here a comparative analysis of Langerin interaction with mannose-rich HIV glycoprotein gp120 and glycosaminoglycan (GAGs), a family of sulfated polysaccharides expressed at the surface of most mammalian cells. Our results first revealed that Langerin bound to these different glycans through very distinct mechanisms and led to the identification of a novel, GAG-specific binding mode within Langerin. In contrast to the canonical lectin domain, this new binding site showed no Ca(2+)-dependency, and could only be detected in entire, trimeric extracellular domains of Langerin. Interestingly binding to GAGs, did not simply rely on a net charge effect, but rather on more discrete saccharide features, such as 6-O-sulfation, or iduronic acid content. Using molecular modelling simulations, we proposed a model of Langerin/heparin complex, which located the GAG binding site at the interface of two of the three Carbohydrate-recognition domains of the protein, at the edge of the a-helix coiled-coil. To our knowledge, the binding properties that we have highlighted here for Langerin, have never been reported for C-type lectins before. These findings provide new insights towards the understanding of Langerin biological functions.

Interaction of albumin with perylene-diimides with aromatic substituents

NASA Astrophysics Data System (ADS)

Farooqi, Mohammed; Penick, Mark; Burch, Jessica; Negrete, George; Brancaleon, Lorenzo

2015-03-01

Polyaromatic hydrocarbons (PAH) binding to proteins remains one of the fundamental aspects of research in biophysics. Ligand binding can regulate the function of proteins. Binding to small ligands remains a very important aspect in the study of the function of many proteins. Perylene diimide or PDI derivatives have attracted initial interest as industrial dyes and pigments. Recently, much attention has been focused on their strong π - π stacks resulting from the large PDI aromatic core. These PDI stacks have distinct optical properties, and provide informative models that mimic the light-harvesting system and initial charge separation and charge transfer in the photosynthetic system. The absorption property of PDI derivatives may be largely tuned from visible to near-infrared region by chemical modifications at the bay-positions. We are currently studying a new class of PDI derivatives with substituents made of the side chains of aromatic amino acids (Tyrosine, Tryptophan and Phenylalanine). We have looked at the fluorescence absorption and emission of these PDIs in water and other organic solvents. PDIs show evidence of dimerization and possible aggregation. We also present binding studies of these PDIs with Human Serum Albumin (HSA). The binding was studied using fluorescence emission quenching of the HSA Tryptophan residue. Stern-Volmer equation is used to derive the quenching constants. PDI binding to HSA also has an effect on the fluorescence emission of the PDIs themselves by red shifting the spectra. Funded by RCMI grant.

Receptor binding properties and antinociceptive effects of chimeric peptides consisting of a micro-opioid receptor agonist and an ORL1 receptor antagonist.

PubMed

Kawano, Susumu; Ito, Risa; Nishiyama, Miharu; Kubo, Mai; Matsushima, Tomoko; Minamisawa, Motoko; Ambo, Akihiro; Sasaki, Yusuke

2007-07-01

Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100< pmol/mouse) than when administered i.t. (ED(50): 1.34-4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

PubMed

Vashisht, Kapil; Verma, Sonia; Gupta, Sunita; Lynn, Andrew M; Dixit, Rajnikant; Mishra, Neelima; Valecha, Neena; Hamblin, Karleigh A; Maytum, Robin; Pandey, Kailash C; van der Giezen, Mark

2017-01-24

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

The structural basis for function in diamond-like carbon binding peptides.

PubMed

Gabryelczyk, Bartosz; Szilvay, Géza R; Linder, Markus B

2014-07-29

The molecular structural basis for the function of specific peptides that bind to diamond-like carbon (DLC) surfaces was investigated. For this, a competition assay that provided a robust way of comparing relative affinities of peptide variants was set up. Point mutations of specific residues resulted in significant effects, but it was shown that the chemical composition of the peptide was not sufficient to explain peptide affinity. More significantly, rearrangements in the sequence indicated that the binding is a complex recognition event that is dependent on the overall structure of the peptide. The work demonstrates the unique properties of peptides for creating functionality at interfaces via noncovalent binding for potential applications in, for example, nanomaterials, biomedical materials, and sensors.

On the atomic-number similarity of the binding energies of electrons in filled shells of elements of the periodic table

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

Karpov, V. Ya.; Shpatakovskaya, G. V., E-mail: shpagalya@yandex.ru

An expression for the binding energies of electrons in the ground state of an atom is derived on the basis of the Bohr–Sommerfeld quantization rule within the Thomas–Fermi model. The validity of this relation for all elements from neon to uranium is tested within a more perfect quantum-mechanical model with and without the inclusion of relativistic effects, as well as with experimental binding energies. As a result, the ordering of electronic levels in filled atomic shells is established, manifested in an approximate atomic-number similarity. It is proposed to use this scaling property to analytically estimate the binding energies of electronsmore » in an arbitrary atom.« less

Quantum confined stark effect on the binding energy of exciton in type II quantum heterostructure

NASA Astrophysics Data System (ADS)

Suseel, Rahul K.; Mathew, Vincent

2018-05-01

In this work, we have investigated the effect of external electric field on the strongly confined excitonic properties of CdTe/CdSe/CdTe/CdSe type-II quantum dot heterostructures. Within the effective mass approximation, we solved the Poisson-Schrodinger equations of the exciton in nanostructure using relaxation method in a self-consistent iterative manner. We changed both the external electric field and core radius of the quantum dot, to study the behavior of binding energy of exciton. Our studies show that the external electric field destroys the positional flipped state of exciton by modifying the confining potentials of electron and hole.

Deciphering the mechanism of interaction of edifenphos with calf thymus DNA

NASA Astrophysics Data System (ADS)

Ahmad, Ajaz; Ahmad, Masood

2018-01-01

Edifenphos is an important organophosphate pesticide with many antifungal and anti-insecticidal properties but it may cause potential hazards to human health. In this work, we have tried to explore the binding mode of action and mechanism of edifenphos to calf thymus DNA (CT-DNA). Several experiments such as ultraviolet-visible absorption spectra and emission spectroscopy showed complex formation between edifenphos and CT-DNA and low binding constant values supporting groove binding mode. These results were further confirmed by circular dichroism (CD), CT-DNA melting studies, viscosity measurements, density functional theory and molecular docking. CD study suggests that edifenphos does not alter native structure of CT-DNA. Isothermal calorimetry reveals that binding of edifenphos with CT-DNA is enthalpy driven process. Competitive binding assay and effect of ionic strength showed that edifenphos binds to CT-DNA via groove binding manner. Hence, edifenphos is a minor groove binder preferably interacting with A-T regions with docking score - 6.84 kJ/mol.

Binding of alkylphenols and alkylated non-phenolics to the rainbow trout (Oncorhynchus mykiss) plasma sex steroid-binding protein.

PubMed

Tollefsen, K-E

2007-09-01

Alkylphenols are well-known endocrine disrupters, mediating effects through the estrogen receptor (ER). Although the estrogenic properties of the alkylphenols are well documented, alternative mechanisms of action are poorly described. In the present work, the interaction of a range of alkyl-substituted phenols and alkyl-substituted non-phenolics with the rainbow trout (Oncorhynchus mykiss) sex steroid-binding protein (rtSBP) were determined by competitive ligand-binding studies. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure were assessed. The results showed that the rtSBP binds to most chemical structures tested, although the highest affinity was obtained for mono-substituted alkylphenols with a chain length of four to eight methyl groups. Interestingly, rtSBP binding was also observed for non-phenolic compounds such as 4-t-butylcyclohexanol and 4-t-butylnitrobenzene suggesting that the rtSBP has a broad binding specificity for alkylphenols and alkylated non-phenolics.

FRET study of G-quadruplex forming fluorescent oligonucleotide probes at the lipid monolayer interface.

PubMed

Swiatkowska, Angelika; Kosman, Joanna; Juskowiak, Bernard

2016-01-05

Spectral properties and G-quadruplex folding ability of fluorescent oligonucleotide probes at the cationic dioctadecyldimethylammonium bromide (DODAB) monolayer interface are reported. Two oligonucleotides, a 19-mer bearing thrombin binding aptamer sequence and a 21-mer with human telomeric sequence, were end-labeled with fluorescent groups (FAM and TAMRA) to give FRET probes F19T and F21T, respectively. The probes exhibited abilities to fold into a quadruplex structure and to bind metal cations (Na(+) and K(+)). Fluorescence spectra of G-quadruplex FRET probes at the monolayer interface are reported for the first time. Investigations included film balance measurements (π-A isotherms) and fluorescence spectra recording using a fiber optic accessory interfaced with a spectrofluorimeter. The effect of the presence of DODAB monolayer, metal cations and the surface pressure of monolayer on spectral behavior of FRET probes were examined. Adsorption of probe at the cationic monolayer interface resulted in the FRET signal enhancement even in the absence of metal cations. Variation in the monolayer surface pressure exerted rather modest effect on the spectral properties of probes. The fluorescence energy transfer efficiency of monolayer adsorbed probes increased significantly in the presence of sodium or potassium ion in subphase, which indicated that the probes retained their cation binding properties when adsorbed at the monolayer interface. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of the DNA binding properties of polyomavirus capsid protein

NASA Technical Reports Server (NTRS)

Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

Boron doping effect on the interface interaction and mechanical properties of graphene reinforced copper matrix composite

NASA Astrophysics Data System (ADS)

Fang, Bingcheng; Li, Jiajun; Zhao, Naiqin; Shi, Chunsheng; Ma, Liying; He, Chunnian; He, Fang; Liu, Enzuo

2017-12-01

In order to explore an efficient way of modifying graphene to improve the Cu/graphene interfacial bonding and remain the excellent mechanical and physical properties of graphene, the interaction between Cu and the pristine, atomic oxygen functionalized and boron- or nitrogen-doped graphene with and without defects was systematically investigated by density functional theory calculation. The electronic structure analysis revealed that the chemically active oxygen can enhance the binding energy Eb of Cu with graphene by forming strong covalent bonds, supporting the experimental study suggesting an vital role of intermediate oxygen in the improvement of the mechanical properties of graphene/Cu composites. Due to the strong hybridization between Cu-3d electron states and the 2p states of both boron and carbon atoms, the boron-doping effect is comparable to or even better than the chemical bridging role of oxygen in the reduced graphene oxide reinforced Cu matrix composite. Furthermore, we evidenced an enhancement of mechanical properties including bulk modulus, shear modulus and Young modulus of graphene/Cu composite after boron doping, which closely relates to the increased interfacial binding energy between boron-doped graphene and Cu surfaces.

Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme.

PubMed

Basu, Anirban; Suresh Kumar, Gopinatha

2017-02-16

Interaction of two food colorant dyes, amaranth and tartrazine, with lysozyme was studied employing multiple biophysical techniques. The dyes exhibited hypochromic changes in the presence of lysozyme. The intrinsic fluorescence of lysozyme was quenched by both dyes; amaranth was a more efficient quencher than tartrazine. The equilibrium constant of amaranth was higher than that of tartarzine. From FRET analysis, the binding distances for amaranth and tartrazine were calculated to be 4.51 and 3.93 nm, respectively. The binding was found to be dominated by non-polyelectrolytic forces. Both dyes induced alterations in the microenvironment surrounding the tryptophan and tyrosine residues of the protein, with the alterations being comparatively higher for the tryptophans than the tyrosines. The interaction caused significant loss in the helicity of lysozyme, the change being higher with amaranth. The binding of both dyes was exothermic. The binding of amaranth was enthalpy driven, while that of tartrazine was predominantly entropy driven. Amaranth delayed lysozyme fibrillation at 25 μM, while tartrazine had no effect even at 100 μM. Nevertheless, both dyes had a significant inhibitory effect on fibrillogenesis. The present study explores the potential antiamyloidogenic property of these azo dyes used as food colorants.

Interrogating the relationship between rat in vivo tissue distribution and drug property data for >200 structurally unrelated molecules

PubMed Central

Harrell, Andrew W; Sychterz, Caroline; Ho, May Y; Weber, Andrew; Valko, Klara; Negash, Kitaw

2015-01-01

The ability to explain distribution patterns from drug physicochemical properties and binding characteristics has been explored for more than 200 compounds by interrogating data from quantitative whole body autoradiography studies (QWBA). These in vivo outcomes have been compared to in silico and in vitro drug property data to determine the most influential properties governing drug distribution. Consistent with current knowledge, in vivo distribution was most influenced by ionization state and lipophilicity which in turn affected phospholipid and plasma protein binding. Basic and neutral molecules were generally better distributed than acidic counterparts demonstrating weaker plasma protein and stronger phospholipid binding. The influence of phospholipid binding was particularly evident in tissues with high phospholipid content like spleen and lung. Conversely, poorer distribution of acidic drugs was associated with stronger plasma protein and weaker phospholipid binding. The distribution of a proportion of acidic drugs was enhanced, however, in tissues known to express anionic uptake transporters such as the liver and kidney. Greatest distribution was observed into melanin containing tissues of the eye, most likely due to melanin binding. Basic molecules were consistently better distributed into parts of the eye and skin containing melanin than those without. The data, therefore, suggest that drug binding to macromolecules strongly influences the distribution of total drug for a large proportion of molecules in most tissues. Reducing lipophilicity, a strategy often used in discovery to optimize pharmacokinetic properties such as absorption and clearance, also decreased the influence of nonspecific binding on drug distribution. PMID:26516585

The effects of structural variations of thiophene-containing Ru(II) complexes on the acid-base and DNA binding properties.

PubMed

Yuan, Cui-Li; Zhang, An-Guo; Zheng, Ze-Bo; Wang, Ke-Zhi

2013-03-01

A phenylthiophenyl-bearing Ru(II) complex of [Ru(bpy)₂(Hbptip)](PF₆)₂ {bpy = 2,2'-bipyridine, Hbptip = 2-(4-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline} was synthesized and characterized by elemental analysis, ¹H NMR spectroscopy, and electrospray ionization mass spectrometry. The ground- and excited-state acid-base properties of the complex were studied by UV-visible absorption and photoluminescence spectrophotometric pH titrations and the negative logarithm values of the ground-state acid ionization constants were derived to be pK(a1) = 1.31 ± 0.09 and pK(a2) = 5.71 ± 0.11 with the pK(a2) associated deprotonation/protonation process occurring over 3 pK(a) units more acidic than thiophenyl-free parent complex of [Ru(bpy)₂(Hpip)]²⁺ {Hpip = 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline}. The calf thymus DNA-binding properties of [Ru(bpy)₂(Hbptip)]²⁺ in Tris-HCl buffer (pH 7.1 and 50 mM NaCl) were investigated by DNA viscosities and density functional theoretical calculations as well as UV-visible and emission spectroscopy techniques of UV-visible and luminescence titrations, steady-state emission quenching by [Fe(CN)₆]⁴⁻, DNA competitive binding with ethidium bromide, DNA melting experiments, and reverse salt effects. The complex was evidenced to bind to the DNA intercalatively with binding affinity being greater than those for previously reported analogs of [Ru(bpy)₂(Hip)]²⁺, [Ru(bpy)₂(Htip)]²⁺, and [Ru(bpy)₂(Haptip)]²⁺ {Hip = 1H-imidazo[4,5-f][1,10]phenanthroline, Htip = 2-thiophenimidazo[4,5-f][1,10]phenanthroline, Haptip = 2-(5-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline}.

Linked magnolol dimer as a selective PPARγ agonist - Structure-based rational design, synthesis, and bioactivity evaluation.

PubMed

Dreier, Dominik; Latkolik, Simone; Rycek, Lukas; Schnürch, Michael; Dymáková, Andrea; Atanasov, Atanas G; Ladurner, Angela; Heiss, Elke H; Stuppner, Hermann; Schuster, Daniela; Mihovilovic, Marko D; Dirsch, Verena M

2017-10-20

The nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and its hetero-dimerization partner retinoid X receptor α (RXRα) are considered as drug targets in the treatment of diseases like the metabolic syndrome and diabetes mellitus type 2. Effort has been made to develop new agonists for PPARγ to obtain ligands with more favorable properties than currently used drugs. Magnolol was previously described as dual agonist of PPARγ and RXRα. Here we show the structure-based rational design of a linked magnolol dimer within the ligand binding domain of PPARγ and its synthesis. Furthermore, we evaluated its binding properties and functionality as a PPARγ agonist in vitro with the purified PPARγ ligand binding domain (LBD) and in a cell-based nuclear receptor transactivation model in HEK293 cells. We determined the synthesized magnolol dimer to bind with much higher affinity to the purified PPARγ ligand binding domain than magnolol (K i values of 5.03 and 64.42 nM, respectively). Regarding their potency to transactivate a PPARγ-dependent luciferase gene both compounds were equally effective. This is likely due to the PPARγ specificity of the newly designed magnolol dimer and lack of RXRα-driven transactivation activity by this dimeric compound.

Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer

PubMed Central

Pasternak, Anna; Hernandez, Frank J.; Rasmussen, Lars M.; Vester, Birte; Wengel, Jesper

2011-01-01

A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15–0.50 kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by Tm versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation. PMID:20870750

Uncertainty analysis of the nonideal competitive adsorption-donnan model: effects of dissolved organic matter variability on predicted metal speciation in soil solution.

PubMed

Groenenberg, Jan E; Koopmans, Gerwin F; Comans, Rob N J

2010-02-15

Ion binding models such as the nonideal competitive adsorption-Donnan model (NICA-Donnan) and model VI successfully describe laboratory data of proton and metal binding to purified humic substances (HS). In this study model performance was tested in more complex natural systems. The speciation predicted with the NICA-Donnan model and the associated uncertainty were compared with independent measurements in soil solution extracts, including the free metal ion activity and fulvic (FA) and humic acid (HA) fractions of dissolved organic matter (DOM). Potentially important sources of uncertainty are the DOM composition and the variation in binding properties of HS. HS fractions of DOM in soil solution extracts varied between 14 and 63% and consisted mainly of FA. Moreover, binding parameters optimized for individual FA samples show substantial variation. Monte Carlo simulations show that uncertainties in predicted metal speciation, for metals with a high affinity for FA (Cu, Pb), are largely due to the natural variation in binding properties (i.e., the affinity) of FA. Predictions for metals with a lower affinity (Cd) are more prone to uncertainties in the fraction FA in DOM and the maximum site density (i.e., the capacity) of the FA. Based on these findings, suggestions are provided to reduce uncertainties in model predictions.

Influence of freezing and thawing on the hydration characteristics, quality, and consumer acceptance of whole muscle beef injected with solutions of salt and phosphate.

PubMed

Pietrasik, Z; Janz, J A M

2009-03-01

Effects of salt/phosphate injection level (112% or 125% pump), salt level (0.5% or 1.5% salt), and freezing/thawing on hydration characteristics, quality, and consumer acceptance of beef semitendinosus were investigated. All enhancement treatments decreased shear force by 25-35%, but negatively affected colour. Increased salt concentration yielded lower purge and cooking losses, and higher water holding capacity. The higher injection level reduced water binding properties, however, the loss in functionality with higher water addition was overcome with increased salt content. Freezing and subsequent thawing was generally detrimental to colour and water binding properties and tended to increase shear force. Freezing and subsequent thawing did not affect fluid release in steaks held for 1 day before analysis, but resulted in decreased water retention in samples held for 7 days. Holding vacuum packaged steaks for 7 days generally increased package purge and negatively affected colour parameters, although water binding characteristics were improved. Consumer panel results demonstrated a negative effect on juiciness and tenderness where meat subject to low salt/high injection was frozen then thawed - the low salt level was insufficient to maintain any positive effect of injection treatment. In general, salt/phosphate injection improved product acceptability and increased willingness to purchase.

Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules.

PubMed

Wasylyk, Christine; Zheng, Hong; Castell, Christelle; Debussche, Laurent; Multon, Marie-Christine; Wasylyk, Bohdan

2008-03-01

Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ

PubMed Central

FAULHAMMER, DIRK; ESCHGFÄLLER, BERND; STARK, SANDRA; BURGSTALLER, PETRA; ENGLBERGER, WERNER; ERFURTH, JEANNETTE; KLEINJUNG, FRANK; RUPP, JOHANNA; VULCU, SEBASTIAN DAN; SCHRÖDER, WERNER; VONHOFF, STEFAN; NAWRATH, HERMANN; GILLEN, CLEMENS; KLUSSMANN, SVEN

2004-01-01

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ binding biostable RNA aptamers (Spiegelmers) using a mirror-image in vitro selection approach. Spiegelmers are l-enantiomeric oligonucleotide ligands that display high affinity and specificity to their targets and high resistance to enzymatic degradation compared to d-oligonucleotides. A representative Spiegelmer from the selections performed was size-minimized to two distinct sequences capable of high affinity binding to N/OFQ. The Spiegelmers were shown to antagonize binding of N/OFQ to the ORL1 receptor in a binding-competition assay. The calculated IC50 values for the Spiegelmers NOX 2149 and NOX 2137a/b were 110 nM and 330 nM, respectively. The competitive antagonistic properties of these Spiegelmers were further demonstrated by their effective and specific inhibition of G-protein activation in two additional models. The Spiegelmers antagonized the N/OFQ-induced GTPγS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor. In oocytes from Xenopus laevis, NOX 2149 showed an antagonistic effect to the N/OFQ-ORL 1 receptor system that was functionally coupled with G-protein-regulated inwardly rectifying K+ channels. PMID:14970396

Impact of mutations on the allosteric conformational equilibrium

PubMed Central

Weinkam, Patrick; Chen, Yao Chi; Pons, Jaume; Sali, Andrej

2012-01-01

Allostery in a protein involves effector binding at an allosteric site that changes the structure and/or dynamics at a distant, functional site. In addition to the chemical equilibrium of ligand binding, allostery involves a conformational equilibrium between one protein substate that binds the effector and a second substate that less strongly binds the effector. We run molecular dynamics simulations using simple, smooth energy landscapes to sample specific ligand-induced conformational transitions, as defined by the effector-bound and unbound protein structures. These simulations can be performed using our web server: http://salilab.org/allosmod/. We then develop a set of features to analyze the simulations and capture the relevant thermodynamic properties of the allosteric conformational equilibrium. These features are based on molecular mechanics energy functions, stereochemical effects, and structural/dynamic coupling between sites. Using a machine-learning algorithm on a dataset of 10 proteins and 179 mutations, we predict both the magnitude and sign of the allosteric conformational equilibrium shift by the mutation; the impact of a large identifiable fraction of the mutations can be predicted with an average unsigned error of 1 kBT. With similar accuracy, we predict the mutation effects for an 11th protein that was omitted from the initial training and testing of the machine-learning algorithm. We also assess which calculated thermodynamic properties contribute most to the accuracy of the prediction. PMID:23228330

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

NASA Technical Reports Server (NTRS)

Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

2003-01-01

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

RNA-binding properties and mapping of the RNA-binding domain from the movement protein of Prunus necrotic ringspot virus.

PubMed

Herranz, M Carmen; Pallás, Vicente

2004-03-01

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is involved in intercellular virus transport. In this study, putative RNA-binding properties of the PNRSV MP were studied. The PNRSV MP was produced in Escherichia coli using an expression vector. Electrophoretic mobility shift assays (EMSAs) using DIG-labelled riboprobes demonstrated that PNRSV MP bound ssRNA cooperatively without sequence specificity. Two different ribonucleoprotein complexes were found to be formed depending on the molar MP : PNRSV RNA ratio. The different responses of the complexes to urea treatment strongly suggested that they have different structural properties. Deletion mutagenesis followed by Northwestern analysis allowed location of a nucleic acid binding domain to aa 56-88. This 33 aa RNA-binding motif is the smallest region delineated among members of the family Bromoviridae for which RNA-binding properties have been demonstrated. This domain is highly conserved within all phylogenetic subgroups previously described for PNRSV isolates. Interestingly, the RNA-binding domain described here and the one described for Alfamovirus are located at the N terminus of their corresponding MPs, whereas similar domains previously characterized in members of the genera Bromovirus and Cucumovirus are present at the C terminus, strongly reflecting their corresponding phylogenetic relationships. The evolutionary implications of this observation are discussed.

Binding mechanism and electrochemical properties of M13 phage-sulfur composite.

PubMed

Dong, Dexian; Zhang, Yongguang; Sutaria, Sanjana; Konarov, Aishuak; Chen, Pu

2013-01-01

Self-assembly of nanostructured materials has been proven a powerful technique in material design and synthesis. By phage display screening, M13 phage was found to strongly bind sulfur particles. Fourier transform infrared and X-ray photoelectron spectroscopy measurements indicated that the strong sulfur-binding ability of M13 phage derives from newly generated S-O and C-S bonds. Using this phage assembled sulfur composite in a lithium battery, the first discharge capacity reached 1117 mAh g(-1), which is more than twice that of the sulfur only cathode. Besides, the negative polysulfide shuttle effect in a lithium-sulfur battery was significantly suppressed.

Binding Mechanism and Electrochemical Properties of M13 Phage-Sulfur Composite

PubMed Central

Dong, Dexian; Zhang, Yongguang; Sutaria, Sanjana; Konarov, Aishuak; Chen, Pu

2013-01-01

Self-assembly of nanostructured materials has been proven a powerful technique in material design and synthesis. By phage display screening, M13 phage was found to strongly bind sulfur particles. Fourier transform infrared and X-ray photoelectron spectroscopy measurements indicated that the strong sulfur-binding ability of M13 phage derives from newly generated S-O and C-S bonds. Using this phage assembled sulfur composite in a lithium battery, the first discharge capacity reached 1117 mAh g-1, which is more than twice that of the sulfur only cathode. Besides, the negative polysulfide shuttle effect in a lithium-sulfur battery was significantly suppressed. PMID:24324560

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data

PubMed Central

Wang, Xiangdan; McKay, Patrick; Dutina, George; Hass, Philip E.; Nijem, Ihsan; Allison, David; Cowan, Kyra J.; Lin, Kevin; Quarmby, Valerie; Yang, Jihong

2017-01-01

ABSTRACT Binding interactions with the neonatal Fc receptor (FcRn) are one determinant of pharmacokinetic properties of recombinant human monoclonal antibody (rhumAb) therapeutics, and a conserved binding motif in the crystallizable fragment (Fc) region of IgG molecules interacts with FcRn. Surface plasmon resonance (SPR) biosensor assays are often used to characterize interactions between FcRn and rhumAb therapeutics. In such assays, generally either the rhumAb (format 1) or the FcRn protein (format 2) is immobilized on a biosensor chip. However, because evidence suggests that, in some cases, the variable domains of a rhumAb may also affect FcRn binding, we evaluated the effect of SPR assay configuration on binding data. We sought to assess FcRn binding properties of 2 rhumAbs (rhumAb1 and rhumAb2) to FcRn proteins using these 2 biosensor assay formats. The two rhumAbs have greater than 99% sequence identity in the Fc domain but differ in their Fab regions. rhumAb2 contains a positively charged patch in the variable domain that is absent in rhumAb1. Our results showed that binding of rhumAb1 to FcRn was independent of biosensor assay configuration, while binding of rhumAb2 to FcRn was highly SPR assay configuration dependent. Further investigations revealed that the format dependency of rhumAb2-FcRn binding is linked to the basic residues that form a positively charged patch in the variable domain of rhumAb2. Our work highlights the importance of analyzing rhumAb-FcRn binding interactions using 2 alternate SPR biosensor assay configurations. This approach may also provide a simple way to identify the potential for non-Fc-driven FcRn binding interactions in otherwise typical IgGs. PMID:28001487

Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta

PubMed Central

Damsgaard, Christian; Storz, Jay F.; Hoffmann, Federico G.

2013-01-01

When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O2 transport before turtles become anoxic. Here, we report the effects of protons, anionic cofactors, and temperature on the O2-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O2 affinity between untreated hemolysates from turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O2 affinity compared with HbA, whereas Bohr and temperature effects, as well as thiol reactivity, are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site positions, we find high ATP affinities for both Hb isoforms, suggesting an alternative and stronger binding site for ATP. The high ATP affinities indicate that, although ATP levels decrease in red blood cells of turtles acclimating to anoxia, the O2 affinity would remain largely unchanged, as confirmed by O2-binding measurements of untreated hemolysates from normoxic and anoxic turtles. Thus, the increase in blood-O2 affinity that accompanies winter acclimation is mainly attributable to a decrease in temperature rather than in concentrations of organic phosphates. This is the first extensive study on freshwater turtle Hb isoforms, providing molecular evidence for adaptive changes in O2 transport associated with acclimation to severe hypoxia. PMID:23986362

Effect of Li Adsorption on the Electronic and Hydrogen Storage Properties of Acenes: A Dispersion-Corrected TAO-DFT Study

PubMed Central

Seenithurai, Sonai; Chai, Jeng-Da

2016-01-01

Due to the presence of strong static correlation effects and noncovalent interactions, accurate prediction of the electronic and hydrogen storage properties of Li-adsorbed acenes with n linearly fused benzene rings (n = 3–8) has been very challenging for conventional electronic structure methods. To meet the challenge, we study these properties using our recently developed thermally-assisted-occupation density functional theory (TAO-DFT) with dispersion corrections. In contrast to pure acenes, the binding energies of H2 molecules on Li-adsorbed acenes are in the ideal binding energy range (about 20 to 40 kJ/mol per H2). Besides, the H2 gravimetric storage capacities of Li-adsorbed acenes are in the range of 9.9 to 10.7 wt%, satisfying the United States Department of Energy (USDOE) ultimate target of 7.5 wt%. On the basis of our results, Li-adsorbed acenes can be high-capacity hydrogen storage materials for reversible hydrogen uptake and release at ambient conditions. PMID:27609626

Binding properties of chiral ruthenium(II) complexes Λ- and Δ-[Ru(bpy)2dppz-11-CO2Me]2+ toward the triplex RNA poly(U)•poly(A)*poly(U).

PubMed

Ni, Wen; Liu, Xiaohua; Tan, Lifeng

2018-05-24

Two chiral ruthenium(II) complexes containing ligand dppz-CO 2 Me (dppz-11-CO 2 Me = dipyrido[3,2-a,2',3'-c]phenazine-11-carboxylic acid methyl ester), Δ-[Ru(bpy) 2 dppz-11-CO 2 Me] 2+ (bpy = 2,2'-bipyridine; Δ-1) and Λ-[Ru(bpy) 2 dppz-11-CO 2 Me] 2+ (Λ-1), were synthesized and characterized. The binding of the two enantiomers with the triplex RNA poly(U)•poly(A)*poly(U) was carried out by various biophysical techniques. Analysis of the absorption and fluorescence features indicates that the binding strengths of the two enantiomers toward the triplex RNA differ only slightly from each other. The total increase in viscosity and shape of the curves for the triplex RNA with Λ-1 is similar to that with Δ-1, suggesting the binding modes of two enantiomers with the triplex RNA are intercalation. Thermal melting measurements indicate that the stabilization effects clearly depended on the concentrations of Λ-1 and Δ-1. However, the third-strand stabilizing effect of Δ-1 dramatically differs from that of Λ-1 when they interact with the chiral environment of the RNA triple at pH = 7.0 and [Na + ] = 35 mM. Combined with the CD (CD = circular dichroism) variations of the triplex RNA with either Λ-1 or Δ-1, the reason for their different triplex stabilization effects may originate from the two enantiomers through different orientations intercalating into nucleobases of the triplex. In addition, effects of higher ionic strengths on the triplex stabilization in the absence and presence of the two enantiomers have also been studied. The results presented here may be useful for understanding the binding properties of the triplex RNA with small molecule, particularly chiral ruthenium(II) complexes. Copyright © 2018 Elsevier Inc. All rights reserved.

Nonlinear effects in evolution - an ab initio study: A model in which the classical theory of evolution occurs as a special case.

PubMed

Clerc, Daryl G

2016-07-21

An ab initio approach was used to study the molecular-level interactions that connect gene-mutation to changes in an organism׳s phenotype. The study provides new insights into the evolutionary process and presents a simplification whereby changes in phenotypic properties may be studied in terms of the binding affinities of the chemical interactions affected by mutation, rather than by correlation to the genes. The study also reports the role that nonlinear effects play in the progression of organs, and how those effects relate to the classical theory of evolution. Results indicate that the classical theory of evolution occurs as a special case within the ab initio model - a case having two attributes. The first attribute: proteins and promoter regions are not shared among organs. The second attribute: continuous limiting behavior exists in the physical properties of organs as well as in the binding affinity of the associated chemical interactions, with respect to displacements in the chemical properties of proteins and promoter regions induced by mutation. Outside of the special case, second-order coupling contributions are significant and nonlinear effects play an important role, a result corroborated by analyses of published activity levels in binding and transactivation assays. Further, gradations in the state of perfection of an organ may be small or large depending on the type of mutation, and not necessarily closely-separated as maintained by the classical theory. Results also indicate that organs progress with varying degrees of interdependence, the likelihood of successful mutation decreases with increasing complexity of the affected chemical system, and differences between the ab initio model and the classical theory increase with increasing complexity of the organism. Copyright © 2016 The Author. Published by Elsevier Ltd.. All rights reserved.

Cyclic mu-opioid receptor ligands containing multiple N-methylated amino acid residues.

PubMed

Adamska-Bartłomiejczyk, Anna; Janecka, Anna; Szabó, Márton Richárd; Cerlesi, Maria Camilla; Calo, Girolamo; Kluczyk, Alicja; Tömböly, Csaba; Borics, Attila

2017-04-15

In this study we report the in vitro activities of four cyclic opioid peptides with various sequence length/macrocycle size and N-methylamino acid residue content. N-Methylated amino acids were incorporated and cyclization was employed to enhance conformational rigidity to various extent. The effect of such modifications on ligand structure and binding properties were studied. The pentapeptide containing one endocyclic and one exocyclic N-methylated amino acid displayed the highest affinity to the mu-opioid receptor. This peptide was also shown to be a full agonist, while the other analogs failed to activate the mu opioid receptor. Results of molecular docking studies provided rationale for the explanation of binding properties on a structural basis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure-Activity Relationships of Bifunctional Cyclic Disulfide Peptides Based on Overlapping Pharmacophores at Opioid and Cholecystokinin Receptors

PubMed Central

Agnes, Richard S.; Ying, Jinfa; Kövér, Katalin E.; Lee, Yeon Sun; Davis, Peg; Ma, Shou-wu; Badghisi, Hamid; Porreca, Frank; Lai, Josephine; Hruby, Victor J.

2008-01-01

Prolonged opioid exposure increases the expression of cholecystokinin (CCK) and its receptors in the central nervous system, where CCK may attenuate the antinociceptive effects of opioids. The complex interactions between opioid and CCK may play a role in the development of opioid tolerance. We designed and synthesized cyclic disulfide peptides and determined their agonist properties at opioid receptors and antagonist properties at CCK receptors. Compound 1 (Tyr-c[D-Cys-Gly-Trp-Cys]-Asp-Phe-NH2) showed potent binding and agonist activities at δ and µ opioid receptors while displaying some binding to CCK receptors. The NMR structure of the lead compound displayed similar conformational features of opioid and CCK ligands. PMID:18502541

Electrostatic steering and ionic tethering in enzyme-ligand binding: insights from simulations.

PubMed

Wade, R C; Gabdoulline, R R; Lüdemann, S K; Lounnas, V

1998-05-26

To bind at an enzyme's active site, a ligand must diffuse or be transported to the enzyme's surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also influence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-influenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and beta-lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as "ionic tethering." We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme's surroundings even when the substrate is nonpolar.

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

PubMed Central

Kang, Hyeran; Bradley, Michael J.; McCullough, Brannon R.; Pierre, Anaëlle; Grintsevich, Elena E.; Reisler, Emil; De La Cruz, Enrique M.

2012-01-01

The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments. PMID:23027950

Analysis of factors influencing hydration site prediction based on molecular dynamics simulations.

PubMed

Yang, Ying; Hu, Bingjie; Lill, Markus A

2014-10-27

Water contributes significantly to the binding of small molecules to proteins in biochemical systems. Molecular dynamics (MD) simulation based programs such as WaterMap and WATsite have been used to probe the locations and thermodynamic properties of hydration sites at the surface or in the binding site of proteins generating important information for structure-based drug design. However, questions associated with the influence of the simulation protocol on hydration site analysis remain. In this study, we use WATsite to investigate the influence of factors such as simulation length and variations in initial protein conformations on hydration site prediction. We find that 4 ns MD simulation is appropriate to obtain a reliable prediction of the locations and thermodynamic properties of hydration sites. In addition, hydration site prediction can be largely affected by the initial protein conformations used for MD simulations. Here, we provide a first quantification of this effect and further indicate that similar conformations of binding site residues (RMSD < 0.5 Å) are required to obtain consistent hydration site predictions.

EFFECTS ON HEPATIC GENE EXPRESSION IN MALE RATS ADMINISTERED PBDES IN HOUSEHOLD DUST

EPA Science Inventory

Studies show that polybrominated diphenyl ethers (PBDEs) decrease thyroid hormone concentrations via induction of hepatic uridinediphosphate-glucoronosyltransferase (UGTs) and transthyretin (Ttr) binding. Because PBDEs exhibit endocrine disrupting properties and are present in h...

X-Ray Structure of the Human Calreticulin Globular Domain Reveals a Peptide-Binding Area and Suggests a Multi-Molecular Mechanism

PubMed Central

Chouquet, Anne; Païdassi, Helena; Ling, Wai Li; Frachet, Philippe; Houen, Gunnar; Arlaud, Gérard J.; Gaboriaud, Christine

2011-01-01

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca2+-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 Å resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism. PMID:21423620

Computational scheme for the prediction of metal ion binding by a soil fulvic acid

USGS Publications Warehouse

Marinsky, J.A.; Reddy, M.M.; Ephraim, J.H.; Mathuthu, A.S.

1995-01-01

The dissociation and metal ion binding properties of a soil fulvic acid have been characterized. Information thus gained was used to compensate for salt and site heterogeneity effects in metal ion complexation by the fulvic acid. An earlier computational scheme has been modified by incorporating an additional step which improves the accuracy of metal ion speciation estimates. An algorithm is employed for the prediction of metal ion binding by organic acid constituents of natural waters (once the organic acid is characterized in terms of functional group identity and abundance). The approach discussed here, currently used with a spreadsheet program on a personal computer, is conceptually envisaged to be compatible with computer programs available for ion binding by inorganic ligands in natural waters.

Alternative Affinity Ligands for Immunoglobulins.

PubMed

Kruljec, Nika; Bratkovič, Tomaž

2017-08-16

The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

Analysis of CD44-Hyaluronan Interactions in an Artificial Membrane System

PubMed Central

Wolny, Patricia M.; Banerji, Suneale; Gounou, Céline; Brisson, Alain R.; Day, Anthony J.; Jackson, David G.; Richter, Ralf P.

2010-01-01

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan. PMID:20663884

Potential of goat probiotic to bind mutagens.

PubMed

Apás, Ana Lidia; González, Silvia Nelina; Arena, Mario Eduardo

2014-08-01

The mutagen binding ability of the goat probiotics (Lactobacillus reuteri DDL 19, Lactobacillus alimentarius DDL 48, Enterococcus faecium DDE 39, and Bifidobacterium bifidum DDBA) was evaluated. The oral administration of these probiotics reduced fecal mutagens and intestinal cancer markers in goats. Secondly, the effects of probiotics against the mutagenesis induced by sodium azide (SA), and Benzopyrene (B[α]P) by performing the modified Ames test using Salmonella typhimurium TA 100 was investigated. The capacity to bind benzopyrene and the stability of the bacterial-mutagen complex was analyzed by HPLC. The dismutagenic potential against both mutagens was proportional to probiotic concentration. Results showed that probiotic antimutagenic capacity against SA was ranging from 13 to 78%. The mixture of four goat probiotics (MGP) displayed higher antimutagenic activity against SA than any individual strains at the same cell concentration. This study shows that the highest diminution of mutagenicity in presence of B[α]P (74%) was observed in presence of MGP. The antimutagenic activity of nearly all the individual probiotic and the MGP were in concordance with the B[α]P binding determined by HPLC. According to our results, the B[α]P binding to probiotic was irreversible still after being washed with DMSO solution. The stability of the toxic compounds-bacterial cell binding is a key consideration when probiotic antimutagenic property is evaluated. MGP exhibits the ability to bind and detoxify potent mutagens, and this property can be useful in supplemented foods for goats since it can lead to the removal of potent mutagens and protect and enhance ruminal health and hence food safety of consumers. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dielectric properties of proteins from simulation: the effects of solvent, ligands, pH, and temperature.

PubMed

Pitera, J W; Falta, M; van Gunsteren, W F

2001-06-01

We have used a standard Fröhlich-Kirkwood dipole moment fluctuation model to calculate the static dielectric permittivity, epsilon(0), for four different proteins, each of which was simulated under at least two different conditions of pH, temperature, solvation, or ligand binding. For the range of proteins and conditions studied, we calculate values for epsilon(0) between 15 and 40. Our results show, in agreement with prior work, that the behavior of charged residues is the primary determinant of the effective permittivity. Furthermore, only environmental changes that alter the properties of charged residues exert a significant effect on epsilon. In contrast, buried water molecules or ligands have little or no effect on protein dielectric properties.

Cholinergic Neurotransmission: Function and Dysfunction, International Cholinergic Symposium (8th) Held at Montreal (Quebec) on 26-30 July 1992

DTIC Science & Technology

1992-12-31

receptor were decreased. In the presence of nicotine 1.0pM, the Kd values of rat cerebral muscarinic receptor bound with its agonist P3H] oxotremorine -M...inhibitory effects of GTPrS on [1 3H] oxotremorine -M binding were potentiated.It is suggsted that the binding properties of brain muscarinic receptor...interval) the dose-response curves of M-agonists arecoline and oxotremorine for producing salivation shifted leftward. Above demonstrated phenomena

Evaluation of Ochratoxin Recognition by Peptides Using Explicit Solvent Molecular Dynamics

PubMed Central

Thyparambil, Aby A.; Bazin, Ingrid; Guiseppi-Elie, Anthony

2017-01-01

Biosensing platforms based on peptide recognition provide a cost-effective and stable alternative to antibody-based capture and discrimination of ochratoxin-A (OTA) vs. ochratoxin-B (OTB) in monitoring bioassays. Attempts to engineer peptides with improved recognition efficacy require thorough structural and thermodynamic characterization of the binding-competent conformations. Classical molecular dynamics (MD) approaches alone do not provide a thorough assessment of a peptide’s recognition efficacy. In this study, in-solution binding properties of four different peptides, a hexamer (SNLHPK), an octamer (CSIVEDGK), NFO4 (VYMNRKYYKCCK), and a 13-mer (GPAGIDGPAGIRC), which were previously generated for OTA-specific recognition, were evaluated using an advanced MD simulation approach involving accelerated configurational search and predictive modeling. Peptide configurations relevant to ochratoxin binding were initially generated using biased exchange metadynamics and the dynamic properties associated with the in-solution peptide–ochratoxin binding were derived from Markov State Models. Among the various peptides, NFO4 shows superior in-solution OTA sensing and also shows superior selectivity for OTA vs. OTB due to the lower penalty associated with solvating its bound complex. Advanced MD approaches provide structural and energetic insights critical to the hapten-specific recognition to aid the engineering of peptides with better sensing efficacies. PMID:28505090

Monoclonal immunotoxin acting on the Na/sup +/ channel, with properties similar to those of a scorpion toxin

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

Barhanin, J.; Meiri, H.; Romey, G.

1985-03-01

The authors describe the properties of a monoclonal antibody against the Na/sup +/ channel. The antibody, 72.38, competitively inhibited the binding of an /sup 125/I-labeled toxin from the Brazilian scorpion Tityus serrulatus (/sup 125/I-TiTX..gamma..) to Na/sup +/ channels of rat brain membranes. The inhibition of /sup 125/I-TiTX..gamma.. binding also was observed with the solubilized Na/sup +/ channel from rat brain membranes. Antibody 72.38 antagonized /sup 125/I-TiTX..gamma.. binding to Na/sup +/ channels from different animal species (fish, avian, and mammalian) and from different tissues (electroplax, brain, heart, and muscle). Moreover, 72.38 has been used for immunofluorescence labeling of Na/sup +/ channelsmore » in rat sciatic nodes of Ranvier and cultured dorsal root ganglion cells. Electrophysiological experiments on rat muscle cells fully confirmed the similarity between TiTX..gamma.. and 72.38 seen in binding experiments. TiTX..gamma.. and 72.38 are without effect on the ion selectivity of the Na/sup +/ channel, but they both drastically change the voltage-dependence of activation and inactivation of the Na/sup +/ channel.« less

Multiple Ion Binding Equilibria, Reaction Kinetics, and Thermodynamics in Dynamic Models of Biochemical Pathways

PubMed Central

Vinnakota, Kalyan C.; Wu, Fan; Kushmerick, Martin J.; Beard, Daniel A.

2009-01-01

The operation of biochemical systems in vivo and in vitro is strongly influenced by complex interactions between biochemical reactants and ions such as H+, Mg2+, K+, and Ca2+. These are important second messengers in metabolic and signaling pathways that directly influence the kinetics and thermodynamics of biochemical systems. Herein we describe the biophysical theory and computational methods to account for multiple ion binding to biochemical reactants and demonstrate the crucial effects of ion binding on biochemical reaction kinetics and thermodynamics. In simulations of realistic systems, the concentrations of these ions change with time due to dynamic buffering and competitive binding. In turn, the effective thermodynamic properties vary as functions of cation concentrations and important environmental variables such as temperature and overall ionic strength. Physically realistic simulations of biochemical systems require incorporating all of these phenomena into a coherent mathematical description. Several applications to physiological systems are demonstrated based on this coherent simulation framework. PMID:19216922

Divers models of divalent cation interaction to calcium-binding proteins: techniques and anthology.

PubMed

Cox, Jos A

2013-01-01

Intracellular Ca(2+)-binding proteins (CaBPs) are sensors of the calcium signal and several of them even shape the signal. Most of them are equipped with at least two EF-hand motifs designed to bind Ca(2+). Their affinities are very variable, can display cooperative effects, and can be modulated by physiological Mg(2+) concentrations. These binding phenomena are monitored by four major techniques: equilibrium dialysis, fluorimetry with fluorescent Ca(2+) indicators, flow dialysis, and isothermal titration calorimetry. In the last quarter of the twentieth century reports on the ion-binding characteristics of several abundant wild-type CaBPs were published. With the advent of recombinant CaBPs it became possible to determine these properties on previously inaccessible proteins. Here I report on studies by our group carried out in the last decade on eight families of recombinant CaBPs, their mutants, or truncated domains. Moreover this chapter deals with the currently used methods for quantifying the binding of Ca(2+) and Mg(2+) to CaBPs.

Characterization of particulate matter binding peptides screened from phage display.

PubMed

Liang Alvin, Aw Wei; Tanaka, Masayoshi; Okochi, Mina

2017-05-01

Particulate matter (PM), especially particulates with diameters of less than 2.5 μm, can penetrate the alveolar region and increase the risk of respiratory diseases. This has stimulated research efforts to develop detection methods so that counter measures can be taken. In this study, four PM binding peptides were obtained by phage display and binding characteristics of these peptides were investigated using the peptide array. The strongest binding peptide, WQDFGAVRSTRS, displayed a binding property, measured in terms of spot intensity, 11.4 times higher than that of the negative control, AAAAA. Inductively coupled plasma mass spectrometry (ICPMS) analysis of the transition metal compounds in the PM bound to the peptide spots was performed, and two peptides showed higher binding towards Cu and Zn compounds in PM. These results suggest that the screened peptides could serve as an indicator of transition metal compounds, which are related to adverse health effects, contained in PM. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na/sup +/ channel interaction

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

Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

1988-01-12

The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinicallymore » induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.« less

Using engineered single-chain antibodies to correlate molecular binding properties and nanoparticle adhesion dynamics.

PubMed

Haun, Jered B; Pepper, Lauren R; Boder, Eric T; Hammer, Daniel A

2011-11-15

Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that bear them.

Effect of resistant starch and aging conditions on the physicochemical properties of frozen soy yogurt.

PubMed

Rezaei, Rahil; Khomeiri, Morteza; Kashaninejad, Mahdi; Mazaheri-Tehrani, Mostafa; Aalami, Mehran

2015-12-01

The present study investigated the effects of resistant starch concentration (0, 1, 2 %), aging time (2, 13, 24 h) and aging temperature (2, 4, 6 °C) on the physicochemical properties of frozen soy yogurt. The results showed that resistant starch increased viscosity because of its water binding properties. Resistant starch also increased foam stability, fat destabilization, and hardness, but it decreased overrun and meltdown rate. Viscosity, hardness and fat destabilization increased as aging time increased. An increase in aging temperature decreased viscosity, overrun, hardness and fat destabilization of frozen yoghurt, but increased the meltdown rate.

Research on structures, mechanical properties, and mechanical responses of TKX-50 and TKX-50 based PBX with molecular dynamics.

PubMed

Ma, Song; Li, Yajin; Li, Yang; Luo, Yunjun

2016-02-01

To improve the practicality and safety of a novel explosive dihydroxylamm onium 5,5'-bis (tetrazole)-1,1'-diolate (TKX-50), polyvinylidene difluoride (PVDF) and polychlorotrifluoroe-thylene (PCTFE) were respectively added to the TKX-50, forming the polymer-bonded explosives (PBX). Interfacial and mechanical properties of PBX were investigated through molecular dynamics (MD) method, desensitizing mechanisms of fluorine-polymers for TKX-50 were researched by compression and bulk shear simulations. Results show that the binding energies (E bind ) between polymers (PVDF or PCTFE) and TKX-50 surfaces all rank in order of (011) > (100) > (010), shorter interatomic distance and the resulted higher potentials lead to higher E bind on TKX-50/PVDF interfaces than that on PCTFE/TKX-50 interfaces. Compared with TKX-50, the ductility of PBX is improved due to the isotropic mechanical property and flexibility of fluorine-polymers especially the PCTFE. Desensitizing effect of fluorine-polymers for TKX-50 is found under loading condition, which is attributed to the enhanced compressibility and buffer capacity against external pressure in compression, as well as the improved lubricity to reduce the sliding potentials in bulk shear process. Graphical Abstract Comparisons of the internal stress and slide potentials of the novel explosive,TKX-50 and its based PBX. Desensitizing effects can be found by the adding of fluorine-polymers, it owes to their better flexibility and lubricity as well as the amorphous nature.

Ni doping effect on the electronic and sensing properties of 2D SnO2

NASA Astrophysics Data System (ADS)

Patel, Anjali; Roondhe, Basant; Jha, Prafulla K.

2018-05-01

In the present work using state of art first principles calculations under the frame work of density functional theory the effect of Nickel (Ni) doping on electronic as well as sensing properties of most stable two dimensional (2D) T-SnO2 phase towards ethanol (C2H5OH) has been observed. It has been found that Ni atom when dope on T-SnO2 causes prominent decrement in the band gap from 2.26 eV to 1.48 eV and improves the sensing phenomena of pristine T-SnO2 towards C2H5OH by increasing the binding energy from -0.18eV to -0.93eV. The comparative analysis of binding energy shows that Ni improves the binding of C2H5OH by 5.16 times the values for pristine T-SnO2. The doping of Ni into 2D T-SnO2 reduces the band gap through lowering of the conduction band minimum, thereby increasing the electron affinity which increases the sensing performance of T-SnO2. The variation in the electronic properties after and before the exposure of ethanol reinforced to use Ni:SnO2 nano structure for sensing applications. The results indicate that the Ni doped T-SnO2 can be utilized in improved optoelectronic as well as sensor devices in the future.

High-affinity cannabinoid binding site in brain: A possible marijuana receptor

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

Nye, J.S.

The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one classmore » of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.« less

Effects of plantain and corn starches on the mechanical and disintegration properties of paracetamol tablets.

