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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 HTrsurface 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, This project was financially supported by The Scientific and Technological Research Council of Turkey (TUBITAK) within the scope of 3001-Initial R&D Projects Support Program [project number 114Z664].

O-Acetylation is a common naturally occurring modification of carbohydrates and is especially widespread in sialic acids, a family of nine-carbon acidic monosaccharides. O-Acetyl migration within the exocyclic glycerol-like side chain of mono-O-acetylated sialic acid reported previously was from the C7- to C9-hydroxyl group with or without an 8-O-acetyl intermediate, which resulted in an equilibrium that favors the formation of the 9-O-acetyl sialic acid. Herein, we provide direct experimental evidence demonstrating that O-acetyl migration is bidirectional, and the rate of equilibration is influenced predominantly by the pH of the sample. While the O-acetyl group on sialic acids and sialoglycans is stable under mildly acidic conditions (pH < 5, the rate of O-acetyl migration is extremely low), reversible O-acetyl migration is observed readily at neutral pH and becomes more significant when the pH increases to slightly basic. Sialoglycan microarray studies showed that esterase-inactivated porcine torovirus hemagglutinin-esterase bound strongly to sialoglycans containing a more stable 9-N-acetylated sialic acid analog, but these compounds were less resistant to periodate oxidation treatment compared to their 9-O-acetyl counterparts. Together with prior studies, the results support the possible influence of sialic acid O-acetylation and O-acetyl migration to host-microbe interactions and potential application of the more stable synthetic N-acetyl mimics.

The donut-shaped cucurbit[n]urils (Qn, n = 6-8) are rigid macrocyclic receptors with widespread use in protein recognition. To date, most applications have centred on the encapsulation of N-terminal aromatic residues by Q7 or Q8. Less attention has been placed on Q6, which can recognize lysine side chains due to its high affinity for alkylamines. In this work, we investigated protein-Q6 complexation by using NMR spectroscopy. Attempts to crystallize protein-Q6 complexes were thwarted by the crystallization of Q6. We studied four proteins that vary in size, net charge, and lysine content. In addition to Q6 interactions with specific Lys or dimethylated Lys residues, we report striking evidence for N-terminal recognition. High affinity (micromolar) binding occurred with the N-terminal Met-Lys motif present in one of the four model proteins. Engineering this feature into another model protein yielded a similar high affinity site. We also present evidence for Q8 binding at this N-terminal feature. These data expand the cucurbituril toolkit for protein sensing.

Molecularly imprinted cryogels for protein recognition have received much attention in recent years with the development of supermacroporous polymer systems. Molecularly imprinted cryogels, which o...

Novel gas-responsive imprinting hydrogels were fabricated by combining N,N′-dimethylaminoethyl methacrylate gas-sensitive monomers, N,N′-methylenebis(acrylamide) cross-linkers, and human serum albu...

This study presents a novel computational approach to study molecular recognition and binding kinetics for drug-like compounds dissociating from a flexible protein system. The intermediates and their free energy profile during ligand association and dissociation processes control ligand–protein binding kinetics and bring a more complete picture of ligand–protein binding. The method applied the milestoning theory to extract kinetics and thermodynamics information from running short classical molecular dynamics (MD) simulations for frames from a given dissociation path. High-dimensional ligand-protein motions (3N-6 degrees of freedom) during ligand dissociation were reduced by use of principal component modes for assigning more than 100 milestones, and classical MD runs were allowed to travel multiple milestones to efficiently obtain ensemble distribution of initial structures for MD simulations and estimate the transition time and rate during ligand traveling between milestones. We used five pyrazolourea ligands and cyclin-dependent kinase 8 with cyclin C (CDK8/CycC) as our model system as well as metadynamics and a pathway search method to sample dissociation pathways. With our strategy, we constructed the free energy profile for highly mobile biomolecular systems. The computed binding free energy and residence time correctly ranked the pyrazolourea ligand series, in agreement with experimental data. Guided by a barrier of a ligand passing an αC helix and activation loop, we introduced one hydroxyl group to parent compounds to design our ligands with increased residence time and validated our prediction by experiments. This work provides a novel and robust approach to investigate dissociation kinetics of large and flexible systems for understanding unbinding mechanisms and designing new small-molecule drugs with desired binding kinetics.

