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Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme‐mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer–peptide hybrids. A hybrid macrocyclic nanomolar binder to the C‐lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition. [ABSTRACT FROM AUTHOR]

Computational methodologies are increasingly addressing modeling of the whole cell at the molecular level. Proteins and their interactions are the key component of cellular processes. Techniques for modeling protein interactions, thus far, have included protein docking and molecular simulation. The latter approaches account for the dynamics of the interactions but are relatively slow, if carried out at all-atom resolution, or are significantly coarse grained. Protein docking algorithms are far more efficient in sampling spatial coordinates. However, they do not account for the kinetics of the association (i.e., they do not involve the time coordinate). Our proof-of-concept study bridges the two modeling approaches, developing an approach that can reach unprecedented simulation timescales at all-atom resolution. The global intermolecular energy landscape of a large system of proteins was mapped by the pairwise fast Fourier transform docking and sampled in space and time by Monte Carlo simulations. The simulation protocol was parametrized on existing data and validated on a number of observations from experiments and molecular dynamics simulations. The simulation protocol performed consistently across very different systems of proteins at different protein concentrations. It recapitulated data on the previously observed protein diffusion rates and aggregation. The speed of calculation allows reaching second-long trajectories of protein systems that approach the size of the cells, at atomic resolution. [ABSTRACT FROM AUTHOR]

Moonlighting proteins have at least two independent functions and are widely found in animals, plants and microorganisms. Moonlighting proteins play important roles in signal transduction, cell growth and movement, tumor inhibition, DNA synthesis and repair, and metabolism of biological macromolecules. Moonlighting proteins are difficult to find through biological experiments, so many researchers identify moonlighting proteins through bioinformatics methods, but their accuracies are relatively low. Therefore, we propose a new method. In this study, we select SVMProt-188D as the feature input, and apply a model combining linear discriminant analysis and basic classifiers in machine learning to study moonlighting proteins, and perform bagging ensemble on the best-performing support vector machine. They are identified accurately and efficiently. The model achieves an accuracy of 93.26% and an F-sorce of 0.946 on the MPFit dataset, which is better than the existing MEL-MP model. Meanwhile, it also achieves good results on the other two moonlighting protein datasets. [ABSTRACT FROM AUTHOR]

Human Transferrin (HTr) imprinted composite cryogel (HTr-MIPCC) discs were synthesized with distinctive structure and increased adsorption capacity and specificity for HTr by combining the advantages of the surface imprinting technique. HEMA-based cryogel was embedded with HTr-surface imprinted particles. Thanks to the imprinted particles embedding, both the higher surface area and specific recognition are provided. The structure of the HTr-MIPCC was characterized by FTIR Spectrometer, SEM, swelling studies, flow dynamics, and surface area measurements. The addition of surface imprinted particles into the cryogel disc has resulted in an increased surface area, enhancing the capacity of composite cryogel for HTr adsorption up to 10.96 mg/g and gel fractionation yields reached up to 83%. Selectivity studies were carried out against HIgG and HSA as competitors. The HTr-MIPCC discs were packaged into an FPLC column for selective depletion of HTr. It was calculated that the HTr-MIPCC is 1.071 and 1.062 times selective for HTr than HSA and HIgG, respectively. According to FPLC studies HTr-MIPCC can be considered as an excellent alternative for affinity matrixes used for the detection and selective recognition of HTr directly from human serum. HTr-MIPCC discs can be used several times without any noteworthy reduction in the adsorption capacity of HTr. [ABSTRACT FROM AUTHOR]

Anion exchange affinity-based controllable surface imprinting is an effective approach to overcome low imprinting efficiency and high non-specific binding capacity. The template proteins were first immobilized on the anchored tetraalkylammonium groups of the nanoparticles via anion exchange affinity-based interactions, enabling monolayer sorption using a low template concentration. The combined use of surface-initiated photoiniferter-mediated polymerization to precisely control the imprinted film thickness, allowing the formation of homogeneous binding cavities, and the construction of effective binding sites resulted in a low non-specific binding capacity and high imprinting efficiency. The obtained imprinted films benefited from the anion exchange mechanism, exhibiting a higher imprinting factor and faster binding rate than the reference material. Binding tests revealed that the binding strength and selective recognition properties could be tuned to a certain extent by adjusting the NaCl concentration. Additionally, in contrast to the harsh template elution conditions of the covalent immobilization approach, over 80% of the template molecules were readily removed from the imprinted films using supersonic elution with an aqueous mixture of NaCl and HAc. Introducing template immobilization by anion exchange interactions to the synthesis of imprinted materials may provide a new approach for effective biomacromolecular imprinting. [ABSTRACT FROM AUTHOR]

Rapid, yet accurate and sensitive testing has been shown to be critical in the control of spreading pandemic diseases such as COVID-19. Current methods which are highly sensitive and can differentiate different strains are slow and cannot be conveniently applied at the point of care. Rapid tests, meanwhile, require a high titer and are not sufficiently sensitive to discriminate between strains. Here, we report a rapid and facile potentiometric detection method based on nanoscale, three-dimensional molecular imprints of analytes on a self-assembled monolayer (SAM), which can deliver analyte-specific detection of both whole virions and isolated proteins in microliter amounts of bodily fluids within minutes. The detection substrate with nanoscale inverse surface patterns of analytes formed by a SAM identifies a target analyte by recognizing its surface nano- and molecular structures, which can be monitored by temporal measurement of the change in substrate open-circuit potential. The sensor unambiguously detected and differentiated H1N1 and H3N2 influenza A virions as well as the spike proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle-East respiratory syndrome (MERS) coronavirus in human saliva with limits of detection reaching 200 PFU/mL and 100 pg/mL for the viral particles and spike proteins, respectively. The demonstrated speed and specificity of detection, combined with a low required sample volume, high sensitivity, ease of potentiometric measurement, and simple sample collection and preparation, suggest that the technique can be used as a highly effective point-of-care diagnostic platform for a fast, accurate, and specific detection of various viral pathogens and their variants. [ABSTRACT FROM AUTHOR]