PubMed

Akin-Ajani, Olufunke D; Itiola, Oludele A; Odeku, Oluwatoyin A

2005-10-22

The effects of plantain starch obtained from the unripe fruit of the plant Musa paradisiaca L. (Musaceae) on the mechanical and disintegration properties of paracetamol tablets have been investigated in comparison with the effects of corn starch BP using a 2(3) factorial experimental design. The individual and combined effects of nature of starch binder (N), concentration of starch binder (C), and the relative density of tablet (RD) on the tensile strength (TS), brittle fracture index (BFI), and disintegration time (DT) of the tablets were investigated. The ranking of the individual effects on TS was RD > C > N, on BFI was C > RD > N and on DT was N > C > RD. The ranking for the interaction effects on TS and DT was N-C > N-RD > C-RD, while that on BFI was N-C > C-RD > N-RD. Changing nature of starch from a "low" (plantain starch) to a "high" (corn starch) level, increasing the concentration of starch binding agent from 2.5% to 10.0% wt/wt, and increasing relative density of the tablet from 0.80 to 0.90, led to increase in the values of TS and DT, but a decrease in BFI. Thus, tablets containing plantain starch had lower tensile strength and disintegration time values than those containing corn starch, but showed better ability to reduce the lamination and capping tendency in paracetamol tablet formulation. The interaction between N and C was significantly (P < .001) higher than those between N and RD and between C and RD. There is therefore the need to carefully choose the nature (N) and concentration (C) of starch used as binding agent in tablet formulations to obtain tablets of desired bond strength and disintegration properties. Furthermore, plantain starch could be useful as an alternative binding agent to cornstarch, especially where faster disintegration is required and the problems of lamination and capping are of particular concern.

Effects of organosolv and ammonia pretreatments on lignin properties and its inhibition for enzymatic hydrolysis

DOE PAGES

Yoo, Chang Geun; Li, Mi; Meng, Xianzhi; ...

2017-04-05

Lignin offers structural support and protection for plant cell walls; but, it also contributes to biomass recalcitrance and the costs of biofuel production via the biological pathway. Organosolv and ammonia pretreatments have been developed to reduce biomass recalcitrance and improve sugar release performance during enzymatic hydrolysis. It is believed that lignin properties are related to its inhibition on enzymatic hydrolysis; therefore, understanding the characteristics of lignin is a key for effective biomass conversion to biofuels. In this study, an organosolv pretreatment using 60% ethanol with 1.25% H 2SO 4 significantly deconstructed poplar lignin and reduced its molecular weights due tomore » the cleavage of lignin inter-unit linkages. The organosolv pretreatment increased the contents of phenolic OH units and the lignin residue showed a high cellulase maximum adsorption capacity. Ammonia pretreatment with 5% ammonium hydroxide was not as effective as organosolv pretreatment on lignin deconstruction. Organosolv lignin residue had lower lignin S/G ratio than the untreated one. Furthermore, when compared to the organosolv lignin residue and untreated lignin, ammonia lignin residue had a higher cellulase adsorption affinity. In addition, the effects of lignin on cellulose hydrolysis was investigated and the results suggested that the presence of lignin with cellulose substrates reduced cellulose hydrolysis, and its inhibitory effect was primarily determined by the lignin properties after each pretreatment. The organosolv pretreatment resulted in a slightly lower cellulase binding strength (249.7 mL g -1) on poplar lignin than that on untreated samples (261.1 mL g -1), while ammonia lignin residue showed a higher cellulase binding strength (402.8 mL g -1) and had more significant inhibition effect on cellulose hydrolysis. Our results demonstrated that the binding strength significantly affected the lignin-derived inhibition on enzymatic hydrolysis of cellulose in the cellulose-lignin mixtures.« less

Effects of organosolv and ammonia pretreatments on lignin properties and its inhibition for enzymatic hydrolysis

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

Yoo, Chang Geun; Li, Mi; Meng, Xianzhi

Lignin offers structural support and protection for plant cell walls; but, it also contributes to biomass recalcitrance and the costs of biofuel production via the biological pathway. Organosolv and ammonia pretreatments have been developed to reduce biomass recalcitrance and improve sugar release performance during enzymatic hydrolysis. It is believed that lignin properties are related to its inhibition on enzymatic hydrolysis; therefore, understanding the characteristics of lignin is a key for effective biomass conversion to biofuels. In this study, an organosolv pretreatment using 60% ethanol with 1.25% H 2SO 4 significantly deconstructed poplar lignin and reduced its molecular weights due tomore » the cleavage of lignin inter-unit linkages. The organosolv pretreatment increased the contents of phenolic OH units and the lignin residue showed a high cellulase maximum adsorption capacity. Ammonia pretreatment with 5% ammonium hydroxide was not as effective as organosolv pretreatment on lignin deconstruction. Organosolv lignin residue had lower lignin S/G ratio than the untreated one. Furthermore, when compared to the organosolv lignin residue and untreated lignin, ammonia lignin residue had a higher cellulase adsorption affinity. In addition, the effects of lignin on cellulose hydrolysis was investigated and the results suggested that the presence of lignin with cellulose substrates reduced cellulose hydrolysis, and its inhibitory effect was primarily determined by the lignin properties after each pretreatment. The organosolv pretreatment resulted in a slightly lower cellulase binding strength (249.7 mL g -1) on poplar lignin than that on untreated samples (261.1 mL g -1), while ammonia lignin residue showed a higher cellulase binding strength (402.8 mL g -1) and had more significant inhibition effect on cellulose hydrolysis. Our results demonstrated that the binding strength significantly affected the lignin-derived inhibition on enzymatic hydrolysis of cellulose in the cellulose-lignin mixtures.« less

Effect of solid surface charge on the binding behaviour of a metal-binding peptide

PubMed Central

Donatan, Senem; Sarikaya, Mehmet; Tamerler, Candan; Urgen, Mustafa

2012-01-01

Over the last decade, solid-binding peptides have been increasingly used as molecular building blocks coupling bio- and nanotechnology. Despite considerable research being invested in this field, the effects of many surface-related parameters that define the binding of peptide to solids are still unknown. In the quest to control biological molecules at solid interfaces and, thereby, tailoring the binding characteristics of the peptides, the use of surface charge of the solid surface may probably play an important role, which then can be used as a potential tuning parameter of peptide adsorption. Here, we report quantitative investigation on the viscoelastic properties and binding kinetics of an engineered gold-binding peptide, 3RGBP1, adsorbed onto the gold surface at different surface charge densities. The experiments were performed in aqueous solutions using an electrochemical dissipative quartz crystal microbalance system. Hydrodynamic mass, hydration state and surface coverage of the adsorbed peptide films were determined as a function of surface charge density of the gold metal substrate. Under each charged condition, binding of 3rGBP1 displayed quantitative differences in terms of adsorbed peptide amount, surface coverage ratio and hydration state. Based on the intrinsically disordered structure of the peptide, we propose a possible mechanism for binding of the peptide that can be used for tuning surface adsorption in further studies. Controlled alteration of peptide binding on solid surfaces, as shown here, may provide novel methods for surface functionalization used for bioenabled processing and fabrication of future micro- and nanodevices. PMID:22491974

SO2-Binding Properties of Cationic η6,η1-NCN-Pincer Arene Ruthenium Platinum Complexes: Spectroscopic and Theoretical Studies

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

Bonnet, Sylvestre A.; Van Lenthe, Joop H.; van Dam, Hubertus JJ

2011-03-01

The SO2-binding properties of a series of η6,η1-NCN-pincer ruthenium platinum complexes have been studied by both UV-visible spectroscopy, and theoretical calculations. When an electronwithdrawing [Ru(C5R5)]+ fragment (R = H or Me) is η6-coordinated to the phenyl ring of the NCNpincer platinum fragment (cf. [2]+ and [3]+, see scheme 1), the characteristic orange coloration (pointing to η1- SO2 binding to Pt) of a solution of the parent NCN-pincer platinum complex 1 in dichloromethane upon SO2-bubbling is not observed. However, when the ruthenium center is η6- coordinated to a phenyl substituent linked in para-position to the carbon-to-platinum bond, i.e. complex [4]+, themore » SO2-binding property of the NCN-platinum center seems to be retained, as bubbling SO2 into a solution of the latter complex produces the characteristic orange color. We performed theoretical calculations at the MP2 level of approximation and TD-DFT studies, which enabled us to interpret the absence of color change in the case of [2]+ as an absence of coordination of SO2 to platinum. We analyze this absence or weaker SO2-coordination in dichloromethane to be a consequence of the relative electron-poorness of the platinum center in the respective η6- ruthenium coordinated NCN-pincer platinum complexes, that leads to a lower binding energy and an elongated calculated Pt-S bond distance. We also discuss the effects of electrostatic interactions in these cationic systems, which also seems to play a destabilizing role for complex [2(SO2)]+.« less

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

PubMed

Zhang, Jun; Li, Jing; Craig, Theodore A; Kumar, Rajiv; Gross, Michael L

2017-07-18

Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca 2+ -binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca 2+ but detaches from DRE under Ca 2+ stimulation, allowing gene expression. The Ca 2+ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca 2+ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca 2+ . Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca 2+ -binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca 2+ signal to CREB-mediated gene transcription.

Recent insights into the biological functions of liver fatty acid binding protein 1

PubMed Central

Wang, GuQi; Bonkovsky, Herbert L.; de Lemos, Andrew; Burczynski, Frank J.

2015-01-01

Over four decades have passed since liver fatty acid binding protein (FABP)1 was first isolated. There are few protein families for which most of the complete tertiary structures, binding properties, and tissue occurrences are described in such detail and yet new functions are being uncovered for this protein. FABP1 is known to be critical for fatty acid uptake and intracellular transport and also has an important role in regulating lipid metabolism and cellular signaling pathways. FABP1 is an important endogenous cytoprotectant, minimizing hepatocyte oxidative damage and interfering with ischemia-reperfusion and other hepatic injuries. The protein may be targeted for metabolic activation through the cross-talk among many transcriptional factors and their activating ligands. Deficiency or malfunction of FABP1 has been reported in several diseases. FABP1 also influences cell proliferation during liver regeneration and may be considered as a prognostic factor for hepatic surgery. FABP1 binds and modulates the action of many molecules such as fatty acids, heme, and other metalloporphyrins. The ability to bind heme is another cytoprotective property and one that deserves closer investigation. The role of FABP1 in substrate availability and in protection from oxidative stress suggests that FABP1 plays a pivotal role during intracellular bacterial/viral infections by reducing inflammation and the adverse effects of starvation (energy deficiency). PMID:26443794

Pyrene-nucleobase conjugates: synthesis, oligonucleotide binding and confocal bioimaging studies.

PubMed

Jabłoński, Artur; Fritz, Yannic; Wagenknecht, Hans-Achim; Czerwieniec, Rafał; Bernaś, Tytus; Trzybiński, Damian; Woźniak, Krzysztof; Kowalski, Konrad

2017-01-01

Fluorescent pyrene-linker-nucleobase (nucleobase = thymine, adenine) conjugates with carbonyl and hydroxy functionalities in the linker were synthesized and characterized. X-ray single-crystal structure analysis performed for the pyrene-C(O)CH 2 CH 2 -thymine ( 2 ) conjugate reveals dimers of molecules 2 stabilized by hydrogen bonds between the thymine moieties. The photochemical characterization showed structure-dependent fluorescence properties of the investigated compounds. The conjugates bearing a carbonyl function represent weak emitters as compared to compounds with a hydroxy function in the linker. The self-assembly properties of pyrene nucleobases were investigated in respect to their binding to single and double strand oligonucleotides in water and in buffer solution. In respect to the complementary oligothymidine T 10 template in water, compounds 3 and 5 both show a self-assembling behavior according to canonical base-base pairing. However, in buffer solution, derivative 5 was much more effective than 3 in binding to the T 10 template. Furthermore the adenine derivative 5 binds to the double-stranded (dA) 10 -T 10 template with a self-assembly ratio of 112%. Such a high value of a self-assembly ratio can be rationalized by a triple-helix-like binding, intercalation, or a mixture of both. Remarkably, compound 5 also shows dual staining pattern in living HeLa cells. Confocal microscopy confirmed that 5 predominantly stains mitochondria but it also accumulates in the nucleoli of the cells.

Modular Synthesis of Biologically Active Phosphatidic Acid Probes Using Click Chemistry

PubMed Central

Smith, Matthew D.; Sudhahar, Christopher G.; Gong, Denghuang; Stahelin, Robert V.

2018-01-01

Phosphatidic acid (PA) is an important signaling lipid that plays roles in a range of biological processes including both physiological and pathophysiological events. PA is one of a number of signaling lipids that can act as site-specific ligands for protein receptors in binding events that enforce membrane-association and generally regulate both receptor function and subcellular localization. However, elucidation of the full scope of PA activities has proven problematic, primarily due to the lack of a consensus sequence among PA-binding receptors. Thus, experimental approaches, such as those employing lipid probes, are necessary for characterizing interactions at the molecular level. Herein, we describe an efficient modular approach to the synthesis of a range of PA probes that employs a late stage introduction of reporter groups. This strategy was exploited in the synthesis of PA probes bearing fluorescent and photoaffinity tags as well as a bifunctional probe containing both a photoaffinity moiety and an azide as a secondary handle for purification purposes. To discern the ability of these PA analogues to mimic the natural lipid in protein binding properties, each compound was incorporated into vesicles for binding studies using a known PA receptor, the C2 domain of PKCα. In these studies, each compound exhibited binding properties that were comparable to those of synthetic PA, indicating their viability as probes for effectively studying the activities of PA in cellular processes. PMID:19668861

Modular evolution of phosphorylation-based signalling systems

PubMed Central

Jin, Jing; Pawson, Tony

2012-01-01

Phosphorylation sites are formed by protein kinases (‘writers’), frequently exert their effects following recognition by phospho-binding proteins (‘readers’) and are removed by protein phosphatases (‘erasers’). This writer–reader–eraser toolkit allows phosphorylation events to control a broad range of regulatory processes, and has been pivotal in the evolution of new functions required for the development of multi-cellular animals. The proteins that comprise this system of protein kinases, phospho-binding targets and phosphatases are typically modular in organization, in the sense that they are composed of multiple globular domains and smaller peptide motifs with binding or catalytic properties. The linkage of these binding and catalytic modules in new ways through genetic recombination, and the selection of particular domain combinations, has promoted the evolution of novel, biologically useful processes. Conversely, the joining of domains in aberrant combinations can subvert cell signalling and be causative in diseases such as cancer. Major inventions such as phosphotyrosine (pTyr)-mediated signalling that flourished in the first multi-cellular animals and their immediate predecessors resulted from stepwise evolutionary progression. This involved changes in the binding properties of interaction domains such as SH2 and their linkage to new domain types, and alterations in the catalytic specificities of kinases and phosphatases. This review will focus on the modular aspects of signalling networks and the mechanism by which they may have evolved. PMID:22889906

Rapid surface-biostructure interaction analysis using strong metal-based nanomagnets.

PubMed

Rotzetter, Aline C C; Schumacher, Christoph M; Zako, Tamotsu; Stark, Wendelin J; Maeda, Mizuo

2013-11-19

Nanomaterials are increasingly suggested for the selective adsorption and extraction of complex compounds in biomedicine. Binding of the latter requires specific surface modifications of the nanostructures. However, even complicated macromolecules such as proteins can afford affinities toward basic surface characteristics such as hydrophobicity, topology, and electrostatic charge. In this study, we address these more basic physical interactions. In a model system, the interaction of bovine serum albumin and amyloid β 42 fibrillar aggregates with carbon-coated cobalt nanoparticles, functionalized with various polymers differing in character, was studied. The possibility of rapid magnetic separation upon binding to the surface represents a valuable tool for studying surface interactions and selectivities. We find that the surface interaction of Aβ 42 fibrillar aggregates is mostly hydrophobic in nature. Because bovine serum albumin (BSA) is conformationally adaptive, it is known to bind surfaces with widely differing properties (charge, topology, and hydrophobicity). However, the rate of tight binding (no desorption upon washing) can vary largely depending on the extent of necessary conformational changes for a specific surface. We found that BSA can only bind slowly to polyethylenimine-coated nanomagnets. Under competitive conditions (high excess BSA compared to that for β 42 fibrillar aggregates), this effect is beneficial for targeting the fibrillar species. These findings highlight the possibility of selective extractions from complex media when advantageous basic physical surface properties are chosen.

Spectroscopic and viscometric elucidation of the interaction between a potential chloride channel blocker and calf-thymus DNA: the effect of medium ionic strength on the binding mode.

PubMed

Ganguly, Aniruddha; Ghosh, Soumen; Guchhait, Nikhil

2015-01-07

The present study demonstrates a detailed characterization of the binding interaction of a potential chloride channel blocker 9-methyl anthroate (9-MA) with calf-thymus DNA. The modulated photophysical properties of the emissive molecule within the microheterogeneous bio-assembly have been spectroscopically exploited to monitor the drug-DNA binding interaction. Experimental results based on fluorescence and absorption spectroscopy aided with DNA-melting, viscometric and circular dichroism studies unambiguously establish the binding mode between the drug and DNA to be principally intercalative. Concomitantly, a discernible dependence of the mode of binding between the concerned moieties on the ionic strength of the medium is noteworthy. A dip-and-rise characteristic of the rotational relaxation profile of the drug within the DNA environment has been argued to be originating from a substantial difference in the lifetime as well as amplitude of the free and DNA bound drug molecule. In view of the prospective biological applications of the drug, the issue of facile dissociation of the intercalated drug from the DNA helix via a simple detergent-sequestration technique has also been unveiled. The utility of the present work resides in exploring the potential applicability of the fluorescence properties of 9-MA for studying its interactions with other relevant biological or biomimicking targets.

[Analysis of the binding capacity of the benzodiazepine site of gabaa receptor in mice C57BL/6 and BALB/C pretreated with anxiolytics].

PubMed

Iarkova, M A

2011-01-01

The level of specific 3H-flunitrazepam binding in synaptosomal membranes of C57BL/6 and BALB/c mice brain underwent to the stress of different types has been studied. Mild stress (Elevated Plus Maze) was shown to induce the decrease of benzodiazepine binding in BALB/c mice only, while the strong one (Exposure to a predator) was revealed to cause this decrease in both strains. Behavioral effects of different non-benzodiazepine drugs possessing anxiolytic properties (Afobazol, Ladasten and Noopept) was accompanied with the normalization of the level of benzodiazepine reception, reduced by the stress of both modalities.

Pyrene maleimide as a probe of microenvironmental and dynamics properties of protein binding sites

NASA Astrophysics Data System (ADS)

Benci, S.; Vaccari, S.; Schianchi, G.; Locatelli, Donata; Vaghi, P.; Bottiroli, Giovanni F.

1995-01-01

N-(1-Pyrene)maleimide is highly fluorescent upon covalent binding with sulfhydryl and amino groups of the proteins. Multiexponential fluorescence decays were observed for the dye bound to different proteins even when a single binding site is involved. The lack of information about the fluorescence decay of free dye does not allow to define the variations of fluorescence parameter following the conjugation and their correlation with the binding properties of the fluorophore. In this work, a study of the fluorescence of the probe, free in solution, bound to different antibodies and to the antigen-antibody complex both in solution and in cell, has been performed. The experimental results showed that chemico-physical properties of the medium influence the fluorescence decay of the probe in both the free and bound forms, although to a different extent. The variations of fluorescence decay and anisotropy of the bound probe are related to the electronic characteristics of microenvironment and show an increased stabilization of the probe binding site with the increasing complexity of the substrate. The sensitivity of the fluorescence properties of the probe to the binding site environment opens interesting perspectives concerning the application of Py- maleimide fluorochromization to assess the degree of specificity of immunocytochemical labelling.

Electronic Structures, Bonding Configurations, and Band-Gap-Opening Properties of Graphene Binding with Low-Concentration Fluorine

DOE PAGES

Duan, Yuhua; Stinespring, Charter D.; Chorpening, Benjamin

2015-06-18

To better understand the effects of low-level fluorine in graphene-based sensors, first-principles density functional theory (DFT) with van der Waals dispersion interactions has been employed to investigate the structure and impact of fluorine defects on the electrical properties of single-layer graphene films. The results show that both graphite-2H and graphene have zero band gaps. When fluorine bonds to a carbon atom, the carbon atom is pulled slightly above the graphene plane, creating what is referred to as a CF defect. The lowest-binding energy state is found to correspond to two CF defects on nearest neighbor sites, with one fluorine abovemore » the carbon plane and the other below the plane. Overall this has the effect of buckling the graphene. The results further show that the addition of fluorine to graphene leads to the formation of an energy band (BF) near the Fermi level, contributed mainly from the 2p orbitals of fluorine with a small contribution from the porbitals of the carbon. Among the 11 binding configurations studied, our results show that only in two cases does the BF serve as a conduction band and open a band gap of 0.37 eV and 0.24 eV respectively. The binding energy decreases with decreasing fluorine concentration due to the interaction between neighboring fluorine atoms. The obtained results are useful for sensor development and nanoelectronics.« less

Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

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

Mori, Tomoaki; Nakamura, Kento; Masaoka, Keisuke

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to constructmore » an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms. - Highlights: • A novel RNA restriction enzyme using SNase was developed tor cleave viral RNA. • Our enzyme cleaved influenza RNA with rates >120-fold higher rates a PIN-fusion one. • Our artificial enzyme with the L5 linker showed the highest RNA cleavage rate. • Our artificial enzyme site-selectively cleaved influenza RNA in vitro.« less

The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

DTIC Science & Technology

1996-08-01

J-4030 TITLE: The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle PRINCIPAL...The In Vivo DNA Binding Properties of 5. FUNDING NUMBERS Wild-Type and Mutant p53 Proteins in Mammary Cell Lines DAMD17-94-J-4030 During the Course of...ABSTRACT (Maximum 200 Using a pair of murine cell lines, one lacking p53 and a derivative cell line containing temperature sensitive p53 val 135

The specificity of binding of growth hormone and prolactin to purified plasma membranes from pregnant-rabbit liver.

PubMed Central

Webb, C F; Cadman, H F; Wallis, M

1986-01-01

The binding of 125I-labelled human growth hormone (hGH) to a purified plasma membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction with Triton X-100, was dependent on time, temperature, the cations used and the receptor concentration. Solubilization did not affect the binding properties of the receptors at low concentrations of Triton X-100. Some somatogenic hormones, such as bovine GH, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled hGH from purified plasma membranes and solubilized receptor preparations, but GHs and prolactins from various other species were rather ineffective. The results indicate that although there are binding sites for hGH in these pregnant rabbit liver membranes, few of these are specifically somatogenic or lactogenic. The binding properties of the purified plasma membranes are similar to those of a microsomal preparation studied previously, suggesting that the complex nature of the binding of hGH is not due to the heterogeneity of cellular membranes used to study binding, but is a property of the receptors associated with plasma membranes. PMID:3790086

Synthesis and evaluation of 7-substituted-5,6-dihydrobenzo[c]acridine derivatives as new c-KIT promoter G-quadruplex binding ligands.

PubMed

Guo, Qian-Liang; Su, Hua-Fei; Wang, Ning; Liao, Sheng-Rong; Lu, Yu-Ting; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Huang, Zhi-Shu

2017-04-21

It has been shown that treatment of cancer cells with c-KIT G-quadruplex binding ligands can reduce their c-KIT expression levels thus inhibiting cell proliferation and inducing cell apoptosis. Herein, a series of new 7-substituted-5,6-dihydrobenzo[c]acridine derivatives were designed and synthesized. Subsequent biophysical evaluation demonstrated that the derivatives could effectively bind to and stabilize c-KIT G-quadruplex with good selectivity against duplex DNA. It was found that 12-N-methylated derivatives with a positive charge introduced at 12-position of 5,6-dihydrobenzo[c]acridine ring had similar binding affinity but lower stabilizing ability to c-KIT G-quadruplex DNA, compared with those of nonmethylated derivatives. Further molecular modeling studies showed possible binding modes of G-quadruplex with the ligands. RT-PCR assay and Western blot showed that compound 2b suppressed transcription and translation of c-KIT gene in K562 cells, which was consistent with the property of an effective G-quadruplex binding ligand targeting c-KIT oncogene promoter. Further biological evaluation showed that compound 2b could induce apoptosis through activation of the caspase-3 cascade pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Beyond the detergent effect: a binding site for sodium dodecyl sulfate (SDS) in mammalian apoferritin

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

Liu, Renyu, E-mail: renyu.liu@uphs.upenn.edu; Bu, Weiming; Xi, Jin

2012-05-01

Using X-ray crystallography and isothermal titration calorimetry, we show that sodium dodecyl sulfate (SDS) binds specifically to a pre-formed internal cavity in horse-spleen apoferritin. Although sodium dodecyl sulfate (SDS) is widely used as an anionic detergent, it can also exert specific pharmacological effects that are independent of the surfactant properties of the molecule. However, structural details of how proteins recognize SDS are scarce. Here, it is demonstrated that SDS binds specifically to a naturally occurring four-helix bundle protein: horse apoferritin. The X-ray crystal structure of the apoferritin–SDS complex was determined at a resolution of 1.9 Å and revealed that themore » SDS binds in an internal cavity that has previously been shown to recognize various general anesthetics. A dissociation constant of 24 ± 9 µM at 293 K was determined by isothermal titration calorimetry. SDS binds in this cavity by bending its alkyl tail into a horseshoe shape; the charged SDS head group lies in the opening of the cavity at the protein surface. This crystal structure provides insights into the protein–SDS interactions that give rise to binding and may prove useful in the design of novel SDS-like ligands for some proteins.« less

Binding of polychlorinated biphenyls to aquatic humic substances: The role of substrate and sorbate properties on partitioning

USGS Publications Warehouse

Uhle, M.E.; Chin, Y.-P.; Aiken, G.R.; McKnight, Diane M.

1999-01-01

Two ortho- (2,2',5 and 2,2',5,6') and a non-ortho- (3,3',4,4') substituted polychlorinated biphenyl (PCB) congeners were used to study the effects of sorbate structure in binding processes to two lacustrine fulvic acids. Binding constants were determined by solubility enhancement of the solutes by the fulvic acids. The binding of the ortho-trichlorobiphenyl was significantly less than the non-ortho-substituted tetrachlorobiphenyl to both fulvic acids. Surprisingly, the measured ortho-trichlorobiphenyl binding constant to both fulvic acids was approximately the same as the ortho- substituted tetrachlorobiphenyl. The effect of the chlorines in the ortho position inhibits free rotation around the 1,1' carbon bond, thereby making the molecule less able to interact effectively with the fulvic acid substrate relative to its non-ortho-substituted congeners. Finally, binding of all three PCBs to the Great Dismal Swamp fulvic acid was significantly higher than for the Pony Lake sample. This observation is attributable to the former substrate's higher degree of aromaticity and polarizability, which can potentially interact more favorably with the PCBs through an increase in van der Waals type interactions.Two ortho- (2,2???,5 and 2,2???,5,6???) and a non-ortho- (3,3???,4,4???) substituted polychlorinated biphenyl (PCB) congeners were used to study the effects of sorbate structure in binding processes to two lacustrine fulvic acids. Binding constants were determined by solubility enhancement of the solutes by the fulvic acids. The binding of the ortho-trichlorobiphenyl was significantly less than the non-ortho-substituted tetrachlorobiphenyl to both fulvic acids. Surprisingly, the measured ortho-trichlorobiphenyl binding constant to both fulvic acids was approximately the same as the ortho-substituted tetrachlorobiphenyl. The effect of the chlorines in the ortho position inhibits free rotation around the 1,1??? carbon bond, thereby making the molecule less able to interact effectively with the fulvic acid substrate relative to its non-ortho-substituted congeners. Finally, binding of all three PCBs to the Great Dismal Swamp fulvic acid was significantly higher than for the Pony Lake sample. This observation is attributable to the former substrate's higher degree of aromaticity and polarizability, which can potentially interact more favorably with the PCBs through an increase in van der Waals type interactions.

Development and Application of Explicitly Correlated Wave Function Based Methods for the Investigation of Optical Properties of Semiconductor Nanomaterials

NASA Astrophysics Data System (ADS)

Elward, Jennifer Mary

Semiconductor nanoparticles, or quantum dots (QDs), are well known to have very unique optical and electronic properties. These properties can be controlled and tailored as a function of several influential factors, including but not limited to the particle size and shape, effect of composition and heterojunction as well as the effect of ligand on the particle surface. This customizable nature leads to extensive experimental and theoretical research on the capabilities of these quantum dots for many application purposes. However, in order to be able to understand and thus further the development of these materials, one must first understand the fundamental interaction within these nanoparticles. In this thesis, I have developed a theoretical method which is called electron-hole explicitly correlated Hartee-Fock (eh-XCHF). It is a variational method for solving the electron-hole Schrodinger equation and has been used in this work to study electron-hole interaction in semiconductor quantum dots. The method was benchmarked with respect to a parabolic quantum dot system, and ground state energy and electron-hole recombination probability were computed. Both of these properties were found to be in good agreement with expected results. Upon successful benchmarking, I have applied the eh-XCHF method to study optical properties of several quantum dot systems including the effect of dot size on exciton binding energy and recombination probability in a CdSe quantum dot, the effect of shape on a CdSe quantum dot, the effect of heterojunction on a CdSe/ZnS quantum dot and the effect of quantum dot-biomolecule interaction within a CdSe-firefly Luciferase protein conjugate system. As metrics for assessing the effect of these influencers on the electron-hole interaction, the exciton binding energy, electron-hole recombination probability and the average electron-hole separation distance have been computed. These excitonic properties have been found to be strongly infuenced by the changing composition of the particle. It has also been found through this work that the explicitly correlated method performs very well when computing these properties as it provides a feasible computational route to compare to both experimental and other theoretical results.

Differences in receptor binding affinity of several phytocannabinoids do not explain their effects on neural cell cultures.

PubMed

Rosenthaler, Sarah; Pöhn, Birgit; Kolmanz, Caroline; Huu, Chi Nguyen; Krewenka, Christopher; Huber, Alexandra; Kranner, Barbara; Rausch, Wolf-Dieter; Moldzio, Rudolf

2014-01-01

Phytocannabinoids are potential candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure-affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [(3)H]CP-55,940 and the phytocannabinoids. The resulting Ki values to CB1 range from 23.5 nM (THCA) to 14711 nM (CBDV), whereas Ki values to CB2 range from 8.5 nM (THC) to 574.2 nM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10 μM, whereas CBC and THCA also displayed slightly positive activities. These findings are not linked to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one. [Corrected] Copyright © 2014 Elsevier Inc. All rights reserved.

Metal cofactor modulated folding and target recognition of HIV-1 NCp7.

PubMed

Ren, Weitong; Ji, Dongqing; Xu, Xiulian

2018-01-01

The HIV-1 nucleocapsid 7 (NCp7) plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3) recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.

Cooperative DNA binding of heterologous proteins: Evidence for contact between the cyclic AMP receptor protein and RNA polymerase

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

Ren, Y.L.; Garges, S.; Adhya, S.

1988-06-01

Four cAMP-independent receptor protein mutants (designated CRP* mutants) isolated previously are able to activate in vivo gene transcription in the absence of cAMP and their activity can be enhanced by cAMP or cGMP. One of the four mutant proteins, CRP*598 (Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter. Binding of wild-type CRP to its site on the lac promoter and activation of abortive initiation by RNA polymerase on this promoter are effected by cAMP but not by cGMP. CRP*598 canmore » activate lacP{sup +}-directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide. DNase I protection (footprinting) indicates that cAMP-CRP* binds to its site on the lac promoter whereas unliganded CRP* and cGMP-CRP* form a stable complex with the ({sup 32}P)lacP{sup +} fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins. This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation of transcription. Although cGMP binds to CRP, it cannot replace cAMP in effecting the requisite conformational transition necessary for site-specific promoter binding.« less

Electrostatic steering and ionic tethering in enzyme–ligand binding: Insights from simulations

PubMed Central

Wade, Rebecca C.; Gabdoulline, Razif R.; Lüdemann, Susanna K.; Lounnas, Valère

1998-01-01

To bind at an enzyme’s active site, a ligand must diffuse or be transported to the enzyme’s surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also influence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-influenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and β-lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as “ionic tethering.” We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme’s surroundings even when the substrate is nonpolar. PMID:9600896

Interactions and diffusion in fine-stranded β-lactoglobulin gels determined via FRAP and binding.

PubMed

Schuster, Erich; Hermansson, Anne-Marie; Ohgren, Camilla; Rudemo, Mats; Lorén, Niklas

2014-01-07

The effects of electrostatic interactions and obstruction by the microstructure on probe diffusion were determined in positively charged hydrogels. Probe diffusion in fine-stranded gels and solutions of β-lactoglobulin at pH 3.5 was determined using fluorescence recovery after photobleaching (FRAP) and binding, which is widely used in biophysics. The microstructures of the β-lactoglobulin gels were characterized using transmission electron microscopy. The effects of probe size and charge (negatively charged Na2-fluorescein (376Da) and weakly anionic 70kDa FITC-dextran), probe concentration (50 to 200 ppm), and β-lactoglobulin concentration (9% to 12% w/w) on the diffusion properties and the electrostatic interaction between the negatively charged probes and the positively charged gels or solutions were evaluated. The results show that the diffusion of negatively charged Na2-fluorescein is strongly influenced by electrostatic interactions in the positively charged β-lactoglobulin systems. A linear relationship between the pseudo-on binding rate constant and the β-lactoglobulin concentration for three different probe concentrations was found. This validates an important assumption of existing biophysical FRAP and binding models, namely that the pseudo-on binding rate constant equals the product of the molecular binding rate constant and the concentration of the free binding sites. Indicators were established to clarify whether FRAP data should be analyzed using a binding-diffusion model or an obstruction-diffusion model. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Chimeric Peptides as Implant Functionalization Agents for Titanium Alloy Implants with Antimicrobial Properties

NASA Astrophysics Data System (ADS)

Yucesoy, Deniz T.; Hnilova, Marketa; Boone, Kyle; Arnold, Paul M.; Snead, Malcolm L.; Tamerler, Candan

2015-04-01

Implant-associated infections can have severe effects on the longevity of implant devices and they also represent a major cause of implant failures. Treating these infections associated with implants by antibiotics is not always an effective strategy due to poor penetration rates of antibiotics into biofilms. Additionally, emerging antibiotic resistance poses serious concerns. There is an urge to develop effective antibacterial surfaces that prevent bacterial adhesion and proliferation. A novel class of bacterial therapeutic agents, known as antimicrobial peptides (AMPs), are receiving increasing attention as an unconventional option to treat septic infection, partly due to their capacity to stimulate innate immune responses and for the difficulty of microorganisms to develop resistance towards them. While host and bacterial cells compete in determining the ultimate fate of the implant, functionalization of implant surfaces with AMPs can shift the balance and prevent implant infections. In the present study, we developed a novel chimeric peptide to functionalize the implant material surface. The chimeric peptide simultaneously presents two functionalities, with one domain binding to a titanium alloy implant surface through a titanium-binding domain while the other domain displays an antimicrobial property. This approach gains strength through control over the bio-material interfaces, a property built upon molecular recognition and self-assembly through a titanium alloy binding domain in the chimeric peptide. The efficiency of chimeric peptide both in-solution and absorbed onto titanium alloy surface was evaluated in vitro against three common human host infectious bacteria, Streptococcus mutans, Staphylococcus epidermidis, and Escherichia coli. In biological interactions such as occur on implants, it is the surface and the interface that dictate the ultimate outcome. Controlling the implant surface by creating an interface composed chimeric peptides may therefore open up new possibilities to modify the implant site and tailor it to a desirable bioactivity.

Characterization of the Sterol and Phosphatidylinositol 4-Phosphate Binding Properties of Golgi-Associated OSBP-Related Protein 9 (ORP9)

PubMed Central

Liu, Xinwei; Ridgway, Neale D.

2014-01-01

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus. PMID:25255026

Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9).

PubMed

Liu, Xinwei; Ridgway, Neale D

2014-01-01

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

PubMed Central

Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

2013-01-01

In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

Vitelline envelope of Bufo arenarum: biochemical and biological characterization.

PubMed

Barisone, Gustavo A; Hedrick, Jerry L; Cabada, Marcelo O

2002-04-01

Vitelline envelopes (VEs) of Bufo arenarum were isolated in order to study their composition and their role in fertilization. VEs are composed of four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa. To characterize its biological properties, we quantitatively determined sperm-VE binding and the induction of the acrosome reaction. Heterologous binding of B. arenarum sperm to Xenopus laevis VE components was observed with about one-third the efficiency of homologous binding. Equivalent binding of X. laevis sperm to the B. arenarum VE was observed. When B. arenarum sperm were incubated with fluorescein isothiocyanate-labeled VE, the labeled glycoproteins bound to the anterior end of the sperm head, showing a lateral distribution. Induction of the acrosome reaction was evaluated by incubating sperm in hypotonic saline media with VE glycoproteins. VEs induced the acrosome reaction in a time- and concentration-dependent manner. The acrosome reaction was maximal after 10 min. The half-maximal effect was obtained at a glycoprotein concentration of 1 microg/ml. Specificity was determined using fertilization envelope glycoproteins, which failed to induce the acrosome reaction. The B. arenarum VE is biochemically similar to other egg envelopes. It also seems that its biological properties are similar to other species in regard to sperm binding and induction of the acrosome reaction. However, as far as we are aware, this is the first observation of the VE inducing the sperm acrosome reaction in amphibians. The relatively small differences observed in heterologous sperm-VE binding in X. laevis and B. arenarum are inconsistent with the current paradigm that species specificity in fertilization is regulated at the sperm-VE binding step.

Recognition of U-rich RNA by Hfq from the Gram-positive pathogen Listeria monocytogenes

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

Kovach, Alexander R.; Hoff, Kirsten E.; Canty, John T.

Hfq is a post-transcriptional regulator that binds U- and A-rich regions of sRNAs and their target mRNAs to stimulate their annealing in order to effect translation regulation and, often, to alter their stability. The functional importance of Hfq and its RNA-binding properties are relatively well understood in Gram-negative bacteria, whereas less is known about the RNAbinding properties of this riboregulator in Gram-positive species. Here, we describe the structure of Hfq from the Grampositive pathogen Listeria monocytogenes in its RNA-free form and in complex with a U 6 oligoribonucleotide. As expected, the protein takes the canonical hexameric toroidal shape of allmore » other known Hfq structures. The U 6 RNA binds on the “proximal face” in a pocket formed by conserved residues Q9, N42, F43, and K58. Additionally residues G5 and Q6 are involved in protein-nucleic and inter-subunit contacts that promote uracil specificity. Unlike Staphylococcus aureus (Sa) Hfq, Lm Hfq requires magnesium to bind U 6 with high affinity. In contrast, the longer oligo-uridine, U 16, binds Lm Hfq tightly in the presence or absence of magnesium, thereby suggesting the importance of additional residues on the proximal face and possibly the lateral rim in RNA interaction. Lastly, intrinsic tryptophan fluorescence quenching (TFQ) studies reveal, surprisingly, that Lm Hfq can bind (GU) 3G and U6 on its proximal and distal faces, indicating a less stringent adenine-nucleotide specificity site on the distal face as compared to the Gram-positive Hfq proteins from Sa and Bacillus subtilis and suggesting as yet uncharacterized RNA-binding modes on both faces.« less

Recognition of U-rich RNA by Hfq from the Gram-positive pathogen Listeria monocytogenes

DOE PAGES

Kovach, Alexander R.; Hoff, Kirsten E.; Canty, John T.; ...

2014-08-22

Hfq is a post-transcriptional regulator that binds U- and A-rich regions of sRNAs and their target mRNAs to stimulate their annealing in order to effect translation regulation and, often, to alter their stability. The functional importance of Hfq and its RNA-binding properties are relatively well understood in Gram-negative bacteria, whereas less is known about the RNAbinding properties of this riboregulator in Gram-positive species. Here, we describe the structure of Hfq from the Grampositive pathogen Listeria monocytogenes in its RNA-free form and in complex with a U 6 oligoribonucleotide. As expected, the protein takes the canonical hexameric toroidal shape of allmore » other known Hfq structures. The U 6 RNA binds on the “proximal face” in a pocket formed by conserved residues Q9, N42, F43, and K58. Additionally residues G5 and Q6 are involved in protein-nucleic and inter-subunit contacts that promote uracil specificity. Unlike Staphylococcus aureus (Sa) Hfq, Lm Hfq requires magnesium to bind U 6 with high affinity. In contrast, the longer oligo-uridine, U 16, binds Lm Hfq tightly in the presence or absence of magnesium, thereby suggesting the importance of additional residues on the proximal face and possibly the lateral rim in RNA interaction. Lastly, intrinsic tryptophan fluorescence quenching (TFQ) studies reveal, surprisingly, that Lm Hfq can bind (GU) 3G and U6 on its proximal and distal faces, indicating a less stringent adenine-nucleotide specificity site on the distal face as compared to the Gram-positive Hfq proteins from Sa and Bacillus subtilis and suggesting as yet uncharacterized RNA-binding modes on both faces.« less

The affinity of a major Ca2+ binding site on GRP78 is differentially enhanced by ADP and ATP.

PubMed

Lamb, Heather K; Mee, Christopher; Xu, Weiming; Liu, Lizhi; Blond, Sylvie; Cooper, Alan; Charles, Ian G; Hawkins, Alastair R

2006-03-31

GRP78 is a major protein regulated by the mammalian endoplasmic reticulum stress response, and up-regulation has been shown to be important in protecting cells from challenge with cytotoxic agents. GRP78 has ATPase activity, acts as a chaperone, and interacts specifically with other proteins, such as caspases, as part of a mechanism regulating apoptosis. GRP78 is also reported to have a possible role as a Ca2+ storage protein. In order to understand the potential biological effects of Ca2+ and ATP/ADP binding on the biology of GRP78, we have determined its ligand binding properties. We show here for the first time that GRP78 can bind Ca2+, ATP, and ADP, each with a 1:1 stoichiometry, and that the binding of cation and nucleotide is cooperative. These observations do not support the hypothesis that GRP78 is a dynamic Ca2+ storage protein. Furthermore, we demonstrate that whereas Mg2+ enhances GRP78 binding to ADP and ATP to the same extent, Ca2+ shows a differential enhancement. In the presence of Ca2+, the KD for ATP is lowered approximately 11-fold, and the KD for ADP is lowered around 930-fold. The KD for Ca2+ is lowered approximately 40-fold in the presence of ATP and around 880-fold with ADP. These findings may explain the biological requirement for a nucleotide exchange factor to remove ADP from GRP78. Taken together, our data suggest that the Ca2+-binding property of GRP78 may be part of a signal transduction pathway that modulates complex interactions between GRP78, ATP/ADP, secretory proteins, and caspases, and this ultimately has important consequences for cell viability.

Adaptation of avian influenza A (H6N1) virus from avian to human receptor-binding preference

PubMed Central

Wang, Fei; Qi, Jianxun; Bi, Yuhai; Zhang, Wei; Wang, Min; Zhang, Baorong; Wang, Ming; Liu, Jinhua; Yan, Jinghua; Shi, Yi; Gao, George F

2015-01-01

The receptor-binding specificity of influenza A viruses is a major determinant for the host tropism of the virus, which enables interspecies transmission. In 2013, the first human case of infection with avian influenza A (H6N1) virus was reported in Taiwan. To gather evidence concerning the epidemic potential of H6 subtype viruses, we performed comprehensive analysis of receptor-binding properties of Taiwan-isolated H6 HAs from 1972 to 2013. We propose that the receptor-binding properties of Taiwan-isolated H6 HAs have undergone three major stages: initially avian receptor-binding preference, secondarily obtaining human receptor-binding capacity, and recently human receptor-binding preference, which has been confirmed by receptor-binding assessment of three representative virus isolates. Mutagenesis work revealed that E190V and G228S substitutions are important to acquire the human receptor-binding capacity, and the P186L substitution could reduce the binding to avian receptor. Further structural analysis revealed how the P186L substitution in the receptor-binding site of HA determines the receptor-binding preference change. We conclude that the human-infecting H6N1 evolved into a human receptor preference. PMID:25940072

Differential Effects of Mutations on the Transport Properties of the Na+/H+ Antiporter NhaA from Escherichia coli*

PubMed Central

Mager, Thomas; Braner, Markus; Kubsch, Bastian; Hatahet, Lina; Alkoby, Dudu; Rimon, Abraham; Padan, Etana; Fendler, Klaus

2013-01-01

Na+/H+ antiporters show a marked pH dependence, which is important for their physiological function in eukaryotic and prokaryotic cells. In NhaA, the Escherichia coli Na+/H+ antiporter, specific single site mutations modulating the pH profile of the transporter have been described in the past. To clarify the mechanism by which these mutations influence the pH dependence of NhaA, the substrate dependence of the kinetics of selected NhaA variants was electrophysiologically investigated and analyzed with a kinetic model. It is shown that the mutations affect NhaA activity in quite different ways by changing the properties of the binding site or the dynamics of the transporter. In the first case, pK and/or KDNa are altered, and in the second case, the rate constants of the conformational transition between the inside and the outside open conformation are modified. It is shown that residues as far apart as 15–20 Å from the binding site can have a significant impact on the dynamics of the conformational transitions or on the binding properties of NhaA. The implications of these results for the pH regulation mechanism of NhaA are discussed. PMID:23836890

Spectroscopic investigation of the interaction between G-quadruplex of KRAS promoter sequence and three isoquinoline alkaloids

NASA Astrophysics Data System (ADS)

Wen, Li-Na; Xie, Meng-Xia

2017-01-01

KRAS promoter can form G-quadruplex structure and regulate gene transcription. The drugs which can bind with G-quadruplex of KRAS promoter may be potential remedy for treatment of cancers associated with KRAS mutation. The interaction mechanism between the G-quadruplex of KRAS promoter and three isoquinoline alkaloids (jatrorrhizine, berberine and sanguinarine) has been investigated by UV-visible, fluorescence and circular dichroism spectroscopic methods. The results showed that the three alkaloids can form complexes with G-quadruplex KRAS promoter with the molecular ratio of 1:1, and the binding constants were (0.90 ± 0.16) × 106 L mol- 1, (0.93 ± 0.21) × 106 L mol- 1 and (1.16 ± 0.45) × 106 L mol- 1 for jatrorrhizine, berberine and sanguinarine. The absorption spectra, KI quenching and fluorescence anisotropy and polarization studies suggested jatrorrhizine and berberine interacted with G-quadruplex by not only end-stacking binding mode but also grooves or loops binding mode, while sanguinarine by end-stacking binding mode. Sanguinarine was more beneficial to maintain the stability and parallel conformation of KRAS promoter G-quadruplex. MTT assay was performed to evaluate antiproliferation effects of the three isoquinoline alkaloids on SW620 cells, and the antiproliferation effects of the three alkaloids were sanguinarine > berberine > jatrorrhizine. All the three alkaloids can bind with KRAS promoter G-quadruplex, and sanguinarine had the better binding property and antiproliferation effects on SW620 cells. The results obtained are meaningful to explore potential reagents targeting the parallel G-quadruplex structure of KRAS promoter for gene theraphy of colorectal carcinomas.

A novel actin binding site of myosin required for effective muscle contraction.

PubMed

Várkuti, Boglárka H; Yang, Zhenhui; Kintses, Bálint; Erdélyi, Péter; Bárdos-Nagy, Irén; Kovács, Attila L; Hári, Péter; Kellermayer, Miklós; Vellai, Tibor; Málnási-Csizmadia, András

2012-02-12

F-actin serves as a track for myosin's motor functions and activates its ATPase activity by several orders of magnitude, enabling actomyosin to produce effective force against load. Although actin activation is a ubiquitous property of all myosin isoforms, the molecular mechanism and physiological role of this activation are unclear. Here we describe a conserved actin-binding region of myosin named the 'activation loop', which interacts with the N-terminal segment of actin. We demonstrate by biochemical, biophysical and in vivo approaches using transgenic Caenorhabditis elegans strains that the interaction between the activation loop and actin accelerates the movement of the relay, stimulating myosin's ATPase activity. This interaction results in efficient force generation, but it is not essential for the unloaded motility. We conclude that the binding of actin to myosin's activation loop specifically increases the ratio of mechanically productive to futile myosin heads, leading to efficient muscle contraction.

Fast and automated functional classification with MED-SuMo: an application on purine-binding proteins.

PubMed

Doppelt-Azeroual, Olivia; Delfaud, François; Moriaud, Fabrice; de Brevern, Alexandre G

2010-04-01

Ligand-protein interactions are essential for biological processes, and precise characterization of protein binding sites is crucial to understand protein functions. MED-SuMo is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is based on a 3D representation of macromolecules using specific surface chemical features associating chemical characteristics with geometrical properties. MED-SMA is an automated and fast method to classify binding sites. It is based on MED-SuMo technology, which builds a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are classified. Proteins potentially inhibited or activated through the same mechanism are gathered. Results are analyzed according to PROSITE annotations and to carefully refined functional annotations extracted from the PDB. As expected, binding sites associated with related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein kinases from different Kinome families are also found together, for example, Aurora-A and CDK2 proteins which are inhibited by the same drugs. Representative examples of different clusters are presented. The effectiveness of the MED-SMA approach is demonstrated as it gathers binding sites of proteins with similar structure-activity relationships. Moreover, an efficient new protocol associates structures absent of cocrystallized ligands to the purine clusters enabling those structures to be associated with a specific binding mechanism. Applications of this classification by binding mode similarity include target-based drug design and prediction of cross-reactivity and therefore potential toxic side effects.

Fast and automated functional classification with MED-SuMo: An application on purine-binding proteins

PubMed Central

Doppelt-Azeroual, Olivia; Delfaud, François; Moriaud, Fabrice; de Brevern, Alexandre G

2010-01-01

Ligand–protein interactions are essential for biological processes, and precise characterization of protein binding sites is crucial to understand protein functions. MED-SuMo is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is based on a 3D representation of macromolecules using specific surface chemical features associating chemical characteristics with geometrical properties. MED-SMA is an automated and fast method to classify binding sites. It is based on MED-SuMo technology, which builds a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are classified. Proteins potentially inhibited or activated through the same mechanism are gathered. Results are analyzed according to PROSITE annotations and to carefully refined functional annotations extracted from the PDB. As expected, binding sites associated with related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein kinases from different Kinome families are also found together, for example, Aurora-A and CDK2 proteins which are inhibited by the same drugs. Representative examples of different clusters are presented. The effectiveness of the MED-SMA approach is demonstrated as it gathers binding sites of proteins with similar structure-activity relationships. Moreover, an efficient new protocol associates structures absent of cocrystallized ligands to the purine clusters enabling those structures to be associated with a specific binding mechanism. Applications of this classification by binding mode similarity include target-based drug design and prediction of cross-reactivity and therefore potential toxic side effects. PMID:20162627

Cation Effects on the Electron-Acceptor Side of Photosystem II.

PubMed

Khan, Sahr; Sun, Jennifer S; Brudvig, Gary W

2015-06-18

The normal pathway of electron transfer on the electron-acceptor side of photosystem II (PSII) involves electron transfer from quinone A, QA, to quinone B, QB. It is possible to redirect electrons from QA(-) to water-soluble Co(III) complexes, which opens a new avenue for harvesting electrons from water oxidation by immobilization of PSII on electrode surfaces. Herein, the kinetics of electron transfer from QA(-) to [Co(III)(terpy)2](3+) (terpy = 2,2';6',2″-terpyridine) are investigated with a spectrophotometric assay revealing that the reaction follows Michaelis-Menten saturation kinetics, is inhibited by cations, and is not affected by variation of the QA reduction potential. A negatively charged site on the stromal surface of the PSII protein complex, composed of glutamic acid residues near QA, is hypothesized to bind cations, especially divalent cations. The cations are proposed to tune the redox properties of QA through electrostatic interactions. These observations may thus explain the molecular basis of the effect of divalent cations like Ca(2+), Sr(2+), Mg(2+), and Zn(2+) on the redox properties of the quinones in PSII, which has previously been attributed to long-range conformational changes propagated from divalent cations binding to the Ca(II)-binding site in the oxygen-evolving complex on the lumenal side of the PSII complex.

