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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]

In this work, we present the case of the G-quadruplex(G4)-forming aptamers we recently identified for the recognition of HMGB1, protein involved in inflammation, autoimmune diseases and cancer. These aptamers were previously analyzed, without annealing them, after proper dilution of the stock solution in a pseudo-physiological buffer mimicking the extracellular environment where the protein exerts its pathological activity, and showed high thermal stability and nuclease resistance, good protein affinity and remarkable in vitro activity. These features were more marked for the aptamers forming dimeric, parallel G4 structures in solution. Herein, we fully characterized the same anti-HMGB1 aptamers after a standard annealing procedure performed on diluted samples. Notably, upon a thermal unfolding/folding cycle, these aptamers, and particularly the best ones in the not-annealed form, showed significant conformational switches compared to the same systems analyzed without annealing, forming exclusively monomeric G4 structures, featured by poor thermal and enzymatic stabilities, along with lower protein affinities. These results prove that, for these aptamers, analyzed in the chosen conditions, annealing at low concentration does not produce a beneficial effect in terms of favouring the most bioactive species. [Display omitted] • G4-forming anti-HMGB1 aptamers were analyzed as low concentrated samples after a typical annealing procedure. • Their functional properties and HMGB1-induced cell migration inhibitory activity were evaluated. • The annealed aptamers showed poor thermal and enzymatic stability and low affinity for HMGB1. • Upon annealing the aptamers lost the dimeric forms, exclusively forming monomeric G4 structures. • Annealing at low conc. leads to irreversible denaturing of the most bioactive conformations of the anti-HMGB1 aptamers. [ABSTRACT FROM AUTHOR]

Here, a novel corrole-based conjugated monomer, pyrrole-corrole (Py-Cor) was synthesized to design a functional interface for affinity interactions, which dramatically increases the sensor signal towards model proteins of immunoglobulin G, albumin, and lysozyme. First, the monomer was characterized by nuclear magnetic resonance spectroscopy (NMR), high-resolution mass spectrometry (HRMS), and Fourier transform infrared spectroscopy (FTIR). Then, the functional monomer was electrochemically polymerized on a gold electrode in the presence of pristine pyrrole as a comonomer. The polymeric film was characterized via both chemical and electrochemical routes by conducting cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), and Raman spectroscopy measurements. Due to the metal chelating feature of the corrole ring, copper(II) [Cu(II)] ions were utilized to design a metal chelate affinity film for demonstrating their interaction capability with various proteins. Cu(II) immobilization process was achieved by voltammetry whereas the metal chelate affinity studies were performed using EIS measurements. In order to show the effect of the presence of Cu(II) ions, the sensor performance in terms of the protein recognition capabilities was carried out both in the presence/absence of Cu(II) ions. The presence of Cu(II) ions in the structure significantly improved the recognition performance of the interfaces as well as enhanced the resulted semi-circuit diameters. [Display omitted] • Novel pyrrole based corrole monomer was synthesized. • poly[pyrrole-(pyrrole-corrole)] affinity interface for the first time in the literature. • A comparative evaluation of sensing performances in the presence and absence of Cu(II) ions. • Significantly lowered LOD and LOQ values in the presence of Cu(II) ions. [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]

• A novel molecularly imprinted polymer has been prepared for the separation and purification of hemoglobin. • Silicon nanoparticles were used as materials to prepare molecularly imprinted polymers. • The molecularly imprinted polymer was successfully used to separate hemoglobin from the blood. Molecularly imprinted polymers (MIPs) are promising for precise protein separation and purification. However, challenges persist due to their large size, variable configuration, and instability during preparation. Here, a simple silicon self-assembly program was designed to synthesize MIPs without any organic reagents and acid-base catalysis, avoiding the structural damage of protein under severe conditions. In this method, employing hemoglobin (Hb) as a model protein, with tween-20 in emulsification, and tetraethyl orthosilicate (TEOS) as the cross-linking agent, along with co-functional monomers 3-aminopropyltriethoxysilane (APTES) and benzyl(triethoxy)silane (BnTES), enhanced binding efficacy was achieved. Successful imprinting was evidenced through surface morphology observation and physical/chemical property evaluations of the synthesized MIPs. A series of adsorption experiments were performed to investigate the recognition performance of Hb-MIPs. The Hb-MIPs not only exhibited large adsorption capacity (400 μg/mg) and good imprinting factor (6.09) toward template protein, but also showed satisfactory selectivity for reference proteins. Five cycles of adsorption proved that the Hb-MIPs had good reusability. In addition, the successful isolation of HB from bovine blood indicated that Hb-MIPs were an excellent separation and purification material. The mild preparation conditions and good adsorption capacity demonstrated the potential value of this method in separation and purification research. [ABSTRACT FROM AUTHOR]