Evaluation of the molecular mechanisms by which an antibody recognizes a specific antigen could help in better understanding of the protein recognition mechanisms. We previously showed that anti-hen egg lysozyme (HEL) monoclonal antibody, HyC1, recognized the structural and hydrodynamic change in HEL. Here, we generated HyC1 single-chain Fv (scFv), and characterized it using different structural and biophysical methods. Similar to HyC1 monoclonal antibody, HyC1 scFv could recognize native HEL from carboxymethylated Cys6 and Cys127 HEL (CM6,127-HEL). Comparison of the binding thermodynamics of HyC1 scFv between HEL and CM6,127-HEL showed that the binding enthalpy change was different, while the binding entropy was remained unchanged. The results indicated that the fluctuation of the residual native structure in both HEL and CM6,127-HEL was similar. The NMR experiments for 15N-labeled HyC1 scFv indicated that the flexibility of HyC1 scFv decreased upon the binding to HEL.

Small molecule spectrophotometric probes for DNA/RNA and proteins are of the utmost importance for diagnostics in biochemical and biomedical research. Both, naturally occurring and synthetic probes, often include peptide sequence responsible for the selectivity toward the particular target; however, commercially available dyes are restricted to single point attachment to the peptide (having one reactive group). Here presented are our recent advances in the development of novel amino acidfluorophore probes, with the unique characteristic of free N- and C-terminus available for incorporation at any peptide backbone position. Intriguingly, already monomeric amino acid-fluorophores showed recognition among various DNA/RNA, whereby steric impact and contribution of halogens is systematically studied. Moreover, some dyes revealed intracellular mitochondria specificity. Further, several hetero-dimeric chromophore systems were prepared, demonstrating that synergistic effect can lead to simultaneous DNA, RNA and protein fluorimetric recognition, combined with enzyme inhibition. Also, homodimeric cyanines equipped with chlorine revealed intriguing DNA/RNA selectivity in respect to well-known parent TOTO and YOYO dyes.

Microcavity-supported lipid bilayers (MSLBs) are contact-free membranes suspended across aqueous-filled pores that maintain the lipid bilayer in a highly fluidic state, free from frictional interactions with substrate. Such platforms offer the prospect of liposome-like fluidity with the compositional versatility and addressability of supported lipid bilayers and thus offer a significant opportunity to model membrane asymmetry, protein-membrane interactions, and aggregation at the membrane interface. Herein we evaluate their performance by studying the effect of transmembrane lipid asymmetry on lipid diffusivity, membrane viscosity, and cholera toxin-ganglioside recognition across six symmetric and asymmetric membranes including binary compositions containing both fluid and gel phases, and ternary phase-separated membrane compositions. Fluorescence lifetime correlation spectroscopy was used to determine the lateral mobility of the lipid and the protein, and electrochemical impedance spectroscopy enabled the detection of the protein-membrane assembly over the nanomolar range. Transmembrane leaflet asymmetry was observed to have a profound impact on membrane electrochemical resistance, where the resistance of a ternary symmetric phase-separated bilayer was found to be at least 2.6 times higher than the asymmetric bilayer with analogous composition in the distal leaflet but where the lower leaflet comprised only 1,2-dioleoyl

Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications.

Herein, we describe two homogeneous conversion systems that can convert protein recognition into the release of predesigned output DNA for the activation of DNA nanomachines. With high conversion efficiency, this strategy allows us to initiate DNA nanomachines to respond to any proteins, opening up the possibility of applying DNA nanomachines in clinical practice.

We evaluate a series of thin-sheet hydrogel molecularly imprinted polymers (MIPs), using a family of acrylamide-based monomers, selective for the target protein myoglobin (Mb). The simple production of the thin-sheet MIP offers an alternative biorecognition surface that is robust, stable and uniform, and has the potential to be adapted for biosensor applications. The MIP containing the functional monomer N-hydroxymethylacrylamide (NHMAm), produced optimal specific rebinding of the target protein (Mb) with 84.9% (± 0.7) rebinding and imprinting and selectivity factors of 1.41 and 1.55, respectively. The least optimal performing MIP contained the functional monomer N,N-dimethylacrylamide (DMAm) with 67.5% (± 0.7) rebinding and imprinting and selectivity factors of 1.11 and 1.32, respectively. Hydrogen bonding effects, within a protein-MIP complex, were investigated using computational methods and Fourier transform infrared (FTIR) spectroscopy. The quantum mechanical calculations predictions of a red shift of the monomer carbonyl peak is borne-out within FTIR spectra, with three of the MIPs, acrylamide, N-(hydroxymethyl) acrylamide, and N-(hydroxyethyl) acrylamide, showing peak downshifts of 4, 11, and 8 cm−1, respectively.