Structures of proteins and protein–protein complexes are determined by the same physical principles and thus share a number of similarities. At the same time, there could be differences because in order to function, proteins interact with other molecules, undergo conformations changes, and so forth, which might impose different restraints on the tertiary versus quaternary structures. This study focuses on structural properties of protein–protein interfaces in comparison with the protein core, based on the wealth of currently available structural data and new structure‐based approaches. The results showed that physicochemical characteristics, such as amino acid composition, residue–residue contact preferences, and hydrophilicity/hydrophobicity distributions, are similar in protein core and protein–protein interfaces. On the other hand, characteristics that reflect the evolutionary pressure, such as structural composition and packing, are largely different. The results provide important insight into fundamental properties of protein structure and function. At the same time, the results contribute to better understanding of the ways to dock proteins. Recent progress in predicting structures of individual proteins follows the advancement of deep learning techniques and new approaches to residue coevolution data. Protein core could potentially provide large amounts of data for application of the deep learning to docking. However, our results showed that the core motifs are significantly different from those at protein–protein interfaces, and thus may not be directly useful for docking. At the same time, such difference may help to overcome a major obstacle in application of the coevolutionary data to docking—discrimination of the intramolecular information not directly relevant to docking. [ABSTRACT FROM AUTHOR]

We present a simple, rapid method for forming supported lipid bilayers on organic electronic devices composed of conducting polymer electrodes using a solvent-assisted lipid bilayer formation method. These supported bilayers present protein recognition elements that are mobile, critical for multivalent binding interactions. Because these polymers are transparent and conducting, we demonstrate, by optical and electrical detection, the specific interactions of proteins with these biomembrane-based bioelectronic devices. This work paves the way for easy formation of biomembrane mimetics for sensing and detection of binding events in a label-free manner on organic electronic devices of more sophisticated architectures. [ABSTRACT FROM AUTHOR]

Comparative docking is based on experimentally determined structures of protein‐protein complexes (templates), following the paradigm that proteins with similar sequences and/or structures form similar complexes. Modeling utilizing structure similarity of target monomers to template complexes significantly expands structural coverage of the interactome. Template‐based docking by structure alignment can be performed for the entire structures or by aligning targets to the bound interfaces of the experimentally determined complexes. Systematic benchmarking of docking protocols based on full and interface structure alignment showed that both protocols perform similarly, with top 1 docking success rate 26%. However, in terms of the models' quality, the interface‐based docking performed marginally better. The interface‐based docking is preferable when one would suspect a significant conformational change in the full protein structure upon binding, for example, a rearrangement of the domains in multidomain proteins. Importantly, if the same structure is selected as the top template by both full and interface alignment, the docking success rate increases 2‐fold for both top 1 and top 10 predictions. Matching structural annotations of the target and template proteins for template detection, as a computationally less expensive alternative to structural alignment, did not improve the docking performance. Sophisticated remote sequence homology detection added templates to the pool of those identified by structure‐based alignment, suggesting that for practical docking, the combination of the structure alignment protocols and the remote sequence homology detection may be useful in order to avoid potential flaws in generation of the structural templates library. [ABSTRACT FROM AUTHOR]

Protein–protein interactions form central elements of almost all cellular processes. Knowledge of the structure of protein–protein complexes but also of the binding affinity is of major importance to understand the biological function of protein–protein interactions. Even weak transient protein–protein interactions can be of functional relevance for the cell during signal transduction or regulation of metabolism. The structure of a growing number of protein–protein complexes has been solved in recent years. Combined with docking approaches or template‐based methods, it is possible to generate structural models of many putative protein–protein complexes or to design new protein–protein interactions. In order to evaluate the functional relevance of putative or predicted protein–protein complexes, realistic binding affinity prediction is of increasing importance. Several computational tools ranging from simple force‐field or knowledge‐based scoring of single protein–protein complexes to ensemble‐based approaches and rigorous binding free energy simulations are available to predict relative and absolute binding affinities of complexes. With a focus on molecular mechanics force‐field approaches the present review aims at presenting an overview on available methods and discussing advantages, approximations, and limitations of the various methods. This article is categorized under:Molecular and Statistical Mechanics > Molecular InteractionsMolecular and Statistical Mechanics > Free Energy MethodsSoftware > Molecular Modeling [ABSTRACT FROM AUTHOR]

In this Minireview, an overview about the past 20 years of the guanidiniocarbonyl‐pyrrole (GCP) binding motif, which was designed, investigated and applied by Prof. Dr. Carsten Schmuck, is presented. Here we highlight the first steps from design and discovery, initial binding studies, applications in material science to advanced biomedical use in protein modulation and delivery of genetic material. [ABSTRACT FROM AUTHOR]

The argonaute protein (Ago) exists in almost all organisms. In eukaryotes, it functions as a regulatory system for gene expression. In prokaryotes, it is a type of defense system against foreign invasive genomes. The Ago system has been engineered for gene silencing and genome editing and plays an important role in biological studies. With an increasing number of genomes and proteomes of various microbes becoming available, computational tools for identifying and annotating argonaute proteins are urgently needed. We introduce AGONOTES (Argonaute Notes). It is a web service especially designed for identifying and annotating Ago. AGONOTES uses the BLASTP similarity search algorithm to categorize all submitted proteins into three groups: prokaryotic argonaute protein (pAgo), eukaryotic argonaute protein (eAgo), and non-argonaute protein (non-Ago). Argonaute proteins can then be aligned to the corresponding standard set of Ago sequences using the multiple sequence alignment program MUSCLE. All functional domains of Ago can further be curated from the alignment results and visualized easily through Bio::Graphic modules in the BioPerl bundle. Compared with existing tools such as CD-Search and available databases such as UniProt and AGONOTES showed a much better performance on domain annotations, which is fundamental in studying the new Ago. AGONOTES can be freely accessed at http://i.uestc.edu.cn/agonotes/. AGONOTES is a friendly tool for annotating Ago domains from a proteome or a series of protein sequences. [ABSTRACT FROM AUTHOR]