Surface imprinted magnetic nanorod for selective extraction of glycoprotein. [Display omitted] In this paper, combining phenylboronic affinity, surface imprinting, and magnetic Fe 3 O 4 , a novel surface imprinted core–shell nanorod (Fe 3 O 4 @SiO 2 /MIPs) was developed for glycoprotein extraction. The Fe 3 O 4 @SiO 2 /MIPs was prepared in water solution using dopamine as functional monomer. The Fe 3 O 4 @SiO 2 /MIPs showed high binding capacity (175.2 mg/g) and fast mass transfer rate (40 min) toward the template protein (ovalbumin, OVA). In addition, the successful extraction of OVA from egg white confirmed the outstanding selectivity of the obtained material. All these results demonstrated that the Fe 3 O 4 @SiO 2 /MIPs had broad application prospects in glycoprotein recognition, biomedical research and clinical diagnosis. [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]

Photoinduced enhanced Raman spectroscopy (PIERS) has emerged as an efficient technique for enhancing the vibrational modes of analyte molecules adsorbed on a plasmonic nanoparticle-semiconductor hybrid material through chemical enhancement governed by electron transfer from the semiconductor to the plasmonic nanoparticles under an additional ultraviolet (UV) preirradiation step. The increase in chemical enhancement is imperative in analyzing and detecting pharmaceutically important moieties, such as amino acids and proteins, with a low Raman scattering cross section, even in complex biological environments. Herein, we demonstrate that UV preirradiation induced the creation of additional oxygen vacancies by introducing a low concentration (≈1%) of Ni as a dopant in the 2D platelike morphology of the BiOCl semiconductor; i.e., defect states in the semiconductor can induce charge transfer from the semiconductor to the plasmonic nanoparticles. This phenomenon facilitates electron transfer to the adsorbed analyte on the plasmonic surface. Additionally, we have shown the usefulness of this method in protein immobilization on the substrate surface, followed by the identification of a specific protein in the mixture of proteins. Proteins containing cysteine residues capture these electrons to form a surface-bound thiol group via a transient disulfide electron adduct radical. This allows differential binding of the protein molecules to the semiconductor plasmonic hybrid depending on the concentration of surface cysteine residues in proteins. Through PIERS and principal component analysis, we demonstrate the possibility of probing and distinguishing biomolecules based on their surface composition and secondary structure components even in their mixtures, thus paving the way for efficient analysis of complex biological systems.

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]

Selective protein recognition is critical in molecular biology techniques such as Western blotting and ELISA. Successful detection of the target proteins in these methods relies on the specific interaction of the antibodies, which often bring a high production cost and require a long incubation time. Aptamers represent an alternative class of simple and affordable affinity reagents for protein recognition, and replacing antibodies with aptamers in Western blotting would potentially be more time- and cost-effective. In this work, multiple fluorescent DNA aptamers were isolated by in vitro selection to selectively label commonly used tag proteins including GST, MBP, and His-tag. The generated aptamers G1, M1, and H1 specifically bound to their cognate target proteins with nanomolar affinities, respectively. Compared with conventional antibody-based immunoblotting, such aptamer-based procedure gave a cleaner background and was able to selectively label target protein in a complex mixture. Lastly, the identified aptamers were also effective in recognition of different fusion proteins with the same tag, thus greatly expanding the scope of the potential applications of these aptamers. This work provided aptamers as useful molecular tools for selective protein recognition in Western blotting analysis.