Self-energy effect and Coulomb potential modulation of the exciton in monolayer MoS2 on polar substrate

NASA Astrophysics Data System (ADS)

Wang, Zi-Wu; Xiao, Yao; Li, Run-Ze; Li, Wei-Ping; Li, Zhi-Qing

2017-11-01

We theoretically investigate the correction of exciton binding energy in monolayer MoS2 resulting from the exciton couples with surface optical (SO) phonons induced by polar substrate. The total correction of binding energy can be divided into the self-energy effect and modification of Coulomb potential using the unitary transformation method. We find that both the self-energy and Coulomb potential vary from tens of meV to several hundreds of meV depending on the cut-off wave vector of SO phonon modes, polarizability of substrate materials and internal distance between the monolayer MoS2 and polar substrate. An effective Coulomb potential is obtained by combining the modified term into the Coulomb potential. This potentially could be widely used in various two-dimensional materials. Our theoretical results not only propose the ways to externally control the exciton binding energy in experiment, but also enrich the understanding of the exciton properties in the dielectric environment.

Electronic structure and optical properties of triangular GaAs/AlGaAs quantum dots: Exciton and impurity states

NASA Astrophysics Data System (ADS)

Tiutiunnyk, A.; Akimov, V.; Tulupenko, V.; Mora-Ramos, M. E.; Kasapoglu, E.; Ungan, F.; Sökmen, I.; Morales, A. L.; Duque, C. A.

2016-03-01

Electronic structure and optical properties in equilateral triangular GaAs/Al0.3Ga0.7As quantum dots are studied extensively. The effects of donor and acceptor impurity atoms positioned in the orthocenter of the triangle, as well as of the external DC electric field are taken into account. Binding energies of the impurity, exciton energies, interband photoluminescence peak positions as well as linear and non-linear optical properties in THz range caused by transitions between excitonic states are calculated and discussed.

Metallofullerenol Gd@C82(OH)22 distracts the proline-rich-motif from putative binding on the SH3 domain

NASA Astrophysics Data System (ADS)

Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

2013-03-01

Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials.Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr33756a

Evaluation of the binding effect of human serum albumin on the properties of granules.

PubMed

Kristó, Katalin; Bajdik, János; Eros, István; Pintye-Hódi, Klára

2008-11-01

The main objective of this study was the application of a solution of human serum albumin as a granulating fluid. The properties of the granules formed were evaluated and compared with those when a conventional binder was applied in the same concentration. The powder mixture contained a soluble (mannitol) and an insoluble component (different types of cellulose). The protein solution applied exerted an appropriate aggregating effect if the system contained microcrystalline celluloses. Powdered cellulose was not suitable for the granulation with human serum albumin solution. As compared with the same concentration of the conventionally applied cellulose ethers as binder, the prepared granules exhibited a larger particle size, a significantly better compressibility, a higher breaking hardness and a favourable deformation process. These findings mainly reflect the good adhesive properties of the protein. The best compressibility and mechanical behaviour were attained on the application of the microcrystalline cellulose Vivapur type 105. This favourable behaviour may be connected with the wettability of cellulose. These results suggest that the formulation of tablets may be easier from an active agent in the serum that binds to albumin (e.g. interferon) since the amount of additives (binder) can be reduced.

Mutagenesis of the C2 domain of protein kinase C-alpha. Differential roles of Ca2+ ligands and membrane binding residues.

PubMed

Medkova, M; Cho, W

1998-07-10

The C2 domains of conventional protein kinase C (PKC) have been implicated in their Ca2+-dependent membrane binding. The C2 domain of PKC-alpha contains several Ca2+ ligands that bind multiple Ca2+ ions and other putative membrane binding residues. To understand the roles of individual Ca2+ ligands and protein-bound Ca2+ ions in the membrane binding and activation of PKC-alpha, we mutated five putative Ca2+ ligands (D187N, D193N, D246N, D248N, and D254N) and measured the effects of mutations on vesicle binding, enzyme activity, and monolayer penetration of PKC-alpha. Altered properties of these mutants indicate that individual Ca2+ ions and their ligands have different roles in the membrane binding and activation of PKC-alpha. The binding of Ca2+ to Asp187, Asp193, and Asp246 of PKC-alpha is important for the initial binding of protein to membrane surfaces. On the other hand, the binding of another Ca2+ to Asp187, Asp246, Asp248, and Asp254 induces the conformational change of PKC-alpha, which in turn triggers its membrane penetration and activation. Among these Ca2+ ligands, Asp246 was shown to be most essential for both membrane binding and activation of PKC-alpha, presumably due to its coordination to multiple Ca2+ ions. Furthermore, to identify the residues in the C2 domain that are involved in membrane binding of PKC-alpha, we mutated four putative membrane binding residues (Trp245, Trp247, Arg249, and Arg252). Membrane binding and enzymatic properties of two double-site mutants (W245A/W247A and R249A/R252A) indicate that Arg249 and Arg252 are involved in electrostatic interactions of PKC-alpha with anionic membranes, whereas Trp245 and Trp247 participate in its penetration into membranes and resulting hydrophobic interactions. Taken together, these studies provide the first experimental evidence for the role of C2 domain of conventional PKC as a membrane docking unit as well as a module that triggers conformational changes to activate the protein.

Influence of halide binding on the hydrogen bonding network in the active site of Salinibacter sensory rhodopsin I.

PubMed

Reissig, Louisa; Iwata, Tatsuya; Kikukawa, Takashi; Demura, Makoto; Kamo, Naoki; Kandori, Hideki; Sudo, Yuki

2012-11-06

In nature, organisms are subjected to a variety of environmental stimuli to which they respond and adapt. They can show avoidance or attractive behaviors away from or toward such stimuli in order to survive in the various environments in which they live. One such stimuli is light, to which, for example, the receptor sensory rhodopsin I (SRI) has been found to respond by regulating both negative and positive phototaxis in, e.g., the archaeon Halobacterium salinarum. Interestingly, to date, all organisms having SRI-like proteins live in highly halophilic environments, suggesting that salt significantly influences the properties of SRIs. Taking advantage of the discovery of the highly stable SRI homologue from Salinibacter ruber (SrSRI), which maintains its color even in the absence of salt, the importance of the chloride ion for the color tuning and for the slow M-decay, which is thought to be essential for the phototaxis function of SRIs, has been reported previously [Suzuki, D., et al. (2009) J. Mol. Biol.392, 48-62]. Here the effects of the anion binding on the structure and structural changes of SRI during its photocycle are investigated by means of Fourier transform infrared (FTIR) spectroscopy and electrochemical experiments. Our results reveal that, among other things, the structural change and proton movement of a characteristic amino acid residue, Asp102 in SrSRI, is suppressed by the binding of an anion in its vicinity, both in the K- and M-intermediate. The presence of this anion also effects the extent of chromophore distrotion, and tentative results indicate an influence on the number and/or properties of internal water molecules. In addition, a photoinduced proton transfer could only be observed in the absence of the bound anion. Possible proton movement pathways, including the residues Asp102 and the putative Cl binding site His131, are discussed. In conclusion, the results show that the anion binding to SRI is not only important for the color tuning, and for controlling the photocycle kinetics, but also induces some structural changes which facilitate the observed properties.

Agonist properties of a stable hexapeptide analog of neurotensin, N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1).

PubMed

Akunne, H C; Demattos, S B; Whetzel, S Z; Wustrow, D J; Davis, D M; Wise, L D; Cody, W L; Pugsley, T A; Heffner, T G

1995-04-18

The major signal transduction pathway for neurotensin (NT) receptors is the G-protein-dependent stimulation of phospholipase C, leading to the mobilization of intracellular free Ca2+ ([Ca2+]i) and the stimulation of cyclic GMP. We investigated the functional actions of an analog of NT(8-13), N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1), and other NT related analogs by quantitative measurement of the cytosolic free Ca2+ concentration in HT-29 (human colonic adenocarcinoma) cells using the Ca(2+)-sensitive dye fura-2/AM and by effects on cyclic GMP levels in rat cerebellar slices. The NT receptor binding affinities for these analogs to HT-29 cell membranes and newborn (10-day-old) mouse brain membranes were also investigated. Data obtained from HT-29 cell and mouse brain membrane preparations showed saturable single high-affinity sites and binding densities (Bmax) of 130.2 and 87.5 fmol/mg protein, respectively. The respective KD values were 0.47 and 0.39 nM, and the Hill coefficients were 0.99 and 0.92. The low-affinity levocabastine-sensitive site was not present (K1 > 10,000) in either membrane preparation. Although the correlation of binding between HT-29 cell membranes and mouse brain membranes was quite significant (r = 0.92), some of the reference agents had lower binding affinities in the HT-29 cell membranes. The metabolically stable compound NT1 plus other NT analogs and related peptides [NT, NT(8-13), xenopsin, neuromedin N, NT(9-13), kinetensin and (D-Trp11)-NT] increased intracellular Ca2+ levels in HT-29 cells, indicating NT receptor agonist properties. The effect of NT1 in mobilizing [Ca2+]i blocked by SR 48692, a non-peptide NT antagonist. Receptor binding affinities of NT analogs to HT-29 cell membranes were positively correlated with potencies for mobilizing intracellular calcium in the same cells. In addition, NT1 increased cyclic GMP levels in rat cerebellar slices, confirming the latter findings of its NT agonist action. These results substantiate the in vitro NT agonist properties of the hexapeptide NT analog NT1.

Binding effects of Mn²⁺ and Zn²⁺ ions on the vibrational properties of guanine-cytosine base pairs in the Watson-Crick and Hoogsteen configurations.

PubMed

Morari, Cristian; Bogdan, Diana; Muntean, Cristina M

2012-11-01

The binding effects of Mn²⁺ and Zn²⁺ ions on the vibrational properties of guanine-cytosine base pairs have been performed using density functional theory investigations. The calculations were carried out on Watson-Crick and Hoogsteen configurations of the base pairs. We have found, that in Watson-Crick configuration, the metal is coordinated to N7 atom of guanine while, in the case of Hoogsteen configuration, the coordination is at N3 atom of guanine. We have pointed out the vibrational bands that can be used to detect the presence of metallic ions in the Watson-Crick and Hoogsteen structures. Our results show that the vibrational amplitudes of metallic atoms are strong for wavenumbers lower than 600 cm⁻¹. Also, we predict that the distinction between Watson-Crick and Hoogsteen configurations can be seen around 85, 170 and 310 cm⁻¹.

Partially ionized hydrogen plasma in strong magnetic fields.

PubMed

Potekhin, A Y; Chabrier, G; Shibanov, Y A

1999-08-01

We study the thermodynamic properties of a partially ionized hydrogen plasma in strong magnetic fields, B approximately 10(12)-10(13) G, typical of neutron stars. The properties of the plasma depend significantly on the quantum-mechanical sizes and binding energies of the atoms, which are strongly modified by thermal motion across the field. We use new fitting formulas for the atomic binding energies and sizes, based on accurate numerical calculations and valid for any state of motion of the atom. In particular, we take into account decentered atomic states, neglected in previous studies of thermodynamics of magnetized plasmas. We also employ analytic fits for the thermodynamic functions of nonideal fully ionized electron-ion Coulomb plasmas. This enables us to construct an analytic model of the free energy. An ionization equilibrium equation is derived, taking into account the strong magnetic field effects and the nonideality effects. This equation is solved by an iteration technique. Ionization degrees, occupancies, and the equation of state are calculated.

Temporal concordance of anorectic, behavioral, cardiovascular and amphetamine receptor binding activity of phenethylamines in rats

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

Borrelli, A.; Blosser, J.; Barrantes, M.

Although numerous studies have described the anorectic, cardiovascular, and behavioral effects of phenthylamines, a comparison of the pharmacological concordance of these properties in a single species is needed. The objectives of this study were to compare the anorectic potency of 13 phenethylamines following po administration with their effects on spontaneous locomotor activity (SLA) and blood pressure (BP) in vivo and with amphetamine receptor affinity in vitro. The anorectic potencies (ED 50) ranged from 12 umol/kg (fenfluramine) to over 400 umol/kg (d-norephedrine and 1-pseudoephedrine). d-Amphetamine, phentermine, and d-norpseudoephedrine were among the most active and 1-pseudoephedrine and 1-nor-ephedrine the least active inmore » increasing SLA. 1-Norephedrine, and d-norpseudoephedrine were the most active increasing BP while d-norephedrine produced a weak vasodepressor effect. A significant correlation (r = .80) was observed between anorectic potency and affinity (IC 50) for /sup 3/H-amphetamine binding sites in the hypothalamus. However, the stereoselectivity between pairs of enantiomers to inhibit food consumption was not paralleled in binding affinity. The rank order of concordance of phenethylamines in anorectic activity was most apparent in behavior and binding affinity.« less

Effects of polyamines on the DNA-reactive properties of dimeric mithramycin complexed with cobalt(II): implications for anticancer therapy.

PubMed

Hou, Ming-Hon; Lu, Wen-Je; Huang, Chun-Yu; Fan, Ruey-Jane; Yuann, Jeu-Ming P

2009-06-09

Few studies have examined the effects of polyamines on the action of DNA-binding anticancer drugs. Here, a Co(II)-mediated dimeric mithramycin (Mith) complex, (Mith)(2)-Co(II), was shown to be resistant to polyamine competition toward the divalent metal ion when compared to the Fe(II)-mediated drug complexes. Surface plasmon resonance experiments demonstrated that polyamines interfered with the binding capacity and association rates of (Mith)(2)-Co(II) binding to DNA duplexes, while the dissociation rates were not affected. Although (Mith)(2)-Co(II) exhibited the highest oxidative activity under physiological conditions (pH 7.3 and 37 degrees C), polyamines (spermine in particular) inhibited the DNA cleavage activity of the (Mith)(2)-Co(II) in a concentration-dependent manner. Depletion of intracellular polyamines by methylglyoxal bis(guanylhydrazone) (MGBG) enhanced the sensitivity of A549 lung cancer cells to (Mith)(2)-Co(II), most likely due to the decreased intracellular effect of polyamines on the action of (Mith)(2)-Co(II). Our study suggests a novel method for enhancing the anticancer activity of DNA-binding metalloantibiotics through polyamine depletion.

Different effects of color-based and location-based selection on visual working memory.

PubMed

Li, Qi; Saiki, Jun

2015-02-01

In the present study, we investigated how feature- and location-based selection influences visual working memory (VWM) encoding and maintenance. In Experiment 1, cue type (color, location) and cue timing (precue, retro-cue) were manipulated in a change detection task. The stimuli were color-location conjunction objects, and binding memory was tested. We found a significantly greater effect for color precues than for either color retro-cues or location precues, but no difference between location pre- and retro-cues, consistent with previous studies (e.g., Griffin & Nobre in Journal of Cognitive Neuroscience, 15, 1176-1194, 2003). We also found no difference between location and color retro-cues. Experiment 2 replicated the color precue advantage with more complex color-shape-location conjunction objects. Only one retro-cue effect was different from that in Experiment 1: Color retro-cues were significantly less effective than location retro-cues in Experiment 2, which may relate to a structural property of multidimensional VWM representations. In Experiment 3, a visual search task was used, and the result of a greater location than color precue effect suggests that the color precue advantage in a memory task is related to the modulation of VWM encoding rather than of sensation and perception. Experiment 4, using a task that required only memory for individual features but not for feature bindings, further confirmed that the color precue advantage is specific to binding memory. Together, these findings reveal new aspects of the interaction between attention and VWM and provide potentially important implications for the structural properties of VWM representations.

Improved dialytic removal of protein-bound uraemic toxins with use of albumin binding competitors: an in vitro human whole blood study

PubMed Central

Tao, Xia; Thijssen, Stephan; Kotanko, Peter; Ho, Chih-Hu; Henrie, Michael; Stroup, Eric; Handelman, Garry

2016-01-01

Protein-bound uraemic toxins (PBUTs) cause various deleterious effects in end-stage kidney disease patients, because their removal by conventional haemodialysis (HD) is severely limited by their low free fraction in plasma. Here we provide an experimental validation of the concept that the HD dialytic removal of PBUTs can be significantly increased by extracorporeal infusion of PBUT binding competitors. The binding properties of indoxyl sulfate (IS), indole-3-acetic acid (IAA) and hippuric acid (HIPA) and their binding competitors, ibuprofen (IBU), furosemide (FUR) and tryptophan (TRP) were studied in uraemic plasma. The effect of binding competitor infusion on fractional removal of PBUT was then quantified in an ex vivo single-pass HD model using uraemic human whole blood. The infusion of a combination of IBU and FUR increased the fractional removal of IS from 6.4 ± 0.1 to 18.3 ± 0.4%. IAA removal rose from 16.8 ± 0.3 to 34.5 ± 0.7%. TRP infusion increased the removal of IS and IAA to 10.5 ± 0.1% and 27.1 ± 0.3%, respectively. Moderate effects were observed on HIPA removal. Pre-dialyzer infusion of PBUT binding competitors into the blood stream can increase the HD removal of PBUTs. This approach can potentially be applied in current HD settings. PMID:27001248

Ion-binding properties of the ClC chloride selectivity filter

PubMed Central

Lobet, Séverine; Dutzler, Raimund

2006-01-01

The ClC channels are members of a large protein family of chloride (Cl−) channels and secondary active Cl− transporters. Despite their diverse functions, the transmembrane architecture within the family is conserved. Here we present a crystallographic study on the ion-binding properties of the ClC selectivity filter in the close homolog from Escherichia coli (EcClC). The ClC selectivity filter contains three ion-binding sites that bridge the extra- and intracellular solutions. The sites bind Cl− ions with mM affinity. Despite their close proximity within the filter, the three sites can be occupied simultaneously. The ion-binding properties are found conserved from the bacterial transporter EcClC to the human Cl− channel ClC-1, suggesting a close functional link between ion permeation in the channels and active transport in the transporters. In resemblance to K+ channels, ions permeate the ClC channel in a single file, with mutual repulsion between the ions fostering rapid conduction. PMID:16341087

Binding Affinity Effects on Physical Characteristics of a Model Phase-Separated Protein Droplet

NASA Astrophysics Data System (ADS)

Chuang, Sara; Banani, Salman; Rosen, Michael; Brangwynne, Clifford

2015-03-01

Non-membrane bound organelles are associated with a range of biological functions. Several of these structures exhibit liquid-like properties, and may represent droplets of phase-separated RNA and/or proteins. These structures are often enriched in multi-valent molecules, however little is known about the interactions driving the assembly, properties, and function. Here, we address this question using a model multi-valent protein system consisting of repeats of Small Ubiquitin-like Modifier (SUMO) protein and a SUMO-interacting motif (SIM). These proteins undergo phase separation into liquid-like droplets. We combine microrheology and quantitative microscopy to determine affect of binding affinity on the viscosity, density and surface tension of these droplets. We also use fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and partitioning experiments to probe the structure and dynamics within these droplets. Our results shed light on how inter-molecular interactions manifests in droplet properties, and lay the groundwork for a comprehensive biophysical picture of intracellular RNA/protein organelles.

Simulation of a model nanopore sensor: Ion competition underlies device behavior.

PubMed

Mádai, Eszter; Valiskó, Mónika; Dallos, András; Boda, Dezső

2017-12-28

We study a model nanopore sensor with which a very low concentration of analyte molecules can be detected on the basis of the selective binding of the analyte molecules to the binding sites on the pore wall. The bound analyte ions partially replace the current-carrier cations in a thermodynamic competition. This competition depends both on the properties of the nanopore and the concentrations of the competing ions (through their chemical potentials). The output signal given by the device is the current reduction caused by the presence of the analyte ions. The concentration of the analyte ions can be determined through calibration curves. We model the binding site with the square-well potential and the electrolyte as charged hard spheres in an implicit background solvent. We study the system with a hybrid method in which we compute the ion flux with the Nernst-Planck (NP) equation coupled with the Local Equilibrium Monte Carlo (LEMC) simulation technique. The resulting NP+LEMC method is able to handle both strong ionic correlations inside the pore (including finite size of ions) and bulk concentrations as low as micromolar. We analyze the effect of bulk ion concentrations, pore parameters, binding site parameters, electrolyte properties, and voltage on the behavior of the device.

Simulation of a model nanopore sensor: Ion competition underlies device behavior

NASA Astrophysics Data System (ADS)

Mádai, Eszter; Valiskó, Mónika; Dallos, András; Boda, Dezső

2017-12-01

We study a model nanopore sensor with which a very low concentration of analyte molecules can be detected on the basis of the selective binding of the analyte molecules to the binding sites on the pore wall. The bound analyte ions partially replace the current-carrier cations in a thermodynamic competition. This competition depends both on the properties of the nanopore and the concentrations of the competing ions (through their chemical potentials). The output signal given by the device is the current reduction caused by the presence of the analyte ions. The concentration of the analyte ions can be determined through calibration curves. We model the binding site with the square-well potential and the electrolyte as charged hard spheres in an implicit background solvent. We study the system with a hybrid method in which we compute the ion flux with the Nernst-Planck (NP) equation coupled with the Local Equilibrium Monte Carlo (LEMC) simulation technique. The resulting NP+LEMC method is able to handle both strong ionic correlations inside the pore (including finite size of ions) and bulk concentrations as low as micromolar. We analyze the effect of bulk ion concentrations, pore parameters, binding site parameters, electrolyte properties, and voltage on the behavior of the device.

Approaching Pharmacological Space: Events and Components.

PubMed

Vistoli, Giulio; Pedretti, Alessandro; Mazzolari, Angelica; Testa, Bernard

2018-01-01

With a view to introducing the concept of pharmacological space and its potential applications in investigating and predicting the toxic mechanisms of xenobiotics, this opening chapter describes the logical relations between conformational behavior, physicochemical properties and binding spaces, which are seen as the three key elements composing the pharmacological space. While the concept of conformational space is routinely used to encode molecular flexibility, the concepts of property spaces and, particularly, of binding spaces are more innovative. Indeed, their descriptors can find fruitful applications (a) in describing the dynamic adaptability a given ligand experiences when inserted into a specific environment, and (b) in parameterizing the flexibility a ligand retains when bound to a biological target. Overall, these descriptors can conveniently account for the often disregarded entropic factors and as such they prove successful when inserted in ligand- or structure-based predictive models. Notably, and although binding space parameters can clearly be derived from MD simulations, the chapter will illustrate how docking calculations, despite their static nature, are able to evaluate ligand's flexibility by analyzing several poses for each ligand. Such an approach, which represents the founding core of the binding space concept, can find various applications in which the related descriptors show an impressive enhancing effect on the statistical performances of the resulting predictive models.

Theoretical studies of Pt-Ti nanoparticles for potential use as PEMFC electrocatalysts.

PubMed

Jennings, Paul C; Pollet, Bruno G; Johnston, Roy L

2012-03-07

A theoretical investigation is presented of alloying platinum with titanium to form binary Pt-Ti nanoalloys as an alternative to the expensive pure platinum catalysts commonly used for Proton Exchange Membrane Fuel Cell cathode electrocatalysts. Density Functional Theory calculations are performed to investigate compositional effects on structural properties as well as Oxygen Reduction Reaction kinetics and poisoning effects. High symmetry A(32)-B(6) clusters are studied to investigate structural properties. From these structures binding energies of hydroxyl and carbon monoxide are studied on a range of sites on the surface of the clusters. Promising results are obtained suggesting that the bimetallic Pt-Ti nanoalloys may exhibit enhanced properties compared to pure platinum catalysts.

Effect of Dioxygen on Copper(II) Binding to α-Synuclein

PubMed Central

Lucas, Heather R.; Lee, Jennifer C.

2010-01-01

Using the fluorescent amino acid tryptophan (Trp), we have characterized the copper(II) binding of F4W α-synuclein in the presence and absence of dioxygen at neutral pH. Variations in Trp fluorescence indicate that copper(II) binding is enhanced by the presence of dioxygen, with the apparent dissociation constant (Kd(app)) changing from 100 nM (anaerobic) to 10 nM (aerobic). To investigate the possible role of methionine oxidation, complementary work focused on synthetic peptide models of the N-terminal Cu(II)-α-syn site, MDV(F/W) and M*DV(F/W), where M*= methionine sulfoxide. Furthermore, we employed circular dichroism (CD) spectroscopy to demonstrate that the phenyl-to-indole (F→W) substitution does not alter copper(II) binding properties and to confirm the 1:1 metal-peptide binding stoichiometry. CD comparisons also revealed that Met1 oxidation does not affect the copper-peptide conformation and further suggested the possible existence of a CuII-Trp/Phe (cation-π) interaction. PMID:20064662

Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding.

PubMed

Churn, Severn B; Rana, Aniruddha; Lee, Kangmin; Parsons, J Travis; De Blas, Angel; Delorenzo, Robert J

2002-09-01

gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.

Facile characterization of aptamer kinetic and equilibrium binding properties using surface plasmon resonance

PubMed Central

Chang, Andrew L.; McKeague, Maureen; Smolke, Christina D.

2015-01-01

Nucleic acid aptamers find widespread use as targeting and sensing agents in nature and biotechnology. Their ability to bind an extensive range of molecular targets, including small molecules, proteins, and ions, with high affinity and specificity enables their use in diverse diagnostic, therapeutic, imaging, and gene-regulatory applications. Here, we describe methods for characterizing aptamer kinetic and equilibrium binding properties using a surface plasmon resonance-based platform. This aptamer characterization platform is broadly useful for studying aptamer–ligand interactions, comparing aptamer properties, screening functional aptamers during in vitro selection processes, and prototyping aptamers for integration into nucleic acid devices. PMID:25432760

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

Analysis of Factors Influencing Hydration Site Prediction Based on Molecular Dynamics Simulations

PubMed Central

2015-01-01

Water contributes significantly to the binding of small molecules to proteins in biochemical systems. Molecular dynamics (MD) simulation based programs such as WaterMap and WATsite have been used to probe the locations and thermodynamic properties of hydration sites at the surface or in the binding site of proteins generating important information for structure-based drug design. However, questions associated with the influence of the simulation protocol on hydration site analysis remain. In this study, we use WATsite to investigate the influence of factors such as simulation length and variations in initial protein conformations on hydration site prediction. We find that 4 ns MD simulation is appropriate to obtain a reliable prediction of the locations and thermodynamic properties of hydration sites. In addition, hydration site prediction can be largely affected by the initial protein conformations used for MD simulations. Here, we provide a first quantification of this effect and further indicate that similar conformations of binding site residues (RMSD < 0.5 Å) are required to obtain consistent hydration site predictions. PMID:25252619

D-glucose derived novel gemini surfactants: synthesis and study of their surface properties, interaction with DNA, and cytotoxicity.

PubMed

Kumar, Vikash; Chatterjee, Amrita; Kumar, Nupur; Ganguly, Anasuya; Chakraborty, Indranil; Banerjee, Mainak

2014-10-09

Four new D-glucose derived m-s-m type gemini surfactants with variable spacer and tail length have been synthesized by a simple and efficient synthetic methodology utilizing the free C-3 hydroxy group of diisopropylidene glucose. The synthetic route to these gemini surfactants with a quaternary ammonium group as polar head group involves a sequence of simple reactions including alkylation, imine formation, quaternization of amine etc. The surface properties of the new geminis were evaluated by surface tension and conductivity measurements. These gemini surfactants showed low cytotoxicity by MTT assay on HeLa cell line. The DNA binding capabilities of these surfactants were determined by agarose gel electrophoresis, fluorescence titration, and DLS experiments. The preliminary studies by agarose gel electrophoresis indicated chain length dependent DNA binding abilities, further supported by ethidium bromide exclusion experiments. Two of the D-glucose derived gemini surfactants showed effective binding with pET-28a plasmid DNA (pDNA) at relatively low N/P ratio (i.e., cationic nitrogen/DNA phosphate molar ratio). Copyright © 2014 Elsevier Ltd. All rights reserved.

Predicting Binding Free Energy Change Caused by Point Mutations with Knowledge-Modified MM/PBSA Method.

PubMed

Petukh, Marharyta; Li, Minghui; Alexov, Emil

2015-07-01

A new methodology termed Single Amino Acid Mutation based change in Binding free Energy (SAAMBE) was developed to predict the changes of the binding free energy caused by mutations. The method utilizes 3D structures of the corresponding protein-protein complexes and takes advantage of both approaches: sequence- and structure-based methods. The method has two components: a MM/PBSA-based component, and an additional set of statistical terms delivered from statistical investigation of physico-chemical properties of protein complexes. While the approach is rigid body approach and does not explicitly consider plausible conformational changes caused by the binding, the effect of conformational changes, including changes away from binding interface, on electrostatics are mimicked with amino acid specific dielectric constants. This provides significant improvement of SAAMBE predictions as indicated by better match against experimentally determined binding free energy changes over 1300 mutations in 43 proteins. The final benchmarking resulted in a very good agreement with experimental data (correlation coefficient 0.624) while the algorithm being fast enough to allow for large-scale calculations (the average time is less than a minute per mutation).

A Model for Predicting Thermoelectric Properties of Bi2Te3

NASA Technical Reports Server (NTRS)

Lee, Seungwon; VonAllmen, Paul

2009-01-01

A parameterized orthogonal tight-binding mathematical model of the quantum electronic structure of the bismuth telluride molecule has been devised for use in conjunction with a semiclassical transport model in predicting the thermoelectric properties of doped bismuth telluride. This model is expected to be useful in designing and analyzing Bi2Te3 thermoelectric devices, including ones that contain such nano - structures as quantum wells and wires. In addition, the understanding gained in the use of this model can be expected to lead to the development of better models that could be useful for developing other thermoelectric materials and devices having enhanced thermoelectric properties. Bi2Te3 is one of the best bulk thermoelectric materials and is widely used in commercial thermoelectric devices. Most prior theoretical studies of the thermoelectric properties of Bi2Te3 have involved either continuum models or ab-initio models. Continuum models are computationally very efficient, but do not account for atomic-level effects. Ab-initio models are atomistic by definition, but do not scale well in that computation times increase excessively with increasing numbers of atoms. The present tight-binding model bridges the gap between the well-scalable but non-atomistic continuum models and the atomistic but poorly scalable ab-initio models: The present tight-binding model is atomistic, yet also computationally efficient because of the reduced (relative to an ab-initio model) number of basis orbitals and flexible parameterization of the Hamiltonian.

Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

PubMed

Abdullah, Nazish; Padmanarayana, Murugesh; Marty, Naomi J; Johnson, Colin P

2014-01-21

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Differential plastic changes in synthesis and binding in the mouse somatostatin system after electroconvulsive stimulation.

PubMed

Olesen, Mikkel Vestergaard; Gøtzsche, Casper René; Christiansen, Søren Hofman; Woldbye, David Paul Drucker

2018-03-21

Electroconvulsive therapy (ECT) is regularly used to treat patients with severe major depression, but the mechanisms underlying the beneficial effects remain uncertain. Electroconvulsive stimulation (ECS) regulates diverse neurotransmitter systems and induces anticonvulsant effects, properties implicated in mediating therapeutic effects of ECT. Somatostatin (SST) is a candidate for mediating these effects because it is upregulated by ECS and exerts seizure-suppressant effects. However, little is known about how ECS might affect the SST receptor system. The present study examined effects of single and repeated ECS on the synthesis of SST receptors (SSTR1-4) and SST, and SST receptor binding ([125I]LTT-SST28) in mouse hippocampal regions and piriform/parietal cortices. A complex pattern of plastic changes was observed. In the dentate gyrus, SST and SSTR1 expression and the number of hilar SST immunoreactive cells were significantly increased at 1 week after repeated ECS while SSTR2 expression was downregulated by single ECS, and SSTR3 mRNA and SST binding were elevated 24 h after repeated ECS. In hippocampal CA1 and parietal/piriform cortices, we found elevated SST mRNA levels 1 week after repeated ECS and elevated SST binding after single ECS and 24 h after repeated ECS. In hippocampal CA3, repeated ECS increased SST expression 1 week after and SST binding 24 h after. In the parietal cortex, SSTR2 mRNA expression was downregulated after single ECS while SSTR4 mRNA expression was upregulated 24 h after repeated ECS. Considering the known anticonvulsant effects of SST, it is likely that these ECS-induced neuroplastic changes in the SST system could participate in modulating neuronal excitability and potentially contribute to therapeutic effects of ECT.

Impact of chitosan application technique on refrigerated catfish fillet quality

USDA-ARS?s Scientific Manuscript database

Chitosan has been reported to have functional properties such as antimicrobial activity, antioxidant activity, and binding action. Several studies reported an extension in the shelf life of various food products when chitosan was applied. However, there is limited study on the effect of chitosan ap...

Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics.

PubMed

Datta-Mannan, Amita; Thangaraju, Arunkumar; Leung, Donmienne; Tang, Ying; Witcher, Derrick R; Lu, Jirong; Wroblewski, Victor J

2015-01-01

Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ∼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition.

Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics

PubMed Central

Datta-Mannan, Amita; Thangaraju, Arunkumar; Leung, Donmienne; Tang, Ying; Witcher, Derrick R; Lu, Jirong; Wroblewski, Victor J

2015-01-01

Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ∼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition. PMID:25695748

Multifunctional receptor model for dioxin and related compound toxic action: possible thyroid hormone-responsive effector-linked site.

PubMed Central

McKinney, J D

1989-01-01

Molecular/theoretical modeling studies have revealed that thyroid hormones and toxic chlorinated aromatic hydrocarbons of environmental significance (for which dioxin or TCDD is the prototype) have similar structural properties that could be important in molecular recognition in biochemical systems. These molecular properties include a somewhat rigid, sterically accessible and polarizable aromatic ring and size-limited, hydrophobic lateral substituents, usually contained in opposite adjoining rings of a diphenyl compound. These molecular properties define the primary binding groups thought to be important in molecular recognition of both types of structures in biochemical systems. Similar molecular reactivities are supported by the demonstration of effective specific binding of thyroid hormones and chlorinated aromatic hydrocarbons with four different proteins, enzymes, or receptor preparations that are known or suspected to be involved in the expression of thyroid hormone activity. These binding interactions represent both aromatic-aromatic (stacking) and molecular cleft-type recognition processes. A multiple protein or multifunctional receptor-ligand binding mechanism model is proposed as a way of visualizing the details and possible role of both the stacking and cleft type molecular recognition factors in the expression of biological activity. The model suggests a means by which hormone-responsive effector-linked sites (possible protein-protein-DNA complexes) can maintain highly structurally specific control of hormone action. Finally, the model also provides a theoretical basis for the design and conduct of further biological experimentation on the molecular mechanism(s) of action of toxic chlorinated aromatic hydrocarbons and thyroid hormones. Images FIGURE 3. A FIGURE 3. B FIGURE 3. C FIGURE 3. D PMID:2551666

Guiding lead optimization with GPCR structure modeling and molecular dynamics.

PubMed

Heifetz, Alexander; James, Tim; Morao, Inaki; Bodkin, Michael J; Biggin, Philip C

2016-10-01

G-protein coupled receptor (GPCR) modeling approaches are widely used in the hit-to-lead and lead optimization stages of drug discovery. Modern protocols that involve molecular dynamics simulation can address key issues such as the free energy of binding (affinity), ligand-induced GPCR flexibility, ligand binding kinetics, conserved water positions and their role in ligand binding and the effects of mutations. The goals of these calculations are to predict the structures of the complexes between existing ligands and their receptors, to understand the key interactions and to utilize these insights in the design of new molecules with improved binding, selectivity or other pharmacological properties. In this review we present a brief survey of various computational approaches illustrated through a hierarchical GPCR modeling protocol and its prospective application in three industrial drug discovery projects. Copyright © 2016 Elsevier Ltd. All rights reserved.

Binding energies and modelling of nuclei in semiclassical simulations

NASA Astrophysics Data System (ADS)

Pérez-García, M. Ángeles; Tsushima, K.; Valcarce, A.

2008-03-01

We study the binding energies of spin isospin saturated nuclei with nucleon number 8⩽A⩽100 in semiclassical Monte Carlo many-body simulations. The model Hamiltonian consists of (i) nucleon kinetic energy, (ii) a nucleon nucleon interaction potential, and (iii) an effective Pauli potential which depends on density. The basic ingredients of the nucleon nucleon potential are a short-range repulsion, and a medium-range attraction. Our results demonstrate that one can always expect to obtain the empirical binding energies for a set of nuclei by introducing a proper density dependent Pauli potential in terms of a single variable, the nucleon number, A. The present work shows that in the suggested procedure there is a delicate counterbalance of kinetic and potential energetic contributions allowing a good reproduction of the experimental nuclear binding energies. This type of calculations may be of interest in further reproduction of other properties of nuclei such as radii and also exotic nuclei.

Identification of glycosaminoglycans using high-performance liquid chromatography on a hydroxyapatite column.

PubMed

Narita, H; Takeda, Y; Takagaki, K; Nakamura, T; Harata, S; Endo, M

1995-11-20

Glycosaminoglycans (heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate, and hyaluronic acid) were labeled with a fluorescent reagent, 2-aminopyridine. The fluoro-labeled glycosaminoglycans were subjected to high-performance liquid chromatography on a hydroxyapatite column. The binding property of each glycosaminoglycan to hydroxyapatite was different. The structural properties of glycosaminoglycans bound to hydroxyapatite were then investigated using chemical desulfated or enzymic depolymerized glycosaminoglycans. This revealed that the sulfate content and molecular weight of the glycosaminoglycans correlated with their binding properties to hydroxyapatite. Desulfated dermatan sulfate but not desulfated chondroitin 6-sulfate bound to the hydroxyapatite. These data indicate that iduronic acid residues of glycosaminoglycans are important for the binding property. The method described which uses hydroxyapatite columns facilitates rapid separation and microanalysis of the glycosaminoglycans, especially dermatan sulfate and chondroitin sulfate.

3-[(E)-(acridin-9‧-ylmethylidene)amino]-1-substituted thioureas and their biological activity

NASA Astrophysics Data System (ADS)

Bečka, Michal; Vilková, Mária; Salem, Othman; Kašpárková, Jana; Brabec, Viktor; Kožurková, Mária

2017-06-01

This paper describes the synthesis of a novel series of acridine thiosemicarbazones through a two-step reaction between various isothiocyanates and hydrazine followed by treatment with acridin-9-carbaldehyde. The properties of this series of seven new derivatives are studied using NMR and biochemical techniques, and the DNA-binding properties of the compounds are determined using spectrophotometric studies (UV-vis absorption, fluorescence, and circular/linear dichroism) and viscometry. The binding constants K are estimated as being in the range of 2.2 to 7.8 × 104 M- 1 and the percentage of hypochromism was found to be 22.11-49.75% (from UV-vis spectral titration). Electrophoretic experiments prove that the novel compounds demonstrate moderate inhibitory effects against Topo I activity at a concentration of 60 × 10- 6 M.

Pyrene–nucleobase conjugates: synthesis, oligonucleotide binding and confocal bioimaging studies

PubMed Central

Jabłoński, Artur; Fritz, Yannic; Wagenknecht, Hans-Achim; Czerwieniec, Rafał; Bernaś, Tytus; Trzybiński, Damian; Woźniak, Krzysztof

2017-01-01

Fluorescent pyrene–linker–nucleobase (nucleobase = thymine, adenine) conjugates with carbonyl and hydroxy functionalities in the linker were synthesized and characterized. X-ray single-crystal structure analysis performed for the pyrene–C(O)CH2CH2–thymine (2) conjugate reveals dimers of molecules 2 stabilized by hydrogen bonds between the thymine moieties. The photochemical characterization showed structure-dependent fluorescence properties of the investigated compounds. The conjugates bearing a carbonyl function represent weak emitters as compared to compounds with a hydroxy function in the linker. The self-assembly properties of pyrene nucleobases were investigated in respect to their binding to single and double strand oligonucleotides in water and in buffer solution. In respect to the complementary oligothymidine T10 template in water, compounds 3 and 5 both show a self-assembling behavior according to canonical base–base pairing. However, in buffer solution, derivative 5 was much more effective than 3 in binding to the T10 template. Furthermore the adenine derivative 5 binds to the double-stranded (dA)10–T10 template with a self-assembly ratio of 112%. Such a high value of a self-assembly ratio can be rationalized by a triple-helix-like binding, intercalation, or a mixture of both. Remarkably, compound 5 also shows dual staining pattern in living HeLa cells. Confocal microscopy confirmed that 5 predominantly stains mitochondria but it also accumulates in the nucleoli of the cells. PMID:29259662

Surface binding properties of aged and fresh (recently excreted) Toxoplasma gondii oocysts

USDA-ARS?s Scientific Manuscript database

The surface properties of aged (stored for 10 years) and fresh (recently excreted) oocysts of Toxoplasma gondii were investigated using monoclonal antibody (mAb) and lectin-binding assays. Fresh oocysts bound a wall-specific mAb labeled with fluorescein isothiocyanate while aged oocysts did not. In ...

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

PubMed Central

Stracke, Jan; Emrich, Thomas; Rueger, Petra; Schlothauer, Tilman; Kling, Lothar; Knaupp, Alexander; Hertenberger, Hubert; Wolfert, Andreas; Spick, Christian; Lau, Wilma; Drabner, Georg; Reiff, Ulrike; Koll, Hans; Papadimitriou, Apollon

2014-01-01

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels. PMID:25517308

Identification and characterization of a salivary-pellicle-binding peptide by phage display.

PubMed

Cukkemane, Nivedita; Bikker, Floris J; Nazmi, Kamran; Brand, Henk S; Veerman, Enno C I

2014-05-01

Dental biofilms are associated with oral diseases, making their control necessary. One way to control them is to prevent initial bacterial adherence to the salivary pellicle and thereby eventually decrease binding of late colonizing potential pathogens. The goal of this study was to generate a salivary-pellicle-binding peptide (SPBP) with antifouling activity towards primary colonizing bacteria. In order to achieve this goal we aimed to: (i) identify novel SPBPs by phage display; (ii) characterize the binding and antifouling properties of the selected SPBPs. A library of 2×10(9) phages displaying a random sequence of 12-mer peptides was used to identify peptides that bound selectively to the in vitro salivary pellicle. Three rounds of panning resulted in the selection of 10 pellicle-binding phages, each displaying a novel peptide sequence. The peptides were synthesized and their binding to the in vitro salivary pellicle was characterized in the presence and absence of calcium ions and Tween-20. The antifouling property of hydroxyapatite (HA) and saliva-coated HA discs treated with and without SPBPs were evaluated against Streptococcus gordonii. Ten unique SPBPs were identified using the phage display. One of these peptides, SPBP 10 (NSAAVRAYSPPS), exhibited significant binding to the in vitro salivary pellicle which was neither influenced by calcium ions, nor affected by up to 0.5% Tween-20. Its antifouling property against S. gordonii was significantly higher on the treated surfaces than on untreated surfaces. Use of the phage display library enabled us to find a specific SPBP with antifouling property towards S. gordonii. Copyright © 2014 Elsevier Ltd. All rights reserved.

Regulation of the Activity of Lactate Dehydrogenases from Four Lactic Acid Bacteria*

PubMed Central

Feldman-Salit, Anna; Hering, Silvio; Messiha, Hanan L.; Veith, Nadine; Cojocaru, Vlad; Sieg, Antje; Westerhoff, Hans V.; Kreikemeyer, Bernd; Wade, Rebecca C.; Fiedler, Tomas

2013-01-01

Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs. PMID:23720742

Effect of disorder on the optical properties of short period superlattices

NASA Technical Reports Server (NTRS)

Strozier, J. A.; Zhang, Y. A.; Horton, C.; Ignatiev, A.; Shih, H. D.

1993-01-01

The optical properties of disordered short period superlattices are studied using a one-dimensional tight-binding model. A difference vector and disorder structure factor are proposed to characterize the disordered superlattice. The density of states, participation number, and optical absorption coefficients for both ordered and disordered superlattices are calculated as a function of energy. The results show that introduction of disorder into an indirect band gap material enhances the optical transition near the indirect band edge.

Deciphering the groove binding modes of tau-fluvalinate and flumethrin with calf thymus DNA

NASA Astrophysics Data System (ADS)

Tao, Mo; Zhang, Guowen; Pan, Junhui; Xiong, Chunhong

2016-02-01

Tau-fluvalinate (TFL) and flumethrin (FL), widely used in agriculture and a class of synthetic pyrethroid pesticides with a similar structure, may cause a potential security risk. Herein, the modes of binding in vitro of TFL and FL with calf thymus DNA (ctDNA) were characterized by fluorescence, UV-vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy with the aid of viscosity measurements, melting analyses and molecular docking studies. The fluorescence titration indicated that both TFL and FL bound to ctDNA forming complexes through hydrogen bonding and van der Waals forces. The binding constants of TFL and FL with ctDNA were in the range of 104 L mol- 1, and FL exhibited a higher binding propensity than TFL. The iodide quenching effect, single/double-stranded DNA effects, and ctDNA melting and viscosity measurements demonstrated that the binding of both TFL and FL to ctDNA was groove mode. The FT-IR analyses suggested the A-T region of the minor groove of ctDNA as the preferential binding for TFL and FL, which was confirmed by the displacement assays with Hoechst 33258 probe, and the molecular docking visualized the specific binding. The changes in CD spectra indicated that both FL and TFL induced the perturbation on the base stacking and helicity of B-DNA, but the disturbance caused by FL was more obvious. Gel electrophoresis analyses indicated that both TFL and FL did not cause significant DNA cleavage. This study provides novel insights into the binding properties of TFL/FL with ctDNA and its toxic mechanisms.

Binding of plasma proteins to titanium dioxide nanotubes with different diameters

PubMed Central

Kulkarni, Mukta; Flašker, Ajda; Lokar, Maruša; Mrak-Poljšak, Katjuša; Mazare, Anca; Artenjak, Andrej; Čučnik, Saša; Kralj, Slavko; Velikonja, Aljaž; Schmuki, Patrik; Kralj-Iglič, Veronika; Sodin-Semrl, Snezna; Iglič, Aleš

2015-01-01

Titanium and titanium alloys are considered to be one of the most applicable materials in medical devices because of their suitable properties, most importantly high corrosion resistance and the specific combination of strength with biocompatibility. In order to improve the biocompatibility of titanium surfaces, the current report initially focuses on specifying the topography of titanium dioxide (TiO2) nanotubes (NTs) by electrochemical anodization. The zeta potential (ζ-potential) of NTs showed a negative value and confirmed the agreement between the measured and theoretically predicted dependence of ζ-potential on salt concentration, whereby the absolute value of ζ-potential diminished with increasing salt concentrations. We investigated binding of various plasma proteins with different sizes and charges using the bicinchoninic acid assay and immunofluorescence microscopy. Results showed effective and comparatively higher protein binding to NTs with 100 nm diameters (compared to 50 or 15 nm). We also showed a dose-dependent effect of serum amyloid A protein binding to NTs. These results and theoretical calculations of total available surface area for binding of proteins indicate that the largest surface area (also considering the NT lengths) is available for 100 nm NTs, with decreasing surface area for 50 and 15 nm NTs. These current investigations will have an impact on increasing the binding ability of biomedical devices in the body leading to increased durability of biomedical devices. PMID:25733829

GDP-GTP exchange processes of G{alpha}i1 protein are accelerated/decelerated depending on the type and the concentration of added detergents.

PubMed

Kubota, Makoto; Tanaka, Takeshi; Kohno, Toshiyuki; Wakamatsu, Kaori

2009-12-01

Although detergents have been widely used in G-protein studies to increase solubility and stability of the protein, we noticed that detergents modulate the nucleotide-binding properties of G-proteins. Hence, we analysed the effects of detergents on guanine nucleotide exchange reactions of Galpha(i1). Lubrol PX, a non-ionic detergent, which has been widely used in nucleotide dissociation/binding assays, was found to accelerate both GDP dissociation and GTPgammaS binding from/to Galpha in parallel at above its critical micelle concentration (cmc). Sodium cholate, an anionic detergent, which have been used to extract G-proteins from animal tissues, decelerated and accelerated GDP dissociation below and above its cmc, respectively. Surprisingly, micellar cholate decelerated GTPgammaS binding, and the binding rate constant was decreased by three orders of magnitude in the presence of 2% cholate. These results demonstrate that the guanine nucleotide exchange reactions of Galpha(i1) are drastically modulated by detergents differently depending on the type and the state (monomeric or micellar) of the detergents and that dissociation of GDP from Galpha(i1) does not necessarily lead to immediate binding of GTP to Galpha(i1) in some cases. These effects of detergents on G-proteins must be taken into account in G-protein experiments.

Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding.

PubMed

Ailte, Ieva; Lingelem, Anne Berit Dyve; Kavaliauskiene, Simona; Bergan, Jonas; Kvalvaag, Audun Sverre; Myrann, Anne-Grethe; Skotland, Tore; Sandvig, Kirsten

2016-07-26

Shiga toxin (Stx), an AB5 toxin, binds specifically to the neutral glycosphingolipid Gb3 at the cell surface before being transported into cells. We here demonstrate that addition of conical lysophospholipids (LPLs) with large head groups inhibit Stx binding to cells whereas LPLs with small head groups do not. Lysophosphatidylinositol (LPI 18:0), the most efficient LPL with the largest head group, was selected for in-depth investigations to study how the binding of Stx is regulated. We show that the inhibition of Stx binding by LPI is reversible and possibly regulated by cholesterol since addition of methyl-β-cyclodextrin (mβCD) reversed the ability of LPI to inhibit binding. LPI-induced inhibition of Stx binding is independent of signalling and membrane turnover as it occurs in fixed cells as well as after depletion of cellular ATP. Furthermore, data obtained with fluorescent membrane dyes suggest that LPI treatment has a direct effect on plasma membrane lipid packing with shift towards a liquid disordered phase in the outer leaflet, while lysophosphoethanolamine (LPE), which has a small head group, does not. In conclusion, our data show that cellular treatment with conical LPLs with large head groups changes intrinsic properties of the plasma membrane and modulates Stx binding to Gb3.

Effects of the amphiphilic peptides mastoparan and adenoregulin on receptor binding, G proteins, phosphoinositide breakdown, cyclic AMP generation, and calcium influx.

PubMed

Shin, Y; Moni, R W; Lueders, J E; Daly, J W

1994-04-01

1. The amphiphilic peptide mastoparan is known to affect phosphoinositide breakdown, calcium influx, and exocytosis of hormones and neurotransmitters and to stimulate the GTPase activity of guanine nucleotide-binding regulatory proteins. Another amphiphilic peptide, adenoregulin was recently identified based on stimulation of agonist binding to A1-adenosine receptors. 2. A comparison of the effects of mastoparan and adenoregulin reveals that these peptides share many properties. Both stimulate binding of agonists to receptors and binding of GTP gamma S to G proteins in brain membranes. The enhanced guanyl nucleotide exchange may be responsible for the complete conversion of receptors to a high-affinity state, complexed with guanyl nucleotide-free G proteins. 3. Both peptides increase phosphoinositide breakdown in NIH 3T3 fibroblasts. Pertussis toxin partially inhibits the phosphoinositide breakdown elicited by mastoparan but has no effect on the response to adenoregulin. N-Ethylmaleimide inhibits the response to both peptides. 4. In permeabilized 3T3 cells, both adenoregulin and mastoparan inhibit GTP gamma S-stimulated phosphoinositide breakdown. Mastoparan slightly increases basal cyclic AMP levels in cultured cells, followed at higher concentrations by an inhibition, while adenoregulin has minimal effects. 5. Both peptides increase calcium influx in cultured cells and release of norepinephrine in pheochromocytoma PC12 cells. The calcium influx elicited by the peptides in 3T3 cells is not markedly altered by N-ethylmaleimide. 6. Multiple sites of action appear likely to underlie the effects of mastoparan/adenoregulin on receptors, G proteins, phospholipase C, and calcium.

Nanoparticles engineered to bind cellular motors for efficient delivery.

PubMed

Dalmau-Mena, Inmaculada; Del Pino, Pablo; Pelaz, Beatriz; Cuesta-Geijo, Miguel Ángel; Galindo, Inmaculada; Moros, María; de la Fuente, Jesús M; Alonso, Covadonga

2018-03-30

Dynein is a cytoskeletal molecular motor protein that transports cellular cargoes along microtubules. Biomimetic synthetic peptides designed to bind dynein have been shown to acquire dynamic properties such as cell accumulation and active intra- and inter-cellular motion through cell-to-cell contacts and projections to distant cells. On the basis of these properties dynein-binding peptides could be used to functionalize nanoparticles for drug delivery applications. Here, we show that gold nanoparticles modified with dynein-binding delivery sequences become mobile, powered by molecular motor proteins. Modified nanoparticles showed dynamic properties, such as travelling the cytosol, crossing intracellular barriers and shuttling the nuclear membrane. Furthermore, nanoparticles were transported from one cell to another through cell-to-cell contacts and quickly spread to distant cells through cell projections. The capacity of these motor-bound nanoparticles to spread to many cells and increasing cellular retention, thus avoiding losses and allowing lower dosage, could make them candidate carriers for drug delivery.