Structural characterization of protein‐protein interactions is essential for our ability to study life processes at the molecular level. Computational modeling of protein complexes (protein docking) is important as the source of their structure and as a way to understand the principles of protein interaction. Rapidly evolving comparative docking approaches utilize target/template similarity metrics, which are often based on the protein structure. Although the structural similarity, generally, yields good performance, other characteristics of the interacting proteins (eg, function, biological process, and localization) may improve the prediction quality, especially in the case of weak target/template structural similarity. For the ranking of a pool of models for each target, we tested scoring functions that quantify similarity of Gene Ontology (GO) terms assigned to target and template proteins in three ontology domains—biological process, molecular function, and cellular component (GO‐score). The scoring functions were tested in docking of bound, unbound, and modeled proteins. The results indicate that the combined structural and GO‐terms functions improve the scoring, especially in the twilight zone of structural similarity, typical for protein models of limited accuracy. [ABSTRACT FROM AUTHOR]

The classical crystal structure of insulin was determined in 1969 by D.C. Hodgkin et al. following a 35‐year program of research. This structure depicted a hexamer remarkable for its self‐assembly as a zinc‐coordinated trimer of dimer. Prominent at the dimer interface was an “aromatic triplet” of conserved residues at consecutive positions in the B chain: PheB24, PheB25 and TyrB26. The elegance of this interface inspired the Oxford team to poetry: “A thing of beauty is a joy forever” (John Keats as quoted by Blundell, T.L., et al. Advances in Protein Chemistry 26:279‐286 [1972]). Here, we revisit this aromatic triplet in light of recent advances in the structural biology of insulin bound as a monomer to fragments of the insulin receptor. Such co‐crystal structures have defined how these side chains pack at the primary hormone‐binding surface of the receptor ectodomain. On receptor binding, the B‐chain β‐strand (residues B24‐B28) containing the aromatic triplet detaches from the α‐helical core of the hormone. Whereas TyrB26 lies at the periphery of the receptor interface and may functionally be replaced by a diverse set of substitutions, PheB24 and PheB25 engage invariant elements of receptor domains L1 and αCT. These critical contacts were anticipated by the discovery of diabetes‐associated mutations at these positions by Donald Steiner et al. at the University of Chicago. Conservation of PheB24, PheB25 and TyrB26 among vertebrate insulins reflects the striking confluence of structure‐based evolutionary constraints: foldability, protective self‐assembly and hormonal activity. [ABSTRACT FROM AUTHOR]

Abstract: The paper presents analysis of our template‐based and free docking predictions in the joint CASP12/CAPRI37 round. A new scoring function for template‐based docking was developed, benchmarked on the D ockground resource, and applied to the targets. The results showed that the function successfully discriminates the incorrect docking predictions. In correctly predicted targets, the scoring function was complemented by other considerations, such as consistency of the oligomeric states among templates, similarity of the biological functions, biological interface relevance, etc. The scoring function still does not distinguish well biological from crystal packing interfaces, and needs further development for the docking of bundles of α‐helices. In the case of the trimeric targets, sequence‐based methods did not find common templates, despite similarity of the structures, suggesting complementary use of structure‐ and sequence‐based alignments in comparative docking. The results showed that if a good docking template is found, an accurate model of the interface can be built even from largely inaccurate models of individual subunits. Free docking however is very sensitive to the quality of the individual models. However, our newly developed contact potential detected approximate locations of the binding sites. [ABSTRACT FROM AUTHOR]

Characterization of life processes at the molecular level requires structural details of protein interactions. The number of experimentally determined structures of protein-protein complexes accounts only for a fraction of known protein interactions. This gap in structural description of the interactome has to be bridged by modeling. An essential part of the development of structural modeling/docking techniques for protein interactions is databases of protein-protein complexes. They are necessary for studying protein interfaces, providing a knowledge base for docking algorithms, and developing intermolecular potentials, search procedures, and scoring functions. Development of protein-protein docking techniques requires thorough benchmarking of different parts of the docking protocols on carefully curated sets of protein-protein complexes. We present a comprehensive description of the D ockground resource () for structural modeling of protein interactions, including previously unpublished unbound docking benchmark set 4, and the X-ray docking decoy set 2. The resource offers a variety of interconnected datasets of protein-protein complexes and other data for the development and testing of different aspects of protein docking methodologies. Based on protein-protein complexes extracted from the PDB biounit files, D ockground offers sets of X-ray unbound, simulated unbound, model, and docking decoy structures. All datasets are freely available for download, as a whole or selecting specific structures, through a user-friendly interface on one integrated website. [ABSTRACT FROM AUTHOR]

ABSTRACT Structural characterization of proteins is essential for understanding life processes at the molecular level. However, only a fraction of known proteins have experimentally determined structures. This fraction is even smaller for protein-protein complexes. Thus, structural modeling of protein-protein interactions (docking) primarily has to rely on modeled structures of the individual proteins, which typically are less accurate than the experimentally determined ones. Such 'double' modeling is the Grand Challenge of structural reconstruction of the interactome. Yet it remains so far largely untested in a systematic way. We present a comprehensive validation of template-based and free docking on a set of 165 complexes, where each protein model has six levels of structural accuracy, from 1 to 6 Å Cα RMSD. Many template-based docking predictions fall into acceptable quality category, according to the CAPRI criteria, even for highly inaccurate proteins (5-6 Å RMSD), although the number of such models (and, consequently, the docking success rate) drops significantly for models with RMSD > 4 Å. The results show that the existing docking methodologies can be successfully applied to protein models with a broad range of structural accuracy, and the template-based docking is much less sensitive to inaccuracies of protein models than the free docking. Proteins 2017; 85:470-478. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Structural characterization of protein-protein interactions is essential for understanding life processes at the molecular level. However, only a fraction of protein interactions have experimentally resolved structures. Thus, reliable computational methods for structural modeling of protein interactions (protein docking) are important for generating such structures and understanding the principles of protein recognition. Template-based docking techniques that utilize structural similarity between target protein-protein interaction and cocrystallized protein-protein complexes (templates) are gaining popularity due to generally higher reliability than that of the template-free docking. However, the template-based approach lacks explicit penalties for intermolecular penetration, as opposed to the typical free docking where such penalty is inherent due to the shape complementarity paradigm. Thus, template-based docking models are commonly assumed to require special treatment to remove large structural penetrations. In this study, we compared clashes in the template-based and free docking of the same proteins, with crystallographically determined and modeled structures. The results show that for the less accurate protein models, free docking produces fewer clashes than the template-based approach. However, contrary to the common expectation, in acceptable and better quality docking models of unbound crystallographically determined proteins, the clashes in the template-based docking are comparable to those in the free docking, due to the overall higher quality of the template-based docking predictions. This suggests that the free docking refinement protocols can in principle be applied to the template-based docking predictions as well. Proteins 2016; 85:39-45. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