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]

A high-performance molecularly imprinted sensing platform inspired by natural recognition mechanisms was fabricated to detect protein by employing a linear electro-polymerizable molecularly imprinted polymer as macromonomer. This was achieved via the combination of a biosensor fabrication with a self-assembly imprinting technique without the use of chemical labels. An amphipathic electroactive copolymer was designed as macro-monomer to maintain structural integrity of the protein template via self-assembly, resulting in generation of a 3D construction around the protein molecule to form imprinted sites. Electro-polymerization was utilized not only to anchor imprinted sites but also to enhance electron transfer. The adaptable sensing platform was based on a strengthened recognition reaction between the MIP layer and template protein after the generation of an electroactive network. Bovine serum albumin (BSA) and trypsin were used as model proteins to investigate the method's generality, which gave broad detection ranges of 10−14–10−5 mg mL−1 for BSA and 10−13–10−8 mg mL−1 for trypsin. These results indicate that the proposed fabrication offers an effective and versatile strategy for protein recognition. Image 1 • A novel electroactive molecularly imprinted interface was developed as an abiotic recognition with high generality. • A linear amphipathic macromolecule was designed as macro-monomer to maintain structural integrity of the protein template. • The sensing platform was based on an enhanced electron transfer mechanism after the generation of an electroactive network. • Bovine serum albumin (BSA) and trypsin were used as model proteins to investigate the method's generality. [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]

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]

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]

We describe a strategy for constructing atomic resolution dynamical ensembles of RNA molecules, spanning up to millisecond timescales, that combines molecular dynamics (MD) simulations with NMR residual dipolar couplings (RDC) measured in elongated RNA. The ensembles are generated via a Monte Carlo procedure by selecting snap-shot from an MD trajectory that reproduce experimentally measured RDCs. Using this approach, we construct ensembles for two variants of the transactivation response element (TAR) containing three (HIV-1) and two (HIV-2) nucleotide bulges. The HIV-1 TAR ensemble reveals significant mobility in bulge residues C24 and U25 and to a lesser extent U23 and neighboring helical residue A22 that give rise to large amplitude spatially correlated twisting and bending helical motions. Omission of bulge residue C24 in HIV-2 TAR leads to a significant reduction in both the local mobility in and around the bulge and amplitude of inter-helical bending motions. In contrast, twisting motions of the helices remain comparable in amplitude to HIV-1 TAR and spatial correlations between them increase significantly. Comparison of the HIV-1 TAR dynamical ensemble and ligand bound TAR conformations reveals that several features of the binding pocket and global conformation are dynamically preformed, providing support for adaptive recognition via a ‘conformational selection’ type mechanism.

The industry transfer of laboratory-use magnetic separation is still hampered by the lack of suitable nanoparticles, both in terms of their features and large-scale availability. Surface Active Maghemite Nanoparticles (SAMNs) characterized by a unique surface chemistry, low environmental impact, scalable synthesis and functionalization were used to develop a bio-inspired lactoferrin (LF) recognition system. Based on the LF affinity for DNA, a self-assembly process was optimized for obtaining a SAMN@DNA hybrid displaying chemical and colloidal stability and LF specificity. SAMN@DNA was successfully tested for the affinity purification of LF from crude bovine whey. Advantages, such as high selectivity and loading capacity, nanoparticle re-usability, outstanding purity (96 ± 1%), preservation of protein conformation and short operational time, were highlighted. Finally, scalability was demonstrated by an automatic system performing continuous purification of LF from 100 liters day−1 of whey. This study responds to essential prerequisites, such as efficiency, re-usability and industrialization feasibility. [Display omitted] • A nano-hybrid recognition site is revealed by molecular modelling on lactoferrin surface. • ɣ-Fe2O3-DNA-lactoferrin hybrid is prepared by self-assembly and characterized. • ɣ-Fe2O3-DNA nanomaterial is optimized for lactoferrin affinity purification. • High-purity and high-yield lactoferrin is attained in two hours by magnetic isolation. • An automatic pilot-plant purifying 100 L day-1 lactoferrin from whey is presented. [ABSTRACT FROM AUTHOR]