Quantum transport properties of carbon nanotube field-effect transistors with electron-phonon coupling

NASA Astrophysics Data System (ADS)

Ishii, Hiroyuki; Kobayashi, Nobuhiko; Hirose, Kenji

2007-11-01

We investigated the electron-phonon coupling effects on the electronic transport properties of metallic (5,5)- and semiconducting (10,0)-carbon nanotube devices. We calculated the conductance and mobility of the carbon nanotubes with micron-order lengths at room temperature, using the time-dependent wave-packet approach based on the Kubo-Greenwood formula within a tight-binding approximation. We investigated the scattering effects of both longitudinal acoustic and optical phonon modes on the transport properties. The electron-optical phonon coupling decreases the conductance around the Fermi energy for the metallic carbon nanotubes, while the conductance of semiconductor nanotubes is decreased around the band edges by the acoustic phonons. Furthermore, we studied the Schottky-barrier effects on the mobility of the semiconducting carbon nanotube field-effect transistors for various gate voltages. We clarified how the electron mobilities of the devices are changed by the acoustic phonon.

Beware of ligand efficiency (LE): understanding LE data in modeling structure-activity and structure-economy relationships.

PubMed

Polanski, Jaroslaw; Tkocz, Aleksandra; Kucia, Urszula

2017-09-11

On the one hand, ligand efficiency (LE) and the binding efficiency index (BEI), which are binding properties (B) averaged versus the heavy atom count (HAC: LE) or molecular weight (MW: BEI), have recently been declared a novel universal tool for drug design. On the other hand, questions have been raised about the mathematical validity of the LE approach. In fact, neither the critics nor the advocates are precise enough to provide a generally understandable and accepted chemistry of the LE metrics. In particular, this refers to the puzzle of the LE trends for small and large molecules. In this paper, we explain the chemistry and mathematics of the LE type of data. Because LE is a weight metrics related to binding per gram, its hyperbolic decrease with an increasing number of heavy atoms can be easily understood by its 1/MW dependency. Accordingly, we analyzed how this influences the LE trends for ligand-target binding, economic big data or molecular descriptor data. In particular, we compared the trends for the thermodynamic ∆G data of a series of ligands that interact with 14 different target classes, which were extracted from the BindingDB database with the market prices of a commercial compound library of ca. 2.5 mln synthetic building blocks. An interpretation of LE and BEI that clearly explains the observed trends for these parameters are presented here for the first time. Accordingly, we show that the main misunderstanding of the chemical meaning of the BEI and LE parameters is their interpretation as molecular descriptors that are connected with a single molecule, while binding is a statistical effect in which a population of ligands limits the formation of ligand-receptor complexes. Therefore, LE (BEI) should not be interpreted as a molecular (physicochemical) descriptor that is connected with a single molecule but as a property (binding per gram). Accordingly, the puzzle of the surprising behavior of LE is explained by the 1/MW dependency. This effect clearly explains the hyperbolic LE trend not as a real increase in binding potency but as a physical limitation due to the different population of ligands with different MWs in a 1 g sample available for the formation of ligand-receptor complexes. Graphical abstract .

Effective field theory description of halo nuclei

NASA Astrophysics Data System (ADS)

Hammer, H.-W.; Ji, C.; Phillips, D. R.

2017-10-01

Nuclear halos emerge as new degrees of freedom near the neutron and proton driplines. They consist of a core and one or a few nucleons which spend most of their time in the classically-forbidden region outside the range of the interaction. Individual nucleons inside the core are thus unresolved in the halo configuration, and the low-energy effective interactions are short-range forces between the core and the valence nucleons. Similar phenomena occur in clusters of 4He atoms, cold atomic gases near a Feshbach resonance, and some exotic hadrons. In these weakly-bound quantum systems universal scaling laws for s-wave binding emerge that are independent of the details of the interaction. Effective field theory (EFT) exposes these correlations and permits the calculation of non-universal corrections to them due to short-distance effects, as well as the extension of these ideas to systems involving the Coulomb interaction and/or binding in higher angular-momentum channels. Halo nuclei exhibit all these features. Halo EFT, the EFT for halo nuclei, has been used to compute the properties of single-neutron, two-neutron, and single-proton halos of s-wave and p-wave type. This review summarizes these results for halo binding energies, radii, Coulomb dissociation, and radiative capture, as well as the connection of these properties to scattering parameters, thereby elucidating the universal correlations between all these observables. We also discuss how Halo EFT's encoding of the long-distance physics of halo nuclei can be used to check and extend ab initio calculations that include detailed modeling of their short-distance dynamics.

The transition in hemoglobin proton-binding characteristics within the basal actinopterygian fishes.

PubMed

Regan, Matthew Daniel; Brauner, Colin J

2010-04-01

Carbon dioxide (CO(2)) transport in the blood of fishes is aided by the proton-binding properties of hemoglobin (Hb) through either a high-intrinsic buffer value and small oxylabile proton binding (Haldane effect), or a low buffer value and large Haldane effect. Primitive species, such as elasmobranchs and sarcopterygians have been shown to rely on the former, while derived species, such as teleosts rely on the latter. Both strategies are effective in the transport of CO(2) in the blood. However, there is a paucity of information on the nature of the transition between these two strategies that appears to occur within the intermediate group of fishes, the basal actinopterygians. The objective of the present study was to simultaneously assess the intrinsic Hb buffer values and Haldane effects of species within the basal actinopterygian lineage to characterize the transition in Hb-proton-binding strategy seen among the fishes. Expressed in order of most basal to most derived, the species investigated included American paddlefish (Polyodon spathula), white sturgeon (Acipenser transmontanus), spotted gar (Lepisosteus oculatus), alligator gar (Atractosteus spatula), bowfin (Amia calva), and mooneye (Hiodon tergisus). Hemolysates from these species were prepared and Hb titrations (under oxygenated and deoxygenated conditions) were performed in both the presence and absence of saturating levels of organic phosphates (GTP). The findings suggest that the nature of the Hb-proton-binding transition may have been punctuated rather than gradual, with the Hb buffer value decreasing and the Haldane effect increasing significantly in bowfin from fairly steady ancestral levels in the four more basal species. This change is coupled with the initial appearance of the choroid rete, as well as an increase in the magnitude and onset pH of the Root effect in bowfin, suggesting that the change in Hb-proton-binding strategy may be associated with the evolution of enhanced O(2) delivery to the eye and an in vivo operational Root effect.

Quantitative determination of cesium binding to ferric hexacyanoferrate: Prussian blue.

PubMed

Faustino, Patrick J; Yang, Yongsheng; Progar, Joseph J; Brownell, Charles R; Sadrieh, Nakissa; May, Joan C; Leutzinger, Eldon; Place, David A; Duffy, Eric P; Houn, Florence; Loewke, Sally A; Mecozzi, Vincent J; Ellison, Christopher D; Khan, Mansoor A; Hussain, Ajaz S; Lyon, Robbe C

2008-05-12

Ferric hexacyanoferrate (Fe4III[FeII(CN)6]3), also known as insoluble Prussian blue (PB) is the active pharmaceutical ingredient (API) of the drug product, Radiogardase. Radiogardase is the first FDA approved medical countermeasure for the treatment of internal contamination with radioactive cesium (Cs) or thallium in the event of a major radiological incident such as a "dirty bomb". A number of pre-clinical and clinical studies have evaluated the use of PB as an investigational decorporation agent to enhance the excretion of metal cations. There are few sources of published in vitro data that detail the binding capacity of cesium to insoluble PB under various chemical and physical conditions. The study objective was to determine the in vitro binding capacity of PB APIs and drug products by evaluating certain chemical and physical factors such as medium pH, particle size, and storage conditions (temperature). In vitro experimental conditions ranged from pH 1 to 9, to cover the range of pH levels that PB may encounter in the gastrointestinal (GI) tract in humans. Measurements of cesium binding were made between 1 and 24h, to cover gastric and intestinal tract residence time using a validated atomic emission spectroscopy (AES) method. The results indicated that pH, exposure time, storage temperature (affecting moisture content) and particle size play significant roles in the cesium binding to both the PB API and the drug product. The lowest cesium binding was observed at gastric pH of 1 and 2, whereas the highest cesium binding was observed at physiological pH of 7.5. It was observed that dry storage conditions resulted in a loss of moisture from PB, which had a significant negative effect on the PB cesium binding capacity at time intervals consistent with gastric residence. Differences were also observed in the binding capacity of PB with different particle sizes. Significant batch to batch differences were also observed in the binding capacity of some PB API and drug products. Our results suggest that certain physiochemical properties affect the initial binding capacity and the overall binding capacity of PB APIs and drug products during conditions that simulated gastric and GI residence time. These physiochemical properties can be utilized as quality attributes to monitor and predict drug product quality under certain manufacturing and storage conditions and may be utilized to enhance the clinical efficacy of PB.

Disturbances of Ligand Potency and Enhanced Degradation of the Human Glycine Receptor at Affected Positions G160 and T162 Originally Identified in Patients Suffering from Hyperekplexia

PubMed Central

Atak, Sinem; Langlhofer, Georg; Schaefer, Natascha; Kessler, Denise; Meiselbach, Heike; Delto, Carolyn; Schindelin, Hermann; Villmann, Carmen

2015-01-01

Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GlyR) undergoes direct interaction with the incoming ligand via a cation-π interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GlyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GlyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, T162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof. PMID:26733802

Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

PubMed Central

De Henau, Olivier; Degroot, Gaetan-Nagim; Imbault, Virginie; Robert, Virginie; De Poorter, Cédric; Mcheik, Saria; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

2016-01-01

Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties. PMID:27716822

Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2.

PubMed

De Henau, Olivier; Degroot, Gaetan-Nagim; Imbault, Virginie; Robert, Virginie; De Poorter, Cédric; Mcheik, Saria; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

2016-01-01

Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.

Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies.

PubMed

Fong, Monica; Berrin, Jean-Guy; Paës, Gabriel

2016-01-01

Enzymes degrading plant biomass polymers are widely used in biotechnological applications. Their efficiency can be limited by non-specific interactions occurring with some chemical motifs. In particular, the lignin component is known to bind enzymes irreversibly. In order to determine interactions of enzymes with their substrates, experiments are usually performed on isolated simple polymers which are not representative of plant cell wall complexity. But when using natural plant substrates, the role of individual chemical and structural features affecting enzyme-binding properties is also difficult to decipher. We have designed and used lignified model assemblies of plant cell walls as templates to characterize binding properties of multi-modular cellulases. These three-dimensional assemblies are modulated in their composition using the three principal polymers found in secondary plant cell walls (cellulose, hemicellulose, and lignin). Binding properties of enzymes are obtained from the measurement of their mobility that depends on their interactions with the polymers and chemical motifs of the assemblies. The affinity of the multi-modular GH45 cellulase was characterized using a statistical analysis to determine the role played by each assembly polymer. Presence of hemicellulose had much less impact on affinity than cellulose and model lignin. Depending on the number of CBMs appended to the cellulase catalytic core, binding properties toward cellulose and lignin were highly contrasted. Model assemblies bring new insights into the molecular determinants that are responsible for interactions between enzymes and substrate without the need of complex analysis. Consequently, we believe that model bioinspired assemblies will provide relevant information for the design and optimization of enzyme cocktails in the context of biorefineries.

The microtubule-active antitumor compound TTI-237 has both paclitaxel-like and vincristine-like properties.

PubMed

Beyer, Carl F; Zhang, Nan; Hernandez, Richard; Vitale, Danielle; Nguyen, Thai; Ayral-Kaloustian, Semiramis; Gibbons, James J

2009-09-01

To compare TTI-237 (5-chloro-6-[2,6-difluoro-4-[3-(methylamino)propoxy]phenyl]-N-[(1S)-2,2,2-trifluoro-1-methylethyl]-[1, 2, 4]triazolo[1,5-a]pyrimidin-7-amine butanedioate) with paclitaxel and vincristine in order to better understand the properties of this new anti-microtubule agent. Tubulin polymerization and depolymerization were followed by turbidimetric assays. Effects of compounds on the binding of [(3)H]guanosine triphosphate ([(3)H]GTP) to tubulin were studied by competition binding assays. Effects of compounds on the phosphorylation of a panel of intracellular proteins were determined by flow cytometry using phosphoprotein-specific antibodies. At low molar ratios of TTI-237:tubulin heterodimer (about 1:30), TTI-237 enhanced depolymerization kinetics in response to low temperature, but stabilized the aggregates at higher ratios (about 1:4). Similarly, the aggregates induced in microtubule protein by TTI-237 were depolymerized by excess Ca(++) at low TTI-237:tubulin-heterodimer molar ratios, but were stable at higher ratios. TTI-237 inhibited the exchange of [(3)H]GTP at the exchangeable nucleotide site of the tubulin heterodimer, and was similar to vincristine in its effects on the phosphorylation of eight intracellular proteins in HeLa cells. TTI-237 has properties that distinguish it from typical vinca-site and taxoid-site ligands, and therefore it may exemplify a new class of microtubule-active compounds.

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma.

PubMed

Ikonomopoulou, M P; Bradley, A J; Whittier, J M; Ibrahim, K

2006-11-01

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4 degrees C with high affinity (K (a) = 1.49 +/- 0.09 x 10(9) M(-1); 0.17 +/- 0.02 x 10(7) M(-1)) and low binding capacity (B (max) = 3.24 +/- 0.84 x 10(-5) M; 0.33 +/- 0.06 x 10(-4) M). The binding affinity and capacity of testosterone at 23 and 36 degrees C, respectively were similar to those determined at 4 degrees C. However, oestradiol showed no binding activity at 36 degrees C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36 degrees C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.

Intermediate activity of midge antifreeze protein is due to a tyrosine-rich ice-binding site and atypical ice plane affinity.

PubMed

Basu, Koli; Wasserman, Samantha S; Jeronimo, Paul S; Graham, Laurie A; Davies, Peter L

2016-04-01

An antifreeze protein (AFP) from a midge (Chironomidae) was recently discovered and modelled as a tightly wound disulfide-braced solenoid with a surface-exposed rank of stacked tyrosines. New isoforms of the midge AFP have been identified from RT-PCR and are fully consistent with the model. Although they differ in the number of 10-residue coils, the row of tyrosines that form the putative ice-binding site is conserved. Recombinant midge AFP has been produced, and the properly folded form purified by ice affinity. This monomeric AFP has a distinct circular dichroism spectrum, a melting temperature between 35 and 50 °C and is fully renaturable on cooling. Mutagenesis of the middle tyrosine in the rank of seven eliminates antifreeze activity, whereas mutation of a tyrosine off this predicted ice-binding face had no such effect. This AFP has unusual properties compared to other known AFPs. First, its freezing-point depression activity is intermediate between that of the hyperactive and moderately active AFPs. As with hyperactive AFPs, when midge AFP-bound ice crystals exceed their freezing-point depression, ice grows explosively perpendicular to the c-axis. However, midge AFP does not bind to the basal plane of ice as do hyperactive AFPs, but rather to a pyramidal plane that is at a shallower angle relative to the basal plane than binding planes of moderate AFPs. These properties distinguish midge AFP from all other ice-binding proteins and the intermediate activity level fits well to the modest challenge of protecting newly emerged adult insects from late spring frosts. Nucleotide sequences of new midge AFP isoforms are available in the GenBank database under accession numbers KU094814-8. Sequences will be released after publication. © 2016 Federation of European Biochemical Societies.

Serotonergic and dopaminergic distinctions in the behavioral pharmacology of (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD).

PubMed

Schindler, Emmanuelle A D; Dave, Kuldip D; Smolock, Elaine M; Aloyo, Vincent J; Harvey, John A

2012-03-01

After decades of social stigma, hallucinogens have reappeared in the clinical literature demonstrating unique benefits in medicine. The precise behavioral pharmacology of these compounds remains unclear, however. Two commonly studied hallucinogens, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD), were investigated both in vivo and in vitro to determine the pharmacology of their behavioral effects in an animal model. Rabbits were administered DOI or LSD and observed for head bob behavior after chronic drug treatment or after pretreatment with antagonist ligands. The receptor binding characteristics of DOI and LSD were studied in vitro in frontocortical homogenates from naïve rabbits or ex vivo in animals receiving an acute drug injection. Both DOI- and LSD-elicited head bobs required serotonin(2A) (5-HT(2A)) and dopamine(1) (D(1)) receptor activation. Serotonin(2B/2C) receptors were not implicated in these behaviors. In vitro studies demonstrated that LSD and the 5-HT(2A/2C) receptor antagonist, ritanserin, bound frontocortical 5-HT(2A) receptors in a pseudo-irreversible manner. In contrast, DOI and the 5-HT(2A/2C) receptor antagonist, ketanserin, bound reversibly. These binding properties were reflected in ex vivo binding studies. The two hallucinogens also differed in that LSD showed modest D(1) receptor binding affinity whereas DOI had negligible binding affinity at this receptor. Although DOI and LSD differed in their receptor binding properties, activation of 5-HT(2A) and D(1) receptors was a common mechanism for eliciting head bob behavior. These findings implicate these two receptors in the mechanism of action of hallucinogens. Copyright © 2011 Elsevier Inc. All rights reserved.

Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles

PubMed Central

2013-01-01

The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein–protein interaction prediction and design methods. PMID:24250278

Protein Allostery and Conformational Dynamics.

PubMed

Guo, Jingjing; Zhou, Huan-Xiang

2016-06-08

The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change affects not only thermodynamic properties but also dynamic properties, including the amplitudes of motions on different time scales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases are highlighted where NMR spectroscopy and molecular dynamics simulation have been used as complementary approaches to identify residues possibly involved in allosteric communication. Perspectives on contentious issues, for example, the relationship between picosecond-nanosecond local and microsecond-millisecond conformational exchange dynamics, are presented.

Spectroscopic investigation of the effect of salt on binding of tartrazine with two homologous serum albumins: quantification by use of the Debye-Hückel limiting law and observation of enthalpy-entropy compensation.

PubMed

Bolel, Priyanka; Datta, Shubhashis; Mahapatra, Niharendu; Halder, Mintu

2012-08-30

Formation of ion pair between charged molecule and protein can lead to interesting biochemical phenomena. We report the evolution of thermodynamics of the binding of tartrazine, a negatively charged azo colorant, and serum albumins with salt. The dye binds predominantly electrostatically in low buffer strengths; however, on increasing salt concentration, affinity decreases considerably. The calculated thermodynamic parameters in high salt indicate manifestation of nonelectrostatic interactions, namely, van der Waals force and hydrogen bonding. Site-marker competitive binding studies and docking simulations indicate that the dye binds with HSA in the warfarin site and with BSA at the interface of warfarin and ibuprofen binding sites. The docked poses indicate nearby amino acid positive side chains, which are possibly responsible for electrostatic interaction. Using the Debye-Hückel interionic attraction theory for binding equilibria, it is shown that, for electrostatic binding the calculated free energy change increases linearly with square root of ionic strength. Also UV-vis, fluorescence, CD data indicate a decrease of interaction with salt concentration. This study quantitatively relates how ionic strength modulates the strength of the protein-ligand electrostatic interaction. The binding enthalpy and entropy have been found to compensate one another. The enthalpy-entropy compensation (EEC), general property of weak intermolecular interactions, has been discussed.

Further Insights into Metal-DOM Interaction: Consideration of Both Fluorescent and Non-Fluorescent Substances

PubMed Central

Xu, Huacheng; Zhong, Jicheng; Yu, Guanghui; Wu, Jun; Jiang, Helong; Yang, Liuyan

2014-01-01

Information on metal binding with fluorescent substances has been widely studied. By contrast, information on metal binding with non-fluorescent substances remains lacking despite the dominance of these substances in aquatic systems. In this study, the metal binding properties of both fluorescent and non-fluorescent substances were investigated by using metal titration combined with two-dimensional correlation spectroscopy (2D–COS) analysis. The organic matters in the eutrophic algae-rich lake, including natural organic matters (NOM) and algae-induced extracellular polymeric substances (EPS), both contained fluorescent and non-fluorescent substances. The peaks in the one-dimensional spectra strongly overlapped, while 2D–COS can decompose the overlapped peaks and thus enhanced the spectral resolution. Moreover, 2D FTIR COS demonstrated that the binding susceptibility of organic ligands in both NOM and algal EPS matrices followed the order: 3400>1380>1650 cm−1, indicative the significant contribution of non-fluorescent ligands in metal binding. The modified Stern-Volmer equation also revealed a substantial metal binding potential for the non-fluorescent substances (logKM: 3.57∼4.92). As for the effects of organic ligands on metal binding, EPS was characterized with higher binding ability than NOM for both fluorescent and non-fluorescent ligands. Algae-induced EPS and the non-fluorescent substances in eutrophic algae-rich lakes should not be overlooked because of their high metal binding potential. PMID:25380246

Glycosylation of Cblns attenuates their receptor binding.

PubMed

Rong, Yongqi; Bansal, Parmil K; Wei, Peng; Guo, Hong; Correia, Kristen; Parris, Jennifer; Morgan, James I

2018-05-18

Cbln1 is the prototype of a family (Cbln1-Cbln4) of secreted glycoproteins and is essential for normal synapse structure and function in cerebellum by bridging presynaptic Nrxn to postsynaptic Grid2. Here we report the effects of glycosylation on the in vitro receptor binding properties of Cblns. Cbln1, 2 and 4 harbor two N-linked glycosylation sites, one at the N-terminus is in a region implicated in Nrxn binding and the second is in the C1q domain, a region involved in Grid2 binding. Mutation (asparagine to glutamine) of the N-terminal site, increased neurexin binding whereas mutation of the C1q site markedly increased Grid2 binding. These mutations did not influence subunit composition of Cbln trimeric complexes (mediated through the C1q domain) nor their assembly into hexamers (mediated by the N-terminal region). Therefore, glycosylation likely masks the receptor binding interfaces of Cblns. As Cbln4 has undetectable Grid2 binding in vitro we assessed whether transgenic expression of wild type Cbln4 or its glycosylation mutants rescued the Cbln1-null phenotype in vivo. Cbln4 partially rescued and both glycosylation mutants completely rescued ataxia in cbln1-null mice. Thus Cbln4 has intrinsic Grid2 binding that is attenuated by glycosylation, and glycosylation mutants exhibit gain of function in vivo. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Elucidation of Lipid Binding Sites on Lung Surfactant Protein A Using X-ray Crystallography, Mutagenesis, and Molecular Dynamics Simulations.

PubMed

Goh, Boon Chong; Wu, Huixing; Rynkiewicz, Michael J; Schulten, Klaus; Seaton, Barbara A; McCormack, Francis X

2016-07-05

Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions.

Probing the Interaction between Cyclic ADTC1 Ac-CADTPPVC-NH2) Peptide with EC1-EC2 domain of E-cadherin using Molecular Docking Approach

NASA Astrophysics Data System (ADS)

Siahaan, P.; Wuning, S.; Manna, A.; Prasasty, V. D.; Hudiyanti, D.

2018-04-01

Deeply understanding that intermolecular interaction between molecules on the paracellular pathway has given insight to its microscopic and macroscopic properties. In the paracellular pathway, synthetic cyclic ADTC1 (Ac-CADTPPVC-NH2) peptide has been studied to modulate EC1-EC2 domain, computationally using molecular docking method. The aim of this research is to probe the effect of amino acid alanine (A) of ADTC1 on its interaction properties. The study carried out in two steps: 1. the optimization using GROMACS v4.6.5 program and; 2. Determination of the interaction properties using AutoDock 4.2 program. The interaction was done for A-J box, and the best position of the binding site and binding energy on the OC and CC ADTC1 peptides against the EC1-EC2 domain of E-cadherin was selected. The result showed that the CC of the F box ADTC1 has the best interaction with binding energy of - 26.36 kJ/mol and its energy was lower than ADTC5 without alanine amino acid. ADTC1 interacted with EC1 of EC1-EC2 on Asp1, Trp2, Val3, Ile4, Ile24, Lys25, Ser26, Asn27, and Met92 residues.

Size-dependent impact of CNTs on dynamic properties of calmodulin

NASA Astrophysics Data System (ADS)

Gao, Jian; Wang, Liming; Kang, Seung-Gu; Zhao, Lina; Ji, Mingjuan; Chen, Chunying; Zhao, Yuliang; Zhou, Ruhong; Li, Jingyuan

2014-10-01

There are growing concerns about the biosafety of nanomaterials such as carbon nanotubes (CNTs) as their applications become more widespread. We report here a theoretical and experimental study of the binding of various sizes of CNTs [CNT (4,4), (5,5), (6,6) and (7,7)] to calmodulin (CaM) protein and, in particular, their impact on the Ca2+-dependent dynamic properties of CaM. Our simulations show that all the CNTs can plug into the hydrophobic binding pocket of Ca2+-bound CaM with binding affinities comparable with the native substrate M13 peptide. Even though CNT (4,4) shows a similar behavior to the M13 peptide in its dissociation from Ca2+-free CaM, wider CNTs still bind firmly to CaM, indicating a potential failure of Ca2+ regulation. Such a size-dependent impact of CNTs on the dynamic properties of CaM is a result of the excessively strong hydrophobic interactions between the wider CNTs and CaM. These simulation results were confirmed by circular dichroism spectroscopy, which showed that the secondary structures of CaM become insensitive to Ca2+ concentrations after the addition of CNTs. Our findings indicate that the cytotoxicity of nanoparticles to proteins arises not only from the inhibition of static protein structures (binding pockets), but also from impacts on their dynamic properties.There are growing concerns about the biosafety of nanomaterials such as carbon nanotubes (CNTs) as their applications become more widespread. We report here a theoretical and experimental study of the binding of various sizes of CNTs [CNT (4,4), (5,5), (6,6) and (7,7)] to calmodulin (CaM) protein and, in particular, their impact on the Ca2+-dependent dynamic properties of CaM. Our simulations show that all the CNTs can plug into the hydrophobic binding pocket of Ca2+-bound CaM with binding affinities comparable with the native substrate M13 peptide. Even though CNT (4,4) shows a similar behavior to the M13 peptide in its dissociation from Ca2+-free CaM, wider CNTs still bind firmly to CaM, indicating a potential failure of Ca2+ regulation. Such a size-dependent impact of CNTs on the dynamic properties of CaM is a result of the excessively strong hydrophobic interactions between the wider CNTs and CaM. These simulation results were confirmed by circular dichroism spectroscopy, which showed that the secondary structures of CaM become insensitive to Ca2+ concentrations after the addition of CNTs. Our findings indicate that the cytotoxicity of nanoparticles to proteins arises not only from the inhibition of static protein structures (binding pockets), but also from impacts on their dynamic properties. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01623h

Binding properties of SUMO-interacting motifs (SIMs) in yeast.

PubMed

Jardin, Christophe; Horn, Anselm H C; Sticht, Heinrich

2015-03-01

Small ubiquitin-like modifier (SUMO) conjugation and interaction play an essential role in many cellular processes. A large number of yeast proteins is known to interact non-covalently with SUMO via short SUMO-interacting motifs (SIMs), but the structural details of this interaction are yet poorly characterized. In the present work, sequence analysis of a large dataset of 148 yeast SIMs revealed the existence of a hydrophobic core binding motif and a preference for acidic residues either within or adjacent to the core motif. Thus the sequence properties of yeast SIMs are highly similar to those described for human. Molecular dynamics simulations were performed to investigate the binding preferences for four representative SIM peptides differing in the number and distribution of acidic residues. Furthermore, the relative stability of two previously observed alternative binding orientations (parallel, antiparallel) was assessed. For all SIMs investigated, the antiparallel binding mode remained stable in the simulations and the SIMs were tightly bound via their hydrophobic core residues supplemented by polar interactions of the acidic residues. In contrary, the stability of the parallel binding mode is more dependent on the sequence features of the SIM motif like the number and position of acidic residues or the presence of additional adjacent interaction motifs. This information should be helpful to enhance the prediction of SIMs and their binding properties in different organisms to facilitate the reconstruction of the SUMO interactome.

Excitonic effects and related properties in semiconductor nanostructures: roles of size and dimensionality

NASA Astrophysics Data System (ADS)

Wu, Shudong; Cheng, Liwen; Wang, Qiang

2017-08-01

The size- and dimensionality-dependence of excitonic effects and related properties in semiconductor nanostructures are theoretically studied in detail within the effective-mass approximation. When nanostructure sizes become smaller than the bulk exciton Bohr radius, excitonic effects are significantly enhanced with reducing size or dimensionality. This is as a result of quantum confinement in more directions leading to larger exciton binding energies and normalized exciton oscillator strengths. These excitonic effects originate from electron-hole Coulombic interactions, which strongly enhance the oscillator strength between the electron and hole. It is also established that the universal scaling of exciton binding energy versus the inverse of the exciton Bohr radius follows a linear scaling law. Herein, we propose a stretched exponential law for the size scaling of optical gap, which is in good agreement with the calculated data. Due to differences in the confinement dimensionality, the radiative lifetime of low-dimensional excitons becomes shorter than that of bulk excitons. The size dependence of the exciton radiative lifetimes is in good agreement with available experimental data. This strongly enhanced electron-hole exchange interaction is expected in low-dimensional structures due to enriched excitonic effects. The main difference in nanostructures compared to the bulk can be interpreted in terms of the enhanced excitonic effects induced by exciton localization. The enhanced excitonic effects are expected to be of importance in developing stable and high-efficiency nanoscale excitonic optoelectronic devices.

The influence of silanisation on the mechanical and degradation behaviour of PLGA/HA composites.

PubMed

Naik, Ashutosh; Best, Serena M; Cameron, Ruth E

2015-03-01

This study investigates the influence of silanisation on the mechanical and degradation behaviour of PLGA/HA composites. Three different silanes (mercaptopropyl trimethoxy silane (MPTMS), aminopropyl trimethoxy silane (APTMS) and aminopropyltriethoxy silane (APTES)) were applied to HA substrates in order to study the effect of head group (which binds to the polymer) and tail group (which binds to the surface hydroxyl groups in HA). A composite of hydroxyapatite (HA) and poly(d,l lactide-co-glycolide (50:50)) (PLGA) was investigated. The influence of concentration, the reaction time, drying temperature and substrate surface on silanisation was examined. TGA was used to detect the degree of silanisation. HA with MPTMS (1wt.% MPTMS with reaction time of 1h) was used as filler in PLGA-30wt.% HA composites for an in-vitro degradation study carried out in PBS. In addition, the mechanical properties of the composites were studied. Silanisation affects the properties of the composite by improving the bonding at the interface and hence it was found to influence the plastic mechanical properties rather than the elastic mechanical properties or the degradation profile of the composite. Copyright © 2014. Published by Elsevier B.V.

The N54-αs Mutant Has Decreased Affinity for βγ and Suggests a Mechanism for Coupling Heterotrimeric G Protein Nucleotide Exchange with Subunit Dissociation.

PubMed

Cleator, John H; Wells, Christopher A; Dingus, Jane; Kurtz, David T; Hildebrandt, John D

2018-05-01

Ser54 of G s α binds guanine nucleotide and Mg 2+ as part of a conserved sequence motif in GTP binding proteins. Mutating the homologous residue in small and heterotrimeric G proteins generates dominant-negative proteins, but by protein-specific mechanisms. For α i/o , this results from persistent binding of α to βγ , whereas for small GTP binding proteins and α s this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of βγ interactions in mediating the properties of the Ser54-like mutants of G α subunits. Unexpectedly, WT- α s or N54- α s coexpressed with α 1B -adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT- α s was more effective than the mutant. One explanation for this result would be that α s , like Ser47 α i/o , blocks receptor activation by sequestering βγ ; implying that N54- α S has reduced affinity for βγ since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT- α s was more effective than the mutant in inhibiting βγ activation of phospholipase C β 2. Further, in vitro synthesized N54- α s bound biotinylated- βγ with lower apparent affinity than did WT- α s The Cys54 mutation also decreased βγ binding but less effectively than N54- α s Substitution of the conserved Ser in α o with Cys or Asn increased βγ binding, with the Cys mutant being more effective. This suggests that Ser54 of α s is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Supported Lipid Bilayer/Carbon Nanotube Hybrids

NASA Astrophysics Data System (ADS)

Zhou, Xinjian; Moran-Mirabal, Jose; Craighead, Harold; McEuen, Paul

2007-03-01

We form supported lipid bilayers on single-walled carbon nanotubes and use this hybrid structure to probe the properties of lipid membranes and their functional constituents. We first demonstrate membrane continuity and lipid diffusion over the nanotube. A membrane-bound tetanus toxin protein, on the other hand, sees the nanotube as a diffusion barrier whose strength depends on the diameter of the nanotube. Finally, we present results on the electrical detection of specific binding of streptavidin to biotinylated lipids with nanotube field effect transistors. Possible techniques to extract dynamic information about the protein binding events will also be discussed.

DFTB+ and lanthanides

NASA Astrophysics Data System (ADS)

Hourahine, B.; Aradi, B.; Frauenheim, T.

2010-07-01

DFTB+ is a recent general purpose implementation of density-functional based tight binding. One of the early motivators to develop this code was to investigate lanthanide impurities in nitride semiconductors, leading to a series of successful studies into structure and electrical properties of these systems. Here we describe our general framework to treat the physical effects needed for these problematic impurities within a tight-binding formalism, additionally discussing forces and stresses in DFTB. We also present an approach to evaluate the general case of Slater-Koster transforms and all of their derivatives in Cartesian coordinates. These developments are illustrated by simulating isolated Gd impurities in GaN.

Insights into an original pocket-ligand pair classification: a promising tool for ligand profile prediction.

PubMed

Pérot, Stéphanie; Regad, Leslie; Reynès, Christelle; Spérandio, Olivier; Miteva, Maria A; Villoutreix, Bruno O; Camproux, Anne-Claude

2013-01-01

Pockets are today at the cornerstones of modern drug discovery projects and at the crossroad of several research fields, from structural biology to mathematical modeling. Being able to predict if a small molecule could bind to one or more protein targets or if a protein could bind to some given ligands is very useful for drug discovery endeavors, anticipation of binding to off- and anti-targets. To date, several studies explore such questions from chemogenomic approach to reverse docking methods. Most of these studies have been performed either from the viewpoint of ligands or targets. However it seems valuable to use information from both ligands and target binding pockets. Hence, we present a multivariate approach relating ligand properties with protein pocket properties from the analysis of known ligand-protein interactions. We explored and optimized the pocket-ligand pair space by combining pocket and ligand descriptors using Principal Component Analysis and developed a classification engine on this paired space, revealing five main clusters of pocket-ligand pairs sharing specific and similar structural or physico-chemical properties. These pocket-ligand pair clusters highlight correspondences between pocket and ligand topological and physico-chemical properties and capture relevant information with respect to protein-ligand interactions. Based on these pocket-ligand correspondences, a protocol of prediction of clusters sharing similarity in terms of recognition characteristics is developed for a given pocket-ligand complex and gives high performances. It is then extended to cluster prediction for a given pocket in order to acquire knowledge about its expected ligand profile or to cluster prediction for a given ligand in order to acquire knowledge about its expected pocket profile. This prediction approach shows promising results and could contribute to predict some ligand properties critical for binding to a given pocket, and conversely, some key pocket properties for ligand binding.

Insights into an Original Pocket-Ligand Pair Classification: A Promising Tool for Ligand Profile Prediction

PubMed Central

Reynès, Christelle; Spérandio, Olivier; Miteva, Maria A.; Villoutreix, Bruno O.; Camproux, Anne-Claude

2013-01-01

Pockets are today at the cornerstones of modern drug discovery projects and at the crossroad of several research fields, from structural biology to mathematical modeling. Being able to predict if a small molecule could bind to one or more protein targets or if a protein could bind to some given ligands is very useful for drug discovery endeavors, anticipation of binding to off- and anti-targets. To date, several studies explore such questions from chemogenomic approach to reverse docking methods. Most of these studies have been performed either from the viewpoint of ligands or targets. However it seems valuable to use information from both ligands and target binding pockets. Hence, we present a multivariate approach relating ligand properties with protein pocket properties from the analysis of known ligand-protein interactions. We explored and optimized the pocket-ligand pair space by combining pocket and ligand descriptors using Principal Component Analysis and developed a classification engine on this paired space, revealing five main clusters of pocket-ligand pairs sharing specific and similar structural or physico-chemical properties. These pocket-ligand pair clusters highlight correspondences between pocket and ligand topological and physico-chemical properties and capture relevant information with respect to protein-ligand interactions. Based on these pocket-ligand correspondences, a protocol of prediction of clusters sharing similarity in terms of recognition characteristics is developed for a given pocket-ligand complex and gives high performances. It is then extended to cluster prediction for a given pocket in order to acquire knowledge about its expected ligand profile or to cluster prediction for a given ligand in order to acquire knowledge about its expected pocket profile. This prediction approach shows promising results and could contribute to predict some ligand properties critical for binding to a given pocket, and conversely, some key pocket properties for ligand binding. PMID:23840299

From selective to highly selective SSRIs: a comparison of the antinociceptive properties of fluoxetine, fluvoxamine, citalopram and escitalopram.

PubMed

Schreiber, Shaul; Pick, Chaim G

2006-08-01

Most Serotonin Selective Reuptake Inhibitors (SSRIs) have been found to possess secondary binding properties, while citalopram and its S-enantiomer (escitalopram) have been reconfirmed "purest SSRIs". Using the mouse model of acute pain hotplate analgesia meter, we evaluated the antinociceptive properties of fluoxetine, fluvoxamine, citalopram and escitalopram, injected i.p. Fluvoxamine induced a dose-dependent clear antinociceptive effect (with an ED(50) value of 6.4 mg/kg). Both fluoxetine and citalopram induced (separately) only a weak antinociceptive effect with an inverse "U" shape curve. All three drug's effects were not abolished by naloxone. Escitalopram did not elicit any effect at quasi-equipotent doses. These findings show that fluoxetine, fluvoxamine and citalopram given i.p. are weak antinociceptors, (not mediated through opioid mechanisms), while escitalopram possesses no antinociceptive properties when injected i.p. This difference between citalopram and escitalopram calls for further studies in order to assess the various differences between the two enantiomers of citalopram, and between each enantiomer and the racemic mixture.

Effect of micronization on the physicochemical properties of insoluble dietary fiber from citrus (Citrus junos Sieb. ex Tanaka) pomace.

PubMed

Ye, Fayin; Tao, Bingbing; Liu, Jia; Zou, Yan; Zhao, Guohua

2016-04-01

The aim of this work was to study the effect of micronization (mechanical and jet grindings) on the physicochemical properties of the insoluble dietary fiber from citrus pomace in comparison with ordinary grinding. The results showed that micronization treatment effectively pulverized the IDF-CP powders to micron scale and significantly increased the soluble dietary fiber content (p < 0.05). Compared with mechanical grinding, jet grinding was more effective in size reduction and resulted in IDF-CP powders with narrower particle size distributions. Micronized IDF-CP powders had smaller particle size, smoother surface, higher fluidity, cation-exchange capacity, and metal cation binding capacity values, but lower water holding capacity, oil holding capacity, and swelling capacity values. These functional properties were significantly dependent on surface area and particle size (D0.5). The present study suggested that micronization treatments could modify functional properties of IDF-CP powders, which promotes their use in food applications. © The Author(s) 2015.

Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins.

PubMed

Sosic, Alice; Saccone, Irene; Carraro, Caterina; Kenderdine, Thomas; Gamba, Elia; Caliendo, Giuseppe; Corvino, Angela; Di Vaio, Paola; Fiorino, Ferdinando; Magli, Elisa; Perissutti, Elisa; Santagada, Vincenzo; Severino, Beatrice; Spada, Valentina; Fabris, Dan; Frecentese, Francesco; Gatto, Barbara

2018-06-12

The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of anti-retrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins.

A novel β-carotene-associated carrot (Daucus carota L.) pectic polysaccharide.

PubMed

Natarajmurthy, Sindhuja H; Askari, Marjan; Pullabhatla, Srinivas; Dharmesh, Shylaja M

2016-01-01

Carrot (Daucus carota L.) is a nutritional source with enriched β-carotene (0.01%) and pectins (0.8%). Although studies have highlighted the association between β-carotene and carrot pectic polysaccharide (CRPP), the precise binding of β-carotene to CRPP and its biological implications were not yet clearly understood. The aim of the present study is to report for the first time a natural carbohydrate polymer-CRPP-with associated β-carotene. The presence of β-carotene in CRPP and its binding is demonstrated by various studies including differential extraction followed by liquid chromatography (LC), liquid chromatography mass spectrometry (LC MS), and Fourier transform infrared spectroscopy (FTIR). Further, we show the binding via its release from CRPP using LC, fermentation studies and scanning electron microscopy (SEM) studies. The role of β-carotene in induction of apoptosis and the galactic-3 inhibitory property was also studied. The presence of β-carotene in CRPP was demonstrated, as well as an association between β-carotene and CRPP, apoptotic and galectin-3 inhibitory properties of CRPP, and upregulation of connexin 43 by CRPP. The data highlights that with the combination of β-carotene with CRPP, the prooxidant effect of β-carotene may be reduced, an apoptotic effect may be enhanced, and the anticancer potential of CRPP thus may be strengthened via galectin-3 inhibition. Copyright © 2016 Elsevier Inc. All rights reserved.

PatchSurfers: Two methods for local molecular property-based binding ligand prediction.

PubMed

Shin, Woong-Hee; Bures, Mark Gregory; Kihara, Daisuke

2016-01-15

Protein function prediction is an active area of research in computational biology. Function prediction can help biologists make hypotheses for characterization of genes and help interpret biological assays, and thus is a productive area for collaboration between experimental and computational biologists. Among various function prediction methods, predicting binding ligand molecules for a target protein is an important class because ligand binding events for a protein are usually closely intertwined with the proteins' biological function, and also because predicted binding ligands can often be directly tested by biochemical assays. Binding ligand prediction methods can be classified into two types: those which are based on protein-protein (or pocket-pocket) comparison, and those that compare a target pocket directly to ligands. Recently, our group proposed two computational binding ligand prediction methods, Patch-Surfer, which is a pocket-pocket comparison method, and PL-PatchSurfer, which compares a pocket to ligand molecules. The two programs apply surface patch-based descriptions to calculate similarity or complementarity between molecules. A surface patch is characterized by physicochemical properties such as shape, hydrophobicity, and electrostatic potentials. These properties on the surface are represented using three-dimensional Zernike descriptors (3DZD), which are based on a series expansion of a 3 dimensional function. Utilizing 3DZD for describing the physicochemical properties has two main advantages: (1) rotational invariance and (2) fast comparison. Here, we introduce Patch-Surfer and PL-PatchSurfer with an emphasis on PL-PatchSurfer, which is more recently developed. Illustrative examples of PL-PatchSurfer performance on binding ligand prediction as well as virtual drug screening are also provided. Copyright © 2015 Elsevier Inc. All rights reserved.

Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity.

PubMed

Korkuć, Paula; Walther, Dirk

2015-01-01

To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous system) suggesting specific molecular and physiological roles of promiscuous metabolites.

Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity

PubMed Central

Korkuć, Paula; Walther, Dirk

2015-01-01

To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous system) suggesting specific molecular and physiological roles of promiscuous metabolites. PMID:26442281

Thermoelectric properties of a trilayer graphene nanoribbon

NASA Astrophysics Data System (ADS)

Orellana, Pedro; Cortes, Natalia; Rosales, Luis; Pacheco, Monica; Chico, Leonor

2015-03-01

In this work the electronic and thermoelectric properties of a three-layer graphene with AAA stacking type are studied. By using a tight-binding model analytical expressions for the transmission and density of states are obtained. Thermoelectric properties are analyzed by numerical integration and results for thermopower and figure of merit, electronic conductance and thermal conductance are obtained. The results show that the interference effects present in this system, like Fano effect, directly affect the behavior of these thermoelectric properties and as well as the Wiedemann-Franz law. There is an enhancement of the thermopower of the system and a violation of the Wiedemann-Franz law in the region of energies close the Fano antiresonances and this has as a consequence an enhancement of the figure of merit of the system. FONDECYT 1140571, 1140388, CONICYT ACT 1204, DGIP/ USM internal Grant 11.14.68.

Positive selection in octopus haemocyanin indicates functional links to temperature adaptation.

PubMed

Oellermann, Michael; Strugnell, Jan M; Lieb, Bernhard; Mark, Felix C

2015-07-05

Octopods have successfully colonised the world's oceans from the tropics to the poles. Yet, successful persistence in these habitats has required adaptations of their advanced physiological apparatus to compensate impaired oxygen supply. Their oxygen transporter haemocyanin plays a major role in cold tolerance and accordingly has undergone functional modifications to sustain oxygen release at sub-zero temperatures. However, it remains unknown how molecular properties evolved to explain the observed functional adaptations. We thus aimed to assess whether natural selection affected molecular and structural properties of haemocyanin that explains temperature adaptation in octopods. Analysis of 239 partial sequences of the haemocyanin functional units (FU) f and g of 28 octopod species of polar, temperate, subtropical and tropical origin revealed natural selection was acting primarily on charge properties of surface residues. Polar octopods contained haemocyanins with higher net surface charge due to decreased glutamic acid content and higher numbers of basic amino acids. Within the analysed partial sequences, positive selection was present at site 2545, positioned between the active copper binding centre and the FU g surface. At this site, methionine was the dominant amino acid in polar octopods and leucine was dominant in tropical octopods. Sites directly involved in oxygen binding or quaternary interactions were highly conserved within the analysed sequence. This study has provided the first insight into molecular and structural mechanisms that have enabled octopods to sustain oxygen supply from polar to tropical conditions. Our findings imply modulation of oxygen binding via charge-charge interaction at the protein surface, which stabilize quaternary interactions among functional units to reduce detrimental effects of high pH on venous oxygen release. Of the observed partial haemocyanin sequence, residue 2545 formed a close link between the FU g surface and the active centre, suggesting a role as allosteric binding site. The prevalence of methionine at this site in polar octopods, implies regulation of oxygen affinity via increased sensitivity to allosteric metal binding. High sequence conservation of sites directly involved in oxygen binding indicates that functional modifications of octopod haemocyanin rather occur via more subtle mechanisms, as observed in this study.

Phosphorylcholine Allows for Evasion of Bactericidal Antibody by Haemophilus influenzae

PubMed Central

Clark, Sarah E.; Snow, Julian; Li, Jianjun; Zola, Tracey A.; Weiser, Jeffrey N.

2012-01-01

The human pathogen Haemophilus influenzae has the ability to quickly adapt to different host environments through phase variation of multiple structures on its lipooligosaccharide (LPS), including phosphorylcholine (ChoP). During colonization with H. influenzae, there is a selection for ChoP+ phase variants. In a murine model of nasopharyngeal colonization, this selection is lost in the absence of adaptive immunity. Based on previous data highlighting the importance of natural antibody in limiting H. influenzae colonization, the effect of ChoP expression on antibody binding and its bactericidal activity was investigated. Flow cytometric analysis revealed that ChoP+ phase variants had decreased binding of antibody to LPS epitopes compared to ChoP− phase variants. This difference in antibody binding correlated with increased survival of ChoP+ phase variants in the presence of antibody-dependent, complement-mediated killing. ChoP+ phase variants were also more resistant to trypsin digestion, suggesting a general effect on the physical properties of the outer membrane. Moreover, ChoP-mediated protection against antibody binding correlated with increased resilience of outer membrane integrity. Collectively, these data suggest that ChoP expression provides a selective advantage during colonization through ChoP-mediated effects on the accessibility of bactericidal antibody to the cell surface. PMID:22396641

Photophysical, photochemical and BSA binding/BQ quenching properties of quaternizable coumarin containing water soluble zinc phthalocyanine complexes

NASA Astrophysics Data System (ADS)

Esenpınar, Aliye Aslı; Durmuş, Mahmut; Bulut, Mustafa

2011-08-01

The non-peripherally ( np-QZnPc) and peripherally ( p-QZnPc) tetrakis-[7-oxo-(3-[(2-diethylaminomethyliodide)ethyl)]-4-methylcoumarin]-phthalocyaninatozinc complexes have been prepared by quaternization of non-peripherally and peripherally tetrakis[7-oxo-(3-[(2-diethylamino)ethyl)]-methylcoumarin] phthalocyaninato zinc complexes with methyliodide in dimethylsulfoxide (DMSO). The new quaternized zinc phthalocyanine complex ( np-QZnPc) has been characterized by elementel analysis, MALDI-TOF, IR and UV-vis spectral data. The photophysical and photochemical properties of the peripherally and non-peripherally quaternized tetrakis-3-[(2-diethylamino)ethyl]-7-oxo-4-methylcoumarin substituted zinc phthalocyanines are reported. The effects of the position of the substituents and the aggregation of the phthalocyanine molecules on the photophysical and photochemical properties are also investigated. General trends are described for photodegradation, singlet oxygen and fluorescence quantum yields, and fluorescence lifetimes for complexes np-ZnPc/ p-ZnPc in DMSO and for complexes np-QZnPc/p-QZnPc in DMSO, phosphate buffered solution (PBS) and PBS+Triton-X 100 solutions. The fluorescence of the tetra-substituted quaternized zinc phthalocyanine complexes ( np-QZnPc/ p-QZnPc) are effectively quenched addition of 1,4-benzoquinone (BQ) and this study also presented the ionic zinc phthalocyanine complexes strongly bind to bovine serum albumin (BSA).

Comparative in silico analyses of Cannabis sativa, Prunella vulgaris and Withania somnifera compounds elucidating the medicinal properties against rheumatoid arthritis.

PubMed

Zaka, Mehreen; Sehgal, Sheikh Arslan; Shafique, Shagufta; Abbasi, Bilal Haider

2017-06-01

From last decade, there has been progressive improvement in computational drug designing. Several diseases are being cured from different plant extracts and products. Rheumatoid Arthritis (RA) is the most shared disease among auto-inflammatory diseases. Tumour necrosis factor (TNF)-α is associated with RA pathway and has adverse effects. Extensive literature review showed that plant species under study (Cannabis sativa, Prunella vulgaris and Withania somnifera) possess anti-inflammatory, anti-arthritic and anti-rheumatic properties. 13 anti-inflammatory compounds were characterised and filtered out from medicinal plant species and analysed for RA by targeting TNF-α through in silico analyses. By using ligand based pharmacophore generation approach and virtual screening against natural products libraries we retrieved twenty unique molecules that displayed utmost binding affinity, least binding energies and effective drug properties. The docking analyses revealed that Ala-22, Glu-23, Ser-65, Gln-67, Tyr-141, Leu-142, Asp-143, Phe-144 and Ala-145 were critical interacting residues for receptor-ligand interactions. It is proposed that the RA patients should use reported compounds for the prescription of RA by targeting TNF-α. This report is opening new dimensions for designing innovative therapeutic targets to cure RA. Copyright © 2017 Elsevier Inc. All rights reserved.

Flexible strategy for immobilizing redox-active compounds using in situ generation of diazonium salts. Investigations of the blocking and catalytic properties of the layers.

PubMed

Noël, Jean-Marc; Sjöberg, Béatrice; Marsac, Rémi; Zigah, Dodzi; Bergamini, Jean-François; Wang, Aifang; Rigaut, Stéphane; Hapiot, Philippe; Lagrost, Corinne

2009-11-03

A versatile two-step method is developed to covalently immobilize redox-active molecules onto carbon surfaces. First, a robust anchoring platform is grafted onto surfaces by electrochemical reduction of aryl diazonium salts in situ generated. Depending on the nature of the layer termini, -COOH or -NH(2), a further chemical coupling involving ferrocenemethylamine or ferrocene carboxylic acid derivatives leads to the covalent binding of ferrocene centers. The chemical strategy using acyl chloride activation is efficient and flexible, since it can be applied either to surface-reactive end groups or to reactive species in solution. Cyclic voltammetry analyses point to the covalent binding of ferrocene units restricted to the upper layers of the underlying aryl films, while AFM measurements show a lost of compactness of the layers after the chemical attachment of ferrocene centers. The preparation conditions of the anchoring layers were found to determine the interfacial properties of the resulted ferrocenyl-modified electrodes. The ferrocene units promoted effective redox mediation providing that the free redox probes are adequately chosen (i.e., vs size/formal potential) and the underlying layers exhibit strong blocking properties. For anchoring films with weaker blocking effect, the coexistence of two distinct phenomena, redox mediation and ET at pinholes could be evidenced.