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

Biomimetic multilevel structured membrane materials have great potential for energy-efficient chemical separations and biomedical applications. The current study represents a simple, yet efficient, method to obtain the biomimetic protein separation membrane and controllable cell culture substrate with high stability, selectivity, and antibacterial property. Here, a molecular imprinting methodology is reported to introduce the high-biocompatible protein ovalbumin (Ova) to a multilevel Ag/SiO2/organic based molecularly imprinted membranes (ASO-MIMs), which have made significant achievements in protein identification and controllable growth of liver cells in vitro platform. Interestingly, the relative morphological observations of the adhered cells and in vitro viability tests show no significant difference between the ASO-MIMs binding with 13.6 mg g−1 Ova (13.6-ASO-MIMs) and bare glass, indicating the excellent biocompatibility of the 13.6-ASO-MIMs. Here, the results on largely enhanced adsorption capacity, perm-selectivity (β values are more than 2.2), regeneration ability (still maintained 90% of the maximum adsorption capacity after 10 cycling operation), and high-performance cell adhesion system (controlled by the binding amount of template protein) are shown, which clearly demonstrates the potential value of this method in smart biomaterials and biosensors. [ABSTRACT FROM AUTHOR]

ABSTRACT Structural characterization of protein-protein interactions is important for understanding life processes. Because of the inherent limitations of experimental techniques, such characterization requires computational approaches. Along with the traditional protein-protein docking (free search for a match between two proteins), comparative (template-based) modeling of protein-protein complexes has been gaining popularity. Its development puts an emphasis on full and partial structural similarity between the target protein monomers and the protein-protein complexes previously determined by experimental techniques (templates). The template-based docking relies on the quality and diversity of the template set. We present a carefully curated, nonredundant library of templates containing 4950 full structures of binary complexes and 5936 protein-protein interfaces extracted from the full structures at 12 Å distance cut-off. Redundancy in the libraries was removed by clustering the PDB structures based on structural similarity. The value of the clustering threshold was determined from the analysis of the clusters and the docking performance on a benchmark set. High structural quality of the interfaces in the template and validation sets was achieved by automated procedures and manual curation. The library is included in the Dockground resource for molecular recognition studies at . Proteins 2015; 83:1563-1570. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Structural characterization of protein-protein interactions is essential for our ability to understand life processes. However, only a fraction of known proteins have experimentally determined structures. Such structures provide templates for modeling of a large part of the proteome, where individual proteins can be docked by template-free or template-based techniques. Still, the sensitivity of the docking methods to the inherent inaccuracies of protein models, as opposed to the experimentally determined high-resolution structures, remains largely untested, primarily due to the absence of appropriate benchmark set(s). Structures in such a set should have predefined inaccuracy levels and, at the same time, resemble actual protein models in terms of structural motifs/packing. The set should also be large enough to ensure statistical reliability of the benchmarking results. We present a major update of the previously developed benchmark set of protein models. For each interactor, six models were generated with the model-to-native Cα RMSD in the 1 to 6 Å range. The models in the set were generated by a new approach, which corresponds to the actual modeling of new protein structures in the 'real case scenario,' as opposed to the previous set, where a significant number of structures were model-like only. In addition, the larger number of complexes (165 vs. 63 in the previous set) increases the statistical reliability of the benchmarking. We estimated the highest accuracy of the predicted complexes (according to CAPRI criteria), which can be attained using the benchmark structures. The set is available at . Proteins 2015; 83:891-897. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Rational design in combination with a screening process was used to develop affinity polymers for a specific binding site on the surface of immunoglobulin G (IgG) proteins. The concept starts with the identification of critical amino acid residues on the protein interface and their topological arrangement. Appropriate binding monomers were subsequently synthesized. Together with a sugar monomer (2-5 equiv) for water solubility and a dansyl monomer (0.5 equiv) as a fluorescent label, they were subjected in aqueous solution to linear radical copolymerization in various compositions (e.g., azobisisobutyronitrile (AIBN), homogeneous water/DMF mixtures). After ultrafiltration and lyophilization, colorless dry water-soluble powders were obtained. NMR spectroscopic and gel permeation chromatography (GPC) characterization indicated molecular weights between 30 and 500 kD and confirmed retention of monomer composition as well as the absence of monomers. In a competitive enzyme-linked immunosorbent assay (ELISA) screen of the polymer libraries (20-50 members), few copolymers qualified as strong and selective binders for the protein A binding site on the Fc fragment of the antibody. Their monomer composition precisely reflected the critical amino acids found at the interface. The simple combination of a charged and a nonpolar binding monomer sufficed for selective submicromolar IgG recognition by the synthetic polymer. Affinities were confirmed by fluorescence titrations; they increased with decreasing salt load but remained largely unaltered at lowered pH. Other proteins, including those of similar size and isoelectric point (pI), were bound 10-1000 times less tightly. This example indicates that interaction domains in other proteins may also be targeted by synthetic polymers if their comonomer composition reflects the nature and arrangement of amino acid residues on the protein surface. [ABSTRACT FROM AUTHOR]