Protein-protein interaction networks are critical components of cellular regulation. Hub proteins, defined by their ability to interact with numerous protein partners, are the pivots of these networks. A hypothesis that an ensemble of rapidly interconverting conformational states contributes significantly to the ability of hub proteins to interact with diverse partners has been proposed. The master gene regulator p53 is a prototype multidomain hub protein. Its DNA-binding domain alone is involved in interactions with many of its partner proteins. We investigated the dynamics of the p53 DNA-binding domain by 15N-NMR Carr-Purcell-Meiboom-Gill relaxation methods. In the DNA-bound state, we detected conformational exchanges in the domain in the microsecond to millisecond timescale, while dynamics at this timescale was not detectable in the free state. This suggests that the binding of p53 to specific DNA sequences promotes exchange between two or more conformational states, creating a broad conformational repertoire necessary for interacting with many partner proteins.

The PDZ domain of Dishevelled 3 protein belongs to a highly abundant protein recognition motif which typically binds short C-terminal peptides. The affinity of the PDZ towards the peptides could be fine-tuned by a variety of post-translation modifications including phosphorylation. However, how phosphorylations affect the PDZ structure and its interactions with ligands remains elusive. Combining molecular dynamics simulations, NMR titration, and biological experiments, we explored the role of previously reported phosphorylation sites and their mimetics in the Dishevelled PDZ domain. Our observations suggest three major roles for phosphorylations: (1) acting as an on/off PDZ binding switch, (2) allosterically affecting the binding groove, and (3) influencing the secondary binding site. Our simulations indicated that mimetics had similar but weaker effects, and the effects of distinct sites were non-additive. This study provides insight into the Dishevelled regulation by PDZ phosphorylation. Furthermore, the observed effects could be used to elucidate the regulation mechanisms in other PDZ domains.

Epitope imprinting is an effective strategy to prepare molecularly imprinted polymers (MIPs) for protein recognition. Indeed, the idea to use as a template just a fragment of the protein of interest, called the epitope, instead of the whole protein, presents some key advantages for the imprinting process, in particular: cutting the costs for MIP production and avoiding protein unfolding during the imprinting process, so to ultimately improve the quality of the stamped binding sites. How to select an epitope for the imprinting is the strategic question. Here, the bioinformatics tools to search for suitable epitopes for the imprinting process and rational tools to select the most suitable epitope are briefly introduced along with protocols for their practical use.

Small 17(33), 2101066 (2021). doi:10.1002/smll.202101066, Published by Wiley-VCH, Weinheim

Molecularly imprinted polymers (MIPs) have evolved as promising platforms for specific recognition of proteins. However, molecular imprinting of the whole protein molecule is complicated by its large size, conformational instability, and structural complexity. These inherent limitations can be overcome by using epitope imprinting. Significant breakthroughs in the synthesis and application of epitope-imprinted polymers (EIPs) have been achieved and reported. This review highlights recent advances in epitope imprinting, from the selection of epitope peptide sequences and functional monomers to the methods applied in polymerization and template removal. Technological innovations in detection and extraction of proteins by EIPs are also provided.

Carbohydrate-modified peptides (i.e., “glycopeptides”) inspired by natural glycoproteins and proteoglycans are receiving increasing interest as the basis for biomaterials with advanced structural and functional properties. This chapter first introduces the reader to different chemical and enzymatic methods that are used to synthesize glycosylated peptides. Then, the chapter presents examples in which the structure of peptides and peptide-based materials can be varied through glycosylation. Finally, the chapter highlights the emerging use of glycosylated peptide materials for medical and biotechnology applications, including protein recognition, cell scaffolding, drug delivery, vaccines, and disease treatment. Collectively, the examples surveyed in this chapter demonstrate the enormous potential of carbohydrate conjugates to inform the structure of peptide-based biomaterials, as well as to endow them with new functional capabilities.