Alteration of human serum albumin binding properties induced by modifications: A review

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, Małgorzata; Szkudlarek, Agnieszka; Chudzik, Mariola; Pożycka, Jadwiga; Sułkowska, Anna

2018-01-01

Albumin, a major transporting protein in the blood, is the main target of modification that affects the binding of drugs to Sudlow's site I and II. These modification of serum protein moderates its physiological function, and works as a biomarker of some diseases. The main goal of the paper was to explain the possible alteration of human serum albumin binding properties induced by modifications such as glycation, oxidation and ageing, their origin, methods of evaluation and positive and negative meaning described by significant researchers.

Electro-optical properties of zigzag and armchair boron nitride nanotubes under a transverse electric field: Tight binding calculations

NASA Astrophysics Data System (ADS)

Chegel, Raad; Behzad, Somayeh

2012-02-01

The electro-optical properties of zigzag and armchair BNNTs in a uniform transverse electric field are investigated within tight binding approximation. It is found that the electric field modifies the band structure and splits band degeneracy where these effects reflect in the DOS and JDOS spectra. A decrease in the band gap, as a function of the electric field, is observed. This gap reduction increases with the diameter and it is independent of chirality. An analytic function to estimate the electric field needed for band gap closing is proposed which is in good agreement with DFT results. In additional, we show that the larger diameter tubes are more sensitive than small ones. Number and position of peaks in DOS and JDOS spectra for armchair and zigzag tubes with similar radius are dependent on electric field strength.

Warping Armchair Graphene Nanoribbon Curvature Effect on Sensing Properties: A Computational Study

NASA Astrophysics Data System (ADS)

Sakina, S. H.; Johari, Zaharah; Auzar, Zuriana; Alias, N. Ezaila; Mohamad, Azam; Zakaria, N. Aini

2018-02-01

The aim of this paper is to investigate the interaction between gas molecules and warped armchair graphene nanoribbons (AGNRs) using Extended-Huckel Theory. There are two types of warping known as inward and upward. The sensing properties including binding energy, charge transfer and sensitivity were examined for both warped AGNR cases for 3m+1 configuration and were compared with previous work. Through simulation, it was found that a substantial increase in binding energy by more than 50% was achieved when warped at a higher angle. It is also showed that there was a significant difference in sensitivity for both warping cases when reacting with O2 and NH3 molecules. Interestingly, the ability of the inward warped in sensing O2 and NH3 considerably increases upon warping angle. By applying back gate bias, this shows that current conductivity of the inward warped is twice as high as the upward warped AGNR.

Engineered Antibodies for Monitoring of Polynuclear Aromatic Hydrocarbons

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

Alexander E. Karu Ph.D; Victoria A. Roberts Ph.D.; Qing X. Li, Ph.D.

2002-01-17

This project was undertaken to fill needs in ODE's human and ecosystem health effects research, site remediation, rapid emergency response, and regulatory compliance monitoring programs. Doe has greatly stimulated development and validation of antibody-based, rapid, field-portable detection systems for small hazardous compounds. These range from simple dipsticks, microplate enzyme-linked immunosorbent assays (ELISAs), and hand-held colorimeters, to ultrasensitive microfluidic reactors, fiber-optic sensors and microarrays that can identify multiple analytes from patterns of cross-reactivity. Unfortunately, the technology to produce antibodies with the most desirable properties did not keep pace. Lack of antibodies remains a limiting factor in production and practical use ofmore » such devices. The goals of our project were to determine the chemical and structural bases for the antibody-analyte binding interactions using advanced computational chemistry, and to use this information to create useful new binding properties through in vitro genetic engineering and combinatorial library methods.« less

Performance evaluation of phage-displayed synthetic human single-domain antibody libraries: A retrospective analysis.

PubMed

Henry, Kevin A; Tanha, Jamshid

2018-05-01

Fully human synthetic single-domain antibodies (sdAbs) are desirable therapeutic molecules but their development is a considerable challenge. Here, using a retrospective analysis of in-house historical data, we examined the parameters that impact the outcome of screening phage-displayed synthetic human sdAb libraries to discover antigen-specific binders. We found no evidence for a differential effect of domain type (V H or V L ), library randomization strategy, incorporation of a stabilizing disulfide linkage or sdAb display format (monovalent vs. multivalent) on the probability of obtaining any antigen-binding human sdAbs, instead finding that the success of library screens was primarily related to properties of target antigens, especially molecular mass. The solubility and binding affinity of sdAbs isolated from successful screens depended both on properties of the sdAb libraries (primarily domain type) and the target antigens. Taking attrition of sdAbs with major manufacturability concerns (aggregation; low expression) and sdAbs that do not recognize native cell-surface antigens as independent probabilities, we calculate the overall likelihood of obtaining ≥1 antigen-binding human sdAb from a single library-target screen as ~24%. Successful library-target screens should be expected to yield ~1.3 human sdAbs on average, each with average binding affinity of ~2 μM. Copyright © 2018 Elsevier B.V. All rights reserved.

Exploration of the effect of sequence variations located inside the binding pocket of HIV-1 and HIV-2 proteases.

PubMed

Triki, Dhoha; Billot, Telli; Visseaux, Benoit; Descamps, Diane; Flatters, Delphine; Camproux, Anne-Claude; Regad, Leslie

2018-04-10

HIV-2 protease (PR2) is naturally resistant to most FDA (Food and Drug Administration)-approved HIV-1 protease inhibitors (PIs), a major antiretroviral class. In this study, we compared the PR1 and PR2 binding pockets extracted from structures complexed with 12 ligands. The comparison of PR1 and PR2 pocket properties showed that bound PR2 pockets were more hydrophobic with more oxygen atoms and fewer nitrogen atoms than PR1 pockets. The structural comparison of PR1 and PR2 pockets highlighted structural changes induced by their sequence variations and that were consistent with these property changes. Specifically, substitutions at residues 31, 46, and 82 induced structural changes in their main-chain atoms that could affect PI binding in PR2. In addition, the modelling of PR1 mutant structures containing V32I and L76M substitutions revealed a cooperative mechanism leading to structural deformation of flap-residue 45 that could modify PR2 flexibility. Our results suggest that substitutions in the PR1 and PR2 pockets can modify PI binding and flap flexibility, which could underlie PR2 resistance against PIs. These results provide new insights concerning the structural changes induced by PR1 and PR2 pocket variation changes, improving the understanding of the atomic mechanism of PR2 resistance to PIs.

The spectral properties of (-)-epigallocatechin 3-O-gallate (EGCG) fluorescence in different solvents: dependence on solvent polarity.

PubMed

Snitsarev, Vladislav; Young, Michael N; Miller, Ross M S; Rotella, David P

2013-01-01

(-)-Epigallocatechin 3-O-gallate (EGCG) a molecule found in green tea and known for a plethora of bioactive properties is an inhibitor of heat shock protein 90 (HSP90), a protein of interest as a target for cancer and neuroprotection. Determination of the spectral properties of EGCG fluorescence in environments similar to those of binding sites found in proteins provides an important tool to directly study protein-EGCG interactions. The goal of this study is to examine the spectral properties of EGCG fluorescence in an aqueous buffer (AB) at pH=7.0, acetonitrile (AN) (a polar aprotic solvent), dimethylsulfoxide (DMSO) (a polar aprotic solvent), and ethanol (EtOH) (a polar protic solvent). We demonstrate that EGCG is a highly fluorescent molecule when excited at approximately 275 nm with emission maxima between 350 and 400 nm depending on solvent. Another smaller excitation peak was found when EGCG is excited at approximately 235 nm with maximum emission between 340 and 400 nm. We found that the fluorescence intensity (FI) of EGCG in AB at pH=7.0 is significantly quenched, and that it is about 85 times higher in an aprotic solvent DMSO. The Stokes shifts of EGCG fluorescence were determined by solvent polarity. In addition, while the emission maxima of EGCG fluorescence in AB, DMSO, and EtOH follow the Lippert-Mataga equation, its fluorescence in AN points to non-specific solvent effects on EGCG fluorescence. We conclude that significant solvent-dependent changes in both fluorescence intensity and fluorescence emission shifts can be effectively used to distinguish EGCG in aqueous solutions from EGCG in environments of different polarity, and, thus, can be used to study specific EGCG binding to protein binding sites where the environment is often different from aqueous in terms of polarity.

Ginsenoside Rc from Panax ginseng exerts anti-inflammatory activity by targeting TANK-binding kinase 1/interferon regulatory factor-3 and p38/ATF-2.

PubMed

Yu, Tao; Yang, Yanyan; Kwak, Yi-Seong; Song, Gwan Gyu; Kim, Mi-Yeon; Rhee, Man Hee; Cho, Jae Youl

2017-04-01

Ginsenoside Rc (G-Rc) is one of the major protopanaxadiol-type saponins isolated from Panax ginseng , a well-known medicinal herb with many beneficial properties including anticancer, anti-inflammatory, antiobesity, and antidiabetic effects. In this study, we investigated the effects of G-Rc on inflammatory responses in vitro and examined the mechanisms of these effects. The in vitro inflammation system used lipopolysaccharide-treated macrophages, tumor necrosis factor-α/interferon-γ-treated synovial cells, and HEK293 cells transfected with various inducers of inflammation. G-Rc significantly inhibited the expression of macrophage-derived cytokines, such as tumor necrosis factor-α and interleukin-1β. G-Rc also markedly suppressed the activation of TANK-binding kinase 1/IκB kinase ε/interferon regulatory factor-3 and p38/ATF-2 signaling in activated RAW264.7 macrophages, human synovial cells, and HEK293 cells. G-Rc exerts its anti-inflammatory actions by suppressing TANK-binding kinase 1/IκB kinase ε/interferon regulatory factor-3 and p38/ATF-2 signaling.

Locking mechanisms in degree-4 vertex origami structures

NASA Astrophysics Data System (ADS)

Fang, Hongbin; Li, Suyi; Xu, Jian; Wang, K. W.

2016-04-01

Origami has emerged as a potential tool for the design of mechanical metamaterials and metastructures whose novel properties originate from their crease patterns. Most of the attention in origami engineering has focused on the wellknown Miura-Ori, a folded tessellation that is flat-foldable for folded sheet and stacked blocks. This study advances the state of the art and expands the research field to investigate generic degree-4 vertex (4-vertex) origami, with a focus on facet-binding. In order to understand how facet-binding attributes to the mechanical properties of 4-vertex origami structures, geometries of the 4-vertex origami cells are analyzed and analytically expressed. Through repeating and stacking 4-vertex cells, origami sheets and stacked origami blocks can be constructed. Geometry analyses discover four mechanisms that will lead to the self-locking of 4-vertex origami cells, sheets, and stacked blocks: in-cell facet-binding, inlayer facet-binding, inter-layer facet binding, and in-layer and inter-layer facet-bindings. These mechanisms and the predicted self-locking phenomena are verified through 3D simulations and prototype experiments. Finally, this paper briefly introduces the unusual mechanical properties caused by the locking of 4-vertex origami structures. The research reported in this paper could foster a new breed of self-locking structures with various engineering applications.

Variation of the net charge, lipophilicity, and side chain flexibility in Dmt(1)-DALDA: Effect on Opioid Activity and Biodistribution.

PubMed

Novoa, Alexandre; Van Dorpe, Sylvia; Wynendaele, Evelien; Spetea, Mariana; Bracke, Nathalie; Stalmans, Sofie; Betti, Cecilia; Chung, Nga N; Lemieux, Carole; Zuegg, Johannes; Cooper, Matthew A; Tourwé, Dirk; De Spiegeleer, Bart; Schiller, Peter W; Ballet, Steven

2012-11-26

The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-d-Arg-Phe-Lys-NH(2) ([Dmt(1)]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt(1)]-DALDA and to investigate the Phe(3) side chain flexibility, the final amide bond was N-methylated and Phe(3) was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (ip and sc) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity, and transport properties. Strikingly, while [Dmt(1)]-DALDA and its N-methyl analogue, Dmt-d-Arg-Phe-NMeLys-NH(2), showed a long-lasting antinociceptive effect (>7 h), the peptides with d-Cit(2) generate potent antinociception more rapidly (maximal effect at 1h postinjection) but also lose their analgesic activity faster when compared to [Dmt(1)]-DALDA and [Dmt(1),NMeLys(4)]-DALDA.

Variation of the net charge, lipophilicity and side chain flexibility in Dmt1-DALDA: effect on opioid activity and biodistribution

PubMed Central

Novoa, Alexandre; Van Dorpe, Sylvia; Wynendaele, Evelien; Spetea, Mariana; Bracke, Nathalie; Stalmans, Sofie; Betti, Cecilia; Chung, Nga N.; Lemieux, Carole; Zuegg, Johannes; Cooper, Matthew A.; Tourwé, Dirk; De Spiegeleer, Bart; Schiller, Peter W.; Ballet, Steven

2012-01-01

The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt1]-DALDA and to investigate the Phe3 side chain flexibility, the final amide bond was N-methylated and Phe3 was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (i.p. and s.c.) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity and transport properties. Strikingly, while [Dmt1]-DALDA and its N-methyl analogue, Dmt-D-Arg-Phe-NMeLys-NH2, showed a long-lasting antinociceptive effect (>7h), the peptides with D-Cit2 generate potent antinociception more rapidly (maximal effect at 1h post-injection) but also lose their analgesic activity faster, when compared to [Dmt1]-DALDA and [Dmt1,NMeLys4]-DALDA. PMID:23102273

Acid-base properties of humic and fulvic acids formed during composting.

PubMed

Plaza, César; Senesi, Nicola; Polo, Alfredo; Brunetti, Gennaro

2005-09-15

The soil acid-base buffering capacity and the biological availability, mobilization, and transport of macro- and micronutrients, toxic metal ions, and xenobiotic organic cations in soil are strongly influenced by the acid-base properties of humic substances, of which humic and fulvic acids are the major fractions. For these reasons, the proton binding behavior of the humic acid-like (HA) and fulvic acid-like (FA) fractions contained in a compost are believed to be instrumental in its successful performance in soil. In this work, the acid-base properties of the HAs and FAs isolated from a mixture of the sludge residue obtained from olive oil mill wastewater (OMW) evaporated in an open-air pond and tree cuttings (TC) at different stages of composting were investigated by a current potentiometric titration method and the nonideal competitive adsorption (NICA)-Donnan model. The NICA-Donnan model provided an excellent description of the acid-base titration data, and pointed out substantial differences in site density and proton-binding affinity between the HAs and FAs examined. With respect to FAs, HAs were characterized by a smaller content of carboxylic- and phenolic-type groups and their larger affinities for proton binding. Further, HAs featured a greater heterogeneity in carboxylic-type groups than FAs. The composting process increased the content and decreased the proton affinity of carboxylic- and phenolic-type groups of HAs and FAs, and increased the heterogeneity of phenolic-type groups of HAs. As a whole, these effects indicated that the composting process could produce HA and FA fractions with greater cation binding capacities. These results suggest that composting of organic materials improves their agronomic and environmental value by increasing their potential to retain and exchange macro- and micronutrients, and to reduce the bioavailability of organic and inorganic pollutants.

The Importance of Being Tyrosine: Lessons in Molecular Recognition from Minimalist Synthetic Binding Proteins

PubMed Central

Koide, Shohei; Sidhu, Sachdev S.

2010-01-01

Summary Combinatorial libraries built with severely restricted chemical diversity have yielded highly functional synthetic binding proteins. Structural analyses of these minimalist binding sites have revealed the dominant role of large tyrosine residues for mediating molecular contacts and of small serine/glycine residues for providing space and flexibility. The concept of using limited residue types to construct optimized binding proteins mirrors findings in the field of small molecule drug development, where it has been proposed that most drugs are built from a limited set of side chains presented by diverse frameworks. The physicochemical properties of tyrosine make it the amino acid that is most effective for mediating molecular recognition, and protein engineers have taken advantage of these characteristics to build tyrosine-rich protein binding sites that outperform natural proteins in terms of affinity and specificity. Knowledge from preceding studies can be used to improve current designs, and thus, synthetic protein libraries will continue to evolve and improve. In the near future, it seems likely that synthetic binding proteins will supersede natural antibodies for most purposes, and moreover, synthetic proteins will enable many new applications beyond the scope of natural proteins. PMID:19298050

Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

PubMed Central

Dedecker, Peter

2017-01-01

Reversibly switchable fluorescent proteins (RSFPs) enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties. PMID:28930199

Nanotube antibody biosensor arrays for the detection of circulating breast cancer cells

NASA Astrophysics Data System (ADS)

Shao, Ning; Wickstrom, Eric; Panchapakesan, Balaji

2008-11-01

Recent reports have shown that nanoscale electronic devices can be used to detect a change in electrical properties when receptor proteins bind to their corresponding antibodies functionalized on the surface of the device, in extracts from as few as ten lysed tumor cells. We hypothesized that nanotube-antibody devices could sensitively and specifically detect entire live cancer cells. We report for the first time a single nanotube field effect transistor array, functionalized with IGF1R-specific and Her2-specific antibodies, which exhibits highly sensitive and selective sensing of live, intact MCF7 and BT474 human breast cancer cells in human blood. Those two cell lines both overexpress IGF1R and Her2, at different levels. Single or small bundle of nanotube devices that were functionalized with IGF1R-specific or Her2-specific antibodies showed 60% decreases in conductivity upon interaction with BT474 or MCF7 breast cancer cells in two µl drops of blood. Control experiments with non-specific antibodies or with MCF10A control breast cells produced a less than 5% decrease in electrical conductivity, illustrating the high sensitivity for whole cell binding by these single nanotube-antibody devices. We postulate that the free energy change due to multiple simultaneous cell-antibody binding events exerted stress along the nanotube surface, decreasing its electrical conductivity due to an increase in band gap. Because the free energy change upon cell-antibody binding, the stress exerted on the nanotube, and the change in conductivity are specific to a specific antigen-antibody interaction; these properties might be used as a fingerprint for the molecular sensing of circulating cancer cells. From optical microscopy observations during sensing, it appears that the binding of a single cell to a single nanotube field effect transistor produced the change in electrical conductivity. Thus we report a nanoscale oncometer with single cell sensitivity with a diameter 1000 times smaller than a cancer cell that functions in a drop of fresh blood.

Insights into the binding interactions of autochthonous dissolved organic matter released from Microcystis aeruginosa with pyrene using spectroscopy.

PubMed

Yang, Chenghu; Liu, Yangzhi; Zhu, Yaxian; Zhang, Yong

2016-03-15

The autochthonous dissolved organic matter (DOM) released by Microcystis aeruginosa (M. aeruginosa-DOM) during its growth period was characterized by spectroscopy. Furthermore, the relationships between the M. aeruginosa-DOM spectroscopic descriptors and the pyrene binding coefficient (KDOC) values were explored. The results showed that the spectroscopic characteristics of the M. aeruginosa-DOM and the binding properties of pyrene were dynamically changed along with the algae growth. Pearson correlation analysis demonstrated that a higher pyrene KDOC value was observed for the M. aeruginosa-DOM that has a higher humification index (HIX) value, a lower biological index (BIX) value and a lower absorption ratio (E2/E3). The presence of protein-like and long-wavelength-excited humic-like components may impose negative and positive effects on binding of pyrene by the M. aeruginosa-DOM, respectively. Principal component analysis (PCA) further supported that the binding affinity of pyrene may be primarily influenced by the humification degree of the M. aeruginosa-DOM. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis and binding affinity analysis of α1-2- and α1-6-O/S-linked dimannosides for the elucidation of sulfur in glycosidic bonds using quartz crystal microbalance sensors.

PubMed

Norberg, Oscar; Wu, Bin; Thota, Niranjan; Ge, Jian-Tao; Fauquet, Germain; Saur, Ann-Kathrin; Aastrup, Teodor; Dong, Hai; Yan, Mingdi; Ramström, Olof

2017-11-27

The role of sulfur in glycosidic bonds has been evaluated using quartz crystal microbalance methodology. Synthetic routes towards α1-2- and α1-6-linked dimannosides with S- or O-glycosidic bonds have been developed, and the recognition properties assessed in competition binding assays with the cognate lectin concanavalin A. Mannose-presenting QCM sensors were produced using photoinitiated, nitrene-mediated immobilization methods, and the subsequent binding study was performed in an automated flow-through instrumentation, and correlated with data from isothermal titration calorimetry. The recorded K d -values corresponded well with reported binding affinities for the O-linked dimannosides with affinities for the α1-2-linked dimannosides in the lower micromolar range. The S-linked analogs showed slightly disparate effects, where the α1-6-linked analog showed weaker affinity than the O-linked dimannoside, as well as positive apparent cooperativity, whereas the α1-2-analog displayed very similar binding compared to the O-linked structure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nanomechanical Contribution of Collagen and von Willebrand Factor A in Marine Underwater Adhesion and Its Implication for Collagen Manipulation.

PubMed

Yoo, Hee Young; Huang, Jun; Li, Lin; Foo, Mathias; Zeng, Hongbo; Hwang, Dong Soo

2016-03-14

Recent works on mussel adhesion have identified a load bearing matrix protein (PTMP1) containing von Willebrand factor (vWF) with collagen binding capability that contributes to the mussel holdfast by manipulating mussel collagens. Using a surface forces apparatus, we investigate for the first time, the nanomechanical properties of vWF-collagen interaction using homologous proteins of mussel byssus, PTMP1 and preCollagens (preCols), as collagen. Mimicking conditions similar to mussel byssus secretion (pH < 5.0) and seawater condition (pH 8.0), PTMP1 and preCol interact weakly in the "positioning" phase based on vWF-collagen binding and strengthen in "locked" phase due to the combined effects of electrostatic attraction, metal binding, and mechanical shearing. The progressive enhancement of binding between PTMP1 with porcine collagen under the aforementioned conditions is also observed. The binding mechanisms of PTMP1-preCols provide insights into the molecular interaction of the mammalian collagen system and the development of an artificial extracellular matrix based on collagens.

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

PubMed

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

2014-07-15

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

Activation of RXR–PPAR heterodimers by organotin environmental endocrine disruptors

PubMed Central

le Maire, Albane; Grimaldi, Marina; Roecklin, Dominique; Dagnino, Sonia; Vivat-Hannah, Valérie; Balaguer, Patrick; Bourguet, William

2009-01-01

The nuclear receptor retinoid X receptor-α (RXR-α)–peroxisome proliferator-activated receptor-γ (PPAR-γ) heterodimer was recently reported to have a crucial function in mediating the deleterious effects of organotin compounds, which are ubiquitous environmental contaminants. However, because organotins are unrelated to known RXR-α and PPAR-γ ligands, the mechanism by which these compounds bind to and activate the RXR-α–PPAR-γ heterodimer at nanomolar concentrations has remained elusive. Here, we show that tributyltin (TBT) activates all three RXR–PPAR-α, -γ, -δ heterodimers, primarily through its interaction with RXR. In addition, the 1.9 Å resolution structure of the RXR-α ligand-binding domain in complex with TBT shows a covalent bond between the tin atom and residue Cys 432 of helix H11. This interaction largely accounts for the high binding affinity of TBT, which only partly occupies the RXR-α ligand-binding pocket. Our data allow an understanding of the binding and activation properties of the various organotins and suggest a mechanism by which these tin compounds could affect other nuclear receptor signalling pathways. PMID:19270714

The Governing Category Parameter in Second Language Acquisition.

ERIC Educational Resources Information Center

Hirakawa, Makiko

This experimental study examined how and to what extent native speakers of Japanese acquire syntactic properties of English reflexives. There is emphasis on the effects of the Governing Category Parameter (Wexler and Manzini, 1987), which relates to Principle A of the Binding Theory (Chomsky, 1981). It is hypothesized that second language (L2)…

Host-guest chemistry of dendrimer-drug complexes. 2. Effects of molecular properties of guests and surface functionalities of dendrimers.

PubMed

Hu, Jingjing; Cheng, Yiyun; Wu, Qinglin; Zhao, Libo; Xu, Tongwen

2009-08-06

The host-guest chemistry of dendrimer-drug complexes is investigated by NMR techniques, including (1)H NMR and 2D-NOESY studies. The effects of molecular properties of drug molecules (protonation ability and spatial steric hindrance of charged groups) and surface functionalities of dendrimers (positively charged amine groups and negatively charged carboxylate groups) on the host-guest interactions are discussed. Different interaction mechanisms between dendrimers and drug molecules are proposed on the basis of NMR results. Primary amine- and secondary amine-containing drugs preferentially bind to negatively charged dendrimers by strong electrostatic interactions, whereas tertiary amine and quaternary ammonium-containing drugs have weak binding ability with dendrimers due to relatively low protonation ability of the tertiary amine group and serious steric hindrance of the quaternary ammonium group. Positively charged drugs locate only on the surface of negatively charged dendrimers, whereas negatively charged drugs locate both on the surface and in the interior cavities of positively charged dendrimers. The host-guest chemistry of dendrimer-drug complexes is promising for the development of new drug delivery systems.

Gotu Kola (Centella Asiatica) extract enhances phosphorylation of cyclic AMP response element binding protein in neuroblastoma cells expressing amyloid beta peptide.

PubMed

Xu, Yanan; Cao, Zhiming; Khan, Ikhlas; Luo, Yuan

2008-04-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that shows cognitive deficits and memory impairment. Extract from the leaves of Gotu Kola (Centella Asiatica) have been used as an alternative medicine for memory improvement in Indian Ayurvedic system of medicine for a long time. Although several studies have revealed its effect in ameliorating the cognitive impairment in rat models of AD and stimulating property on neuronal dendrites of hippocampal region, the molecular mechanism of Gotu Kola on neuroprotection still remains to be elucidated. In this study, we report that phosphorylation of cyclic AMP response element binding protein (CREB) is enhanced in both a neuroblastoma cell line expressing amyloid beta 1-42 (Abeta) and in rat embryonic cortical primary cell culture. In addition, the contribution of two major single components to the enhanced CREB phosphorylatioin was examined. Furthermore, inhibitors were applied in this study revealing that ERK/RSK signaling pathway might mediate this effect of Gotu Kola extract. Taken together, we provide a possible molecular mechanism for memory enhancing property of Gotu Kola extract for the first time.

Effect of the Flexible Regions of the Oncoprotein Mouse Double Minute X on Inhibitor Binding Affinity.

PubMed

Qin, Lingyun; Liu, Huili; Chen, Rong; Zhou, Jingjing; Cheng, Xiyao; Chen, Yao; Huang, Yongqi; Su, Zhengding

2017-11-07

The oncoprotein MdmX (mouse double minute X) is highly homologous to Mdm2 (mouse double minute 2) in terms of their amino acid sequences and three-dimensional conformations, but Mdm2 inhibitors exhibit very weak affinity for MdmX, providing an excellent model for exploring how protein conformation distinguishes and alters inhibitor binding. The intrinsic conformation flexibility of proteins plays pivotal roles in determining and predicting the binding properties and the design of inhibitors. Although the molecular dynamics simulation approach enables us to understand protein-ligand interactions, the mechanism underlying how a flexible binding pocket adapts an inhibitor has been less explored experimentally. In this work, we have investigated how the intrinsic flexible regions of the N-terminal domain of MdmX (N-MdmX) affect the affinity of the Mdm2 inhibitor nutlin-3a using protein engineering. Guided by heteronuclear nuclear Overhauser effect measurements, we identified the flexible regions that affect inhibitor binding affinity around the ligand-binding pocket on N-MdmX. A disulfide engineering mutant, N-MdmX C25-C110/C76-C88 , which incorporated two staples to rigidify the ligand-binding pocket, allowed an affinity for nutlin-3a higher than that of wild-type N-MdmX (K d ∼ 0.48 vs K d ∼ 20.3 μM). Therefore, this mutant provides not only an effective protein model for screening and designing of MdmX inhibitors but also a valuable clue for enhancing the intermolecular interactions of the pharmacophores of a ligand with pronounced flexible regions. In addition, our results revealed an allosteric ligand-binding mechanism of N-MdmX in which the ligand initially interacts with a compact core, followed by augmenting intermolecular interactions with intrinsic flexible regions. This strategy should also be applicable to many other protein targets to accelerate drug discovery.

Unusual dynamic properties of water near the ice-binding plane of hyperactive antifreeze protein

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

Kuffel, Anna; Czapiewski, Dariusz; Zielkiewicz, Jan, E-mail: jaz@chem.pg.gda.pl

2015-10-07

The dynamical properties of solvation water of hyperactive antifreeze protein from Choristoneura fumiferana (CfAFP) are analyzed and discussed in context of its antifreeze activity. The protein comprises of three well-defined planes and one of them binds to the surface of ice. The dynamical properties of solvation water around each of these planes were analyzed separately; the results are compared with the dynamical properties of solvation water of ice around its two crystallographic planes: basal and prism. Three main conclusions are inferred from our investigations. The first one is that the solvation shell of CfAFP does not seem to be particularlymore » far-ranged, at least not beyond what is usually observed for proteins that do not interact with ice. Therefore, it does not appear to us that the antifreeze activity is enhanced by a long-ranged retardation of water mobility. Also the correlation between the collective mobility of water and the collective mobility of protein atoms highly resembles the one measured for the protein that does not interact with ice. Our second conclusion is that the dynamical properties of solvation water of CfAFP are non-uniform. The dynamics of solvation water of ice-binding plane is, in some respects, different from the dynamics of solvation water of the two remaining planes. The feature that distinguishes the dynamics of solvation water of the three planes is the activation energy of diffusion process. The third conclusion is that—from the three analyzed solvation shells of CfAFP—the dynamical properties of solvation water of the ice-binding plane resemble the most the properties of solvation water of ice; note, however, that these properties still clearly differ from the dynamic properties of solvation water of ice.« less

G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

PubMed

Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

2017-09-01

G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural evidence of the species-dependent albumin binding of the modified cyclic phosphatidic acid with cytotoxic properties

PubMed Central

Sekula, Bartosz; Ciesielska, Anna; Rytczak, Przemyslaw; Koziołkiewicz, Maria; Bujacz, Anna

2016-01-01

Cyclic phosphatidic acids (cPAs) are naturally occurring, very active signalling molecules, which are involved in several pathological states, such as cancer, diabetes or obesity. As molecules of highly lipidic character found in the circulatory system, cPAs are bound and transported by the main extracellular lipid binding protein–serum albumin. Here, we present the detailed interactions between human serum albumin (HSA) and equine serum albumin (ESA) with a derivative of cPA, 1-O-myristoyl-sn-glycerol-2,3-cyclic phosphorodithioate (Myr-2S-cPA). Initial selection of the ligand used for the structural study was made by the analysis of the therapeutically promising properties of the sulfur containing analogues of cPA in respect to the unmodified lysophospholipids (LPLs). Substitution of one or two non-bridging oxygen atoms in the phosphate group with one or two sulfur atoms increases the cytotoxic effect of cPAs up to 60% on the human prostate cancer (PC) cells. Myr-2S-cPA reduces cancer cell viability in a dose-dependent manner, with IC50 value of 29.0 μM after 24 h incubation, which is almost 30% lower than IC50 of single substituted phosphorothioate cPA. Although, the structural homology between HSA and ESA is big, their crystal complexes with Myr-2S-cPA demonstrate significantly different mode of binding of this LPL analogue. HSA binds three molecules of Myr-2S-cPA, whereas ESA only one. Moreover, none of the identified Myr-2S-cPA binding sites overlap in both albumins. PMID:27129297

Cellular level models as tools for cytokine design.

PubMed

Radhakrishnan, Mala L; Tidor, Bruce

2010-01-01

Cytokines and growth factors are critical regulators that connect intracellular and extracellular environments through binding to specific cell-surface receptors. They regulate a wide variety of immunological, growth, and inflammatory response processes. The overall signal initiated by a population of cytokine molecules over long time periods is controlled by the subtle interplay of binding, signaling, and trafficking kinetics. Building on the work of others, we abstract a simple kinetic model that captures relevant features from cytokine systems as well as related growth factor systems. We explore a large range of potential biochemical behaviors, through systematic examination of the model's parameter space. Different rates for the same reaction topology lead to a dramatic range of biochemical network properties and outcomes. Evolution might productively explore varied and different portions of parameter space to create beneficial behaviors, and effective human therapeutic intervention might be achieved through altering network kinetic properties. Quantitative analysis of the results reveals the basis for tensions among a number of different network characteristics. For example, strong binding of cytokine to receptor can increase short-term receptor activation and signal initiation but decrease long-term signaling due to internalization and degradation. Further analysis reveals the role of specific biochemical processes in modulating such tensions. For instance, the kinetics of cytokine binding and receptor activation modulate whether ligand-receptor dissociation can generally occur before signal initiation or receptor internalization. Beyond analysis, the same models and model behaviors provide an important basis for the design of more potent cytokine therapeutics by providing insight into how binding kinetics affect ligand potency. (c) 2010 American Institute of Chemical Engineers

Equilibrium isotope effects on noncovalent interactions in a supramolecular host-guest system.

PubMed

Mugridge, Jeffrey S; Bergman, Robert G; Raymond, Kenneth N

2012-02-01

The self-assembled supramolecular complex [Ga(4)L(6)](12-) (1; L = 1,5-bis[2,3-dihydroxybenzamido]naphthalene) can act as a molecular host in aqueous solution and bind cationic guest molecules to its highly charged exterior surface or within its hydrophobic interior cavity. The distinct internal cavity of host 1 modifies the physical properties and reactivity of bound guest molecules and can be used to catalyze a variety of chemical transformations. Noncovalent host-guest interactions in large part control guest binding, molecular recognition and the chemical reactivity of bound guests. Herein we examine equilibrium isotope effects (EIEs) on both exterior and interior guest binding to host 1 and use these effects to probe the details of noncovalent host-guest interactions. For both interior and exterior binding of a benzylphosphonium guest in aqueous solution, protiated guests are found to bind more strongly to host 1 (K(H)/K(D) > 1) and the preferred association of protiated guests is driven by enthalpy and opposed by entropy. Deuteration of guest methyl and benzyl C-H bonds results in a larger EIE than deuteration of guest aromatic C-H bonds. The observed EIEs can be well explained by considering changes in guest vibrational force constants and zero-point energies. DFT calculations further confirm the origins of these EIEs and suggest that changes in low-frequency guest C-H/D vibrational motions (bends, wags, etc.) are primarily responsible for the observed EIEs. © 2011 American Chemical Society

Fatty acids bind tightly to the N-terminal domain of angiopoietin-like protein 4 and modulate its interaction with lipoprotein lipase.

PubMed

Robal, Terje; Larsson, Mikael; Martin, Miina; Olivecrona, Gunilla; Lookene, Aivar

2012-08-24

Angiopoietin-like protein 4 (Angptl4), a potent regulator of plasma triglyceride metabolism, binds to lipoprotein lipase (LPL) through its N-terminal coiled-coil domain (ccd-Angptl4) inducing dissociation of the dimeric enzyme to inactive monomers. In this study, we demonstrate that fatty acids reduce the inactivation of LPL by Angptl4. This was the case both with ccd-Angptl4 and full-length Angptl4, and the effect was seen in human plasma or in the presence of albumin. The effect decreased in the sequence oleic acid > palmitic acid > myristic acid > linoleic acid > linolenic acid. Surface plasmon resonance, isothermal titration calorimetry, fluorescence, and chromatography measurements revealed that fatty acids bind with high affinity to ccd-Angptl4. The interactions were characterized by fast association and slow dissociation rates, indicating formation of stable complexes. The highest affinity for ccd-Angptl4 was detected for oleic acid with a subnanomolar equilibrium dissociation constant (K(d)). The K(d) values for palmitic and myristic acid were in the nanomolar range. Linoleic and linolenic acid bound with much lower affinity. On binding of fatty acids, ccd-Angptl4 underwent conformational changes resulting in a decreased helical content, weakened structural stability, dissociation of oligomers, and altered fluorescence properties of the Trp-38 residue that is located close to the putative LPL-binding region. Based on these results, we propose that fatty acids play an important role in modulating the effects of Angptl4.

Specificity of O-glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

PubMed Central

Chen, Liqun; Drake, Matthew R.; Resch, Michael G.; Greene, Eric R.; Himmel, Michael E.; Chaffey, Patrick K.; Beckham, Gregg T.; Tan, Zhongping

2014-01-01

The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16 °C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs. PMID:24821760

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties.

PubMed

Putta, Priya; Rankenberg, Johanna; Korver, Ruud A; van Wijk, Ringo; Munnik, Teun; Testerink, Christa; Kooijman, Edgar E

2016-11-01

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins. Copyright © 2016. Published by Elsevier B.V.

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

PubMed Central

Greibe, Eva; Fedosov, Sergey; Sorensen, Boe S.; Højrup, Peter; Poulsen, Steen S.; Nexo, Ebba

2012-01-01

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins. PMID:22872637

Binding of a cyclic organoselenium compound with gold nanoparticles (GNP) and its effect on electron transfer properties.

PubMed

Kumar, Pavitra V; Singh, Beena G; Maiti, Nandita; Iwaoka, Michio; Priyadarsini, K Indira

2014-12-15

Binding of a cyclic organoselenium compound, DL-trans-3,4-dihydroxy-1-selenolane (DHSred) with gold nanoparticles (GNP) of different sizes was studied by absorption spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), surface enhanced Raman spectroscopy (SERS) and zeta-potential (ζ) measurements. GNP of different size were synthesized by varying the reaction conditions and their size was determined by DLS and TEM techniques. The absorption spectral data showed red shift in the surface plasmon resonance (SPR) band indicating increase in the size of GNP on binding to DHSred. SERS studies confirmed that the binding of DHSred with GNP is through selenium center with planar orientation of DHSred on the GNP surface. The product of the number of binding sites (n) in GNP and the binding constant (K) was estimated for GNP of different particle size. The zeta potential (ζ) value of GNP decreased marginally in the presence of DHSred. Further, the binding of DHSred with GNP was found to enhance its reactivity with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radicals (ABTS(·-)) and the reactivity increased with decrease in the GNP size. Such enhancement in the reducing ability may have a greater impact on the antioxidant activity of DHSred. Copyright © 2014 Elsevier Inc. All rights reserved.

Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

PubMed

Vulfius, Catherine A; Kasheverov, Igor E; Kryukova, Elena V; Spirova, Ekaterina N; Shelukhina, Irina V; Starkov, Vladislav G; Andreeva, Tatyana V; Faure, Grazyna; Zouridakis, Marios; Tsetlin, Victor I; Utkin, Yuri N

2017-01-01

Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by further experiments.

Comparison of solute-binding properties of plastic materials used as pharmaceutical product containers.

PubMed

Jenke, Dennis; Couch, Tom; Gillum, Amy

2010-01-01

Material/water equilibrium binding constants (E(b)) were determined for 11 organic solutes and 2 plastic materials commonly used in pharmaceutical product containers (plasticized polyvinyl chloride and polyolefin). In general, solute binding by the plasticized polyvinyl chloride material was greater, by nearly an order of magnitude, than the binding by the polyolefin (on an equal weight basis). The utilization of the binding constants to facilitate container compatibility assessments (e.g., drug loss by container binding) for drug-containing products is discussed.

Actin-binding proteins sensitively mediate F-actin bundle stiffness

NASA Astrophysics Data System (ADS)

Claessens, Mireille M. A. E.; Bathe, Mark; Frey, Erwin; Bausch, Andreas R.

2006-09-01

Bundles of filamentous actin (F-actin) form primary structural components of a broad range of cytoskeletal processes including filopodia, sensory hair cell bristles and microvilli. Actin-binding proteins (ABPs) allow the cell to tailor the dimensions and mechanical properties of the bundles to suit specific biological functions. Therefore, it is important to obtain quantitative knowledge on the effect of ABPs on the mechanical properties of F-actin bundles. Here we measure the bending stiffness of F-actin bundles crosslinked by three ABPs that are ubiquitous in eukaryotes. We observe distinct regimes of bundle bending stiffness that differ by orders of magnitude depending on ABP type, concentration and bundle size. The behaviour observed experimentally is reproduced quantitatively by a molecular-based mechanical model in which ABP shearing competes with F-actin extension/compression. Our results shed new light on the biomechanical function of ABPs and demonstrate how single-molecule properties determine mesoscopic behaviour. The bending mechanics of F-actin fibre bundles are general and have implications for cytoskeletal mechanics and for the rational design of functional materials.

Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

PubMed

Hill, Darryl J; Griffiths, Natalie J; Borodina, Elena; Andreae, Clio A; Sessions, Richard B; Virji, Mumtaz

2015-01-01

The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

Identification and Therapeutic Potential of a Vitronectin Binding Region of Meningococcal Msf

PubMed Central

Hill, Darryl J.; Griffiths, Natalie J.; Borodina, Elena; Andreae, Clio A.; Sessions, Richard B.; Virji, Mumtaz

2015-01-01

The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen. PMID:25826209

Spectroscopic and molecular docking studies on the charge transfer complex of bovine serum albumin with quinone in aqueous medium and its influence on the ligand binding property of the protein.

PubMed

Satheshkumar, Angupillai; Elango, Kuppanagounder P

2014-09-15

The spectral techniques such as UV-Vis, (1)H NMR and fluorescence and electrochemical experiments have been employed to investigate the interaction between 2-methoxy-3,5,6-trichloro-1,4-benzoquinone (MQ; a water soluble quinone) and bovine serum albumin (BSA) in aqueous medium. The fluorescence of BSA was quenched by MQ via formation of a 1:1 BSA-MQ charge transfer adduct with a formation constant of 3.3×10(8) L mol(-1). Based on the Forster's theory the binding distance between them is calculated as 2.65 nm indicating high probability of binding. For the first time, influence of quinone on the binding property of various types of ligands such as aspirin, ascorbic acid, nicotinimide and sodium stearate has also been investigated. The results indicated that the strong and spontaneous binding existing between BSA and MQ, decreased the intensity of binding of these ligands with BSA. Since Tryptophan (Trp) is the basic residue present in BSA, a comparison between binding property of Trp-MQ adduct with that of BSA-MQ with these ligands has also been attempted. 1H NMR titration study indicated that the Trp forms a charge transfer complex with MQ, which reduces the interaction of Trp with the ligands. Molecular docking study supported the fact that the quinone interacts with the Trp212 unit of the BSA and the free energy change of binding (ΔG) for the BSA-MQ complex was found to be -46 kJ mol(-1), which is comparable to our experimental free energy of binding (-49 kJ mol(-1)) obtained from fluorescence study. Copyright © 2014 Elsevier B.V. All rights reserved.

Spectroscopic and molecular docking studies on the charge transfer complex of bovine serum albumin with quinone in aqueous medium and its influence on the ligand binding property of the protein

NASA Astrophysics Data System (ADS)

Satheshkumar, Angupillai; Elango, Kuppanagounder P.

2014-09-01

The spectral techniques such as UV-Vis, 1H NMR and fluorescence and electrochemical experiments have been employed to investigate the interaction between 2-methoxy-3,5,6-trichloro-1,4-benzoquinone (MQ; a water soluble quinone) and bovine serum albumin (BSA) in aqueous medium. The fluorescence of BSA was quenched by MQ via formation of a 1:1 BSA-MQ charge transfer adduct with a formation constant of 3.3 × 108 L mol-1. Based on the Forster’s theory the binding distance between them is calculated as 2.65 nm indicating high probability of binding. For the first time, influence of quinone on the binding property of various types of ligands such as aspirin, ascorbic acid, nicotinimide and sodium stearate has also been investigated. The results indicated that the strong and spontaneous binding existing between BSA and MQ, decreased the intensity of binding of these ligands with BSA. Since Tryptophan (Trp) is the basic residue present in BSA, a comparison between binding property of Trp-MQ adduct with that of BSA-MQ with these ligands has also been attempted. 1H NMR titration study indicated that the Trp forms a charge transfer complex with MQ, which reduces the interaction of Trp with the ligands. Molecular docking study supported the fact that the quinone interacts with the Trp212 unit of the BSA and the free energy change of binding (ΔG) for the BSA-MQ complex was found to be -46 kJ mol-1, which is comparable to our experimental free energy of binding (-49 kJ mol-1) obtained from fluorescence study.

Using Tryptophan Mutants To Probe the Structural and Functional Status of BsSCO, a Copper Binding, Cytochrome c Oxidase Assembly Protein from Bacillus subtilis.

PubMed

Hussain, Shina; Andrews, Diann; Hill, Bruce C

2017-12-05

The synthesis of cytochrome c oxidase protein from Bacillus subtilis (i.e., BsSCO) binds copper with picomolar affinity, which increases the protein's melting temperature (i.e., T M ) by 20 °C. Here two native tryptophans (i.e., W36 and W101) are identified as major contributors to BsSCO's structural form, and their contributions to the stability, intrinsic fluorescence, and copper binding properties of BsSCO are explored. Single mutations of tryptophan to phenylalanine decrease the T M by 10 °C and the folding free energy by 3-4 kcal/mol. A more severe change to alanine (i.e., W36A BsSCO) decreases the T M by 20 °C and the stability by 9 kcal/mol. However, these mutants bind copper with high affinity and assemble cytochrome c oxidase in vivo. Replacing phenylalanine at a position near (∼5 Å) the copper binding site with tryptophan (i.e., F42W) increases the T M of apo-BsSCO by 3 °C but diminishes the effect of copper binding. When both native tryptophans are changed to alanine, apo-BsSCO is unfolded in vitro and is not functional in cytochrome c oxidase assembly in vivo. A double-mutant of BsSCO in which W36A is combined with F42W exhibits a form of metastability. Apo-W36A/F42W BsSCO melts at 37 °C, which upon binding of copper shifts to 65 °C. B. subtilis expressing W36A/F42W BsSCO and grown at 37 °C does not assemble cytochrome c oxidase. However, when these cells are cooled to 25 °C, cytochrome c oxidase activity is recovered. Our results illustrate the subtle relationship between the structural stability and functional properties of BsSCO in the assembly of cytochrome c oxidase.

DNA condensing effects and sequence selectivity of DNA binding of antitumor noncovalent polynuclear platinum complexes.

PubMed

Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

2014-02-03

The noncovalent analogues of antitumor polynuclear platinum complexes represent a structurally discrete class of platinum drugs. Their chemical and biological properties differ significantly from those of most platinum chemotherapeutics, which bind to DNA in a covalent manner by formation of Pt-DNA adducts. In spite of the fact that these noncovalent polynuclear platinum complexes contain no leaving groups, they have been shown to bind to DNA with high affinity. We report here on the DNA condensation properties of a series of noncovalent analogues of antitumor polynuclear platinum complexes described by biophysical and biochemical methods. The results demonstrate that these polynuclear platinum compounds are capable of inducing DNA condensation at more than 1 order of magnitude lower concentrations than conventional spermine. Atomic force microscopy studies of DNA condensation confined to a mica substrate have revealed that the DNA morphologies become more compact with increasing concentration of the platinum complexes. Moreover, we also found that the noncovalent polynuclear platinum complex [{Pt(NH3)3}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](6+) (TriplatinNC-A) binds to DNA in a sequence-dependent manner, namely, to A/T-rich sequences and A-tract regions, and that noncovalent polynuclear platinum complexes protect DNA from enzymatic cleavage by DNase I. The results suggest that mechanisms of antitumor and cytotoxic activities of these complexes may be associated with their unique ability to condense DNA along with their sequence-specific DNA binding. Owing to their high cellular accumulation, it is also reasonable to suggest that their mechanism of action is based on the competition with naturally occurring DNA condensing agents, such as polyamines spermine, spermidine, and putrescine, for intracellular binding sites, resulting in the disturbance of the correct binding of regulatory proteins initiating the onset of apoptosis.

Structural properties of the intrinsically disordered, multiple calcium ion-binding otolith matrix macromolecule-64 (OMM-64).

PubMed

Poznar, Monika; Hołubowicz, Rafał; Wojtas, Magdalena; Gapiński, Jacek; Banachowicz, Ewa; Patkowski, Adam; Ożyhar, Andrzej; Dobryszycki, Piotr

2017-11-01

Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Environmental Screening Effects in 2D Materials: Renormalization of the Bandgap, Electronic Structure, and Optical Spectra of Few-Layer Black Phosphorus.

PubMed

Qiu, Diana Y; da Jornada, Felipe H; Louie, Steven G

2017-08-09

Few-layer black phosphorus has recently emerged as a promising 2D semiconductor, notable for its widely tunable bandgap, highly anisotropic properties, and theoretically predicted large exciton binding energies. To avoid degradation, it has become common practice to encapsulate black phosphorus devices. It is generally assumed that this encapsulation does not qualitatively affect their optical properties. Here, we show that the contrary is true. We have performed ab initio GW and GW plus Bethe-Salpeter equation (GW-BSE) calculations to determine the quasiparticle (QP) band structure and optical spectrum of one-layer (1L) through four-layer (4L) black phosphorus, with and without encapsulation between hexagonal boron nitride and sapphire. We show that black phosphorus is exceptionally sensitive to environmental screening. Encapsulation reduces the exciton binding energy in 1L by as much as 70% and completely eliminates the presence of a bound exciton in the 4L structure. The reduction in the exciton binding energies is offset by a similarly large renormalization of the QP bandgap so that the optical gap remains nearly unchanged, but the nature of the excited states and the qualitative features of the absorption spectrum change dramatically.

Intra- versus Intermolecular Hydrogen Bonding: Solvent-Dependent Conformational Preferences of a Common Supramolecular Binding Motif from 1 H NMR and Vibrational Circular Dichroism Spectra.

PubMed

Demarque, Daniel P; Merten, Christian

2017-12-19

When predicting binding properties of small molecules or larger supramolecular aggregates, intra- and intermolecular hydrogen bonds are often considered the most important factor. Spectroscopic techniques such as 1 H NMR spectroscopy are typically utilized to characterize such binding events, but interpretation is often qualitative and follows chemical intuition. In this study, we compare the effects of intramolecular hydrogen bonding and solvation on two chiral 2,6-pyridinediyl-dialkylamides. In comparison with 1 H NMR spectroscopy, vibrational circular dichroism (VCD) spectroscopy proved to be more sensitive to conformational changes. In fact, the change of the solvent from CDCl 3 to [D 6 ]DMSO generates mirror-image VCD spectra for the same enantiomer. Here, the common sense that the sterically less hindered group is more prone to solvation proved to be wrong according predicted VCD spectra, which clearly show that both asymmetric amide hydrogens are equally likely to be solvated, but never simultaneously. The competition between intra- and intermolecular hydrogen bonding and their importance for a correct prediction of spectral properties are discussed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

ATP regulation of the ligand-binding properties in temperate and cold-adapted haemoglobins. X-ray structure and ligand-binding kinetics in the sub-Antarctic fish Eleginops maclovinus.

PubMed

Coppola, Daniela; Abbruzzetti, Stefania; Nicoletti, Francesco; Merlino, Antonello; Gambacurta, Alessandra; Giordano, Daniela; Howes, Barry D; De Sanctis, Giampiero; Vitagliano, Luigi; Bruno, Stefano; di Prisco, Guido; Mazzarella, Lelio; Smulevich, Giulietta; Coletta, Massimo; Viappiani, Cristiano; Vergara, Alessandro; Verde, Cinzia

2012-10-30

The major haemoglobin of the sub-Antarctic fish Eleginops maclovinus was structurally and functionally characterised with the aim to compare molecular environmental adaptations in the O(2)-transport system of sub-Antarctic fishes of the suborder Notothenioidei with those of their high-latitude relatives. Ligand-binding kinetics of the major haemoglobin of E. maclovinus indicated strong stabilisation of the liganded quaternary T state, enhanced in the presence of the physiological allosteric effector ATP, compared to that of high-Antarctic Trematomus bernacchii. The activation enthalpy for O(2) dissociation was dramatically lower than that in T. bernacchii haemoglobin, suggesting remarkable differences in temperature sensitivity and structural changes associated with O(2) release and exit from the protein. The haemoglobin functional properties, together with the X-ray structure of the CO form at 1.49 Å resolution, the first of a temperate notothenioid, strongly support the hypothesis that in E. maclovinus, whose life-style varies according to changes in habitat, the mechanisms that regulate O(2) affinity and the ATP-induced Root effect differ from those of high-Antarctic Notothenioids.