Metalloporphyrins are often found in nature as coordination recognition sites within biological process, and synthetically offer the potential for use in therapeutic, catalytic and diagnostic applications. While porphyrin containing biological recognition elements have stability limitations, molecularly imprinted polymers bearing these structures offer an alternative with excellent robustness and the ability to work in extreme conditions. In this work, we synthesised a polymerizable porphyrin and metalloporphyrin and have incorporated these as co-monomers within a hydrogel thin-sheet MIP for the specific recognition of bovine haemoglobin (BHb). The hydrogels were evaluated using Scatchard analysis, with K d values of 10.13 × 10−7, 5.30 × 10−7, and 3.40 × 10−7 M, for the control MIP, porphyrin incorporated MIP and the iron-porphyrin incorporated MIP, respectively. The MIPs also observed good selectivity towards the target protein with 73.8%, 77.4%, and 81.2% rebinding of the BHb target for the control MIP, porphyrin incorporated MIP and the iron-porphyrin incorporated MIP, respectively, compared with the non-imprinted (NIP) counterparts. Specificity was determined against a non-target protein, Bovine Serum Albumin (BSA). The results indicate that the introduction of the metalloporphyrin as a functional co-monomer is significantly beneficial to the recognition of a MIP, further enhancing MIP capabilities at targeting proteins. [Display omitted] • The design synthesis and incorporation of a polymerizable porphyrin and metalloporphyrin into a thin-sheet hydrogel MIP. • Use of metalloporphyrin as a co-monomer within the MIP thin sheet in order to improve binding. • Scatchard analysis showed K d values of 10.13 × 10−7, 5.30 × 10−7, and 3.40 × 10−7 M, for the control MIP, porphyrin incorporated MIP and the iron-porphyrin incorporated MIP, respectively. • The MIP produced excellent selectivity for the target protein with 73.8%, 77.4%, and 81.2% rebinding of the BHb target for the control MIP, porphyrin incorporated MIP and the iron-porphyrin incorporated MIP, respectively. • This shows that the MIP has the potential to be a highly robust and selective material for molecular recognition use. [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]

Abstract A quartz crystal microbalance (QCM) sensor for detecting cytochrome c based on an oriented surface epitope imprinted polymer was fabricated in this paper. By using the palmitic acid-modified epitope of cytochrome c as the template and the 3-aminopropyltriethoxysilane as the monomer, we prepared a new oriented surface epitope imprinted polymer by the reverse microemulsion polymerization. The prepared oriented imprinted polymer had better imprinting effect than the non-oriented imprinted polymer. And compared to previous studies, this polymerization method is simple and could be carried out at room temperature in the presence of oxygen, under regular atmospheric conditions. Then, by combining the advantages of molecularly imprinted polymers and QCM sensors, we used the prepared polymer to establish a QCM sensor. The described sensor showed good sensitivity and selectivity towards cytochrome c. The linear range was from 0.005 μg mL−1 to 0.050 μg mL−1 and the detection limit was 3.6 ng mL−1 which is lower than most of previous works. Besides, it could be used for real sample analysis and had satisfactory reproducibility and accuracy. This work proposed a new way of fabricating oriented surface epitope imprinted polymers-based QCM sensors for selectively detecting proteins at very low concentrations. Graphical abstract fx1 Highlights • A new quartz crystal microbalance sensor was successfully modified by an oriented surface epitope imprinted polymer. • Compared to previous studies, the preparation of the polymer was simpler, easier and without harsh environment. • This oriented imprinted polymer had better imprinting effect than the non-oriented one. • This protocol introduced the hydrolysis of tetraethoxysilane into oriented epitope imprinting for the first time. • This sensor had lower detection limit than most of the previous studies and is promising for real sample analysis. [ABSTRACT FROM AUTHOR]

Abstract This research aims to engineer molecularly imprinted polymer (MIP)-based synthetic receptors for the molecular recognition of neuron specific enolase (NSE) biomarker. The synthetic peptide derived from the NSE was synthesized along with its cysteine and histidine modified versions. The modified peptides were utilized as templates for molecular imprinting, which was achieved by combination of epitope- and electrochemical surface imprinting strategy. The subsequently generated imprinted cavities were used for the detection of the NSE derived peptide and NSE. The imprints created with cysteine (CME) and histidine modified epitopes (HME) could detect the peptide in a concentration range of 2–128 µM and 15.6 nM to 128 µM, respectively. The recognition of NSE was achieved by the same imprints in a linear range of 1–64 ng mL−1 (CME) and 0.25–64 ng mL−1 (HME), respectively. The target molecules bound to the control polymer very weakly, confirming the high selectivity of the MIP cavities. Selectivity studies resulted in imprinting factors of 8.8 and 11 for the CME and HME imprints, respectively. The affinity analyses provided dissociation constants of 2.3 × 10−10 M and 3 × 10–11 M for NSE recognition using the corresponding epitope imprints. Cross-reactivity studies with non-specific molecules proved high specificity of the artificial receptors for the targets. Highlights • First epitope-mediated MIPs synthesized for neuron specific enolase biomarker. • Two different epitope imprinting approaches using cysteine- and histidine-modified peptides. • Highly specific and sensitive electrochemical sensors were manufactured with epitope imprints. • CV, SWV and AFM were employed to reveal template-MIP interactions. • This universal and promising strategy can be employed for a wide range protein markers. [ABSTRACT FROM AUTHOR]