A versatile approach based on nanosphere lithography is proposed to generate surface-imprinted polymers for selective protein recognition. A layer of 750 nm diameter latex bead-protein conjugate is deposited onto the surface of gold-coated quartz crystals followed by the electrosynthesis of a poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) film with thicknesses on the order of the bead radius. The removal of the polymer bead-protein conjugates, facilitated by using a cleavable protein-nanosphere linkage is shown to result in 2D arrays of periodic complementary size cavities. Here it is demonstrated by nanogravimetric measurements that the imprinting proceeds further at molecular level and the protein (avidin) coating of the beads generates selective recognition sites for avidin on the surface of the PEDOT/PSS film. The binding capacity of such surface-imprinted polymer films is ca. 6.5 times higher than that of films imprinted with unmodified beads. They also exhibit excellent selectivity against analogues of avidin, i.e., extravidin, streptavidin, and neutravidin, the latter being in fact undetectable. This methodology, if coupled with properly oriented conjugation of the macromolecular template to the nanoparticles, offers the possibility of site-directed imprinting. [ABSTRACT FROM AUTHOR]

Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand-receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin-based proteins called sbAvd-1 and sbAvd-2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone-binding protein was immobilized on the surface. Repeated formation and rupture of the ligand-receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex-rupturing force. In this way, we obtained the molecular dissociation rate (koff) and energy landscape distances (xβ) of the four possible complexes: sbAvd-1-biotin, sbAvd-1-testosterone, sbAvd-2-biotin and sbAvd-2-testosterone. It was found that the kinetic off-rates for both proteins and both ligands are similar. In contrast, the xβ values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone-binding proteins, implying a decreased cross-reactivity of sbAvd-2. Unravelling the binding behaviour of the investigated testosterone-binding proteins is expected to improve their usability for possible sensing applications. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

ABSTRACT Characterization of life processes at the molecular level requires structural details of protein-protein interactions (PPIs). The number of experimentally determined protein structures accounts only for a fraction of known proteins. This gap has to be bridged by modeling, typically using experimentally determined structures as templates to model related proteins. The fraction of experimentally determined PPI structures is even smaller than that for the individual proteins, due to a larger number of interactions than the number of individual proteins, and a greater difficulty of crystallizing protein-protein complexes. The approaches to structural modeling of PPI (docking) often have to rely on modeled structures of the interactors, especially in the case of large PPI networks. Structures of modeled proteins are typically less accurate than the ones determined by X-ray crystallography or nuclear magnetic resonance. Thus the utility of approaches to dock these structures should be assessed by thorough benchmarking, specifically designed for protein models. To be credible, such benchmarking has to be based on carefully curated sets of structures with levels of distortion typical for modeled proteins. This article presents such a suite of models built for the benchmark set of the X-ray structures from the D ockground resource (http://dockground.bioinformatics.ku.edu) by a combination of homology modeling and Nudged Elastic Band method. For each monomer, six models were generated with predefined Cα root mean square deviation from the native structure (1, 2, ..., 6 Å). The sets and the accompanying data provide a comprehensive resource for the development of docking methodology for modeled proteins. Proteins 2014; 82:278-287. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

In the search of molecules that could recognize sizeable areas of protein surfaces, a series of ten helical aromatic oligoamide foldamers was synthesized on solid phase. The foldamers comprise three to five monomers carrying various proteinogenic side chains, and exist as racemic mixtures of interconverting right-handed and left-handed helices. Functionalization of the foldamers by a nanomolar ligand of human carbonic anhydrase II (HCA) ensured that they would be held in close proximity to the protein surface. Foldamer-protein interactions were screened by circular dichroism (CD). One foldamer displayed intense CD bands indicating that a preferred helix handedness is induced upon interacting with the protein surface. The crystal structure of the complex between this foldamer and HCA could be resolved at 2.1 Å resolution and revealed a number of unanticipated protein-foldamer, foldamer-foldamer, and protein-protein interactions. [ABSTRACT FROM AUTHOR]

ABSTRACT The increasing amount of structural information on protein-protein interactions makes it possible to predict the structure of protein-protein complexes by comparison/alignment of the interacting proteins to the ones in cocrystallized complexes. In the predictions based on structure similarity, the template search is performed by structural alignment of the target interactors with the entire structures or with the interface only of the subunits in cocrystallized complexes. This study investigates the scope of the structural similarity that facilitates the detection of a broad range of templates significantly divergent from the targets. The analysis of the target-template similarity is based on models of protein-protein complexes in a large representative set of heterodimers. The similarity of the biological and crystal packing interfaces, dissimilar interface structural motifs in overall similar structures, interface similarity to the full structure, and local similarity away from the interface were analyzed. The structural similarity at the protein-protein interfaces only was observed in ∼25% of target-template pairs with sequence identity <20% and primarily homodimeric templates. For ∼50% of the target-template pairs, the similarity at the interface was accompanied by the similarity of the whole structure. However, the structural similarity at the interfaces was still stronger than that of the noninterface parts. The study provides insights into structural and functional diversity of protein-protein complexes, and relative performance of the interface and full structure alignment in docking. Proteins 2013; 81:2137-2142. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

A localized surface plasmon resonance (LSPR) sensor surface was fabricated by the deposition of gold nanorods on a glass substrate and subsequent immobilization of the DNA aptamer, which specifically bind to thrombin. This LSPR aptamer sensor showed a response of 6-nm λmax shift for protein binding with the detection limit of at least 10 pM, indicating one of the highest sensitivities achieved for thrombin detection by optical extinction LSPR. We also tested the LSPR sensor fabricated using gold bipyramid, which showed higher refractive index sensitivity than the gold nanorods, but the overall response of gold bipyramid sensor appears to be 25% less than that of the gold nanorod substrate, despite the approximately twofold higher refractive index sensitivity. XPS analysis showed that this is due to the low surface density of aptamers on the gold bipyramid compared with gold nanorods. The low surface density of the aptamers on the gold bipyramid surface may be due to the effect of shape of the nanostructure on the kinetics of aptamer monolayer formation. The small size of aptamers relative to other bioreceptors is the key to achieving high sensitivity by biosensors on the basis of LSPR, demonstrated here for protein binding. The generality of aptamer sensors for protein detection using gold nanorod and gold nanobipyramid substrates is anticipated to have a large impact in the important development of sensors toward biomarkers, environmental toxins, and warfare agents. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The molecular recognition between cholera toxin B and GM1‐functionalized phospholipid/block copolymer hybrid membranes can be controlled by varying the lipid/block copolymer composition. Confocal laser scanning microscopy and fluorescence correlation spectroscopy were used to study the protein–receptor interaction and dynamic processes in the membrane. [ABSTRACT FROM AUTHOR]