Inhibition of human Na(v)1.5 sodium channels by strychnine and its analogs.

PubMed

Yuan, Chunhua; Sun, Lirong; Zhang, Meng; Li, Shuji; Wang, Xuemin; Gao, Tianming; Zhu, Xinhong

2011-08-15

Strychnine and brucine from the seeds of the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors. In this study, we have characterized the pharmacological properties of strychnine and its analogs on human Na(v)1.5 channels to assess their potential therapeutic advantage in certain arrhythmias. Among the eight alkaloids, only strychnine and icajine exhibited inhibition potency on the Na(v)1.5 channel with the half-maximum inhibition (IC(50)) values of 83.1μM and 104.6μM, respectively. Structure-function analysis indicated that the increased bulky methoxy groups on the phenyl ring or the negatively charged oxygen atom may account for this lack of inhibition on the Na(v)1.5 channel. Strychnine and icajine may bind to the channel by cation-π interactions. The substitution with a large side chain on the phenyl ring or the increased molecular volume may alter the optimized position for the compound close to the binding sites of the channel. Strychnine and icajine bind to the Na(v)1.5 channel with a new mechanism that is different from TTX and local anesthetics. They bind to the outer vestibule of the channel pore with fast association and dissociation rates at resting state. Strychnine and icajine had little effect on steady-state fast inactivation but markedly shifted the slow inactivation of Na(v)1.5 currents toward more hyperpolarized potentials. The property of icajine influencing slow-inactivated state of Na(v)1.5 channel would be potential therapeutic advantages in certain arrhythmias. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of temperature acclimation on red blood cell oxygen affinity in Pacific bluefin tuna (Thunnus orientalis) and yellowfin tuna (Thunnus albacares).

PubMed

Lilly, Laura E; Bonaventura, Joseph; Lipnick, Michael S; Block, Barbara A

2015-03-01

Hemoglobin-oxygen (Hb-O2) binding properties are central to aerobic physiology, and must be optimized for an animal's aerobic requirements and environmental conditions, both of which can vary widely with seasonal changes or acutely with diving. In the case of tunas, the matter is further complicated by large regional temperature differences between tissues within the same animal. This study investigates the effects of thermal acclimation on red blood cell Hb-O2 binding in Pacific bluefin tuna (T. orientalis) and yellowfin tuna (T. albacares) maintained in captive tanks at acclimation temperatures of 17°, 20° and 24 °C. Oxygen binding properties of acclimated tuna isolated red blood cells were examined under varying experimental temperatures (15°-35 °C) and CO2 levels (0%, 0.5% and 1.5%). Results for Pacific bluefin tuna produced temperature-independence at 17 °C- and 20 °C-acclimation temperatures and significant reverse temperature-dependence at 24 °C-acclimation in the absence of CO2, with instances of reverse temperature-dependence in 17 °C- and 24 °C-acclimations at 0.5% and 1.5% CO2. In contrast, yellowfin tuna produced normal temperature-dependence at each acclimation temperature at 0% CO2, temperature-independence at 0.5% and 1.5% CO2, and significant reverse temperature-dependence at 17 °C-acclimation and 0.5% CO2. Thermal acclimation of Pacific bluefin tuna increased O2 binding affinity of the 17 °C-acclimation group, and produced a significantly steeper oxygen equilibrium curve slope (nH) at 24 °C-acclimation compared to the other acclimation temperatures. We discuss the potential implications of these findings below. Copyright © 2014 Elsevier Inc. All rights reserved.

Size-dependent impact of CNTs on dynamic properties of calmodulin.

PubMed

Gao, Jian; Wang, Liming; Kang, Seung-gu; Zhao, Lina; Ji, Mingjuan; Chen, Chunying; Zhao, Yuliang; Zhou, Ruhong; Li, Jingyuan

2014-11-07

There are growing concerns about the biosafety of nanomaterials such as carbon nanotubes (CNTs) as their applications become more widespread. We report here a theoretical and experimental study of the binding of various sizes of CNTs [CNT (4,4), (5,5), (6,6) and (7,7)] to calmodulin (CaM) protein and, in particular, their impact on the Ca(2+)-dependent dynamic properties of CaM. Our simulations show that all the CNTs can plug into the hydrophobic binding pocket of Ca(2+)-bound CaM with binding affinities comparable with the native substrate M13 peptide. Even though CNT (4,4) shows a similar behavior to the M13 peptide in its dissociation from Ca(2+)-free CaM, wider CNTs still bind firmly to CaM, indicating a potential failure of Ca(2+) regulation. Such a size-dependent impact of CNTs on the dynamic properties of CaM is a result of the excessively strong hydrophobic interactions between the wider CNTs and CaM. These simulation results were confirmed by circular dichroism spectroscopy, which showed that the secondary structures of CaM become insensitive to Ca(2+) concentrations after the addition of CNTs. Our findings indicate that the cytotoxicity of nanoparticles to proteins arises not only from the inhibition of static protein structures (binding pockets), but also from impacts on their dynamic properties.

Cation induced electrochromism in 2,4-dinitrophenylhydrazine (DNPH): Tuning optical properties of aromatic rings

NASA Astrophysics Data System (ADS)

Sanader, Željka; Brunet, Claire; Broyer, Michel; Antoine, Rodolphe; Dugourd, Philippe; Mitrić, Roland; Bonačić-Koutecký, Vlasta

2013-05-01

We have theoretically investigated the influence of protons and noble metal cations on optical properties of 2,4-dinitrophenylhydrazine (DNPH). We show that optical properties of aromatic rings can be tuned by cation-induced electrochromism in DNPH due to binding to specific NO2 groups. Our findings on cation-induced electrochromism in DNPH may open new routes in two different application areas, due to the fact that DNPH can easily bind to biological molecules and surface materials through carbonyl groups.

Complex Actions of Thyroid Hormone Receptor Antagonist NH-3 on Gene Promoters in Different Cell Lines

PubMed Central

Shah, Vanya; Nguyen, Phuong; Nguyen, Ngoc-Ha; Togashi, Marie; Scanlan, Thomas S.; Baxter, John D.; Webb, Paul

2014-01-01

It is desirable to obtain new antagonists for thyroid hormone (TRs) and other nuclear receptors (NRs). We previously used X-ray structural models of TR ligand binding domains (LBDs) to design compounds, such as NH-3, that impair coactivator binding to activation function 2 (AF-2) and block thyroid hormone (triiodothyronine, T3) actions. However, TRs bind DNA and are transcriptionally active without ligand. Thus, NH-3 could modulate TR activity via effects on other coregulator interaction surfaces, such as activation function (AF-1) and corepressor binding sites. Here, we find that NH-3 blocks TR-LBD interactions with coactivators and corepressors and also inhibits activities of AF-1 and AF-2 in transfections. While NH-3 lacks detectable agonist activity at T3-activated genes in GC pituitary cells it nevertheless activates spot 14 (S14) in HTC liver cells with the latter effect accompanied by enhanced histone H4 acetylation and coactivator recruitment at the S14 promoter. Surprisingly, T3 promotes corepressor recruitment to target promoters. NH-3 effects vary; we observe transient recruitment of N-CoR to S14 in GC cells and dismissal and rebinding of N-CoR to the same promoter in HTC cells. We propose that NH-3 will generally behave as an antagonist by blocking AF-1 and AF-2 but that complex effects on coregulator recruitment may result in partial/mixed agonist effects that are independent of blockade of T3 binding in some contexts. These properties could ultimately be utilized in drug design and development of new selective TR modulators. PMID:18930112

From the Arctic to fetal life: physiological importance and structural basis of an 'additional' chloride-binding site in haemoglobin.

PubMed

De Rosa, M Cristina; Castagnola, Massimo; Bertonati, Claudia; Galtieri, Antonio; Giardina, Bruno

2004-06-15

Haemoglobins from mammals of sub-Arctic and Arctic species, as well as fetal human Hb, are all characterized by a significantly lower Delta H of oxygenation compared with the majority of mammalian haemoglobins from temperate species (exceptions are represented by some cold-resistant species, such as cow, horse and pig). This has been interpreted as an adaptive mechanism of great importance from a physiological point of view. To date, the molecular basis of this thermodynamic characteristic is still not known. In the present study, we show that binding of extra chloride (with respect to adult human Hb) ions to Hb would significantly contribute to lowering the overall heat of oxygenation, thus providing a molecular basis for the low effect of temperature on the oxygenation-deoxygenation cycle. To this aim, the oxygen binding properties of bovine Hb, bear (Ursus arctos) Hb and horse Hb, which are representative of this series of haemoglobins, have been studied with special regard to the effect of heterotropic ligands, such as organic phosphates (namely 2,3-diphosphoglycerate) and chloride. Functional results are consistent with a mechanism for ligand binding that involves an additional binding site for chloride ion. Analysis of computational chemistry results, obtained by the GRID program, further confirm the hypothesis that the reason for the lower Delta H of oxygenation is mainly due to an increase in the number of the oxygen-linked chloride-binding sites.

Three-dimensional structure-activity relationship modeling of cocaine binding to two monoclonal antibodies by comparative molecular field analysis.

PubMed

Paula, Stefan; Tabet, Michael R; Keenan, Susan M; Welsh, William J; Ball, W James

2003-01-17

Successful immunotherapy of cocaine addiction and overdoses requires cocaine-binding antibodies with specific properties, such as high affinity and selectivity for cocaine. We have determined the affinities of two cocaine-binding murine monoclonal antibodies (mAb: clones 3P1A6 and MM0240PA) for cocaine and its metabolites by [3H]-radioligand binding assays. mAb 3P1A6 (K(d) = 0.22 nM) displayed a 50-fold higher affinity for cocaine than mAb MM0240PA (K(d) = 11 nM) and also had a greater specificity for cocaine. For the systematic exploration of both antibodies' binding specificities, we used a set of approximately 35 cocaine analogues as structural probes by determining their relative binding affinities (RBAs) using an enzyme-linked immunosorbent competition assay. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models on the basis of comparative molecular field analysis (CoMFA) techniques correlated the binding data with structural features of the ligands. The analysis indicated that despite the mAbs' differing specificities for cocaine, the relative contributions of the steric (approximately 80%) and electrostatic (approximately 20%) field interactions to ligand-binding were similar. Generated three-dimensional CoMFA contour plots then located the specific regions about cocaine where the ligand/receptor interactions occurred. While the overall binding patterns of the two mAbs had many features in common, distinct differences were observed about the phenyl ring and the methylester group of cocaine. Furthermore, using previously published data, a 3D-QSAR model was developed for cocaine binding to the dopamine reuptake transporter (DAT) that was compared to the mAb models. Although the relative steric and electrostatic field contributions were similar to those of the mAbs, the DAT cocaine-binding site showed a preference for negatively charged ligands. Besides establishing molecular level insight into the interactions that govern cocaine binding specificity by biopolymers, the three-dimensional images obtained reflect the properties of the mAbs binding pockets and provide the initial information needed for the possible design of novel antibodies with properties optimized for immunotherapy. Copyright 2003 Elsevier Science Ltd.

What Is the True Color of Fresh Meat? A Biophysical Undergraduate Laboratory Experiment Investigating the Effects of Ligand Binding on Myoglobin Using Optical, EPR, and NMR Spectroscopy

ERIC Educational Resources Information Center

Linenberger, Kimberly; Bretz, Stacey Lowery; Crowder, Michael W.; McCarrick, Robert; Lorigan, Gary A.; Tierney, David L.

2011-01-01

With an increased focus on integrated upper-level laboratories, we present an experiment integrating concepts from inorganic, biological, and physical chemistry content areas. Students investigate the effects of ligand strength on the spectroscopic properties of the heme center in myoglobin using UV-vis, [superscript 1]H NMR, and EPR…

n-Dodecyl β-D-maltoside specifically competes with general anesthetics for anesthetic binding sites.

PubMed

Xu, Longhe; Matsunaga, Felipe; Xi, Jin; Li, Min; Ma, Jingyuan; Liu, Renyu

2014-01-01

We recently demonstrated that the anionic detergent sodium dodecyl sulfate (SDS) specifically interacts with the anesthetic binding site in horse spleen apoferritin, a soluble protein which models anesthetic binding sites in receptors. This raises the possibility of other detergents similarly interacting with and occluding such sites from anesthetics, thereby preventing the proper identification of novel anesthetic binding sites. n-Dodecyl β-D-maltoside (DDM) is a non-ionic detergent commonly used during protein-anesthetic studies because of its mild and non-denaturing properties. In this study, we demonstrate that SDS and DDM occupy anesthetic binding sites in the model proteins human serum albumin (HSA) and horse spleen apoferritin and thereby inhibit the binding of the general anesthetics propofol and isoflurane. DDM specifically interacts with HSA (Kd = 40 μM) with a lower affinity than SDS (Kd = 2 μM). DDM exerts all these effects while not perturbing the native structures of either model protein. Computational calculations corroborated the experimental results by demonstrating that the binding sites for DDM and both anesthetics on the model proteins overlapped. Collectively, our results indicate that DDM and SDS specifically interact with anesthetic binding sites and may thus prevent the identification of novel anesthetic sites. Special precaution should be taken when undertaking and interpreting results from protein-anesthetic investigations utilizing detergents like SDS and DDM.

Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay.

PubMed

Rupesh, Kanchi Ravi; Smith, Aaron; Boehmer, Paul E

2014-11-28

We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1°C for (dT)5 to a maximum of 9°C with oligomers ⩾10 nucleotides, with an apparent Kd of <1μM for (dT)20. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9°C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner. Copyright © 2014 Elsevier Inc. All rights reserved.

Potential antidepressant properties of IDN 5491 (hyperforin-trimethoxybenzoate), a semisynthetic ester of hyperforin.

PubMed

Cervo, Luigi; Mennini, Tiziana; Rozio, Marco; Ekalle-Soppo, Charlotte Blanche; Canetta, Alessandro; Burbassi, Silvia; Guiso, Giovanna; Pirona, Lorenza; Riva, Antonella; Morazzoni, Paolo; Caccia, Silvio; Gobbi, Marco

2005-03-01

Hyperforin is one of the possible active principles mediating the antidepressant activity of Hypericum perforatum L. extracts. The ester derivative IDN 5491 (hyperforin-trimethoxybenzoate) showed antidepressant-like properties in the forced swimming test (FST) in rats, with no effect on open-field activity, when given as three intraperitoneal injections in 24 h at 3.125 and 6.25 mg/kg. The plasma concentrations of IDN 5491 were 30-50 microM, and those of hyperforin much lower but still close to those after effective doses of hyperforin-dicyclohexylammonium and Hypericum extract. This suggests that hyperforin plays a role in the antidepressant-like effect of the ester and of Hypericum extract. In vitro binding and uptake data showed that IDN 5491 is inactive on a wide panel of CNS targets at a concentration (14 microM) much higher than that measured in the brain of treated rats (0.3 microM). Like the extract, the antidepressant-like effect of IDN 5491 was blocked by (-)-sulpiride, a selective D2 receptor antagonist and by BD-1047, a selective sigma1 antagonist. Ex-vivo binding studies showed that brain sigma1 receptors are occupied after in vivo treatment with IDN 5491, possibly by an unknown metabolite or by endogenous ligand induced by hyperforin.

Effect of the degree of disorder on electronic and optical properties in random superlattices

NASA Technical Reports Server (NTRS)

Wang, E. G.; Su, W. P.; Ting, C. S.

1994-01-01

A three-dimensional tight-binding calculation is developed and used to study disorder effects in a realistic random superlattice. With increasing disorder, a tendency of possible indirect-direct band-gap transition is suggested. Direct evidence of mobility edges between localized and extended states in three-dimensional random systems is given. As system disorder increases, the optical absorption intensities increase dramatically from five to forty-five times stronger than the ordered (GaAs)(sub 1)/(AlAs)(sub 1) superlattice. It is believed that the degree of disorder significantly affects electronic and optical properties of GaAs/AlAs random superlattices.

Retention and loss of water extractable carbon in soils: effect of clay properties.

PubMed

Nguyen, Trung-Ta; Marschner, Petra

2014-02-01

Clay sorption is important for organic carbon (C) sequestration in soils, but little is known about the effect of different clay properties on organic C sorption and release. To investigate the effect of clay content and properties on sorption, desorption and loss of water extractable organic C (WEOC), two experiments were conducted. In experiment 1, a loamy sand alone (native) or mixed with clay isolated from a surface or subsoil (78 and 96% clay) resulting in 90, 158 and 175 g clay kg(-1) soil. These soil treatments were leached with different WEOC concentrations, and then CO2 release was measured for 28 days followed by leaching with reverse osmosis water at the end of experiment. The second experiment was conducted to determine WEOC sorption and desorption of clays isolated from the loamy sand (native), surface soil and subsoil. Addition of clays isolated from surface and subsoil to sandy loam increased WEOC sorption and reduced C leaching and cumulative respiration in percentage of total organic C and WEOC added when expressed per g soil and per g clay. Compared to clays isolated from the surface and subsoil, the native clay had higher concentrations of illite and exchangeable Ca(2+), total organic C and a higher CEC but a lower extractable Fe/Al concentration. This indicates that compared to the clay isolated from the surface and the subsoil, the native clay had fewer potential WEOC binding sites because it had lower Fe/Al content thus lower number of binding sites and the existing binding sites are already occupied native organic matter. The results of this study suggest that in the soils used here, the impact of clay on WEOC sorption and loss is dependent on its indigenous organic carbon and Fe and/or Al concentrations whereas clay mineralogy, CEC, exchangeable Ca(2+) and surface area are less important. © 2013.

Chimeric peptides as implant functionalization agents for titanium alloy implants with antimicrobial properties.

PubMed

Yucesoy, Deniz T; Hnilova, Marketa; Boone, Kyle; Arnold, Paul M; Snead, Malcolm L; Tamerler, Candan

2015-04-01

Implant-associated infections can have severe effects on the longevity of implant devices and they also represent a major cause of implant failures. Treating these infections associated with implants by antibiotics is not always an effective strategy due to poor penetration rates of antibiotics into biofilms. Additionally, emerging antibiotic resistance poses serious concerns. There is an urge to develop effective antibacterial surfaces that prevent bacterial adhesion and proliferation. A novel class of bacterial therapeutic agents, known as antimicrobial peptides (AMP's), are receiving increasing attention as an unconventional option to treat septic infection, partly due to their capacity to stimulate innate immune responses and for the difficulty of microorganisms to develop resistance towards them. While host- and bacterial- cells compete in determining the ultimate fate of the implant, functionalization of implant surfaces with antimicrobial peptides can shift the balance and prevent implant infections. In the present study, we developed a novel chimeric peptide to functionalize the implant material surface. The chimeric peptide simultaneously presents two functionalities, with one domain binding to a titanium alloy implant surface through a titanium-binding domain while the other domain displays an antimicrobial property. This approach gains strength through control over the bio-material interfaces, a property built upon molecular recognition and self-assembly through a titanium alloy binding domain in the chimeric peptide. The efficiency of chimeric peptide both in-solution and absorbed onto titanium alloy surface was evaluated in vitro against three common human host infectious bacteria, S. mutans, S. epidermidis , and E. coli . In biological interactions such as occurs on implants, it is the surface and the interface that dictate the ultimate outcome. Controlling the implant surface by creating an interface composed chimeric peptides may therefore open up new possibilities to cover the implant site and tailor it to a desirable bioactivity.

Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

PubMed Central

King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; Mcdougal, Owen M.

2017-01-01

Computational molecular docking is a fast and effective in silico method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The DockoMatic tutorial described herein provides a framework by which instructors can guide students through a drug screening exercise. Using receptor models derived from readily available protein crystal structures, docking programs have the ability to predict ligand binding properties, such as preferential binding orientations and binding affinities. The use of computational studies can significantly enhance complimentary wet chemical experimentation by providing insight into the important molecular interactions within the system of interest, as well as guide the design of new candidate ligands based on observed binding motifs and energetics. In this laboratory tutorial, the graphical user interface, DockoMatic, facilitates docking job submissions to the docking engine, AutoDock 4.2. The purpose of this exercise is to successfully dock a 17-amino acid peptide, α-conotoxin TxIA, to the acetylcholine binding protein from Aplysia californica-AChBP to determine the most stable binding configuration. Each student will then propose two specific amino acid substitutions of α-conotoxin TxIA to enhance peptide binding affinity, create the mutant in DockoMatic, and perform docking calculations to compare their results with the class. Students will also compare intermolecular forces, binding energy, and geometric orientation of their prepared analog to their initial α-conotoxin TxIA docking results. PMID:26537635

Differential effects of grape ( Vitis vinifera ) skin polyphenolics on human platelet aggregation and low-density lipoprotein oxidation.

PubMed

Shanmuganayagam, Dhanansayan; Beahm, Mark R; Kuhns, Melissa A; Krueger, Christian G; Reed, Jess D; Folts, John D

2012-06-13

Antioxidant and antiplatelet properties of grape products are thought to be responsible for observed antiatherosclerotic effects. Diverse classes of phenolics are derived from the seed and skin (GSK) of grapes. The relative contributions of the classes of phenolics to observed properties of grape products are unknown. In this paper, GSK fractions were used to examine effects on platelet aggregation, low-density lipoprotein (LDL) oxidation in vitro, and relative binding of phenolics to LDL. GSK was separated into six fractions (fractions 1-6), and primary phenolics were characterized using high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Fractions 4, 5, and 6, enriched in polygalloyl polyflavan-3-ols (PGPFs) with 3-6, 4-8, and 6-15 degrees of polymerization, respectively, inhibited platelet aggregation. Fractions 1-3, containing various amounts of oligosaccharides, hydroxycinnamic acids, anthocyanins, flavanols, and low molecular weight PGPFs, significantly increased platelet aggregation. Fractions 4-6 were most effective in binding LDL and inhibiting LDL oxidation. Fractions 5 and 6 exhibited the greatest inhibition of platelet aggregation and LDL oxidation, suggesting that polymeric PGPFs are responsible for the beneficial effects of grape products. Conversely, phenolics in fractions 1-3 may reduce the net biological potency of the grape products and have undesirable effects on cardiovascular disease risk factors.

The impact of low and no-caloric sweeteners on glucose absorption, incretin secretion, and glucose tolerance.

PubMed

Chan, Catherine B; Hashemi, Zohre; Subhan, Fatheema B

2017-08-01

The consumption of non-nutritive, low, or no-calorie sweeteners (LCS) is increasing globally. Previously thought to be physiologically inert, there is a growing body of evidence that LCS not only provide a sweet taste but may also elicit metabolic effects in the gastrointestinal tract. This review provides a brief overview of the chemical and receptor-binding properties and effects on chemosensation of different LCS but focuses on the extent to which LCS stimulates glucose transport, incretin and insulin secretion, and effects on glucose tolerance. Aspartame and sucralose both bind to a similar region of the sweet receptor. For sucralose, the data are contradictory regarding effects on glucose tolerance in humans and may depend on the food or beverage matrix and the duration of administration, as suggested by longer term rodent studies. For aspartame, there are fewer data. On the other hand, acesulfame-potassium (Ace-K) and saccharin have similar binding characteristics to each other but, while Ace-K may increase incretin secretion and glucose responses in humans, there are no data on saccharin except in rats, which show impaired glucose tolerance after chronic administration. Additional research, particularly of the effects of chronic consumption, is needed to provide concrete evidence for beneficial or detrimental effects of LCS on blood glucose regulation in humans.

Whey protein hydrolysates decrease IL-8 secretion in lipopolysaccharide (LPS)-stimulated respiratory epithelial cells by affecting LPS binding to Toll-like receptor 4.

PubMed

Iskandar, Michèle M; Dauletbaev, Nurlan; Kubow, Stan; Mawji, Nadir; Lands, Larry C

2013-07-14

Whey proteins (WP) exert anti-inflammatory and antioxidant effects. Hyperbaric pressurisation of whey increases its digestibility and changes the spectrum of peptides released during digestion. We have shown that dietary supplementation with pressurised whey improves nutritional status and systemic inflammation in patients with cystic fibrosis (CF). Both clinical indices are largely affected by airway processes, to which respiratory epithelial cells actively contribute. Here, we tested whether peptides released from the digestion of pressurised whey can attenuate the inflammatory responses of CF respiratory epithelial cells. Hydrolysates of pressurised WP (pWP) and native WP (nWP, control) were generated in vitro and tested for anti-inflammatory properties judged by the suppression of IL-8 production in CF and non-CF respiratory epithelial cell lines (CFTE29o- and 1HAEo-, respectively). We observed that, in both cell lines, pWP hydrolysate suppressed IL-8 production stimulated by lipopolysaccharide (LPS) to a greater magnitude compared with nWP hydrolysate. Neither hydrolysate suppressed IL-8 production induced by TNF-α or IL-1β, suggesting an effect on the Toll-like receptor (TLR) 4 pathway, the cellular sensor for LPS. Further, neither hydrolysate affected TLR4 expression or neutralised LPS. Both pWP and nWP hydrolysates similarly reduced LPS binding to surface TLR4, while pWP tended to more potently increase extracellular antioxidant capacity. (1) anti-inflammatory properties of whey are enhanced by pressurisation; (2) suppression of IL-8 production may contribute to the clinical effects of pressurised whey supplementation on CF; (3) this effect may be partly explained by a combination of reduced LPS binding to TLR4 and enhanced extracellular antioxidant capacity.

Serum albumin forms a lactoferrin-like soluble iron-binding complex in presence of hydrogen carbonate ions.

PubMed

Ueno, Hiroshi M; Urazono, Hiroshi; Kobayashi, Toshiya

2014-02-15

The iron-lactoferrin complex is a common food ingredient because of its iron-solubilizing capability in the presence of hydrogen carbonate ions. However, it is unclear whether the formation of a stable iron-binding complex is limited to lactoferrin. In this study, we investigated the effects of bovine serum albumin (BSA) on iron solubility and iron-catalyzed lipid oxidation in the presence of hydrogen carbonate ions. BSA could solubilize >100-fold molar equivalents of iron at neutral pH, exceeding the specific metal-binding property of BSA. This iron-solubilizing capability of BSA was impaired by thermally denaturing BSA at ≥ 70 °C for 10 min at pH 8.5. The resulting iron-BSA complex inhibited iron-catalyzed oxidation of soybean oil in a water-in-oil emulsion measured using the Rancimat test. Our study is the first to show that BSA, like lactoferrin, forms a soluble iron-binding complex in the presence of hydrogen carbonate ions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Linear Scaling of the Exciton Binding Energy versus the Band Gap of Two-Dimensional Materials

NASA Astrophysics Data System (ADS)

Choi, Jin-Ho; Cui, Ping; Lan, Haiping; Zhang, Zhenyu

2015-08-01

The exciton is one of the most crucial physical entities in the performance of optoelectronic and photonic devices, and widely varying exciton binding energies have been reported in different classes of materials. Using first-principles calculations within the G W -Bethe-Salpeter equation approach, here we investigate the excitonic properties of two recently discovered layered materials: phosphorene and graphene fluoride. We first confirm large exciton binding energies of, respectively, 0.85 and 2.03 eV in these systems. Next, by comparing these systems with several other representative two-dimensional materials, we discover a striking linear relationship between the exciton binding energy and the band gap and interpret the existence of the linear scaling law within a simple hydrogenic picture. The broad applicability of this novel scaling law is further demonstrated by using strained graphene fluoride. These findings are expected to stimulate related studies in higher and lower dimensions, potentially resulting in a deeper understanding of excitonic effects in materials of all dimensionalities.

Investigating isoindoline, tetrahydroisoquinoline, and tetrahydrobenzazepine scaffolds for their sigma receptor binding properties.

PubMed

Linkens, Kathryn; Schmidt, Hayden R; Sahn, James J; Kruse, Andrew C; Martin, Stephen F

2018-05-10

Substituted norbenzomorphans are known to display high affinity and selectivity for the two sigma receptor (σR) subtypes. In order to study the effects of simplifying the structures of these compounds, a scaffold hopping strategy was used to design several novel sets of substituted isoindolines, tetrahydroisoquinolines and tetrahydro-2-benzazepines. The binding affinities of these new compounds for the sigma 1 (σ1R) and sigma 2 (σ2R) receptors were determined, and some analogs were identified that exhibit high affinity (K i  ≤ 25 nM) and significant selectivity (>10-fold) for σ1R or σ2R. The preferred binding modes of selected compounds for the σ1R are predicted by modeling studies, and the nature of substituents on the aromatic ring and the nitrogen atom of the bicyclic skeleton appears to affect the preferred binding orientation of σ1R-preferring ligands. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit Vps25.

PubMed

Emerman, Amy B; Blower, Michael

2018-06-14

RNA-binding proteins (RBPs) are critical regulators of gene expression. Recent studies have uncovered hundreds of mRNA-binding proteins that do not contain annotated RNA-binding domains and have well-established roles in other cellular processes. Investigation of these nonconventional RBPs is critical for revealing novel RNA-binding domains and may disclose connections between RNA regulation and other aspects of cell biology. Endosomal sorting complex required for transport II (ESCRT-II) is a nonconventional RNA-binding complex that has a canonical role in multivesicular body formation. ESCRT-II previously has been identified as an RNA-binding complex in Drosophila oocytes, but whether its RNA-binding properties extend beyond Drosophila is unknown. In this study, we found that the RNA-binding properties of ESCRT-II are conserved in Xenopus eggs, where ESCRT-II interacted with hundreds of mRNAs. Using a UV-crosslinking approach, we demonstrated that ESCRT-II binds directly to RNA through its subunit Vps25. UV-crosslinking and immunoprecipitation (CLIP)-Seq revealed that Vps25 specifically recognizes a polypurine (i.e. GA-rich) motif in RNA. Using purified components, we could reconstitute the selective Vps25-mediated binding of the polypurine motif in vitro. Our results provide insight into the mechanism by which ESCRT-II selectively binds to mRNAs and also suggest an unexpected link between endosome biology and RNA regulation. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanically tunable actin networks using programmable DNA based cross-linkers

NASA Astrophysics Data System (ADS)

Schnauss, Joerg; Lorenz, Jessica; Schuldt, Carsten; Kaes, Josef; Smith, David

Cells employ multiple cross-linkers with very different properties. Studies of the entire phase space, however, were infeasible since they were restricted to naturally occurring cross-linkers. These components cannot be controllably varied and differ in many parameters. We resolve this limitation by forming artificial actin cross-linkers, which can be controllably varied. The basic building block is DNA enabling a well-defined length variation. DNA can be attached to actin binding peptides with known binding affinities. We used bulk rheology to investigate mechanical properties of these networks. We were able to reproduce mechanical features of actin networks cross-linked by fascin by using a short version of our artificial complex with a high binding affinity. Additionally, we were able to resemble findings for the cross-linker alpha-actinin by employing a long cross-linker with a low binding affinity. Between these natural limits we investigated three different cross-linker lengths each with two different binding affinities. With these controlled variations we are able to precisely screen the phase space of cross-linked actin networks by changing only one specific parameter and not the entire set of properties as in the case of naturally occurring cross-linking complexes.

The Identity-Location Binding Problem.

PubMed

Howe, Piers D L; Ferguson, Adam

2015-09-01

The binding problem is fundamental to visual perception. It is the problem of associating an object's visual properties with itself and not with some other object. The problem is made particular difficult because different properties of an object, such as its color, shape, size, and motion, are often processed independently, sometimes in different cortical areas. The results of these separate analyses have to be combined before the object can be seen as a single coherent entity as opposed to a collection of unconnected features. Visual bindings are typically initiated and updated in a serial fashion, one object at a time. Here, we show that one type of binding, location-identity bindings, can be updated in parallel. We do this by using two complementary techniques, the simultaneous-sequential paradigm and systems factorial technology. These techniques make different assumptions and rely on different behavioral measures, yet both came to the same conclusion. Copyright © 2014 Cognitive Science Society, Inc.

In vitro evidence for RNA binding properties of the coat protein of prunus necrotic ringspot ilarvirus and their comparison to related and unrelated viruses.

PubMed

Pallás, V; Sánchez-Navarro, J A; Díez, J

1999-01-01

The RNA binding properties of the prunus necrotic ringspot virus (PNRSV) coat protein (CP) were demonstrated by northwestern and dot-blot analyses. The capability to bind PNRSV RNA 4 was compared with viruses representing three different interactions prevailing in the assembly and architecture of virions. The results showed that cucumber mosaic virus (CMV) and PNRSV CPs, which stabilise their virions mainly through RNA-protein interactions bound PNRSV RNA 4 even at very high salt concentrations. The CP of cherry leaf roll nepovirus, whose virions are predominantly stabilised by protein-protein interactions did not bind even at the lowest salt concentration tested. Finally the CP of carnation mottle carmovirus, that has an intermediate position in which both RNA-protein and protein-protein interactions are equally important showed a salt-dependent RNA binding.

Interlayer coupling and electronic structure of misfit-layered bismuth-based cobaltites

NASA Astrophysics Data System (ADS)

Takakura, Sho-ichi; Yamamoto, Isamu; Tanaka, Eishi; Azuma, Junpei; Maki, Makoto

2017-05-01

The [Bi2M2O4] pCoO2 materials (M =Ca , Sr, and Ba) were studied to clarify the effect of the lattice incommensurability on electronic properties using angle-resolved photoemission spectroscopy and transmission electron microscopy (TEM). Results show that the insulating behavior is characterized by a spectral weight for binding energies higher than 2.0 eV. Moreover, the spectral shape is modified as a function of the incident photon energy, demonstrating a close relationship between the electrical properties and interlayer coupling. TEM results show that the effect of the lattice mismatch differs for different misfit parameters p . We therefore conclude that the carrier concentration and the chemical environment at the misfit interface, which depend on the degree of incommensurability, mutually determine the electronic properties of the system.

The β subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin

PubMed Central

Hanner, Markus; Schmalhofer, William A.; Munujos, Petraki; Knaus, Hans-Günther; Kaczorowski, Gregory J.; Garcia, Maria L.

1997-01-01

Transient expression of either α or α+β subunits of the high-conductance Ca2+-activated K+ (maxi-K) channel has been achieved in COS-1 cells. Expression has been studied using charybdotoxin (ChTX), a peptidyl inhibitor that binds in the pore on the α subunit. Although some properties of monoiodotyrosine-ChTX (125I-ChTX) binding to membranes derived from each type of transfected cells appear to be identical, other parameters of the binding reaction are markedly different. Under low ionic strength conditions, the affinity constant for 125I-ChTX measured under equilibrium binding conditions is increased ca. 50-fold in the presence of the β subunit. The rate constant for 125I-ChTX association is enhanced ca. 5-fold, whereas the dissociation rate constant is decreased more than 7-fold when the β subunit is present. These data indicate that functional coassembly of maxi-K channel subunits can be obtained in a transient expression system, and that the β subunit has profound effects on 125I-ChTX binding. We postulate that certain negatively charged residues in the large extracellular loop of β attract the positively charged 125I-ChTX to its binding site on α through electrostatic interactions, and account for effects observed on ligand association kinetics. Moreover, another residue(s) in the loop of β must contribute to stabilization of the toxin-bound state, either by a direct interaction with toxin, or through an allosteric effect on the α subunit. Certain regions in the extracellular loop of the β subunit may be in close proximity to the pore of the channel, and could play an important role in maxi-K channel function. PMID:9096310

Salivary proline-rich protein may reduce tannin-iron chelation: a systematic narrative review.

PubMed

Delimont, Nicole M; Rosenkranz, Sara K; Haub, Mark D; Lindshield, Brian L

2017-01-01

Tannins are often cited for antinutritional effects, including chelation of non-heme iron. Despite this, studies exploring non-heme iron bioavailability inhibition with long-term consumption have reported mixed results. Salivary proline-rich proteins (PRPs) may mediate tannin-antinutritional effects on non-heme iron bioavailability. To review evidence regarding biochemical binding mechanisms and affinity states between PRPs and tannins, as well as effects of PRPs on non-heme iron bioavailability with tannin consumption in vivo. Narrative systematic review and meta-analysis. Common themes in biochemical modeling and affinity studies were collated for summary and synthesis; data were extracted from in vivo experiments for meta-analysis. Thirty-two studies were included in analysis. Common themes that positively influenced tannin-PRP binding included specificity of tannin-PRP binding, PRP and tannin stereochemistry. Hydrolyzable tannins have different affinities than condensed tannins when binding to PRPs. In vivo, hepatic iron stores and non-heme iron absorption are not significantly affected by tannin consumption ( d  = -0.64-1.84; -2.7-0.13 respectively), and PRP expression may increase non-heme iron bioavailability with tannin consumption. In vitro modeling suggests that tannins favor PRP binding over iron chelation throughout digestion. Hydrolyzable tannins are not representative of tannin impact on non-heme iron bioavailability in food tannins because of their unique structural properties and PRP affinities. With tannin consumption, PRP production is increased, and may be an initial line of defense against tannin-non-heme iron chelation in vivo . More research is needed to compare competitive binding of tannin-PRP to tannin-non-heme iron complexes, and elucidate PRPs' role in adaption to non-heme iron bioavailability in vivo.

Recognition and binding of β-lactam antibiotics to bovine serum albumin by frontal affinity chromatography in combination with spectroscopy and molecular docking.

PubMed

Li, Qian; Zhang, Tianlong; Bian, Liujiao

2016-03-01

Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical β-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV-vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22×10(1), 4.86×10(2) and 3.32×10(3) (L/mol), respectively. All the three β-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with β-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties. Copyright © 2016 Elsevier B.V. All rights reserved.

Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

PubMed

Yang, Danlin; Giragossian, Craig; Castellano, Steven; Lasaro, Marcio; Xiao, Haiguang; Saraf, Himanshu; Hess Kenny, Cynthia; Rybina, Irina; Huang, Zhong-Fu; Ahlberg, Jennifer; Bigwarfe, Tammy; Myzithras, Maria; Waltz, Erica; Roberts, Simon; Kroe-Barrett, Rachel; Singh, Sanjaya

2017-10-01

Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

PubMed

Carbone, V; Kim, H; Huang, J X; Baker, M A; Ong, C; Cooper, M A; Li, J; Rockman, S; Velkov, T

2013-01-01

Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

Effect of Stapling Architecture on Physiochemical Properties and Cell Permeability of Stapled α-Helical Peptides: A Comparative Study.

PubMed

Tian, Yuan; Jiang, Yanhong; Li, Jingxu; Wang, Dongyuan; Zhao, Hui; Li, Zigang

2017-11-02

Stapled peptides have emerged as a new class of targeting molecules with high binding affinity and specificity for intracellular undruggable targets. Their ability to penetrate cell membranes is exceptionally intriguing but remains elusively and controversially discussed. To understand the effect of stapling architectures on their physiochemical properties and to aid in promoting their cell permeability, we report herein a comparative study on the physiochemical properties and cell permeability of stapled α-helical peptides with different types of crosslinks. We highlight the decisive impact of the intrinsic properties of the crosslinks on cell permeability rather than the helical contents of the peptides in model amphipathic sequences targeting estrogen receptor-coactivator interaction. We envision this finding to shed further light on the chemical optimization of stapled α-helical peptides or macrocyclic cell-penetrating peptides for enhanced cell penetration. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of rare earth elements and REE-binding proteins on physiological responses in plants.

PubMed

Liu, Dongwu; Wang, Xue; Chen, Zhiwei

2012-02-01

Rare earth elements (REEs), which include 17 elements in the periodic table, share chemical properties related to a similar external electronic configuration. REEs enriched fertilizers have been used in China since the 1980s. REEs could enter the cell and cell organelles, influence plant growth, and mainly be bound with the biological macromolecules. REE-binding proteins have been found in some plants. In addition, the chlorophyll activities and photosynthetic rate can be regulated by REEs. REEs could promote the protective function of cell membrane and enhance the plant resistance capability to stress produced by environmental factors, and affect the plant physiological mechanism by regulating the Ca²⁺ level in the plant cells. The focus of present review is to describe how REEs and REE-binding proteins participate in the physiological responses in plants.

A single residue mutation in Hha preserving structure and binding to H-NS results in loss of H-NS mediated gene repression properties.

PubMed

Cordeiro, Tiago N; Garcia, Jesús; Pons, José-Ignacio; Aznar, Sonia; Juárez, Antonio; Pons, Miquel

2008-09-03

In this study, we report that a single mutation of cysteine 18 to isoleucine (C18I) in Escherichia coli Hha abolishes the repression of the hemolysin operon observed in the wild-type protein. The phenotype also includes a significant decrease in the growth rate of E. coli cells at low ionic strength. Other substitutions at this position (C18A, C18S) have no observable effects in E. coli growth or hemolysin repression. All mutants are stable and well folded and bind H-NS in vitro with similar affinities suggesting that Cys 18 is not directly involved in H-NS binding but this position is essential for the activity of the H-NS/Hha heterocomplexes in the regulation of gene expression.

Close binding of identity and location in visual feature perception

NASA Technical Reports Server (NTRS)

Johnston, J. C.; Pashler, H.

1990-01-01

The binding of identity and location information in disjunctive feature search was studied. Ss searched a heterogeneous display for a color or a form target, and reported both target identity and location. To avoid better than chance guessing of target identity (by choosing the target less likely to have been seen), the difficulty of the two targets was equalized adaptively; a mathematical model was used to quantify residual effects. A spatial layout was used that minimized postperceptual errors in reporting location. Results showed strong binding of identity and location perception. After correction for guessing, no perception of identity without location was found. A weak trend was found for accurate perception of target location without identity. We propose that activated features generate attention-calling "interrupt" signals, specifying only location; attention then retrieves the properties at that location.

Point defects in Cd(Zn)Te and TlBr: Theory

NASA Astrophysics Data System (ADS)

Lordi, Vincenzo

2013-09-01

The effects of various crystal defects on the performances of CdTe, CdZnxTe (CZT), and TlBr for room-temperature high-energy radiation detection are examined using first-principles theoretical methods. The predictive, parameter-free, atomistic approaches used provide fundamental understanding of defect properties that are difficult to measure and also allow rapid screening of possibilities for material engineering, such as optimal doping and annealing conditions. Several recent examples from the author's work are reviewed, including: (i) accurate calculations of the thermodynamic and electronic properties of native point defects and point defect complexes in CdTe and CZT; (ii) the effects of Zn alloying on the native point defect properties in CZT; (iii) point defect diffusion and binding leading to Te clustering in Cd(Zn)Te; (iv) the profound effect of native point defects—principally vacancies—on the intrinsic material properties of TlBr, particularly its electronic and ionic conductivity; and (v) a study on doping TlBr to independently control the electronic and ionic conductivity.

Effects of the Amino Acid Linkers on the Melanoma-Targeting and Pharmacokinetic Properties of Indium-111-labeled Lactam Bridge-Cyclized α-MSH Peptides

PubMed Central

Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

2011-01-01

The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma targeting and pharmacokinetic properties of novel 111In-labeled lactam bridge-cyclized DOTA-[X]-CycMSHhex {1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2, X=GlyGlyNle, GlyGluNle or NleGlyGlu} peptides. Methods Three novel DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex and DOTA-NleGE-CycMSHhex peptides were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. Results DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed 2.1 and 11.5 nM MC1 receptor binding affinities, whereas DOTA-NleGE-CycMSHhex showed 873.4 nM MC1 receptor binding affinity. The introduction of the -GlyGly- linker maintained high melanoma uptake while decreased the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex. The tumor uptake values of 111In-DOTA-GGNle-CycMSHhex were 19.05 ± 5.04 and 18.6 ± 3.56 % injected dose/gram (%ID/g) at 2 and 4 h post-injection. 111In-DOTA-GGNle-CycMSHhex exhibited 28, 32 and 42% less renal uptake values than 111In-DOTA-Nle-CycMSHhex we reported previously, and 61, 65 and 68% less liver uptake values than 111In-DOTA-Nle-CycMSHhex at 2, 4 and 24 h post-injection, respectively. Conclusion The amino acid linkers exhibited the profound effects on the melanoma targeting and pharmacokinetic properties of the 111In-labeled lactam bridge-cyclized α-MSH peptides. Introduction of the -GlyGly- linker maintained high melanoma uptake while reducing the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide. PMID:21421725

Effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of 111In-labeled lactam bridge-cyclized alpha-MSH peptides.

PubMed

Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

2011-04-01

The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of (111)In-labeled lactam bridge-cyclized DOTA-[X]-CycMSH(hex) {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH(2); X = GGNle, GENle, or NleGE; GG = -Gly-Gly- and GE = -Gly-Glu-} peptides. Three novel peptides (DOTA-GGNle-CycMSH(hex), DOTA-GENle-CycMSH(hex), and DOTA-NleGE-CycMSH(hex)) were designed and synthesized. The melanocortin-1 (MC1) receptor-binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma-targeting and pharmacokinetic properties of (111)In-DOTA-GGNle-CycMSH(hex) and (111)In-DOTA-GENle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. DOTA-GGNle-CycMSH(hex) and DOTA-GENle-CycMSH(hex) displayed 2.1 and 11.5 nM MC1 receptor-binding affinities, whereas DOTA-NleGE-CycMSH(hex) showed 873.4 nM MC1 receptor-binding affinity. The introduction of the -GG- linker maintained high melanoma uptake while decreasing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex). The tumor uptake of (111)In-DOTA-GGNle-CycMSH(hex) was 19.05 ± 5.04 and 18.6 ± 3.56 percentage injected dose per gram at 2 and 4 h after injection, respectively. (111)In-DOTA-GGNle-CycMSH(hex) exhibited 28%, 32%, and 42% less kidney uptake than (111)In-DOTA-Nle-CycMSH(hex) we reported previously, and 61%, 65%, and 68% less liver uptake than (111)In-DOTA-Nle-CycMSH(hex) at 2, 4, and 24 h after injection, respectively. The amino acid linkers exhibited profound effects on the melanoma-targeting and pharmacokinetic properties of the (111)In-labeled lactam bridge-cyclized α-melanocyte-stimulating hormone peptides. Introduction of the -GG- linker maintained high melanoma uptake while reducing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex), highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide.

Interface Schottky barrier engineering via strain in metal-semiconductor composites

NASA Astrophysics Data System (ADS)

Ma, Xiangchao; Dai, Ying; Yu, Lin; Huang, Baibiao

2016-01-01

The interfacial carrier transfer property, which is dominated by the interface Schottky barrier height (SBH), plays a crucial role in determining the performance of metal-semiconductor heterostructures in a variety of applications. Therefore, artificially controlling the interface SBH is of great importance for their industrial applications. As a model system, the Au/TiO2 (001) heterostructure is studied using first-principles calculations and the tight-binding method in the present study. Our investigation demonstrates that strain can be an effective way to decrease the interface SBH and that the n-type SBH can be more effectively decreased than the p-type SBH. Astonishingly, strain affects the interface SBH mainly by changing the intrinsic properties of Au and TiO2, whereas the interfacial potential alignment is almost independent of strain due to two opposite effects, which are induced by strain at the interfacial region. These observed trends can be understood on the basis of the general free-electron gas model of typical metals, the tight-binding theory and the crystal-field theory, which suggest that similar trends may be generalized for many other metal-semiconductor heterostructures. Given the commonness and tunability of strain in typical heterostructures, we anticipate that the tunability of the interface SBH with strain described here can provide an alternative effective way for realizing more efficient applications of relevant heterostructures.The interfacial carrier transfer property, which is dominated by the interface Schottky barrier height (SBH), plays a crucial role in determining the performance of metal-semiconductor heterostructures in a variety of applications. Therefore, artificially controlling the interface SBH is of great importance for their industrial applications. As a model system, the Au/TiO2 (001) heterostructure is studied using first-principles calculations and the tight-binding method in the present study. Our investigation demonstrates that strain can be an effective way to decrease the interface SBH and that the n-type SBH can be more effectively decreased than the p-type SBH. Astonishingly, strain affects the interface SBH mainly by changing the intrinsic properties of Au and TiO2, whereas the interfacial potential alignment is almost independent of strain due to two opposite effects, which are induced by strain at the interfacial region. These observed trends can be understood on the basis of the general free-electron gas model of typical metals, the tight-binding theory and the crystal-field theory, which suggest that similar trends may be generalized for many other metal-semiconductor heterostructures. Given the commonness and tunability of strain in typical heterostructures, we anticipate that the tunability of the interface SBH with strain described here can provide an alternative effective way for realizing more efficient applications of relevant heterostructures. Electronic supplementary information (ESI) available: The changes of Au 5d DOS, valence bands of TiO2, the interfacial bond length and interfacial energy with strain, and the local DOS results for the change of SBH with strain. See DOI: 10.1039/c5nr05583k

Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

PubMed

Muchima, Kaname; Todaka, Taro; Shinchi, Hiroyuki; Sato, Ayaka; Tazoe, Arisa; Aramaki, Rikiya; Kakitsubata, Yuhei; Yokoyama, Risa; Arima, Naomichi; Baba, Masanori; Wakao, Masahiro; Ito, Yuji; Suda, Yasuo

2018-04-01

Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.

Effect of van der Waals interactions on the structural and binding properties of GaSe

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

Sarkisov, Sergey Y., E-mail: sarkisov@mail.tsu.ru; Kosobutsky, Alexey V., E-mail: kosobutsky@kemsu.ru; Kemerovo State University, Krasnaya 6, 650043 Kemerovo

The influence of van der Waals interactions on the lattice parameters, band structure, elastic moduli and binding energy of layered GaSe compound has been studied using projector-augmented wave method within density functional theory. We employed the conventional local/semilocal exchange-correlation functionals and recently developed van der Waals functionals which are able to describe dispersion forces. It is found that application of van der Waals density functionals allows to substantially increase the accuracy of calculations of the lattice constants a and c and interlayer distance in GaSe at ambient conditions and under hydrostatic pressure. The pressure dependences of the a-parameter, Ga–Ga, Ga–Semore » bond lengths and Ga–Ga–Se bond angle are characterized by a relatively low curvature, while c(p) has a distinct downward bowing due to nonlinear shrinking of the interlayer spacing. From the calculated binding energy curves we deduce the interlayer binding energy of GaSe, which is found to be in the range 0.172–0.197 eV/layer (14.2–16.2 meV/Å{sup 2}). - Highlights: • Effects of van der Waals interactions are analyzed using advanced density functionals. • Calculations with vdW-corrected functionals closely agree with experiment. • Interlayer binding energy of GaSe is estimated to be 14.2–16.2 meV/Å{sup 2}.« less

( sup 3 H)-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and ( sup 3 H) ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

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

Branchek, T.; Adham, N.; Macchi, M.

1990-11-01

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to (3H)ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding themore » serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both (3H)DOB and (3H)ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p) to this system caused a rightward shift and steepening of agonist competition curves for (3H) ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity (3H)DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that (3H)DOB and (3H)ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein.« less

Gel Filtration Of Dilute Human Embryonic Hemoglobins Reveals Basis For Their Increased Oxygen Binding

PubMed Central

Manning, Lois R.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, James M.

2016-01-01

This report establishes a correlation between two known properties of the human embryonic hemoglobins-- their weak subunit assemblies as demonstrated here by gel filtration at very dilute protein concentrations and their high oxygen affinities and reduced cooperativities reported previously by others but without a mechanistic basis. We demonstrate here that their high oxygen affinities are a consequence of their weak assemblies. Weak vs strong hemoglobin tetramers represent a regulatory mechanism to modulate oxygen binding capacity by altering the equilibrium between the various steps in the assembly process that can be described as an inverse allosteric effect. PMID:27965062

Gel filtration of dilute human embryonic hemoglobins reveals basis for their increased oxygen binding.

PubMed

Manning, Lois R; Popowicz, Anthony M; Padovan, Julio C; Chait, Brian T; Manning, James M

2017-02-15

This report establishes a correlation between two known properties of the human embryonic hemoglobins-- their weak subunit assemblies as demonstrated here by gel filtration at very dilute protein concentrations and their high oxygen affinities and reduced cooperativities reported previously by others but without a mechanistic basis. We demonstrate here that their high oxygen affinities are a consequence of their weak assemblies. Weak vs strong hemoglobin tetramers represent a regulatory mechanism to modulate oxygen binding capacity by altering the equilibrium between the various steps in the assembly process that can be described as an inverse allosteric effect. Copyright © 2016 Elsevier Inc. All rights reserved.