Abstract In this study, we have reported a novel fabrication technique for human serum albumin (HSA) imprinted composite bacterial cellulose nanofibers (MIP-cBCNFs) used for the depletion of HSA selectively from artificial blood plasma for proteomic applications. Molecular imprinting was achieved by using metal ion coordination interactions of N ‑methacryloyl‑(l)‑histidinemethylester (MAH) monomer and Cu(II) ions. MAH-Cu(II)-HSA complex was polymerized with bacterial cellulose nanofibers (BCNFs) under constant stirring at room temperature. The characterization of the MIP-cBCNFs was carried out by FTIR-ATR, SEM, contact angle measurements and surface area measurements. The adsorption experiments of HSA onto the MIP-BCNFs and NIP-BCNFs from aqueous HSA solutions were investigated in a batch system. The selectivity of the MIP-cBCNFs was investigated by using non-template human transferrin (HTR), and myoglobin (Myo). The relative selectivity coefficients of the MIP-cBCNFs were calculated as 4.73 and 3.02 for HSA/HTR and HSA/Myo molecules, respectively. In addition, the depletion of HSA from artificial human plasma was confirmed by SDS-PAGE and 2-D gel electrophoresis. As a result, it has been shown that metal ion coordination interactions contribute to specific binding of template when preparing MIP-cBCNFs for the depletion of HSA with a high adsorption capacity, significant selectivity and reusability. [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]

A novel dual-template epitope imprinting polymer coated on magnetic carbon nanotubes (MCNTs@D-EMIP) was successfully prepared for specific recognition of porcine serum albumin (PSA) via dual-template epitope imprinting, metal chelation imprinting and distillation-precipitation polymerization (DPP). C-terminal peptides and N-terminal peptides of PSA were selected as templates simultaneously, and zinc acrylate and ethylene glycol dimethacrylate (EGDMA) were used as functional monomer and cross-linker, respectively. The epitope templates were immobilized by metal chelation and six-membered ring formed with zinc acrylate. Finally, MCNTs@D-EMIP was synthesized by DPP in only 30 min, which was much shorter than those of other polymerization methods. The prepared MCNTs@D-EMIP displayed specific recognition ability toward PSA and its adsorption amount and imprinting factor were 45.05 mg g −1 and 4.50, which were much higher than those of single template epitope imprinting polymers. Besides, high-performance liquid chromatography (HPLC) analysis of PSA in porcine blood serum real sample indicated that the specificity was not affected by other competitive proteins, which forcefully stated that the MCNTs@D-EMIP had potential to be applied in bio-separation area. In addition, the results of cross-reactivity experiment proved that this strategy had generality to prepare dual-template epitope imprinting polymer for recognition of target protein. In summary, this study provided an efficient protocol to recognize target protein in complex sample via dual-template epitope imprinting approach, metal chelation imprinting and distillation-precipitation polymerization. [ABSTRACT FROM AUTHOR]

Molecularly imprinted polymers (MIPs) are tailor made recognition materials that can mimic biological receptors. If used as recognition units for chemosensors fabrication, they outperform natural receptors with their durability, chemical stability, and low production costs. Novel techniques of MIP deposition as thin films, surface development, and introduction of additional properties are very much demanded in terms of selective and sensitive chemosensors fabrication. Therefore, in recent years a particular attention has been paid to syntheses of nanostructured MIP films and MIP nanoparticles. The present brief review surveys novel achievements in the field of MIP nanostructures and their application for determination of protein analytes. [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]