Traditional approaches to protein-protein docking sample the binding modes with no regard to similar experimentally determined structures (templates) of protein-protein complexes. Emerging template-based docking approaches utilize such similar complexes to determine the docking predictions. The docking problem assumes the knowledge of the participating proteins' structures. Thus, it provides the possibility of aligning the structures of the proteins and the template complexes. The progress in the development of template-based docking and the vast experience in template-based modeling of individual proteins show that, generally, such approaches are more reliable than the free modeling. The key aspect of this modeling paradigm is the availability of the templates. The current common perception is that due to the difficulties in experimental structure determination of protein-protein complexes, the pool of docking templates is insignificant, and thus a broad application of template-based docking is possible only at some future time. The results of our large scale, systematic study show that, surprisingly, in spite of the limited number of protein-protein complexes in the Protein Data Bank, docking templates can be found for complexes representing almost all the known protein-protein interactions, provided the components themselves have a known structure or can be homology-built. About one-third of the templates are of good quality when they are compared to experimental structures in test sets extracted from the Protein Data Bank and would be useful starting points in modeling the complexes. This finding dramatically expands our ability to model protein interactions, and has far-reaching implications for the protein docking field in general. [ABSTRACT FROM AUTHOR]

Deformation of lipid membranes into curved structures such as buds and tubules is essential to many cellular structures including endocytic pits and filopodia. Binding of specific proteins to lipid membranes has been shown to promote membrane bending during endocytosis and transport vesicle formation. Additionally. specific lipid species are found to colocalize with many curved membrane structures, inspiring ongoing exploration of a variety of roles for lipid domains in membrane bending. However, the specific mechanisms by which lipids and proteins collaborate to induce curvature remain unknown. Here we demonstrate a new mechanism for induction and amplification of lipid membrane curvature that relies on steric confinement of protein binding on membrane surfaces. Using giant lipid vesicles that contain domains with high affinity for his-tagged proteins, we show that protein crowding on lipid domain surfaces creates a protein layer that buckles outward, spontaneously bending the domain into stable buds and tubules. In contrast to previously described bending mechanisms relying on local steric interactions between proteins and lipids (i.e. helix insertion into membranes), this mechanism produces tubules whose dimensions are defined by global parameters: domain size and membrane tension. Our results suggest the intriguing possibility that confining structures, such as lipid domains and protein lattices, can amplify membrane bending by concentrating the steric interactions between bound proteins. This observation highlights a fundamental physical mechanism for initiation and control of membrane bending that may help explain how lipids and proteins collaborate to create the highly curved structures observed in vivo. [ABSTRACT FROM AUTHOR]

Noncovalent binding interactions between proteins are the central physicochemical phenomenon underlying biological signaling and functional control on the molecular level. Here, we perform an extensive structural analysis of a large set of bound and unbound ubiquitin conformers and study the level of residual induced fit after conformational selection in the binding process. We show that the region surrounding the binding site in ubiquitin undergoes conformational changes that are significantly more pronounced compared with the whole molecule on average. We demonstrate that these induced-fit structural adjustments are comparable in magnitude to conformational selection. Our final model of ubiquitin binding blends conformational selection with the subsequent induced fit and provides a quantitative measure of their respective contributions. [ABSTRACT FROM AUTHOR]

A compound exhibits tautomerism if it can be represented by two structures that are related by an intramolecular movement of hydrogen from one atom to another. The different tautomers of a molecule usually have different molecular fingerprints, hydrophobicities and p Ka’s as well as different 3D shape and electrostatic properties; additionally, proteins frequently preferentially bind a tautomer that is present in low abundance in water. As a result, the proper treatment of molecules that can tautomerize, ~25% of a database, is a challenge for every aspect of computer-aided molecular design. Library design that focuses on molecular similarity or diversity might inadvertently include similar molecules that happen to be encoded as different tautomers. Physical property measurements might not establish the properties of individual tautomers with the result that algorithms based on these measurements may be less accurate for molecules that can tautomerize—this problem influences the accuracy of filtering for library design and also traditional QSAR. Any 2D or 3D QSAR analysis must involve the decision of if or how to adjust the observed Ki or IC50 for the tautomerization equilibria. QSARs and recursive partitioning methods also involve the decision as to which tautomer(s) to use to calculate the molecular descriptors. Docking virtual screening must involve the decision as to which tautomers to include in the docking and how to account for tautomerization in the scoring. All of these decisions are more difficult because there is no extensive database of measured tautomeric ratios in both water and non-aqueous solvents and there is no consensus as to the best computational method to calculate tautomeric ratios in different environments. [ABSTRACT FROM AUTHOR]

Caspase-mediated apoptosis has important roles in normal cell differentiation and aging and in many diseases including cancer, neuromuscular disorders and neurodegenerative diseases. Therefore, modulation of caspase activity and conformational states is of therapeutic importance. We report crystal structures of a new unliganded conformation of caspase-7 and the inhibited caspase-7 with the tetrapeptide Ac-YVAD-Cho. Different conformational states and mechanisms for substrate recognition have been proposed based on unliganded structures of the redundant apoptotic executioner caspase-3 and -7. The current study shows that the executioner caspase-3 and -7 have similar conformations for the unliganded active site as well as the inhibitor-bound active site. The new unliganded caspase-7 structure exhibits the tyrosine flipping mechanism in which the Tyr230 has rotated to block entry to the S2 binding site similar to the active site conformation of unliganded caspase-3. The inhibited structure of caspase-7/YVAD shows that the P4 Tyr binds the S4 region specific to polar residues at the expense of a main chain hydrogen bond between the P4 amide and carbonyl oxygen of caspase-7 Gln 276, which is similar to the caspase-3 complex. This new knowledge of the structures and conformational states of unliganded and inhibited caspases will be important for the design of drugs to modulate caspase activity and apoptosis. [ABSTRACT FROM AUTHOR]