[11C]Harmine Binding to Brain Monoamine Oxidase A: Test-Retest Properties and Noninvasive Quantification.

PubMed

Zanderigo, Francesca; D'Agostino, Alexandra E; Joshi, Nandita; Schain, Martin; Kumar, Dileep; Parsey, Ramin V; DeLorenzo, Christine; Mann, J John

2018-02-08

Inhibition of the isoform A of monoamine oxidase (MAO-A), a mitochondrial enzyme catalyzing deamination of monoamine neurotransmitters, is useful in treatment of depression and anxiety disorders. [ 11 C]harmine, a MAO-A PET radioligand, has been used to study mood disorders and antidepressant treatment. However, [ 11 C]harmine binding test-retest characteristics have to date only been partially investigated. Furthermore, since MAO-A is ubiquitously expressed, no reference region is available, thus requiring arterial blood sampling during PET scanning. Here, we investigate [ 11 C]harmine binding measurements test-retest properties; assess effects of using a minimally invasive input function estimation on binding quantification and repeatability; and explore binding potentials estimation using a reference region-free approach. Quantification of [ 11 C]harmine distribution volume (V T ) via kinetic models and graphical analyses was compared based on absolute test-retest percent difference (TRPD), intraclass correlation coefficient (ICC), and identifiability. The optimal procedure was also used with a simultaneously estimated input function in place of the measured curve. Lastly, an approach for binding potentials quantification in absence of a reference region was evaluated. [ 11 C]harmine V T estimates quantified using arterial blood and kinetic modeling showed average absolute TRPD values of 7.7 to 15.6 %, and ICC values between 0.56 and 0.86, across brain regions. Using simultaneous estimation (SIME) of input function resulted in V T estimates close to those obtained using arterial input function (r = 0.951, slope = 1.073, intercept = - 1.037), with numerically but not statistically higher test-retest difference (range 16.6 to 22.0 %), but with overall poor ICC values, between 0.30 and 0.57. Prospective studies using [ 11 C]harmine are possible given its test-retest repeatability when binding is quantified using arterial blood. Results with SIME of input function show potential for simplifying data acquisition by replacing arterial catheterization with one arterial blood sample at 20 min post-injection. Estimation of [ 11 C]harmine binding potentials remains a challenge that warrants further investigation.

Modulation of mood and cognitive performance following acute administration of single doses of Melissa officinalis (Lemon balm) with human CNS nicotinic and muscarinic receptor-binding properties.

PubMed

Kennedy, D O; Wake, G; Savelev, S; Tildesley, N T J; Perry, E K; Wesnes, K A; Scholey, A B

2003-10-01

Melissa officinalis (Lemon balm) is a herbal medicine that has traditionally been attributed with memory-enhancing properties, but which is currently more widely used as a mild sedative and sleep aid. In a previous study it was demonstrated that a commercial Melissa extract led to dose-specific increases in calmness, and dose-dependent decrements in timed memory task performance. However, the extract utilized in that study did not exhibit in vitro cholinergic receptor-binding properties. The current study involved an initial screening of samples of M. officinalis for human acetylcholinesterase inhibition and cholinergic receptor-binding properties. The cognitive and mood effects of single doses of the most cholinergically active dried leaf were then assessed in a randomized, placebo-controlled, double-blind, balanced crossover study. Following the in vitro analysis, 20 healthy, young participants received single doses of 600, 1000, and 1600 mg of encapsulated dried leaf, or a matching placebo, at 7-day intervals. Cognitive performance and mood were assessed predose and at 1, 3, and 6 h postdose using the Cognitive Drug Research computerized assessment battery and Bond-Lader visual analog scales, respectively. In vitro analysis of the chosen extract established IC(50) concentrations of 0.18 and 3.47 mg ml(-1), respectively, for the displacement of [(3)H]-(N)-nicotine and [(3)H]-(N)-scopolamine from nicotinic and muscarinic receptors in the human cerebral cortex tissue. However, no cholinesterase inhibitory properties were detected. The most notable cognitive and mood effects were improved memory performance and increased 'calmness' at all postdose time points for the highest (1600 mg) dose. However, while the profile of results was overwhelmingly favorable for the highest dose, decrements in the speed of timed memory task performance and on a rapid visual information-processing task increased with decreasing dose. These results suggest that doses of Melissa officinalis at or above the maximum employed here can improve cognitive performance and mood and may therefore be a valuable adjunct in the treatment of Alzheimer's disease. The results also suggest that different preparations derived from the same plant species may exhibit different properties depending on the process used for the sample preparation.

Engineering Tocopherol Selectivity in α-TTP: A Combined In Vitro/In Silico Study

PubMed Central

Helbling, Rachel E.; Aeschimann, Walter; Simona, Fabio; Stocker, Achim; Cascella, Michele

2012-01-01

We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of -tocopherol transfer protein (-TTP) towards -tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for -tocopherol to -TTP 8.262.13 kcal mol lower than that of -tocopherol. Our calculations show that -tocopherol binds to -TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of -TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to -tocopherol, with striking structural similarities to the wild-type--tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of -TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover, the identification of a -tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for -tocopherol selection in omnivorous animals. PMID:23152872

Redox process is crucial for inhibitory properties of aurintricarboxylic acid against activity of YopH: virulence factor of Yersinia pestis

PubMed Central

Kuban-Jankowska, Alicja; Gorska, Magdalena; Tuszynski, Jack A; Ossowski, Tadeusz; Wozniak, Michal

2015-01-01

YopH is a bacterial protein tyrosine phosphatase, which is essential for the viability and pathogenic virulence of the plague-causing Yersinia sp. bacteria. Inactivation of YopH activity would lead to the loss of bacterial pathogenicity. We have studied the inhibitory properties of aurintricarboxylic acid (ATA) against YopH phosphatase and found that at nanomolar concentrations ATA reversibly decreases the activity of YopH. Computational docking studies indicated that in all binding poses ATA binds in the YopH active site. Molecular dynamics simulations showed that in the predicted binding pose, ATA binds to the essential Cys403 and Arg409 residues in the active site and has a stronger binding affinity than the natural substrate (pTyr). The cyclic voltammetry experiments suggest that ATA reacts remarkably strongly with molecular oxygen. Additionally, the electrochemical reduction of ATA in the presence of a negative potential from −2.0 to 2.5 V generates a current signal, which is observed for hydrogen peroxide. Here we showed that ATA indicates a unique mechanism of YopH inactivation due to a redox process. We proposed that the potent inhibitory properties of ATA are a result of its strong binding in the YopH active site and in situ generation of hydrogen peroxide near catalytic cysteine residue. PMID:26286963

Physicochemical properties of low sodium frankfurter with added walnut: effect of transglutaminase combined with caseinate, KCl and dietary fibre as salt replacers.

PubMed

Colmenero, F Jiménez; Ayo, M J; Carballo, J

2005-04-01

This study compares the effects of combinations of microbial transglutaminase (TGase) and various non-meat ingredients (caseinate, KCl and wheat fibre) used as salt replacers, with the effects of NaCl on the physicochemical properties (cooking loss, emulsion stability, texture and colour) of frankfurters with added walnuts. The combination of TGase with caseinate, KCl or fibre led to harder, springier and chewier (P<0.05) frankfurters with better water- and fat-binding properties (emulsion stability and cooking loss) (P<0.05) than those made with TGase only. Ranking of ingredient efficiency in combination with TGase showed that caseinate>KCl>fibre. Frankfurters with caseinate presented the highest lightness and the lowest redness values. Frankfurter with NaCl had a harder, springier and chewier gel/emulsion network with lower cooking loss than those NaCl free.

Intermetallic Growth and Interfacial Properties of the Grain Refiners in Al Alloys.

PubMed

Li, Chunmei; Cheng, Nanpu; Chen, Zhiqian; Xie, Zhongjing; Hui, Liangliang

2018-04-20

Al₃TM(TM = Ti, Zr, Hf, Sc) particles acting as effective grain refiners for Al alloys have been receiving extensive attention these days. In order to judge their nucleation behaviors, first-principles calculations are used to investigate their intermetallic and interfacial properties. Based on energy analysis, Al₃Zr and Al₃Sc are more suitable for use as grain refiners than the other two intermetallic compounds. Interfacial properties show that Al/Al₃TM(TM = Ti, Zr, Hf, Sc) interfaces in I-ter interfacial mode exhibit better interface wetting effects due to larger Griffith rupture work and a smaller interface energy. Among these, Al/Al₃Sc achieves the lowest interfacial energy, which shows that Sc atoms should get priority for occupying interfacial sites. Additionally, Sc-doped Al/Al₃(Zr, Sc) interfacial properties show that Sc can effectively improve the Al/Al₃(Zr, Sc) binding strength with the Al matrix. By combining the characteristics of interfaces with the properties of intermetallics, the core-shell structure with Al₃Zr-core or Al₃Zr(Sc1-1)-core encircled with an Sc-rich shell forms.

Allosteric control in a metalloprotein dramatically alters function

PubMed Central

Baxter, Elizabeth Leigh; Zuris, John A.; Wang, Charles; Vo, Phu Luong T.; Axelrod, Herbert L.; Cohen, Aina E.; Paddock, Mark L.; Nechushtai, Rachel; Onuchic, Jose N.; Jennings, Patricia A.

2013-01-01

Metalloproteins (MPs) comprise one-third of all known protein structures. This diverse set of proteins contain a plethora of unique inorganic moieties capable of performing chemistry that would otherwise be impossible using only the amino acids found in nature. Most of the well-studied MPs are generally viewed as being very rigid in structure, and it is widely thought that the properties of the metal centers are primarily determined by the small fraction of amino acids that make up the local environment. Here we examine both theoretically and experimentally whether distal regions can influence the metal center in the diabetes drug target mitoNEET. We demonstrate that a loop (L2) 20 Å away from the metal center exerts allosteric control over the cluster binding domain and regulates multiple properties of the metal center. Mutagenesis of L2 results in significant shifts in the redox potential of the [2Fe-2S] cluster and orders of magnitude effects on the rate of [2Fe-2S] cluster transfer to an apo-acceptor protein. These surprising effects occur in the absence of any structural changes. An examination of the native basin dynamics of the protein using all-atom simulations shows that twisting in L2 controls scissoring in the cluster binding domain and results in perturbations to one of the cluster-coordinating histidines. These allosteric effects are in agreement with previous folding simulations that predicted L2 could communicate with residues surrounding the metal center. Our findings suggest that long-range dynamical changes in the protein backbone can have a significant effect on the functional properties of MPs. PMID:23271805

Different effect of hydrogelation on anti-fouling and circulation properties of dextran–iron oxide nanoparticles

PubMed Central

Karmali, Priya Prakash; Chao, Ying; Park, Ji-Ho (Joe); Sailor, Michael J.; Ruoslahti, Erkki; Esener, Sadik C.; Simberg, Dmitri

2012-01-01

Premature recognition and clearance of nanoparticulate imaging and therapeutic agents by macrophages in the tissues can dramatically reduce both the nanoparticle half-life and delivery to the diseased tissue. Grafting nanoparticles with hydrogels prevents nanoparticulate recognition by liver and spleen macrophages and greatly prolongs circulation times in vivo. Understanding the mechanisms by which hydrogels achieve this “stealth” effect has implications for the design of long-circulating nanoparticles. Thus, the role of plasma protein absorption in the hydrogel effect is not yet understood. Short-circulating dextran-coated iron oxide nanoparticles could be converted into stealth hydrogel nanoparticles by crosslinking with 1-chloro-2,3-epoxypropane. We show that hydrogelation did not affect the size, shape and zeta potential, but completely prevented the recognition and clearance by liver macrophages in vivo. Hydrogelation decreased the number of hydroxyl groups on the nanoparticle surface and reduced the binding of the anti-dextran antibody. At the same time, hydrogelation did not reduce the absorption of cationic proteins on the nanoparticle surface. Specifically, there was no effect on the binding of kininogen, histidine-rich glycoprotein, and protamine sulfate to the anionic nanoparticle surface. In addition, hydrogelation did not prevent activation of plasma kallikrein on the metal oxide surface. These data suggest that: (a) a stealth hydrogel coating does not mask charge interactions with iron oxide surface and (b) the total blockade of plasma protein absorption is not required for maintaining iron oxide nanoparticles’ long-circulating stealth properties. These data illustrate a novel, clinically promising property of long-circulating stealth nanoparticles. PMID:22243419

Effects of Vacuum Tumbling with Chitosan and Water Soluble Chitosan on the Shelf Life of Catfish Fillets in Refrigerated Storage

USDA-ARS?s Scientific Manuscript database

Chitosan (CH) is mainly made from crustacean shells and has been reported to have a number of functional properties such as its antimicrobial activity, binding action, and antioxidant activity. CH’s water insolubility restricts the use of this compound in some systems. However, when treated with enz...

New polymers for phase partitioning

NASA Technical Reports Server (NTRS)

Harris, J. M.

1981-01-01

The synthesizing of several polyethylene glycols having crown ethers attached is reported. This work led to the identification of three new polymer types which promise to be more effective at selectively binding specific cell types. Work was completed on identification of chemical properties of the new polymer crowns and on development of new techniques for determination of polymer-phase composition.

Molecular dynamic simulations on the structures and properties of epsilon-CL-20(0 0 1)/F 2314 PBX.

PubMed

Xu, Xiaojuan; Xiao, Jijun; Huang, Hui; Li, Jinshan; Xiao, Heming

2010-03-15

Molecular dynamical (MD) simulations with the COMPASS force field were employed to investigate the influences of temperature (T), the concentration of F(2314) binder (W%), and crystal defects on the mechanical properties, binding energy (E(bind)), and detonation properties of epsilon-CL-20(001)/F(2314) PBX (polymer bonded explosives). T was found to have some influences on the mechanical properties, and the PBX at 298 K was considered with better mechanical properties. By radial distribution function g(r) analysis the three types of hydrogen bonds, H...O, H...F, and H...Cl were predicted as the main interaction formats between F(2314) and epsilon-CL-20, and the strength of these interactions changed with temperature changing. The isotropic properties of the PBX increased with W% increasing, but each modulus and E(bind) did not monotonously vary with W% increasing. The detonation properties of the PBX decreased with the increasing W%, and the PBX with 4.69% F(2314) was regarded with good detonation properties. The existence of crystal defects (vacancy or adulteration) might increase the elasticity but destabilize the system to some extent, and the mechanical properties of PBX were chiefly determined by the main body explosive. The above information was thought guidable for practical formulation design of PBX. (c) 2009 Elsevier B.V. All rights reserved.

Rate Constants and Mechanisms of Protein–Ligand Binding

PubMed Central

Pang, Xiaodong; Zhou, Huan-Xiang

2017-01-01

Whereas protein–ligand binding affinities have long-established prominence, binding rate constants and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new experimental techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate constants, can and should be quantitatively determined. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and physical insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its reduction to simple rate equations; computation of binding rate constants; quantitative determination of binding mechanisms; and elucidation of physical factors that control binding rate constants and mechanisms. PMID:28375732

Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes.

PubMed

Peng, X; Katz, M; Gerzanich, V; Anand, R; Lindstrom, J

1994-03-01

The alpha-bungarotoxin-binding acetylcholine receptors from the human neuroblastoma cell line SH-SY5Y were found to cross-react with some monoclonal antibodies to alpha 7 subunits of nicotinic acetylcholine receptors from chicken brain. The human alpha 7 subunit cDNA from SH-SY5Y was cloned, revealing 94% amino acid sequence identity to rat alpha 7 subunits and 92% identity to chicken alpha 7 subunits. Native human alpha 7 receptors showed affinities for some ligands similar to those previously observed with native chicken alpha 7 receptors, but for other ligands there were large species-specific differences in binding affinity. These results paralleled properties of alpha 7 homomers expressed in Xenopus oocytes. Human alpha 7 homomers exhibited rapidly desensitizing, inwardly rectifying, agonist-induced, cation currents that triggered Ca(2+)-sensitive Cl- channels in the oocytes. A change in efficacy from partial agonist for chicken alpha 7 homomers to full agonist for human alpha 7 homomers was exhibited by 1,1-dimethyl-4-phenylpiperazinium. This result reveals a large species-specific pharmacological difference, despite small differences in alpha 7 sequences. This is important for understanding the effects of these drugs in humans and for identifying amino acids that may contribute to the acetylcholine binding site, for analysis by in vitro mutagenesis. These results also characterize properties of native alpha 7 receptors and alpha 7 homomers that will provide criteria for functional properties expected of structural subunits, when these can be identified, cloned, and coexpressed with alpha 7 subunits.

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

Woo, Sung Oh; Teizer, Winfried; WPI-Advanced Institute for Materials Research, Tohoku University, Sendai

We report a deterioration of the electrical transport properties of a graphene field effect transistor due to energetic electron irradiation on a stack of Poly Methyl Methacrylate (PMMA) on graphene (PMMA/graphene bilayer). Prior to electron irradiation, we observed that the PMMA layer on graphene does not deteriorate the carrier transport of graphene but improves its electrical properties instead. As a result of the electron irradiation on the PMMA/graphene bilayer, the Raman “D” band appears after removal of PMMA. We argue that the degradation of the transport behavior originates from the binding of hydrogen generated during the PMMA backbone secession process.

Functionalization of ( n, 0) CNTs ( n = 3-16) by uracil: DFT studies

NASA Astrophysics Data System (ADS)

Mirzaei, Mahmoud; Harismah, Kun; Jafari, Elham; Gülseren, Oğuz; Rad, Ali Shokuhi

2018-01-01

Density functional theory (DFT) calculations were performed to investigate stabilities and properties for uracil (U)-functionalized carbon nanotubes (CNTs). To this aim, the optimized molecular properties were evaluated for ( n, 0) models of CNTs ( n = 3-16) in the original and U-functionalized forms. The results indicated that the dipole moments and energy gaps were independent of tubular diameters whereas the binding energies showed that the U-functionalization could be better achieved for n = 8-11 curvatures of ( n, 0) CNTs. Further studies based on the evaluated atomic-scale properties, including quadrupole coupling constants ( C Q ), indicated that the electronic properties of atoms could detect the effects of diameters variations of ( n, 0) CNTs, in which the effects were very much significant for the atoms around the U-functionalization regions. Finally, the achieved results of singular U, original CNTs, and CNT-U hybrids were compared to each other to demonstrate the stabilities and properties for the U-functionalized ( n, 0) CNTs.

The oligomerization state determines regulatory properties and inhibitor sensitivity of type 4 cAMP-specific phosphodiesterases.

PubMed

Richter, Wito; Conti, Marco

2004-07-16

PDE4 splice variants are classified into long and short forms depending on the presence or absence of two unique N-terminal domains termed upstream conserved regions 1 and 2 (UCR1 and -2). We have shown previously that the UCR module mediates dimerization of PDE4 long forms, whereas short forms, which lack UCR1, behave as monomers. In the present study, we demonstrate that dimerization is an essential structural element that determines the regulatory properties and inhibitor sensitivities of PDE4 enzymes. Comparing the properties of the dimeric wild type PDE4D3 with several monomeric mutant PDE4D3 constructs revealed that disruption of dimerization ablates the activation of PDE4 long forms by either protein kinase A phosphorylation or phosphatidic acid binding. Moreover, the analysis of heterodimers consisting of a catalytically active and a catalytically inactive PDE4D3 subunit indicates that protein kinase A phosphorylation of both subunits is essential to fully activate PDE4 enzymes. In addition to affecting enzyme regulation, disruption of dimerization reduces the sensitivity of the enzymes toward the prototypical PDE4 inhibitor rolipram. Parallel binding assays indicated that this shift in rolipram sensitivity is likely mediated by a decrease in the number of inhibitor binding sites in the high affinity rolipram binding state. Thus, although dimerization is not a requirement for high affinity rolipram binding, it functions to stabilize PDE4 long forms in their high affinity rolipram binding conformation. Taken together, our data indicate that dimerization defines the properties of PDE4 enzymes and suggest a common structural and functional organization for all PDEs.

Physicochemical and tablet properties of Cyperus alulatus rhizomes starch granules.

PubMed

Paramakrishnan, N; Jha, S; Kumar, K Jayaram

2015-05-01

The starch extracted from rhizomes of Cyperus alulatus (CA) was characterized for its physicochemical, morphological and tableting properties. Rhizomes of CA yield a significant quantity of starch granules (CASG) i.e., 11.93%. CASG was characterized in terms of moisture, ash and amylose contents, solubility and swelling power, paste clarity and water retention capacity. The swelling power was found to be significantly improved with the increase in temperature. Scanning electron micrographs revealed that the granule's surface was smooth, the granules were spherical, mostly round, disc like, and the size range was 6.65-12.13 μm. Finger print region in FTIR spectra confirmed its carbohydrate nature. The evaluated micromeritic properties of extracted granule's bulk density, tapped density, Carr's index, Hausner ratio, true density and porosity render unique practicability of CASG being used as an adjuvant in pharmaceutical solid dosage forms. Tablets prepared by using CASG showed higher mechanical strength and more disintegration time, which depicted the characteristic binding nature of the starch granules. As CASG is imparting better binding properties in less concentration and also it can be used in combination with the established starches to get the synergistic effect; this starch can be used commercially in the tablet preparation. Copyright © 2015 Elsevier B.V. All rights reserved.

Mirror energy difference and the structure of loosely bound proton-rich nuclei around A =20

NASA Astrophysics Data System (ADS)

Yuan, Cenxi; Qi, Chong; Xu, Furong; Suzuki, Toshio; Otsuka, Takaharu

2014-04-01

The properties of loosely bound proton-rich nuclei around A =20 are investigated within the framework of the nuclear shell model. In these nuclei, the strength of the effective interactions involving the loosely bound proton s1/2 orbit is significantly reduced in comparison with that of those in their mirror nuclei. We evaluate the reduction of the effective interaction by calculating the monopole-based-universal interaction (VMU) in the Woods-Saxon basis. The shell-model Hamiltonian in the sd shell, such as USD, can thus be modified to reproduce the binding energies and energy levels of the weakly bound proton-rich nuclei around A =20. The effect of the reduction of the effective interaction on the structure and decay properties of these nuclei is also discussed.

Optimized Standard Operating Procedures for the Analysis of Cerebrospinal Fluid Aβ42 and the Ratios of Aβ Isoforms Using Low Protein Binding Tubes.

PubMed

Vanderstichele, Hugo Marcel Johan; Janelidze, Shorena; Demeyer, Leentje; Coart, Els; Stoops, Erik; Herbst, Victor; Mauroo, Kimberley; Brix, Britta; Hansson, Oskar

2016-05-31

Reduced cerebrospinal fluid (CSF) concentration of amyloid-β1-42 (Aβ1-42) reflects the presence of amyloidopathy in brains of subjects with Alzheimer's disease (AD). To qualify the use of Aβ1-42/Aβ1-40 for improvement of standard operating procedures (SOP) for measurement of CSF Aβ with a focus on CSF collection, storage, and analysis. Euroimmun ELISAs for CSF Aβ isoforms were used to set up a SOP with respect to recipient properties (low binding, polypropylene), volume of tubes, freeze/thaw cycles, addition of detergents (Triton X-100, Tween-20) in collection or storage tubes or during CSF analysis. Data were analyzed with linear repeated measures and mixed effects models. Optimization of CSF analysis included a pre-wash of recipients (e.g., tubes, 96-well plates) before sample analysis. Using the Aβ1-42/Aβ1-40 ratio, in contrast to Aβ1-42, eliminated effects of tube type, additional freeze/thaw cycles, or effect of CSF volumes for polypropylene storage tubes. 'Low binding' tubes reduced the loss of Aβ when aliquoting CSF or in function of additional freeze/thaw cycles. Addition of detergent in CSF collection tubes resulted in an almost complete absence of variation in function of collection procedures, but affected the concentration of Aβ isoforms in the immunoassay. The ratio of Aβ1-42/Aβ1-40 is a more robust biomarker than Aβ1-42 toward (pre-) analytical interfering factors. Further, 'low binding' recipients and addition of detergent in collection tubes are able to remove effects of SOP-related confounding factors. Integration of the Aβ1-42/Aβ1-40 ratio and 'low-binding tubes' into guidance criteria may speed up worldwide standardization of CSF biomarker analysis.

Formononetin, a phyto-oestrogen, and its metabolites up-regulate interleukin-4 production in activated T cells via increased AP-1 DNA binding activity

PubMed Central

Park, Jin; Kim, Seung H; Cho, Daeho; Kim, Tae S

2005-01-01

Phyto-oestrogens are polyphenolic non-steroidal plant compounds with oestrogen-like biological activity. Phyto-oestrogens have many biological effects including oestrogen agonist/antagonist properties. However, the effect of phyto-oestrogens on allergic responses remains unclear. In this study we investigated whether formononetin, a phyto-oestrogen, and its metabolites, daidzein and equol, affect production of interleukin-4 (IL-4), a pro-inflammatory cytokine closely associated with allergic immune response, in primary CD4+ T cells and EL4 T lymphoma cells. Formononetin, daidzein and equol significantly enhanced IL-4 production from both CD4+ T cells and EL4 cells in a dose-dependent manner. Formononetin, daidzein and equol also enhanced IL-4 gene promoter activity in EL4 cells transiently transfected with IL-4 gene promoter constructs, but this effect was impaired in EL4 cells transfected with an IL-4 promoter construct deleted of P4 site carrying nuclear factor of activated T cells (NF-AT) and activator protein-1 (AP-1) binding sites. In addition, formononetin, daidzein and equol increased AP-1 DNA binding activities while did not affect NF-AT DNA binding activities. The enhancing effects on IL-4 production and AP-1 DNA binding activities were abrogated by specific inhibitors for phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC) and p38 mitogen-activated protein kinase (MAPK), indicating that formononetin, daidzein and equol might enhance IL-4 production by increased activation of AP-1 through the PI3-K/PKC/p38 MAPK signalling pathway. These results suggest that phyto-oestrogens and some of their metabolites may increase allergic responses via the enhancement of IL-4 production in T cells. PMID:16108819

Modular Integration of Upconverting Nanocrystal-Dendrimer Composites for Folate Receptor-Specific NIR Imaging and Light-Triggered Drug Release.

PubMed

Wong, Pamela T; Chen, Dexin; Tang, Shengzhuang; Yanik, Sean; Payne, Michael; Mukherjee, Jhindan; Coulter, Alexa; Tang, Kenny; Tao, Ke; Sun, Kang; Baker, James R; Choi, Seok Ki

2015-12-02

Upconversion nanocrystals (UCNs) display near-infrared (NIR)-responsive photoluminescent properties for NIR imaging and drug delivery. The development of effective strategies for UCN integration with other complementary nanostructures for targeting and drug conjugation is highly desirable. This study reports on a core/shell-based theranostic system designed by UCN integration with a folate (FA)-conjugated dendrimer for tumor targeting and with photocaged doxorubicin as a cytotoxic agent. Two types of UCNs (NaYF4:Yb/Er (or Yb/Tm); diameter = ≈50 to 54 nm) are described, each displaying distinct emission properties upon NIR (980 nm) excitation. The UCNs are surface modified through covalent attachment of photocaged doxorubicin (ONB-Dox) and a multivalent FA-conjugated polyamidoamine (PAMAM) dendrimer G5(FA)6 to prepare UCN@(ONB-Dox)(G5FA). Surface plasmon resonance experiments performed with G5(FA)6 dendrimer alone show nanomolar binding avidity (KD = 5.9 × 10(-9) M) to the folate binding protein. This dendrimer binding corresponds with selective binding and uptake of UCN@(ONB-Dox)(G5FA) by FAR-positive KB carcinoma cells in vitro. Furthermore, UCN@(ONB-Dox)(G5FA) treatment of FAR(+) KB cells inhibits cell growth in a light dependent manner. These results validate the utility of modularly integrated UCN-dendrimer nanocomposites for cell type specific NIR imaging and light-controlled drug release, thus serving as a new theranostic system. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acid-base properties of humic substances from composted and thermally-dried sewage sludges and amended soils as determined by potentiometric titration and the NICA-Donnan model.

PubMed

Fernández, José M; Plaza, César; Senesi, Nicola; Polo, Alfredo

2007-09-01

The acid-base properties of humic acids (HAs) and fulvic acids (FAs) isolated from composted sewage sludge (CS), thermally-dried sewage sludge (TS), soils amended with either CS or TS at a rate of 80 t ha(-1)y(-1) for 3y and the corresponding unamended soil were investigated by use of potentiometric titrations. The non-ideal competitive adsorption (NICA)-Donnan model for a bimodal distribution of proton binding sites was fitted to titration data by use of a least-squares minimization method. The main fitting parameters of the NICA-Donnan model obtained for each HA and FA sample included site densities, median affinity constants and widths of affinity distributions for proton binding to low and high affinity sites, which were assumed to be, respectively, carboxylic- and phenolic-type groups. With respect to unamended soil HA and FA, the HAs and FAs from CS, and especially TS, were characterized by smaller acidic functional group contents, larger proton binding affinities of both carboxylic- and phenolic-type groups, and smaller heterogeneity of carboxylic and phenolic-type groups. Amendment with CS or TS led to a decrease of acidic functional group contents and a slight increase of proton binding affinities of carboxylic- and phenolic-type groups of soil HAs and FAs. These effects were more evident in the HA and FA fractions from CS-amended soil than in those from TS-amended soil.

In silico design of fragment-based drug targeting host processing α-glucosidase i for dengue fever

NASA Astrophysics Data System (ADS)

Toepak, E. P.; Tambunan, U. S. F.

2017-02-01

Dengue is a major health problem in the tropical and sub-tropical regions. The development of antiviral that targeting dengue’s host enzyme can be more effective and efficient treatment than the viral enzyme. Host enzyme processing α-glucosidase I has an important role in the maturation process of dengue virus envelope glycoprotein. The inhibition of processing α-glucosidase I can become a promising target for dengue fever treatment. The antiviral approach using in silico fragment-based drug design can generate drug candidates with high binding affinity. In this research, 198.621 compounds were obtained from ZINC15 Biogenic Database. These compounds were screened to find the favorable fragments according to Rules of Three and pharmacological properties. The screening fragments were docked into the active site of processing α-glucosidase I. The potential fragment candidates from the molecular docking simulation were linked with castanospermine (CAST) to generate ligands with a better binding affinity. The Analysis of ligand - enzyme interaction showed ligands with code LRS 22, 28, and 47 have the better binding free energy than the standard ligand. Ligand LRS 28 (N-2-4-methyl-5-((1S,3S,6S,7R,8R,8aR)-1,6,7,8-tetrahydroxyoctahydroindolizin-3-yl) pentyl) indolin-1-yl) propionamide) itself among the other ligands has the lowest binding free energy. Pharmacological properties prediction also showed the ligands LRS 22, 28, and 47 can be promising as the dengue fever drug candidates.

Presynaptic Filament Dynamics in Homologous Recombination and DNA Repair

PubMed Central

Liu, Jie; Ehmsen, Kirk T.; Heyer, Wolf-Dietrich; Morrical, Scott W.

2014-01-01

Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity. PMID:21599536

Evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2A protein.

PubMed

Wood, Martyn D; Gillard, Michel

2017-02-01

Brivaracetam (BRV) and levetiracetam (LEV) are effective antiepileptic drugs that bind selectively to the synaptic vesicle 2A (SV2A) protein. However, BRV differs from LEV in that it exhibits more potent and complete seizure suppression in animal models including in amygdala-kindled mice, where BRV afforded nearly complete seizure suppression. This raises the possibility that aside from potency differences, BRV and LEV may interact differently with the SV2A protein, which is not apparent in radioligand-binding competition studies. In this study, we used a recently identified SV2A allosteric modulator, UCB1244283, that appears to induce conformational changes in SV2A, to probe the binding properties of labeled BRV and LEV. Radioligand binding studies were carried out using [ 3 H]BRV and [ 3 H]LEV. Studies were performed in membranes from both recombinant cells expressing human SV2A protein and human brain tissue. The modulator increased the binding of both radioligands but by different mechanisms. For [ 3 H]BRV, the increase was driven mainly by an increase in affinity, whereas for [ 3 H]LEV, the increase was due to an increase in the number of apparent binding sites. Kinetic studies confirmed this differential effect. These studies suggest that LEV and BRV may act at different binding sites or interact with different conformational states of the SV2A protein. It is possible that some of the pharmacologic differences between BRV and LEV could be due to different interactions with the SV2A protein. © 2016 The Authors. Epilepsia published by Wiley Periodicals Inc. on behalf of International League Against Epilepsy.

Many-body effects and excitonic features in 2D biphenylene carbon

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

Lüder, Johann, E-mail: johann.luder@physics.uu.se; Puglia, Carla; Eriksson, Olle

2016-01-14

The remarkable excitonic effects in low dimensional materials in connection to large binding energies of excitons are of great importance for research and technological applications such as in solar energy and quantum information processing as well as for fundamental investigations. In this study, the unique electronic and excitonic properties of the two dimensional carbon network biphenylene carbon were investigated with GW approach and the Bethe-Salpeter equation accounting for electron correlation effects and electron-hole interactions, respectively. Biphenylene carbon exhibits characteristic features including bright and dark excitons populating the optical gap of 0.52 eV and exciton binding energies of 530 meV asmore » well as a technologically relevant intrinsic band gap of 1.05 eV. Biphenylene carbon’s excitonic features, possibly tuned, suggest possible applications in the field of solar energy and quantum information technology in the future.« less

Solution and fluorescence properties of symmetric dipicolylamine-containing dichlorofluorescein-based Zn2+ sensors.

PubMed

Wong, Brian A; Friedle, Simone; Lippard, Stephen J

2009-05-27

The mechanism by which dipicolylamine (DPA) chelate-appended fluorophores respond to zinc was investigated by the synthesis and study of five new analogues of the 2',7'-dichlorofluorescein-based Zn(2+) sensor Zinpyr-1 (ZP1). With the use of absorption and emission spectroscopy in combination with potentiometric titrations, a detailed molecular picture has emerged of the Zn(2+) and H(+) binding properties of the ZP1 family of sensors. The two separate N(3)O donor atom sets on ZP1 converge to form binding pockets in which all four heteroatoms participate in coordination to either Zn(2+) or protons. The position of the pyridyl group nitrogen atom, 2-pyridyl or 4-pyridyl, has a large impact on the fluorescence response of the dyes to protons despite relatively small changes in pK(a) values. The fluorescence quenching effects of such multifunctional electron-donating units are often taken as a whole. Despite the structural complexity of ZP1, however, we provide evidence that the pyridyl arms of the DPA appendages participate in the quenching process, in addition to the contribution from the tertiary nitrogen amine atom. Potentiometric titrations reveal ZP1 dissociation constants (K(d)) for Zn(2+) of 0.04 pM and 1.2 nM for binding to the first and second binding pockets of the ligand, respectively, the second of which correlates with the value observed by fluorescence titration. This result demonstrates that both binding pockets of this symmetric, ditopic sensor need to be occupied in order for full fluorescence turn-on to be achieved. These results have significant implications for the design and implementation of fluorescent sensors for studies of mobile zinc ions in biology.

Acid-base properties of brown seaweed biomass considered as a Donnan gel. A model reflecting electrostatic effects and chemical heterogeneity.

PubMed

Rey-Castro, Carlos; Lodeiro, Pablo; Herrero, Roberto; Sastre de Vicente, Manuel E

2003-11-15

Brown seaweeds are interesting materials to be used as biosorbents for heavy metals due to their high binding ability and low cost. The study of the passive biosorption of protons on this kind of materials and its dependency on pH, ionic strength, and medium composition is essential for the practical application of brown algae in wastewater treatment. This work reports the results of the study of the proton binding equilibria of dead biomass from the seaweeds Sargassum muticum, Cystoseira baccata, and Saccorhiza polyschides by potentiometric titration with a glass electrode in the pH range between 2 and 8. Two different salts, NaCl and KNO3, in concentrations ranging from 0.05 to 2 mol x L(-1), were used as background electrolytes. The influence of the ionic strength was accounted for by means of the Donnan model in combination with the master curve approach. Different empirical expressions to describe the swelling behavior of the biosorbent were tested. On the basis of the intrinsic affinity distribution analysis a unimodal Langmuir-Freundlich isotherm was selected to describe the proton binding properties. The results show very little influence of the type of salt. The ionic strength dependency of the proton binding is very similar for the three species, and average empirical expressions of the Donnan volume are proposed. The maximum proton binding capacities obtained ranged between 2.4 and 2.9 mol x kg(-1), with average intrinsic proton affinity constants between 3.1 and 3.3, and heterogeneity parameters of ca. 0.5 for S. muticum and C. baccata, and slightly higher (ca. 0.7) for S. polyschides. The combined Langmuir-Freundlich equation and Donnan model allowed a good description of the experimental charge vs pH curves obtained.

Twisted cyanines: a non-planar fluorogenic dye with superior photostability and its use in a protein-based fluoromodule.

PubMed

Shank, Nathaniel I; Pham, Ha H; Waggoner, Alan S; Armitage, Bruce A

2013-01-09

The cyanine dye thiazole orange (TO) is a well-known fluorogenic stain for DNA and RNA, but this property precludes its use as an intracellular fluorescent probe for non-nucleic acid biomolecules. Further, as is the case with many cyanines, the dye suffers from low photostability. Here, we report the synthesis of a bridge-substituted version of TO named α-CN-TO, where the central methine hydrogen of TO is replaced by an electron withdrawing cyano group, which was expected to decrease the susceptibility of the dye toward singlet oxygen-mediated degradation. An X-ray crystal structure shows that α-CN-TO is twisted drastically out of plane, in contrast to TO, which crystallizes in the planar conformation. α-CN-TO retains the fluorogenic behavior of the parent dye TO in viscous glycerol/water solvent, but direct irradiation and indirect bleaching studies showed that α-CN-TO is essentially inert to visible light and singlet oxygen. In addition, the twisted conformation of α-CN-TO mitigates nonspecific binding and fluorescence activation by DNA and a previously selected TO-binding protein and exhibits low background fluorescence in HeLa cell culture. α-CN-TO was then used to select a new protein that binds and activates fluorescence from the dye. The new α-CN-TO/protein fluoromodule exhibits superior photostability to an analogous TO/protein fluoromodule. These properties indicate that α-CN-TO will be a useful fluorogenic dye in combination with specific RNA and protein binding partners for both in vitro and cell-based applications. More broadly, structural features that promote nonplanar conformations can provide an effective method for reducing nonspecific binding of cationic dyes to nucleic acids and other biomolecules.

Twisted Cyanines: A Non-Planar Fluorogenic Dye with Superior Photostability and its Use in a Protein-Based Fluoromodule

PubMed Central

Shank, Nathaniel I.; Pham, Ha; Waggoner, Alan S.; Armitage, Bruce A.

2013-01-01

The cyanine dye thiazole orange (TO) is a well-known fluorogenic stain for DNA and RNA, but this property precludes its use as an intracellular fluorescent probe for non-nucleic acid biomolecules. Further, as is the case with many cyanines, the dye suffers from low photostability. Here we report the synthesis of a bridge-substituted version of TO named α-CN-TO, where the central methine hydrogen of TO is replaced by an electron withdrawing cyano group, which was expected to decrease the susceptibility of the dye toward singlet oxygen-mediated degradation. An X-ray crystal structure shows that α-CN-TO is twisted drastically out of plane, in contrast to TO, which crystallizes in the planar conformation. α-CN-TO retains the fluorogenic behavior of the parent dye TO in viscous glycerol/water solvent, but direct irradiation and indirect bleaching studies showed that α-CN-TO is essentially inert to visible light and singlet oxygen. In addition, the twisted conformation of α-CN-TO mitigates non-specific binding and fluorescence activation by DNA and a previously selected TO-binding protein and exhibits low background fluorescence in HeLa cell culture. α-CN-TO was then used to select a new protein that binds and activates fluorescence from the dye. The new α-CN-TO/protein fluoromodule exhibits superior photostability to an analogous TO/protein fluoromodule. These properties indicate that α-CN-TO will be a useful fluorogenic dye in combination with specific RNA and protein binding partners for both in vitro and cell-based applications. More broadly, structural features that promote nonplanar conformations can provide an effective method for reducing nonspecific binding of cationic dyes to nucleic acids and other biomolecules. PMID:23252842

Synthesis and anion binding studies of tris(3-aminopropyl)amine-based tripodal urea and thiourea receptors: Proton transfer-induced selectivity for hydrogen sulfate over sulfate.

PubMed

Khansari, Maryam Emami; Johnson, Corey R; Basaran, Ismet; Nafis, Aemal; Wang, Jing; Leszczynski, Jerzy; Hossain, Md Alamgir

2015-01-01

Tris(3-aminopropyl)amine-based tripodal urea and thiourea receptors, tris([(4-cyanophenyl)amino]propyl)urea ( L1 ) and tris([(4-cyanophenyl)amino]propyl)thiourea ( L2 ), have been synthesized and their anion binding properties have been investigated for halides and oxoanions. As investigated by 1 H NMR titrations, each receptor binds an anion with a 1:1 stoichiometry via hydrogen-bonding interactions (NH⋯anion), showing the binding trend in the order of F - > H 2 PO 4 - > HCO 3 - > HSO 4 - > CH 3 COO - > SO 4 2- > Cl - > Br - > I in DMSO- d 6 . The interactions of the receptors were further studied by 2D NOESY, showing the loss of NOESY contacts of two NH resonances for the complexes of F - , H 2 PO 4 - , HCO 3 - , HSO 4 - or CH 3 COO - due to the strong NH⋯anion interactions. The observed higher binding affinity for HSO 4 - than SO 4 2- is attributed to the proton transfer from HSO 4 - to the central nitrogen of L1 or L2 which was also supported by the DFT calculations, leading to the secondary acid-base interactions. The thiourea receptor L2 has a general trend to show a higher affinity for an anion as compared to the urea receptor L1 for the corresponding anion in DMSO- d 6 . In addition, the compound L2 has been exploited for its extraction properties for fluoride in water using a liquid-liquid extraction technique, and the results indicate that the receptor effectively extracts fluoride from water showing ca. 99% efficiency (based on L2 ).

Synthesis and anion binding studies of tris(3-aminopropyl)amine-based tripodal urea and thiourea receptors: Proton transfer-induced selectivity for hydrogen sulfate over sulfate

PubMed Central

Khansari, Maryam Emami; Johnson, Corey R.; Basaran, Ismet; Nafis, Aemal; Wang, Jing

2015-01-01

Tris(3-aminopropyl)amine-based tripodal urea and thiourea receptors, tris([(4-cyanophenyl)amino]propyl)urea (L1) and tris([(4-cyanophenyl)amino]propyl)thiourea (L2), have been synthesized and their anion binding properties have been investigated for halides and oxoanions. As investigated by 1H NMR titrations, each receptor binds an anion with a 1:1 stoichiometry via hydrogen-bonding interactions (NH⋯anion), showing the binding trend in the order of F− > H2PO4− > HCO3− > HSO4− > CH3COO− > SO42− > Cl− > Br− > I in DMSO-d6. The interactions of the receptors were further studied by 2D NOESY, showing the loss of NOESY contacts of two NH resonances for the complexes of F−, H2PO4−, HCO3−, HSO4− or CH3COO− due to the strong NH⋯anion interactions. The observed higher binding affinity for HSO4− than SO42− is attributed to the proton transfer from HSO4− to the central nitrogen of L1 or L2 which was also supported by the DFT calculations, leading to the secondary acid-base interactions. The thiourea receptor L2 has a general trend to show a higher affinity for an anion as compared to the urea receptor L1 for the corresponding anion in DMSO-d6. In addition, the compound L2 has been exploited for its extraction properties for fluoride in water using a liquid-liquid extraction technique, and the results indicate that the receptor effectively extracts fluoride from water showing ca. 99% efficiency (based on L2). PMID:28184300

Insulin receptor in mouse neuroblastoma cell line N18TG2: binding properties and visualization with colloidal gold.

PubMed

Sartori, C; Stefanini, S; Bernardo, A; Augusti-Tocco, G

1992-08-01

Insulin function in the nervous system is still poorly understood. Possible roles as a neuromodulator and as a growth factor have been proposed (Baskin et al., 1987, Ann. Rev. Physiol. 49, 335-347). Stable cell lines may provide an appropriate experimental system for the analysis of insulin action on the various cellular components of the central nervous system. We report here a study to investigate the presence and the properties of insulin specific binding sites in the murine neuroblastoma line, N18TG2, together with insulin action on cell growth and metabolism. Also, receptor internalization has been studied. Binding experiments, carried out in standard conditions at 20 degrees C, enabled us to demonstrate that these cells bind insulin in a specific manner, thus confirming previous findings on other cell lines. Saturation curves showed the presence of two binding sites with Kd 0.3 and 9.7 nM. Competition experiments with porcine and bovine insulin showed an IC50 of 1 and 10 nM, respectively. Competition did not occur in the presence of the unrelated hormones ACTH and FSH. Dissociation experiments indicated the existence of an internalization process of the ligand-receptor complex; this was confirmed by an ultrastructural study using gold conjugated insulin. As far as the insulin action in N18TG2 cells is concerned, physiological concentrations stimulate cell proliferation, whereas no stimulation of glucose uptake was observed, indicating that insulin action in these cells is not mediated by general metabolic effects. On the basis of these data, N18TG2 line appears to be a very suitable model for further studies of the neuronal type insulin receptors, and possibly insulin specific action on the nervous system.

Structural evidence of the species-dependent albumin binding of the modified cyclic phosphatidic acid with cytotoxic properties.

PubMed

Sekula, Bartosz; Ciesielska, Anna; Rytczak, Przemyslaw; Koziołkiewicz, Maria; Bujacz, Anna

2016-07-01

Cyclic phosphatidic acids (cPAs) are naturally occurring, very active signalling molecules, which are involved in several pathological states, such as cancer, diabetes or obesity. As molecules of highly lipidic character found in the circulatory system, cPAs are bound and transported by the main extracellular lipid binding protein-serum albumin. Here, we present the detailed interactions between human serum albumin (HSA) and equine serum albumin (ESA) with a derivative of cPA, 1-O-myristoyl-sn-glycerol-2,3-cyclic phosphorodithioate (Myr-2S-cPA). Initial selection of the ligand used for the structural study was made by the analysis of the therapeutically promising properties of the sulfur containing analogues of cPA in respect to the unmodified lysophospholipids (LPLs). Substitution of one or two non-bridging oxygen atoms in the phosphate group with one or two sulfur atoms increases the cytotoxic effect of cPAs up to 60% on the human prostate cancer (PC) cells. Myr-2S-cPA reduces cancer cell viability in a dose-dependent manner, with IC50 value of 29.0 μM after 24 h incubation, which is almost 30% lower than IC50 of single substituted phosphorothioate cPA. Although, the structural homology between HSA and ESA is big, their crystal complexes with Myr-2S-cPA demonstrate significantly different mode of binding of this LPL analogue. HSA binds three molecules of Myr-2S-cPA, whereas ESA only one. Moreover, none of the identified Myr-2S-cPA binding sites overlap in both albumins. © 2016 The Author(s).

Identification of the gamma subunit-interacting residues on photoreceptor cGMP phosphodiesterase, PDE6alpha '.

PubMed

Granovsky, A E; Artemyev, N O

2000-12-29

Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the G protein-mediated visual transduction cascade. In the dark, the activity of PDE6 is shut off by the inhibitory gamma subunit (Pgamma). Chimeric proteins between cone PDE6alpha' and cGMP-binding and cGMP-specific PDE (PDE5) have been constructed and expressed in Sf9 cells to study the mechanism of inhibition of PDE6 catalytic activity by Pgamma. Substitution of the segment PDE5-(773-820) by the corresponding PDE6alpha'-(737-784) sequence in the wild-type PDE5 or in a PDE5/PDE6alpha' chimera containing the catalytic domain of PDE5 results in chimeric enzymes capable of inhibitory interaction with Pgamma. The catalytic properties of the chimeric PDEs remained similar to those of PDE5. Ala-scanning mutational analysis of the Pgamma-binding region, PDE6alpha'-(750-760), revealed PDE6alpha' residues essential for the interaction. The M758A mutation markedly impaired and the Q752A mutation moderately impaired the inhibition of chimeric PDE by Pgamma. The analysis of the catalytic properties of mutant PDEs and a model of the PDE6 catalytic domain suggest that residues Met(758) and Gln(752) directly bind Pgamma. A model of the PDE6 catalytic site shows that PDE6alpha'-(750-760) forms a loop at the entrance to the cGMP-binding pocket. Binding of Pgamma to Met(758) would effectively block access of cGMP to the catalytic cavity, providing a structural basis for the mechanism of PDE6 inhibition.

Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.

PubMed

Link, Reet; Veiksina, Santa; Rinken, Ago; Kopanchuk, Sergei

2017-03-15

Melanocortin 4 (MC 4 ) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC 4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC 4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ 1/2 =160min) and UTBC102 (τ 1/2 =7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ 1/2 =224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC 4 receptor specific ligands and could complement each other in these studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin.

PubMed Central

Schilling, D; Reid IV, J D; Hujer, A; Morgan, D; Demoll, E; Bummer, P; Fenstermaker, R A; Kaetzel, D M

1998-01-01

Site-directed mutagenesis of the platelet-derived growth factor (PDGF) B-chain was conducted to determine the importance of cationic amino acid residues (Arg160-Lys161-Lys162; RKK) located within the loop III region in mediating the biological and cell-association properties of the molecule. Binding to both PDGF alpha-and beta-receptors was inhibited by the conversion of all three cationic residues into anionic glutamates (RKK-->EEE), whereas an RKK-->SSS mutant also exhibited a modest loss in affinity for beta-receptors. Replacements with serine at either Arg160 (RKK-->SKK) or at all three positions (RKK-->SSS) had little effect on binding to alpha-receptors. Replacements with either glutamic or serine residues at any of the three positions also resulted in significant inhibition of heparin-binding activity. Furthermore, the RKK-->EEE mutant exhibited decreased association with the cell surface and accumulated in the culture medium as 29-32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK-->EEE mutants of either the A-chain or the B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor-binding activities, each of the loop III mutants retained full mitogenic activity when applied to cultured Swiss 3T3 cells. CD spectrophotometric analysis of the RKK-->EEE mutant revealed a secondary structure indistinguishable from the wild type, with a high degree of beta-sheet structure and random coil content (50% and 43% respectively). These findings indicate an important role of the Arg160-Lys161-Lys162 sequence in mediating the biological and cell-associative activities of the PDGF-BB homodimer, and reveal that the mitogenic activity of PDGF-BB is insufficient to mediate its full oncogenic properties. PMID:9677323

Biochemical and functional characterization of an albumin protein belonging to the hemopexin superfamily from Lens culinaris seeds.

PubMed

Scarafoni, Alessio; Gualtieri, Elisa; Barbiroli, Alberto; Carpen, Aristodemo; Negri, Armando; Duranti, Marcello

2011-09-14

The present paper reports the purification and biochemical characterization of an albumin identified in mature lentil seeds with high sequence similarity to pea PA2. These proteins are found in many edible seeds and are considered potentially detrimental for human health due to the potential allergenicity and lectin-like activity. Thus, the description of their possible presence in food and the assessment of the molecular properties are relevant. The M(r), pI, and N-terminal sequence of this protein have been determined. The work included the study of (i) the binding properties to hemine to assess the presence of hemopexin structural domains and (ii) the binding properties of the protein to thiamin. In addition, the structural changes induced by heating have been evaluated by means of spectroscopic techniques. Denaturation temperature has also been determined. The present work provides new insights about the structural molecular features and the ligand-binding properties and dynamics of this kind of seed albumin.

Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

PubMed

Jobst, Markus A; Milles, Lukas F; Schoeler, Constantin; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Gaub, Hermann E; Nash, Michael A

2015-10-31

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicted dual binding modes across multiple bacterial species, our approach opens up new possibilities for understanding assembly and catalytic properties of a broad range of multi-enzyme complexes.