• A facile method was developed to imprint protein on the surface of flower-like NiO. • The NiO@PDA/MIPs possessed flower-like sandwich-structure. • The NiO@PDA/MIPs exhibited excellent binding capacity, selectivity and reusability. • The NiO@PDA/MIPs could efficiently extraction OVA from egg white samples. In this work, a facile method for synthesis of flower-like sandwich-structured molecularly imprinted polymers (NiO@PDA/MIPs) was proposed for protein recognition. Polydopamine modified flower-like NiO was used as substrate to immobilize the target protein (ovalbumin, OVA), and dopamine was utilized as functional monomer to form the imprinted layer. The whole preparation process was conducted in aqueous solution at room temperature. The key preparation conditions were studied systematically. Owing to the large surface-to-volume of the flower-like structure and the multifunctional groups on the polydopamine layer, the NiO@PDA/MIPs showed large binding capacity (143.2 mg/g), efficient adsorption kinetics (60 min) and excellent selectivity toward OVA. Meanwhile, the NiO@PDA/MIPs possessed satisfactory stability and reusability. Finally, successful capture of OVA from egg white suggested its potential value in practical applications. [ABSTRACT FROM AUTHOR]

The potential of the modified bacterial cellulose (BC) nanofibers was determined bearing metal ion coordination interactions to enhance the protein adsorption and binding capacity. Thus, a household synthesized metal chelating monomer, namely N-methacryloyl- l -histidine methylester (MAH), and a commercial metal chelating monomer, namely 4-vinylimidazole (VIm), were used to complex with metal ions Cu(II) and Ni(II) respectively for the synthesis of the modified BC nanofibers. The modified nanofibers were characterized by FT-IR, SEM and EDX measurements. The protein adsorption tests were carried out using hemoglobin as a model protein and it was determined that the maximum adsorption capacity of hemoglobin onto the modified BC nanofibers was found as 47.40 mg/g. The novel strategy for the preparation of metal chelated nanofibers was developed. [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]

Core-shell structural adenosine-imprinted microspheres were prepared via a two-step procedure. Polystyrene core particles (CP) were firstly prepared via a reversible addition-fragmentation chain transfer (RAFT) polymerization leaving the iniferter on the surface of the cores, then a molecularly imprinted polymer (MIP) shell was synthesized on the surface of the cores by using acrylamide (AAm) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. The formation and growth of the MIP layer were seen dependent on the initiator (AIBN), AAm and the polymerization time used within the polymerization. SEM/TEM images showed that the dimensions of the cores and shells were 2 μM and 44 nm, respectively. The MIP microspheres exhibited a fast rebinding rate within 2 h and a maximum adsorption capacity of 177 μg per gram for adenosine. The adsorption fitted a Langmuir-Freundlich (LF) isotherm model with a K LF value of 41 mL/μg and a q m value of 177 μg/g for the MIP microspheres. The values were larger than those for a non-molecularly imprinted polymer (NIP) particles (5 mL/μg and 88 μg/g) indicating a better adsorption ability towards adenosine. The MIP microspheres showed a good selectivity for adenosine with a higher adsorption (683 nmol/g) for adenosine than that (91 nmol/g, 24 nmol/g and 54 nmol/g) for guanosine, cytidine and uridine respectively. Further experiment proved that the adenosine-imprinted polymer microspheres also had a good selectivity for ADP-ribosylated proteins that the MIP could extract the ADP-ribosylated proteins from the cell extract samples. [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]

Structural characterization of protein interactions is essential for our ability to understand and modulate physiological processes. Computational approaches to modeling of protein complexes provide structural information that far exceeds capabilities of the existing experimental techniques. Protein structure prediction in general, and prediction of protein interactions in particular, has been revolutionized by the rapid progress in Deep Learning techniques. The work of Schweke et al. (Proteomics 2023, 23, 2200323) presents a community-wide study of an important problem of distinguishing physiological protein-protein complexes/interfaces (experimentally determined or modeled) from non-physiological ones. The authors designed and generated a large benchmark set of physiological and non-physiological homodimeric complexes, and evaluated a large set of scoring functions, as well as AlphaFold predictions, on their ability to discriminate the non-physiological interfaces. The problem of separating physiological interfaces from non-physiological ones is very difficult, largely due to the lack of a clear distinction between the two categories in a crowded environment inside a living cell. Still, the ability to identify key physiologically significant interfaces in the variety of possible configurations of a protein-protein complex is important. The study presents a major data resource and methodological development in this important direction for molecular and cellular biology., (© 2023 Wiley-VCH GmbH.)

Abstract Background The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. Method Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). Results The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. Conclusion HisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.