Caspase-3 recognition of various P4 residues in its numerous protein substrates was investigated by crystallography, kinetics, and calculations on model complexes. Asp is the most frequent P4 residue in peptide substrates, although a wide variety of P4 residues are found in the cellular proteins cleaved by caspase-3. The binding of peptidic inhibitors with hydrophobic P4 residues, or no P4 residue, is illustrated by crystal structures of caspase-3 complexes with Ac-IEPD-Cho, Ac-WEHD-Cho, Ac-YVAD-Cho, and Boc-D(OMe)-Fmk at resolutions of 1.9–2.6 Å. The P4 residues formed favorable hydrophobic interactions in two separate hydrophobic regions of the binding site. The side chains of P4 Ile and Tyr form hydrophobic interactions with caspase-3 residues Trp206 and Trp214 within a non-polar pocket of the S4 subsite, while P4 Trp interacts with Phe250 and Phe252 that can also form the S5 subsite. These interactions of hydrophobic P4 residues are distinct from those for polar P4 Asp, which indicates the adaptability of caspase-3 for binding diverse P4 residues. The predicted trends in peptide binding from molecular models had high correlation with experimental values for peptide inhibitors. Analysis of structural models for the binding of 20 different amino acids at P4 in the aldehyde peptide Ac-XEVD-Cho suggested that the majority of hydrophilic P4 residues interact with Phe250, while hydrophobic residues interact with Trp206, Phe250, and Trp214. Overall, the S4 pocket of caspase-3 exhibits flexible adaptation for different residues and the new structures and models, especially for hydrophobic P4 residues, will be helpful for the design of caspase-3 based drugs. [ABSTRACT FROM AUTHOR]

Dysregulation of growth and differentiation factor 5 (GDF-5) signalling, a member of the TGF-β superfamily, is strongly linked to skeletal malformation. GDF-5-mediated signal transduction involves both BMP type I receptors, BMPR-IA and BMPR-IB. However, mutations in either GDF-5 or BMPR-IB lead to similar phenotypes, indicating that in chondrogenesis GDF-5 signalling seems to be exclusively mediated through BMPR-IB. Here, we present structural insights into the GDF-5:BMPR-IB complex revealing how binding specificity for BMPR-IB is generated on a molecular level. In BMPR-IB, a loop within the ligand-binding epitope functions similar to a latch allowing high-affinity binding of GDF-5. In BMPR-IA, this latch is in a closed conformation leading to steric repulsion. The new structural data now provide also a molecular basis of how phenotypically relevant missense mutations in GDF-5 might impair receptor binding and activation. [ABSTRACT FROM AUTHOR]

The development of oligonucleotide-linked small molecules for protein recognition is a worthy undertaking with potential applications in drug discovery and diagnostics. Although the ground work for this novel approach to protein recognition is still being laid, a number of promising systems have recently been developed. Herein, we discuss a selection of these systems focusing on the distinct tactics that can be employed for protein binding. Also reported is a new example that underscores the unique versatility of linking aptamers to protein-binding small molecules. In particular, we discuss the development of a chimeric aptamer that in the 'on' state can bind and signal the presence of a target protein. Furthermore, we demonstrate that the activity of this sensing supramolecule can be switched 'off' on cue. [ABSTRACT FROM AUTHOR]

The binding transition state (TS) is the rate-limiting step for transient molecular interactions. This important step in the molecular recognition process, however, is largely understood only at a qualitative level. To establish a more quantitative picture of the TS structure, we exploit a set of biophysical techniques that have provided major insights in protein folding applications. As a model system representing the large class of "weakly charged" protein-protein interactions, we examine the binding of a variety of human growth hormone (hGH) variants to the human growth hormone receptor (hGHR) and the human prolactin receptor (hPRLR). hGH variants were chosen to probe different features of the TS structure, based on their highly reengineered interfaces. Both Eyring and urea (m value) analyses suggest that the majority of binding surface burial occurs after TS. A comprehensive ɸ4 analysis showed that individual hGH interface residues do not contribute energetically to the stability of the TS, but there is a TS "hot spot" in the receptor. Zinc dependence studies that take advantage of an endogenous tetracoordinated interfacial metal binding demonstrate that surfaces of the molecules have attained a high orientational complementarity by the time the TS is reached. The model that best fits these data are that a "knobs-into-holes" process precisely aligns the two molecular interfaces in forming the TS structure. Surprisingly, most of the thermodynamic character of the binding reaction is focused in the fine-tuning process occurring after TS. [ABSTRACT FROM AUTHOR]

Caspase-3, -6 and -7 cleave many proteins at specific sites to induce apoptosis. Their recognition of the P5 position in substrates has been investigated by kinetics, modeling and crystallography. Caspase-3 and -6 recognize P5 in pentapeptides as shown by enzyme activity data and interactions observed in the crystal structure of caspase-3/LDESD and in a model for caspase-6. In caspase-3 the P5 main-chain was anchored by interactions with Ser209 in loop-3 and the P5 Leu side-chain interacted with Phe250 and Phe252 in loop-4 consistent with 50% increased hydrolysis of LDEVD relative to DEVD. Caspase-6 formed similar interactions and showed a preference for polar P5 in QDEVD likely due to interactions with polar Lys265 and hydrophobic Phe263 in loop-4. Caspase-7 exhibited no preference for P5 residue in agreement with the absence of P5 interactions in the caspase-7/LDESD crystal structure. Initiator caspase-8, with Pro in the P5-anchoring position and no loop-4, had only 20% activity on tested pentapeptides relative to DEVD. Therefore, caspases-3 and -6 bind P5 using critical loop-3 anchoring Ser/Thr and loop-4 side-chain interactions, while caspase-7 and -8 lack P5-binding residues. [ABSTRACT FROM AUTHOR]