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

PubMed Central

Murphy, James M.; Zhang, Qingwei; Young, Samuel N.; Reese, Michael L.; Bailey, Fiona P.; Eyers, Patrick A.; Ungureanu, Daniela; Hammaren, Henrik; Silvennoinen, Olli; Varghese, Leila N.; Chen, Kelan; Tripaydonis, Anne; Jura, Natalia; Fukuda, Koichi; Qin, Jun; Nimchuk, Zachary; Mudgett, Mary Beth; Elowe, Sabine; Gee, Christine L.; Liu, Ling; Daly, Roger J.; Manning, Gerard; Babon, Jeffrey J.; Lucet, Isabelle S.

2017-01-01

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains. PMID:24107129

Identification of curcumin derivatives as human LMTK3 inhibitors for breast cancer: a docking, dynamics, and MM/PBSA approach.

PubMed

Anbarasu, K; Jayanthi, S

2018-05-01

Human lemur tyrosine kinase-3 (LMTK3) is primarily involved in regulation of estrogen receptor-α (ERα) by phosphorylation activity. LMTK3 acts as key biomarker for ERα positive breast cancer and identified as novel drug target for breast cancer. Due to the absence of experimental reports, the computational approach has been followed to screen LMTK3 inhibitors from natural product curcumin derivatives based on rational inhibitor design. The initial virtual screening and re-docking resulted in identification of top three leads with favorable binding energy and strong interactions in critical residues of ATP-binding cavity. ADME prediction confirmed the pharmacological activity of the leads with various properties. The stability and binding affinity of leads were well refined in dynamic system from 25 ns MD simulations. The behavior of protein motion towards closure of ATP-binding cavity was evaluated based on eigenvectors by PCA. In addition, MM/PBSA calculations also confirmed the relative binding free energy of LMTK3-lead complexes in favor of the effective binding. From our study, novel LMTK3 inhibitors tetrahydrocurcumin, curcumin 4,4'-diacetate, and demethoxycurcumin have been proposed with inhibition mechanism. Further experimental evaluation on reported lead candidates might prove its role in breast cancer therapeutics.

Cd and proton adsorption onto bacterial consortia grown from industrial wastes and contaminated geologic settings.

PubMed

Borrok, David M; Fein, Jeremy B; Kulpa, Charles F

2004-11-01

To model the effects of bacterial metal adsorption in contaminated environments, results from metal adsorption experiments involving individual pure stains of bacteria must be extrapolated to systems in which potentially dozens of bacterial species are present. This extrapolation may be made easier because bacterial consortia from natural environments appear to exhibit similar metal binding properties. However, bacteria that thrive in highly perturbed contaminated environments may exhibit significantly different adsorptive behavior. Here we measure proton and Cd adsorption onto a range of bacterial consortia grown from heavily contaminated industrial wastes, groundwater, and soils. We model the results using a discrete site surface complexation approach to determine binding constants and site densities for each consortium. The results demonstrate that bacterial consortia from different contaminated environments exhibit a range of total site densities (approximately a 3-fold difference) and Cd-binding constants (approximately a 10-fold difference). These ranges for Cd binding constants may be small enough to suggest that bacteria-metal adsorption in contaminated environments can be described using relatively few "averaged" bacteria-metal binding constants (in conjunction with the necessary binding constants for competing surfaces and ligands). However, if additional precision is necessary, modeling parameters must be developed separately for each contaminated environment of interest.

Engineering the Pseudomonas aeruginosa II lectin: designing mutants with changed affinity and specificity

NASA Astrophysics Data System (ADS)

Kříž, Zdeněk; Adam, Jan; Mrázková, Jana; Zotos, Petros; Chatzipavlou, Thomais; Wimmerová, Michaela; Koča, Jaroslav

2014-09-01

This article focuses on designing mutations of the PA-IIL lectin from Pseudomonas aeruginosa that lead to change in specificity. Following the previous results revealing the importance of the amino acid triad 22-23-24 (so-called specificity-binding loop), saturation in silico mutagenesis was performed, with the intent of finding mutations that increase the lectin's affinity and modify its specificity. For that purpose, a combination of docking, molecular dynamics and binding free energy calculation was used. The combination of methods revealed mutations that changed the performance of the wild-type lectin and its mutants to their preferred partners. The mutation at position 22 resulted in 85 % in inactivation of the binding site, and the mutation at 23 did not have strong effects thanks to the side chain being pointed away from the binding site. Molecular dynamics simulations followed by binding free energy calculation were performed on mutants with promising results from docking, and also at those where the amino acid at position 24 was replaced for bulkier or longer polar chain. The key mutants were also prepared in vitro and their binding properties determined by isothermal titration calorimetry. Combination of the used methods proved to be able to predict changes in the lectin performance and helped in explaining the data observed experimentally.

Non-ionic detergents facilitate non-specific binding of M13 bacteriophage to polystyrene surfaces.

PubMed

Hakami, Abdulrahim R; Ball, Jonathan K; Tarr, Alexander W

2015-09-01

Phage-displayed random peptide libraries are widely used for identifying peptide interactions with proteins and other substrates. Selection of peptide ligands involves iterative rounds of affinity enrichment. The binding properties of the selected phage clones are routinely tested using immunoassay after propagation to high titre in a bacterial host and precipitation using polyethylene glycol (PEG) and high salt concentration. These immunoassays can suffer from low sensitivity and high background signals. Polysorbate 20 (Tween(®) 20) is a non-ionic detergent commonly used in immunoassay washing buffers to reduce non-specific binding, and is also used as a blocking reagent. We have observed that Tween 20 enhances non-specific M13 library phage binding in a peptide-independent manner. Other non-ionic detergents were also found to promote significant, dose-dependent non-specific phage binding in ELISA. This effect was not observed for assays using phage concentrated by ultracentrifugation, suggesting that interactions occur between detergents and the PEG-precipitated phage, irrespective of the displayed peptide motif. This artefact may impact on successful affinity selection of peptides from phage-display libraries. We propose alternative methods for screening phage libraries for identifying binding interactions with target ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Spectroscopic analysis of the interaction between thiazolo[2,3-b]pyrimidine analogues and bovine serum albumin.

PubMed

Yu, Xianyong; Yang, Ying; Yao, Qing; Tao, Hongwen; Lu, Shiyu; Xie, Jian; Zhou, Hu; Yi, Pinggui

2012-10-01

The interaction between thiazolo[2,3-b]pyrimidine (TZPM) analogues and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy and UV-Vis spectroscopy at two different temperatures (299 and 307K) under imitated physiological conditions. The results indicate that both static quenching and dynamic quenching contribute to the fluorescence quenching of BSA by TZPM. The binding constant (K(a)) and binding sites (n) were calculated from the obtained spectra. Based on the Förster non-radiation energy transfer theory, the average binding distance between BSA and TZPM was estimated. The synchronous fluorescence spectra indicate that the conformation of BSA has been changed. The comparison of binding potency of TZPM and BSA suggests that the substituents on the benzene ring enhance the binding affinity of TZPM and BSA. We investigated the possible sub-domains on BSA that bind TZPM by displacement experiments. Furthermore, to explore the effect of molecular structure on the binding, a study on quantitative structure-property relationship (QSPR) was performed, the quantitative relationship equation of R(0), r and K(a) were obtained. We observed that R(0), r and K(a) between BSA and TZPM is connected with the margin of the highest and the lowest occupied orbital energy (ΔE), dipole moment (μ), Molar Volume (V(m)), Mole Mass (M). Copyright © 2012 Elsevier B.V. All rights reserved.

Detecting Local Ligand-Binding Site Similarity in Non-Homologous Proteins by Surface Patch Comparison

PubMed Central

Sael, Lee; Kihara, Daisuke

2012-01-01

Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. PMID:22275074

Detecting local ligand-binding site similarity in nonhomologous proteins by surface patch comparison.

PubMed

Sael, Lee; Kihara, Daisuke

2012-04-01

Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. Copyright © 2011 Wiley Periodicals, Inc.

Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

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

Nuemket, Nipawan; Tanaka, Yoshikazu; Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810

2011-07-29

Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinummore » neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.« less

Effects of Iron Deficiency on Iron Binding and Internalization into Acidic Vacuoles in Dunaliella salina1[W][OA

PubMed Central

Paz, Yakov; Shimoni, Eyal; Weiss, Meira; Pick, Uri

2007-01-01

Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe3+ ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability. PMID:17513481

Polymorphisms A387P in thrombospondin-4 and N700S in thrombospondin-1 perturb calcium binding sites.

PubMed

Stenina, Olga I; Ustinov, Valentin; Krukovets, Irene; Marinic, Tina; Topol, Eric J; Plow, Edward F

2005-11-01

Recent genetic studies have associated members of the thrombospondin (TSP) gene family with premature cardiovascular disease. The disease-associated polymorphisms lead to single amino acid changes in TSP-4 (A387P) and TSP-1 (N700S). These substitutions reside in adjacent domains of these highly homologous proteins. Secondary structural predictive programs and the homology of the domains harboring these amino acid substitutions to those in other proteins pointed to potential alterations of putative Ca2+ binding sites that reside in close proximity to the polymorphic amino acids. Since Ca2+ binding is critical for the structure and function of TSP family members, direct evidence for differences in Ca2+ binding by the polymorphic forms was sought. Using synthetic peptides and purified recombinant variant fragments bearing the amino acid substitutions, we measured differences in Tb3+ luminescence as an index of Ca2+ binding. The Tb3+ binding constants placed the TSP-1 region affected by N700S polymorphism among other high-affinity Ca2+ binding sites. The affinity of Ca2+ binding was lower for peptides (3.5-fold) and recombinant fragments (10-fold) containing the S700 vs. the N700 form. In TSP-4, the P387 form acquired an additional Ca2+ binding site absent in the A387 form. The results of our study suggest that both substitutions (A387P in TSP-4 and N700S in TSP-1) alter Ca2+ binding properties. Since these substitutions exert the opposite effects on Ca2+ binding, a decrease in TSP-1 and an increase in TSP-4, the two TSP variants are likely to influence cardiovascular functions in distinct but yet pathogenic ways.

Computational exploration of a protein receptor binding space with student proposed peptide ligands.

PubMed

King, Matthew D; Phillips, Paul; Turner, Matthew W; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M

2016-01-01

Computational molecular docking is a fast and effective in silico method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The DockoMatic tutorial described herein provides a framework by which instructors can guide students through a drug screening exercise. Using receptor models derived from readily available protein crystal structures, docking programs have the ability to predict ligand binding properties, such as preferential binding orientations and binding affinities. The use of computational studies can significantly enhance complimentary wet chemical experimentation by providing insight into the important molecular interactions within the system of interest, as well as guide the design of new candidate ligands based on observed binding motifs and energetics. In this laboratory tutorial, the graphical user interface, DockoMatic, facilitates docking job submissions to the docking engine, AutoDock 4.2. The purpose of this exercise is to successfully dock a 17-amino acid peptide, α-conotoxin TxIA, to the acetylcholine binding protein from Aplysia californica-AChBP to determine the most stable binding configuration. Each student will then propose two specific amino acid substitutions of α-conotoxin TxIA to enhance peptide binding affinity, create the mutant in DockoMatic, and perform docking calculations to compare their results with the class. Students will also compare intermolecular forces, binding energy, and geometric orientation of their prepared analog to their initial α-conotoxin TxIA docking results. © 2015 The International Union of Biochemistry and Molecular Biology.

First principles study of carbon nanostructures, transition metal dichalcogenides, and magnetoelectric interfaces

NASA Astrophysics Data System (ADS)

Hammouri, Mahmoud

Perovskite oxides such as lead zirconate titanate, lanthanum manganite and two dimensional, atomically thick materials such as graphene, carbon nanotubes, graphene nanoribbon, and transition-metal dichalcogenides (TMDs) received intensive attention due to their electronic, magnetic, and transport properties. Understanding the properties and structure of these materials in solid state is a longstanding scientific challenge, especially for experimentalists. Using state-of-the-art density functional theory, different properties can be explained with an excellent match with experiments. This thesis presents an Ab initio density functional theory study of the electronic, magnetic, and transport properties of nanostructure systems. Nanostructures studied in this thesis include graphene, carbon nanotubes, graphene nanoribbons, zirconium disulfide, and La0.67Sr0.33MnO3/PbZr 02 Ti0.8O3 (LSMO/PZT) (100) interface. I investigated the mechanism of chemical functionalization of the side walls of carbon nanotubes by benzyne molecules. Binding energies, geometries, and electronic structure changes due to this functionalization are examined in detail. The binding energies between benzyne molecules and carbon nanotubes are found to be inversely proportional to nanotube diameter. We also studied the properties of graphene nanoribbons under compressions. Our study showed that the band gaps of graphene nanoribbons were strongly affected by applied compression. In addition, we found that the effect of compression has a strong influence on the IV-characteristic. We also investigated the effect of uniaxial strain on the electronic and magnetic properties of zirconium disulfide nanoribbons. Our calculation showed that the magnetization of zirconium disulfide nanoribbons can be switched on and off by the applied strain. In the last part, we studied the properties of the interface between two perovskite oxides, lead zirconate titanate and lanthanum strontium manganite. Our study demonstrated that the magnetoelectric coupling observed at this interface can be explained by the magnetic reconstruction of lanthanum strontium manganite.

Effects of Single Nucleotide Polymorphisms on Human N-Acetyltransferase 2 Structure and Dynamics by Molecular Dynamics Simulation

PubMed Central

Rajasekaran, M.; Abirami, Santhanam; Chen, Chinpan

2011-01-01

Background Arylamine N-acetyltransferase 2 (NAT2) is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear. Methodology/Principal Findings We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential. Conclusions/Significance Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms. PMID:21980537

The PP1 binding code: a molecular-lego strategy that governs specificity.

PubMed

Heroes, Ewald; Lesage, Bart; Görnemann, Janina; Beullens, Monique; Van Meervelt, Luc; Bollen, Mathieu

2013-01-01

Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo. © 2012 The Authors Journal compilation © 2012 FEBS.

Cu(II) binding by a pH-fractionated fulvic acid

USGS Publications Warehouse

Brown, G.K.; Cabaniss, S.E.; MacCarthy, P.; Leenheer, J.A.

1999-01-01

The relationship between acidity, Cu(II) binding and sorption to XAD resin was examined using Suwannee River fulvic acid (SRFA). The work was based on the hypothesis that fractions of SRFA eluted from an XAD column at various pH's from 1.0 to 12.0 would show systematic variations in acidity and possibly aromaticity which in turn would lead to different Cu(II) binding properties. We measured equilibrium Cu(II) binding to these fractions using Cu2+ ion-selective electrode (ISE) potentiometry at pH 6.0. Several model ligands were also examined, including cyclopentane-1,2,3,4-tetracarboxylic acid (CP-TCA) and tetrahydrofuran-2,3,4,5-tetracarboxylic acid (THF-TCA), the latter binding Cu(II) much more strongly as a consequence of the ether linkage. The SRFA Cu(II) binding properties agreed with previous work at high ionic strength, and binding was enhanced substantially at lower ionic strength, in agreement with Poisson-Boltzmann predictions for small spheres. Determining Cu binding constants (K(i)) by non-linear regression with total ligand concentrations (L(Ti)) taken from previous work, the fractions eluted at varying pH had K(i) similar to the unfractionated SRFA, with a maximum enhancement of 0.50 log units. We conclude that variable-pH elution from XAD does not isolate significantly strong (or weak) Cu(II)-binding components from the SRFA mixture. Copyright (C) 1999 Elsevier Science B.V.

Density-functional tight-binding investigation of the structure, stability and material properties of nickel hydroxide nanotubes

NASA Astrophysics Data System (ADS)

Jahangiri, Soran; Mosey, Nicholas J.

2018-01-01

Nickel hydroxide is a material composed of two-dimensional layers that can be rolled up to form cylindrical nanotubes belonging to a class of inorganic metal hydroxide nanotubes that are candidates for applications in catalysis, energy storage, and microelectronics. The stabilities and other properties of this class of inorganic nanotubes have not yet been investigated in detail. The present study uses self-consistent-charge density-functional tight-binding calculations to examine the stabilities, mechanical properties, and electronic properties of nickel hydroxide nanotubes along with the energetics associated with the adsorption of water by these systems. The tight-binding model was parametrized for this system based on the results of first-principles calculations. The stabilities of the nanotubes were examined by calculating strain energies and performing molecular dynamics simulations. The results indicate that single-walled nickel hydroxide nanotubes are stable at room temperature, which is consistent with experimental investigations. The nanotubes possess size-dependent mechanical properties that are similar in magnitude to those of other inorganic nanotubes. The electronic properties of the nanotubes were also found to be size-dependent and small nickel oxyhydroxide nanotubes are predicted to be semiconductors. Despite this size-dependence, both the mechanical and electronic properties were found to be almost independent of the helical structure of the nanotubes. The calculations also show that water molecules have higher adsorption energies when binding to the interior of the nickel hydroxide nanotubes when compared to adsorption in nanotubes formed from other two-dimensional materials such as graphene. The increased adsorption energy is due to the hydrophilic nature of nickel hydroxide. Due to the broad applications of nickel hydroxide, the nanotubes investigated here are also expected to be used in catalysis, electronics, and clean energy production.

Development of a sugar-binding residue prediction system from protein sequences using support vector machine.

PubMed

Banno, Masaki; Komiyama, Yusuke; Cao, Wei; Oku, Yuya; Ueki, Kokoro; Sumikoshi, Kazuya; Nakamura, Shugo; Terada, Tohru; Shimizu, Kentaro

2017-02-01

Several methods have been proposed for protein-sugar binding site prediction using machine learning algorithms. However, they are not effective to learn various properties of binding site residues caused by various interactions between proteins and sugars. In this study, we classified sugars into acidic and nonacidic sugars and showed that their binding sites have different amino acid occurrence frequencies. By using this result, we developed sugar-binding residue predictors dedicated to the two classes of sugars: an acid sugar binding predictor and a nonacidic sugar binding predictor. We also developed a combination predictor which combines the results of the two predictors. We showed that when a sugar is known to be an acidic sugar, the acidic sugar binding predictor achieves the best performance, and showed that when a sugar is known to be a nonacidic sugar or is not known to be either of the two classes, the combination predictor achieves the best performance. Our method uses only amino acid sequences for prediction. Support vector machine was used as a machine learning algorithm and the position-specific scoring matrix created by the position-specific iterative basic local alignment search tool was used as the feature vector. We evaluated the performance of the predictors using five-fold cross-validation. We have launched our system, as an open source freeware tool on the GitHub repository (https://doi.org/10.5281/zenodo.61513). Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Photoionization cross section and binding energy of single dopant in hollow cylindrical core/shell quantum dot

NASA Astrophysics Data System (ADS)

Feddi, E.; El-Yadri, M.; Dujardin, F.; Restrepo, R. L.; Duque, C. A.

2017-02-01

In this study, we have investigated the confined donor impurity in a hollow cylindrical-shell quantum dot. The charges are assumed to be completely confined to the interior of the shell with rigid walls. Within the framework of the effective-mass approximation and by using a simple variational approach, we have computed the donor binding energy as a function of the shell sizes in order to study the behavior of the electron-impurity attraction for a very small thickness. Our results show that the binding energy of a donor impurity placed at the center of cylindrical core/shell dots depends strongly on the shell size. The binding energy increases when the shell-wideness becomes smaller and shows the same behavior as in a simple cylindrical quantum dot. A special case has been studied, which corresponds to the ratio between the inner and outer radii near to one (a/b → 1) for which our model gives a non-significant behavior of the impurity binding energy. This fact implies the existence of a critical value (a/b) for which the binding energy of the donor impurity tends to the limit value of 4 effective Rydbergs as in a 2D quantum well. We also analyse the photoionization cross section considering only the in-plane incident radiation polarization. We determine its behavior as a function of photon energy, shell size, and donor position. The measurement of photoionization in such systems would be of great interest to understand the optical properties of carriers in quantum dots.

A method for predicting individual residue contributions to enzyme specificity and binding-site energies, and its application to MTH1.

PubMed

Stewart, James J P

2016-11-01

A new method for predicting the energy contributions to substrate binding and to specificity has been developed. Conventional global optimization methods do not permit the subtle effects responsible for these properties to be modeled with sufficient precision to allow confidence to be placed in the results, but by making simple alterations to the model, the precisions of the various energies involved can be improved from about ±2 kcal mol -1 to ±0.1 kcal mol -1 . This technique was applied to the oxidized nucleotide pyrophosphohydrolase enzyme MTH1. MTH1 is unusual in that the binding and reaction sites are well separated-an advantage from a computational chemistry perspective, as it allows the energetics involved in docking to be modeled without the need to consider any issues relating to reaction mechanisms. In this study, two types of energy terms were investigated: the noncovalent interactions between the binding site and the substrate, and those responsible for discriminating between the oxidized nucleotide 8-oxo-dGTP and the normal dGTP. Both of these were investigated using the semiempirical method PM7 in the program MOPAC. The contributions of the individual residues to both the binding energy and the specificity of MTH1 were calculated by simulating the effect of mutations. Where comparisons were possible, all calculated results were in agreement with experimental observations. This technique provides fresh insight into the binding mechanism that enzymes use for discriminating between possible substrates.

Constraining the surface properties of effective Skyrme interactions

NASA Astrophysics Data System (ADS)

Jodon, R.; Bender, M.; Bennaceur, K.; Meyer, J.

2016-08-01

Background: Deformation energy surfaces map how the total binding energy of a nuclear system depends on the geometrical properties of intrinsic configurations, thereby providing a powerful tool to interpret nuclear spectroscopy and large-amplitude collective-motion phenomena such as fission. The global behavior of the deformation energy is known to be directly connected to the surface properties of the effective interaction used for its calculation. Purpose: The precise control of surface properties during the parameter adjustment of an effective interaction is key to obtain a reliable and predictive description of nuclear properties. The most relevant indicator is the surface-energy coefficient asurf. There are several possibilities for its definition and estimation, which are not fully equivalent and require a computational effort that can differ by orders of magnitude. The purpose of this study is threefold: first, to identify a scheme for the determination of asurf that offers the best compromise between robustness, precision, and numerical efficiency; second, to analyze the correlation between values for asurf and the characteristic energies of the fission barrier of 240Pu; and third, to lay out an efficient and robust procedure for how the deformation properties of the Skyrme energy density functional (EDF) can be constrained during the parameter fit. Methods: There are several frequently used possibilities to define and calculate the surface energy coefficient asurf of effective interactions built for the purpose of self-consistent mean-field calculations. The most direct access is provided by the model system of semi-infinite nuclear matter, but asurf can also be extracted from the systematics of binding energies of finite nuclei. Calculations can be carried out either self-consistently [Hartree-Fock (HF)], which incorporates quantal shell effects, or in one of the semiclassical extended Thomas-Fermi (ETF) or modified Thomas-Fermi (MTF) approximations. The latter is of particular interest because it provides asurf as a numerical integral without the need to solve self-consistent equations. Results for semi-infinite nuclear matter obtained with the HF, ETF, and MTF methods will be compared with one another and with asurf, as deduced from ETF calculations of very heavy fictitious nuclei. Results: The surface energy coefficient of 76 parametrizations of the Skyrme EDF have been calculated. Values obtained with the HF, ETF, and MTF methods are not identical, but differ by fairly constant systematic offsets. By contrast, extracting asurf from the binding energy of semi-infinite matter or of very large nuclei within the same method gives the same result within the numerical uncertainties. Conclusions: Despite having some drawbacks compared to the other methods studied here, the MTF approach provides sufficiently precise values for asurf such that it can be used as a very robust constraint on surface properties during a parameter fit at negligible additional cost. While the excitation energy of superdeformed states and the height of fission barriers is obviously strongly correlated to asurf, the presence of shell effects prevents a one-to-one correspondence between them. As in addition the value of asurf providing realistic fission barriers depends on the choices made for corrections for spurious motion, its "best value" (within a given scheme to calculate it) depends on the fit protocol. Through the construction of a series of eight parametrizations SLy5s1-SLy5s8 of the standard Skyrme EDF with systematically varied asurf value, it is shown how to arrive at a fit with realistic deformation properties.

Spectroscopic characterization of metal bound phytochelatin analogue (Glu-Cys)4-Gly.

PubMed

Cheng, Yongsheng; Yan, Yong-Bin; Liu, Jinyuan

2005-10-01

The metal ion binding properties of a phytochelatin (PC) analogue, (Glu-Cys)4-Gly (named as EC4), have been studied by a divalent metal ion binding assay monitored by UV-visible spectroscopy, circular dichroism and NMR spectroscopy. Spectro- photometric titration with different divalent metal ions have revealed that the stiochoimetry of metal-bound EC4 was 1:1, and its metal binding affinities with different divalent metal ions in the order of Cd(II)>Cu(II)>Zn(II)>Pb(II)>Ni(II)>Co(II). UV-visible spectroscopic analysis of metal complexes indicated that four sulfur atoms in cysteine residues are attributable to ligand-to-metal charge transfer (LMCT) between divalent metal ions and EC4, and further confirmed by 1D H1 NMR study and Circular Dichroism. In addition, Circular Dichroism spectra of both free and metal-bound forms of EC4 revealed that metal coordination drives the nonapeptide chain to fold into a turned conformation. The comprehensive analysis of spectroscopic properties of the nonapeptide complexed with metal ions not only provides a fundamental description of the metal ion binding properties of PC analogue, but also shows a correlation between metal binding affinity of PC analogue and the induction activity of metal ions.

Unconventional binding sites and receptors for VIP and related peptides PACAP and PHI/PHM: an update.

PubMed

Muller, Jean-Marc; Debaigt, Colin; Goursaud, Stéphanie; Montoni, Alicia; Pineau, Nicolas; Meunier, Annie-Claire; Janet, Thierry

2007-09-01

The 28-amino-acid neuropeptide VIP and related peptides PACAP and PHI/PHM modulate virtually all of the vital functions in the body. These peptides are also commonly recognized as major regulators of cell growth and differentiation. Through their trophic and cytoprotective functions, they appear to play major roles in embryonic development, neurogenesis and the progression of a number of cancer types. These peptides bind to three well-characterized subtypes of G-protein coupled receptors: VPAC1 and VPAC2 share a common high affinity in the nanomolar range for VIP and PACAP; a third receptor type, PAC1, has been characterized for its high affinity for PACAP but its low affinity for VIP. Complex effects and pharmacological behaviors of these peptides suggest that multiple subtypes of binding sites may cooperate to mediate their function in target cells and tissues. In this complex response, some of these binding sites correspond to the definition of the conventional receptors cited above, while others display unexpected pharmacological and functional properties. Here we present potential clues that may lead investigators to further characterize the molecular nature and functions of these atypical binding species.

The effects of Q-nuclei on stellar burning

NASA Astrophysics Data System (ADS)

Boyd, R. N.; Turner, R. E.; Sur, B.; Rybarcyk, L.; Joseph, C.

1985-01-01

The effects of anomalous nuclei, Q-nuclei, on stellar burning are examined. The baryon binding energies, beta-decay properties, and thermonuclear reaction rates for the Q-nuclei suggest they could catalyze a cycle in which four protons are combined to form a 4He nucleus. The properties required of the Q-nuclei for them to solve the solar neutrino problem are determined. A solar modelling calculation was performed with Q-nuclei included, and several interesting results therefrom are compared to observations. Finally the solar neutrino detection rates for 71Ga and 115In detectors, in addition to that for 37Cl, are estimated when Q-nuclei are included in the solar burning.

Hole pairing and thermodynamic properties of the two dimensional frustrated t-J model

NASA Astrophysics Data System (ADS)

Roy, K.; Pal, P.; Nath, S.; Ghosh, N. K.

2018-04-01

The frustrated t-J model is investigated by using the exact-diagonalization (ED) method on an 8-site cluster. The effect on next-nearest-neighbor (NNN) exchange interaction J' (frustration) on the hole pairing and the thermodynamic properties of the system is considered. Two holes initially remain unbound at smaller value of J'/t, but tend to bind at larger value. The maximum possibility of pair formation has been observed to be at NNN sites. Entropy calculation shows that the system goes to more disordered state with J'. The specific heat curves show a single peak structure. A decrease in effective exchange energy is observed due to the frustration.

Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

PubMed

Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

2013-06-14

Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

Point Defect Properties of Cd(Zn)Te and TlBr for Room-Temperature Gamma Radiation Detectors

NASA Astrophysics Data System (ADS)

Lordi, Vincenzo

2013-03-01

The effects of various crystal defects in CdTe, Cd1-xZnxTe (CZT), and TlBr are critical for their performance as room-temperature gamma radiation detectors. We use predictive first principles theoretical methods to provide fundamental, atomic scale understanding of the defect properties of these materials to enable design of optimal growth and processing conditions, such as doping, annealing, and stoichiometry. Several recent cases will be reviewed, including (i) accurate calculations of the thermodynamic and electronic properties of native point defects and point defect complexes in CdTe and CZT; (ii) the effects of Zn alloying on the native point defect properties of CZT; (iii) point defect diffusion and binding related to Te clustering in Cd(Zn)Te; (iv) the profound effect of native point defects--principally vacancies--on the intrinsic material properties of TlBr, particularly electronic and ionic conductivity; (v) tailored doping of TlBr to independently control the electronic and ionic conductivity; and (vi) the effects of metal impurities on the electronic properties and device performance of TlBr detectors. Prepared by LLNL under Contract DE-AC52-07NA27344 with support from the National Nuclear Security Administration Office of Nonproliferation and Verification Research and Development NA-22.

Wobbled electronic properties of lithium clusters: Deterministic approach through first principles

NASA Astrophysics Data System (ADS)

Kushwaha, Anoop Kumar; Nayak, Saroj Kumar

2018-03-01

The innate tendency to form dendritic growth promoted through cluster formation leading to the failure of a Li-ion battery system have drawn significant attention of the researchers towards the effective destabilization of the cluster growth through selective implementation of electrolytic media such as acetonitrile (MeCN). In the present work, using first principles density functional theory and continuum dielectric model, we have investigated the origin of oscillatory nature of binding energy per atom of Lin (n ≤ 8) under the influence of MeCN. In the gas phase, we found that static mean polarizability is strongly correlated with binding energy and shows oscillatory nature with cluster size due to the open shell of Lin cluster. However, in acetonitrile medium, the binding energy has been correlated with electrostatic Lin -MeCN interaction and it has been found that both of them possess wobbled behavior characterized by the cluster size.

The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

NASA Astrophysics Data System (ADS)

Zuo, Ping; Manley, James L.

1994-04-01

ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

Bandgap engineering of GaN nanowires

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

Ming, Bang-Ming; Yan, Hui; Wang, Ru-Zhi, E-mail: wrz@bjut.edu.cn, E-mail: yamcy@csrc.ac.cn

2016-05-15

Bandgap engineering has been a powerful technique for manipulating the electronic and optical properties of semiconductors. In this work, a systematic investigation of the electronic properties of [0001] GaN nanowires was carried out using the density functional based tight-binding method (DFTB). We studied the effects of geometric structure and uniaxial strain on the electronic properties of GaN nanowires with diameters ranging from 0.8 to 10 nm. Our results show that the band gap of GaN nanowires depends linearly on both the surface to volume ratio (S/V) and tensile strain. The band gap of GaN nanowires increases linearly with S/V, whilemore » it decreases linearly with increasing tensile strain. These linear relationships provide an effect way in designing GaN nanowires for their applications in novel nano-devices.« less

Clarified Açaí (Euterpe oleracea) Juice as an Anticonvulsant Agent: In Vitro Mechanistic Study of GABAergic Targets.

PubMed

Arrifano, Gabriela P F; Lichtenstein, Mathieu P; Souza-Monteiro, José Rogério; Farina, Marcelo; Rogez, Hervé; Carvalho, José Carlos Tavares; Suñol, Cristina; Crespo-López, Maria Elena

2018-01-01

Seizures affect about 50 million people around the world. Approximately 30% of seizures are refractory to the current pharmacological arsenal, so, the pursuit of new therapeutic alternatives is essential. Clarified Euterpe oleracea (EO) juice showed anticonvulsant properties similar to diazepam in an in vivo model with pentylenetetrazol, a GABA A receptor blocker. This study investigated the effects of EO on the main GABAergic targets for anticonvulsant drugs, analyzing the effect on the GABA receptor's benzodiazepine and picrotoxinin binding sites and the GABA uptake. Primary cultures of cortical neurons and astrocytes were treated with EO (0-25%) for up to 90 min. [ 3 H]Flunitrazepam and [ 3 H]TBOB binding, [ 3 H]GABA uptake, cell viability, and morphology were assayed. Nonlethal concentrations of EO increased agonist binding and decreased antagonist binding in cortical neurons. Low concentrations significantly inhibited GABA uptake, especially in astrocytes, suggesting an accumulation of endogenous GABA in the synaptic cleft. The results demonstrate, for the first time, that EO can improve GABAergic neurotransmission via interactions with GABA A receptor and modulation of GABA uptake. Understanding these molecular mechanisms will help in the treatment of seizures and epilepsy, especially in developing countries where geographic isolation and low purchasing power are the main barriers to access to adequate treatment.

Clarified Açaí (Euterpe oleracea) Juice as an Anticonvulsant Agent: In Vitro Mechanistic Study of GABAergic Targets

PubMed Central

Arrifano, Gabriela P. F.; Lichtenstein, Mathieu P.; Souza-Monteiro, José Rogério; Rogez, Hervé

2018-01-01

Seizures affect about 50 million people around the world. Approximately 30% of seizures are refractory to the current pharmacological arsenal, so, the pursuit of new therapeutic alternatives is essential. Clarified Euterpe oleracea (EO) juice showed anticonvulsant properties similar to diazepam in an in vivo model with pentylenetetrazol, a GABAA receptor blocker. This study investigated the effects of EO on the main GABAergic targets for anticonvulsant drugs, analyzing the effect on the GABA receptor's benzodiazepine and picrotoxinin binding sites and the GABA uptake. Primary cultures of cortical neurons and astrocytes were treated with EO (0–25%) for up to 90 min. [3H]Flunitrazepam and [3H]TBOB binding, [3H]GABA uptake, cell viability, and morphology were assayed. Nonlethal concentrations of EO increased agonist binding and decreased antagonist binding in cortical neurons. Low concentrations significantly inhibited GABA uptake, especially in astrocytes, suggesting an accumulation of endogenous GABA in the synaptic cleft. The results demonstrate, for the first time, that EO can improve GABAergic neurotransmission via interactions with GABAA receptor and modulation of GABA uptake. Understanding these molecular mechanisms will help in the treatment of seizures and epilepsy, especially in developing countries where geographic isolation and low purchasing power are the main barriers to access to adequate treatment. PMID:29743978

Effects of two novel amino acid substitutions on the penicillin binding properties of the PBP5 C‑terminal from Enterococcus faecium.

PubMed

Zhou, Chengjiang; Niu, Haiying; Yu, Hui; Zhou, Lishe; Wang, Zhanli

2015-10-01

The low‑affinity penicillin‑binding protein (PBP)5 is responsible for resistance to β‑lactam antibiotics in Enterococcus faecium. (E. faecium). In order to evaluate more fully the potential of this species for the development of resistance to β-lactam antibiotics, the present study aimed to examine the extent of penicillin-binding protein (PBP) variations in a collection of clinical E. faecium isolates. In the present study, the C‑terminal domain of PBP5 (PBP5‑CD) of 13 penicillin‑resistant clinical isolates of E. faecium were sequenced and the correlation between penicillin resistance and particular amino acid changes were analyzed. The present study identified for the first time, to the best of our knowledge, two novel substitutions (Tyr460Phe and Ala462Thr or Val462Thr) of E. faecium PBP5‑CD. The covalent interaction between penicillin and PBP5‑CD was also investigated using homology modeling and molecular docking methods. The theoretical calculation revealed that Phe460 and Thr462 were involved in penicillin binding, suggesting that substitutions at these positions exert effects on the affinity for penicillin, and this increased affinity translates into lower resistance in vitro.

Expression, subcellular localization and regulation of sigma receptor in retinal Müller cells

PubMed Central

Jiang, Guoliang; Mysona, Barbara; Dun, Ying; Gnana-Prakasam, Jaya P.; Pabla, Navjotsin; Li, Weiguo; Dong, Zheng; Ganapathy, Vadivel; Smith, Sylvia B.

2013-01-01

Purpose Sigma receptors (σR) are non-opioid, non-phencyclidine binding sites with robust neuroprotective properties. σR1 is expressed in brain oligodendrocytes, but its expression and binding capacity have not been analyzed in retinal glial cells. This study examined the expression, subcellular localization, binding activity and regulation of σR1 in retinal Müller cells. Methods Primary mouse Müller cells (1°MC) were analyzed by RT-PCR, immunoblotting and immunocytochemistry for the expression of σR1 and data were compared to the rat Müller cell line, rMC-1 and rat ganglion cell line, RGC-5. Confocal microscopy was used to determine the subcellular σR1 location in 1°MC. Membranes prepared from these cells were used for binding assays using [3H]-pentazocine (PTZ). The kinetics of binding, the ability of various σR1 ligands to compete with σR1 binding and the effects of nitric oxide (NO) and reactive oxygen species (ROS) donors on binding were examined. Results σR1 is expressed in 1°MC and is localized to the nuclear and endoplasmic reticulum membranes. Binding assays showed that in 1°MCs, rMC-1 and RGC-5 cells, the binding of PTZ was saturable. [3H]-PTZ bound with high affinity in RGC-5 and rMC-1 cells and the binding was similarly robust in 1°MC. Competition studies showed marked inhibition of [3H]-PTZ binding in the presence of σR1-specific ligands. Incubation of cells with NO and ROS donors markedly increased σR1 binding activity. Conclusions Müller cells express σR1 and demonstrate robust σR1 binding activity, which is inhibited by σR1 ligands and is stimulated during oxidative stress. The potential of Müller cells to bind σR1 ligands may prove beneficial in retinal degenerative diseases such as diabetic retinopathy. PMID:17122151

Expression, subcellular localization, and regulation of sigma receptor in retinal muller cells.

PubMed

Jiang, Guoliang; Mysona, Barbara; Dun, Ying; Gnana-Prakasam, Jaya P; Pabla, Navjotsin; Li, Weiguo; Dong, Zheng; Ganapathy, Vadivel; Smith, Sylvia B

2006-12-01

Sigma receptors (sigmaRs) are nonopioid, nonphencyclidine binding sites with robust neuroprotective properties. Type 1 sigmaR1 (sigmaR1) is expressed in brain oligodendrocytes, but its expression and binding capacity have not been analyzed in retinal glial cells. This study examined the expression, subcellular localization, binding activity, and regulation of sigmaR1 in retinal Müller cells. Primary mouse Müller cells (MCs) were analyzed by RT-PCR, immunoblotting, and immunocytochemistry for the expression of sigmaR1, and data were compared with those of the rat Müller cell line (rMC-1) and the rat ganglion cell line (RGC-5). Confocal microscopy was used to determine the subcellular sigmaR1 location in primary mouse MCs. Membranes prepared from these cells were used for binding assays with [3H]-pentazocine (PTZ). The kinetics of binding, the ability of various sigmaR1 ligands to compete with sigmaR1 binding, and the effects of donated nitric oxide (NO) and reactive oxygen species (ROS) on binding were examined. sigmaR1 is expressed in primary mouse MCs and is localized to the nuclear and endoplasmic reticulum membranes. Binding assays showed that in primary mouse MCs, rMC-1, and RGC-5, the binding of PTZ was saturable. [3H]-PTZ bound with high affinity in RGC-5 and rMC-1 cells, and the binding was similarly robust in primary mouse MCs. Competition studies showed marked inhibition of [3H]-PTZ binding in the presence of sigmaR1-specific ligands. Incubation of cells with NO and ROS donors markedly increased sigmaR1 binding activity. MCs express sigmaR1 and demonstrate robust sigmaR1 binding activity, which is inhibited by sigmaR1 ligands and is stimulated during oxidative stress. The potential of Müller cells to bind sigmaR1 ligands may prove beneficial in retinal degenerative diseases such as diabetic retinopathy.

HMG I(Y) interferes with the DNA binding of NF-AT factors and the induction of the interleukin 4 promoter in T cells

PubMed Central

Klein-Hessling, Stefan; Schneider, Günter; Heinfling, Annette; Chuvpilo, Sergei; Serfling, Edgar

1996-01-01

HMG I(Y) proteins bind to double-stranded A+T oligonucleotides longer than three base pairs. Such motifs form part of numerous NF-AT-binding sites of lymphokine promoters, including the interleukin 4 (IL-4) promoter. NF-AT factors share short homologous peptide sequences in their DNA-binding domain with NF-κB factors and bind to certain NF-κB sites. It has been shown that HMG I(Y) proteins enhance NF-κB binding to the interferon β promoter and virus-mediated interferon β promoter induction. We show that HMG I(Y) proteins exert an opposite effect on the DNA binding of NF-AT factors and the induction of the IL-4 promoter in T lymphocytes. Introduction of mutations into a high-affinity HMG I(Y)-binding site of the IL-4 promoter, which decreased HMG I(Y)-binding to a NF-AT-binding sequence, the Pu-bB (or P) site, distinctly increased the induction of the IL-4 promoter in Jurkat T leukemia cells. High concentrations of HMG I(Y) proteins are able to displace NF-ATp from its binding to the Pu-bB site. High HMG I(Y) concentrations are typical for Jurkat cells and peripheral blood T lymphocytes, whereas El4 T lymphoma cells and certain T helper type 2 cell clones contain relatively low HMG I(Y) concentrations. Our results indicate that HMG I(Y) proteins do not cooperate, but instead compete with NF-AT factors for the binding to DNA even though NF-AT factors share some DNA-binding properties with NF-kB factors. This competition between HMG I(Y) and NF-AT proteins for DNA binding might be due to common contacts with minor groove nucleotides of DNA and may be one mechanism contributing to the selective IL-4 expression in certain T lymphocyte populations, such as T helper type 2 cells. PMID:8986808

Specific binding of nicergoline on an alpha1-like adrenoreceptor in the rat retina.

PubMed

Lograno, M D; Tricarico, D; Masciopinto, V; Scuderl, A C

2000-02-01

Systemic treatment with nicergoline, an ergoline derivative showing alpha1-antagonist properties, causes vasodilatation in the eye without apparent untoward cardiovascular effects. In the present work we investigated the ability of nicergoline to inhibit the binding of radiolabelled prazosin in the rat retina and cortex. We found that nicergoline inhibited [3H]prazosin binding in both tissues, being more potent than unlabelled prazosin in the retinal tissue. The competition curves of the ergoline derivative were well fitted by a one-site model in the cortical tissue, with an IC50 (concentration of the drugs needed to inhibit the binding of labelled prazosin by 50%) of 2.54 x 10(-8) M, and by a two-site model in the retinal tissue, with IC50 values of 7.08 x 10(-12) M and 1.82 x 10(-5) M. 2-(2,6 dimetoxyphenoxyethyl) aminomethyl-1,4-benzodioxane hydrochloride (WB4101) and phentolamine, selective ligands for the high-affinity binding site for prazosin, in particular the alpha1A-site, fully inhibited prazosin binding in the cortex but only partially inhibited prazosin binding in the retina, being less potent in this tissue than either nicergoline or prazosin. Our results suggest that a binding component of alpha1-adrenoreceptors is expressed to a lesser extent in the retina than the cortex, leading to a reduced response of the retinal tissue to prazosin, and more particularly to WB4101 and phentolamine. The selective binding of the nicergoline on this retinal adrenoreceptor may explain the peculiar efficacy of the drug in ocular pathophysiology.

Molecular mechanisms of immunosuppression.

PubMed

Baumann, G; Zenke, G; Wenger, R; Hiestand, P; Quesniaux, V; Andersen, E; Schreier, M H

1992-04-01

The immunosuppressive drug cyclosporin A (CsA, Sandimmun, SIM) is currently being evaluated in a variety of autoimmune disorders with some remarkable successes. Despite the wide empiric application of CsA, the precise mechanism of action of this drug remains elusive. To identify the molecular mode of action of CsA in the process of T cell activation, we have compared the biological profile of cyclophilin-binding cyclosporin analogues (CBCA), which lack immunosuppressive properties, with CsA. We have found that CsA binding to its intracellular receptor (cyclophilin) is required but not sufficient for immunosuppression. Moreover, inhibition of the peptidyl-prolyl cis-trans isomerase activity of cyclophilin does not seem to be relevant for the inhibitory effects of CsA. In analogy to the immunosuppressants FK506 and rapamycin, a specific structure at the 'effector' domain of the CsA molecule different from the immunophilin 'binding' domain determines the biological activity. Overall, a significant understanding of the structure-activity relationship of CsA has emerged. This will have a major impact on the identification of the precise mechanism of action of CsA and its side effects in the process of immunosuppression.

Investigating the Importance of the Pocket-estimation Method in Pocket-based Approaches: An Illustration Using Pocket-ligand Classification.

PubMed

Caumes, Géraldine; Borrel, Alexandre; Abi Hussein, Hiba; Camproux, Anne-Claude; Regad, Leslie

2017-09-01

Small molecules interact with their protein target on surface cavities known as binding pockets. Pocket-based approaches are very useful in all of the phases of drug design. Their first step is estimating the binding pocket based on protein structure. The available pocket-estimation methods produce different pockets for the same target. The aim of this work is to investigate the effects of different pocket-estimation methods on the results of pocket-based approaches. We focused on the effect of three pocket-estimation methods on a pocket-ligand (PL) classification. This pocket-based approach is useful for understanding the correspondence between the pocket and ligand spaces and to develop pharmacological profiling models. We found pocket-estimation methods yield different binding pockets in terms of boundaries and properties. These differences are responsible for the variation in the PL classification results that can have an impact on the detected correspondence between pocket and ligand profiles. Thus, we highlighted the importance of the pocket-estimation method choice in pocket-based approaches. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

From oleic acid-capped iron oxide nanoparticles to polyethyleneimine-coated single-particle magnetofectins

NASA Astrophysics Data System (ADS)

Cruz-Acuña, Melissa; Maldonado-Camargo, Lorena; Dobson, Jon; Rinaldi, Carlos

2016-09-01

Various inorganic nanoparticle designs have been developed and used as non-viral gene carriers. Magnetic gene carriers containing polyethyleneimine (PEI), a well-known transfection agent, have been shown to improve DNA transfection speed and efficiency in the presence of applied magnetic field gradients that promote particle-cell interactions. Here we report a method to prepare iron oxide nanoparticles conjugated with PEI that: preserves the narrow size distribution of the nanoparticles, conserves magnetic properties throughout the process, and results in efficient transfection. We demonstrate the ability of the particles to electrostatically bind with DNA and transfect human cervical cancer (HeLa) cells by the use of an oscillating magnet array. Their transfection efficiency is similar to that of Lipofectamine 2000™, a commercial transfection reagent. PEI-coated particles were subjected to acidification, and acidification in the presence of salts, before DNA binding. Results show that although these pre-treatments did not affect the ability of particles to bind DNA they did significantly enhanced transfection efficiency. Finally, we show that these magnetofectins (PEI-MNP/DNA) complexes have no effect on the viability of cells at the concentrations used in the study. The systematic preparation of magnetic vectors with uniform physical and magnetic properties is critical to progressing this non-viral transfection technology.

Harmane: an atypical neurotransmitter?

PubMed

Abu Ghazaleh, Haya; Lalies, Maggie D; Nutt, David J; Hudson, Alan L

2015-03-17

Harmane is an active component of clonidine displacing substance and a candidate endogenous ligand for imidazoline binding sites. The neurochemistry of tritiated harmane was investigated in the present study examining its uptake and release properties in the rat brain central nervous system (CNS) in vitro. At physiological temperature, [(3)H]harmane was shown to be taken up in rat brain cortex. Further investigations demonstrated that treatment with monoamine uptake blockers (citalopram, nomifensine and nisoxetine) did not alter [(3)H]harmane uptake implicating that the route of [(3)H]harmane transport was distinct from the monoamine uptake systems. Furthermore, imidazoline ligands (rilmenidine, efaroxan, 2-BFI and idazoxan) showed no prominent effect on [(3)H]harmane uptake suggesting the lack of involvement of imidazoline binding sites. Subsequent analyses showed that disruption of the Na(+) gradient using ouabain or choline chloride did not block [(3)H]harmane uptake suggesting a Na(+)-independent transport mechanism. Moreover, higher temperatures (50°C) failed to impede [(3)H]harmane uptake implying a non-physiological transporter. The failure of potassium to evoke the release of preloaded [(3)H]harmane from rat brain cortex indicates that the properties of this putative endogenous ligand for imidazoline binding sites do not resemble that of a conventional neurotransmitter. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Target-molecule-triggered rupture of aptamer-encapsulated polyelectrolyte microcapsules.

PubMed

Zhang, Xueru; Chabot, Denise; Sultan, Yasir; Monreal, Carlos; DeRosa, Maria C

2013-06-26

Polyelectrolyte microcapsules have great potential for serving as carriers for the delivery of their contents when triggered by an external stimulus. Aptamers are synthetic ssDNA or RNA that can bind to specific targets with high affinity and selectivity. Aptamers may retain these superior molecular recognition properties after encapsulation within polymer microcapsules. In this work, stable polyelectrolyte microcapsules with encapsulated aptamers were obtained by the layer-by-layer (LbL) method. Polyelectrolyte films were deposited onto a CaCO3 template that had been predoped with polystyrene sulfonate (PSS) and aptamer sequences (SA) that have an affinity for the dye sulforhodamine B (SRB). The PSS and aptamers are thought to serve as an internal scaffold supporting the microcapsule walls. These microcapsules would present target-molecule-triggered rupture properties. Microcapsule collapse was triggered by the binding of SRB to the encapsulated aptamer. The specificity of microcapsule collapse was investigated using a similar dye, tetramethylrosamine (TMR), which does not have affinity for SA. A high concentration of TMR did not lead to the collapse of the microcapsules. The effect of target binding on the microcapsules was confirmed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). These microcapsules may have potential applications in targeted delivery systems for the controlled release of drugs, pesticides, or other payloads.

Lecithin retinol acyltransferase and its S175R mutant have a similar secondary structure content and maximum insertion pressure but different enzyme activities.

PubMed

Bussières, Sylvain; Cantin, Line; Salesse, Christian

2011-11-01

Recent work on Lecithin:retinol acyltransferase (LRAT) allowed to gather a large amount of information on its secondary structure, enzymatic properties and membrane binding. A truncated form of LRAT (tLRAT) as well as its S175R mutant leading to retinis pigmentosa, a severe form of retinal dystrophy, were studied to understand the role of this mutation on the dysfunction of this protein. Consistently with previous reports, the S175R-tLRAT mutant was shown to lack enzyme activity. However, very similar secondary structures probed by circular dichroism have been obtained with the S175R-tLRAT mutant and tLRAT. Moreover, similar values of maximum insertion pressure of the S175R-tLRAT mutant and tLRAT have been obtained using Langmuir monolayers, thus suggesting that the S175R mutation has no effect on the membrane binding properties of tLRAT. These findings leave open the possibility that the loss of enzymatic activity associated with the S175R mutant is related to loss of an essential nucleophile near the active site, or alternatively, to steric obstruction of the active site that impedes substrate binding. Copyright © 2011 Elsevier Ltd. All rights reserved.

Binding of pyrene to aquatic and commercial humic substances: The role of molecular weight and aromaticity

USGS Publications Warehouse

Chin, Y.-P.; Aiken, G.R.; Danielsen, K.M.

1997-01-01

The binding of pyrene to a number of humic substances isolated from various aquatic sources and a commercial humic acid was measured using the solubility enhancement method. The humic materials used in this study were characterized by various spectroscopic and liquid chromatography methods. A strong correlation was observed between the pyrene binding coefficient, K(doc), and the molecular weights, molar absorptivities at 280 nm, and aromaticity of the aquatic humic substances. Binding of pyrene to the commercial humic acid, however, was significantly stronger and did not obey the relationships observed between K(doc) and the chemical properties of the aquatic humic substrates. These results suggest that the molecular weight and the aromatic content of the humic substrates exert influences on the binding of nonpolar and planar aromatic molecules and that the physicochemical properties of both humic materials and organic solutes are important in controlling the speciation of nonpolar organic contaminants in natural waters.