Surface glycosylation of polymeric membranes has been inspired by the structure of natural biomembranes. It refers to that glycosyl groups are introduced onto the membrane surface by various strategies, which combine the separation function of the membrane with the biological function of the saccharides in one system. In this review, progress in the surface glycosylation of polymeric membranes is highlighted in two aspects, i.e. the glycosylation methods and the potential applications of the surface-glycosylated membranes. [ABSTRACT FROM AUTHOR]

Understanding protein interactions has broad implications for the mechanism of recognition, protein design, and assigning putative functions to uncharacterized proteins. Studying protein flexibility is a key component in the challenge of describing protein interactions. In this work, we characterize the observed conformational change for a set of 20 proteins that undergo large conformational change upon association (>2 Å Ca RMSD) and ask what features of the motion are successfully reproduced by the normal modes of the system. We demonstrate that normal modes can be used to identify mobile regions and, in some proteins, to reproduce the direction of conformational change. In 35% of the proteins studied, a single low-frequency normal mode was found that describes well the direction of the observed conformational change. Finally, we find that for a set of 134 proteins from a docking benchmark that the characteristic frequencies of normal modes can be used to predict reliably the extent of observed conformational change. We discuss the implications of the results for the mechanics of protein recognition. [ABSTRACT FROM AUTHOR]

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The ADP-ribosylating. toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens t-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-la in complex with the nonhydrolyzable NAD analog βTAD at 2.8 Å resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60- Tyr-62 in the N domain), loop II (active-site loop), loop Ill, loop IV (PN loop), and loop V (ADP-ribosylating turn-turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition. [ABSTRACT FROM AUTHOR]

The retinal phosphodiesterase (PDE6) inhibitory γ-subunit (PDEγ) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic αβ-subunits of PDE6 and the α-subunit of transducin (αt). Detailed structural analysis of PDEγ has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDEγ, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDEγ46-87 when bound to αt in the transition-state complex. NMR mapping of the interaction between PDEγ46-87 and the chimeric PDE5/6 catalytic domain confirmed that C-terminal residues 74-87 of PDEγ are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to αt, is left free at this stage. These results indicate that the interaction between PDEγ and αt during the phototransduction cascade involves the selection of preconfigured transient conformations. [ABSTRACT FROM AUTHOR]

Molecular imprinting is an important tool for generating synthetic receptors with specific recognition sites. The resulting artificial receptor has extensive applications in chromatographic stationary phases, solid phase extraction, catalysis, drug delivery and sensors. The synthesis of molecularly imprinted polymers (MIPs) specific for proteins has been proved challenging due to a number of inherent problems in protein imprinting but potentially rewarding work. Hence, this review discusses recent advances in various synthetic protocols developed to overcome the obstacles, focusing on their advantages, disadvantages and potential future direction. In addition, the case of the selection of the functional monomers has also been reviewed here. [ABSTRACT FROM AUTHOR]

High-throughput screening of protein-protein and protein-peptide interactions is of high interest both for biotechnological and pharmacological applications. Here, we propose the use of the noncoded amino acids o-nitrotyrosine and p-iodophenylalanine as spectroscopic probes in combination with circular dichroism and fluorescence quenching techniques (i.e., collisional quenching and resonance energy transfer) as a means to determine the peptide orientation in complexes with SH3 domains. Proline-rich peptides bind SH3 modules in two alternative orientations, according to their sequence motifs, classified as class I and class II. The method was tested on an SH3 domain from a yeast myosin that is known to recognize specifically class I peptides. We exploited the fluorescence quenching effects induced by o-nitrotyrosine and p-iodophenylalanine on the fluorescence signal of a highly conserved Trp residue, which is the signature of SH3 domains and sits directly in the binding pocket. In particular, we studied how the introduction of the two probes at different positions of the peptide sequence (i.e., N-terminally or C-terminally) influences the spectroscopic properties of the complex. This approach provides clear-cut evidence of the orientation of the binding peptide in the SH3 pocket. The chemical strategy outlined here can be easily extended to other protein modules, known to bind linear sequence motifs in a highly directional manner. [ABSTRACT FROM AUTHOR]

Recent analyses of high-throughput protein interaction data coupled with large-scale investigations of evolutionary properties of interaction networks have left some unanswered questions. To what extent do protein interactions act as constraints during evolution of the protein sequence? How does the type of interaction, specifically transient or obligate, play into these constraints? Are the mutations in the binding site of an interacting protein correlated with mutations in the binding site of its partner? We address these and other questions by relying on a carefully curated dataset of protein complex structures. Results point to the importance of distinguishing between transient and obligate interactions. We conclude that residues in the interfaces of obligate complexes tend to evolve at a relatively slower rate, allowing them to coevolve with their interacting partners. In contrast, the plasticity inherent in transient interactions leads to an increased rate of substitution for the interface residues and leaves little or no evidence of correlated mutations across the interface. [ABSTRACT FROM AUTHOR]

Molecularly imprinted membranes imprinted for a large-molecular-weight protein were realised using a blend of natural and synthetic polymers. Bioartificial membranes of synthetic (poly(ethylene-co-vinyl alcohol)–EVAL, Clarene®) and biological (Dextran) polymers, molecularly imprinted with α-amylase as the template, were prepared and investigated. Dimethyl sulfoxide (DMSO) solutions of the α-amylase template, Clarene and Dextran were mixed under stirring in the desired proportions and dipped in DMSO (solvent)/water (non solvent) mixture, to obtain the phase separation. The release of Clarene, Dextran and α-amylase in the inversion baths was quantified by spectrophotometric methods and final composition of membranes was established. To study the interactions between the polymer components and between polymeric materials and the template, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were carried out. Results indicated that stable and continuous bioartificial membranes of Clarene and Dextran can be obtained, whereby calorimetric analysis suggested the presence of high interaction between α-amylase and the Clarene component. [ABSTRACT FROM AUTHOR]