Using a covalent chemical probe and X-ray crystallography coupled to nuclear magnetic resonance data, we elucidated the dynamic molecular basis of protein recognition between the carrier protein and adenylation domain in pyoluteorin biosynthesis. These findings reveal a unique binding mode, which contrasts previously solved carrier protein and partner protein interfaces.

This chapter enlightens the development and application of dye-based as well as genetically encoded nanosensors for metabolites related to various diseases, their comparison, and usefulness in understanding the metabolic pathways by using novel high throughput assays. Previously dye-based fluorescent probes have been constructed for the in vivo detection of cellular molecules. To study the real-time localization and monitoring of metabolites in live cells, genetically encoded fluorescent nanosensors are steadily used as an elected method in comparison with chemical probes that are intrinsically dependent on effective delivery into the cell and sometimes causes the toxicity in the cells. Several methods have been reported to detect cancer and neurodegenerative diseases. However, FRET-based disease-specific biomarker detection is a preferable method because of its high sensitivity and selectivity. By using FRET-based genetically encoded nanosensor, we can detect some of the metabolites which are related to several diseases like cancer, Alzheimer, and diabetes. In humans, lack of reactive oxygen species (ROS) causes certain autoimmune diseases and excess production of ROS causes cardiovascular and respiratory diseases which can be detected in real time and non-invasively by using a nanosensor. Protein recognition-based genetically encoded sensors are projected to discover many research, medical, and environmental applications. Many human problems can be solved by the utilization of nanosensors which can also be helpful to authenticate the medicines to their principle components. Thus, sensor biology can be revolutionary for human welfare in the future with the help of FRET-based tools.

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.

The highly sequence-selective molecular recognition of proteins and peptides in Nature inspires and informs the development of synthetic receptors to mimic, measure, and modulate these processes. This chapter focuses on the cucurbit[n]urils, which have been particularly powerful in this regard. We describe the discovery and elaboration of the sequence-selective recognition of peptides by cucurbit[n]urils. In addition, we overview the development of this supramolecular chemistry into myriad applications and the extension to protein recognition. Finally, we compare the binding properties of synthetic receptors with those of natural N-recognins, proteases, and other sequence-selective protein receptors. The strong correlation between natural and synthetic receptors in their molecular basis for sequence recognition should serve to improve the design of next-generation compounds.

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. © 2018

© 2018 American Chemical Society. Glycoproteins adhered on the cellular membrane play a pivotal role in a wide range of cellular functions. Their importance is particularly relevant in the recognition process between infectious pathogens (such as viruses, bacteria, toxins) and their host cells. Multivalent interactions at the pathogen-cell interfaces govern binding events and can result in a strong and specific interaction. Here we report an approach to mimic the cell surface presentation of carbohydrate ligands by the multivalent display of sugars on the surface of peptoid nanosheets. The constructs provide a highly organized 2D platform for recognition of carbohydrate-binding proteins. The sugars were displayed using different linker lengths or within loops containing 2-6 hydrophilic peptoid monomers. Both the linkers and the loops contained one alkyne-bearing monomer, to which different saccharides were attached by copper-catalyzed azide-alkyne cycloaddition reactions. Peptoid nanosheets functionalized with different saccharide groups were able to selectively bind multivalent lectins, Concanavalin A and Wheat Germ Agglutinin, as observed by fluorescence microscopy and a homogeneous Förster resonance energy transfer (FRET)-based binding assay. To evaluate the potential of this system as sensor for threat agents, the ability of functionalized peptoid nanosheets to bind Shiga toxin was also studied. Peptoid nanosheets were functionalized with globotriose, the natural ligand of Shiga toxin, and the effective binding of the nanomaterial was verified by the FRET-based binding assay. In all cases, evidence for multivalent binding was observed by systematic variation of the ligand display density on the nanosheet surface. These cell surface mimetic nanomaterials may find utility in the inactivation of pathogens or as selective molecular recognition elements.

Gas-responsive molecularly imprinted polymer (MIP) particles, which have nanocavities in their shell layer for protein recognition, were developed for the first time. The MIP particles showed higher affinity for the target protein inside a CO2-treated aqueous medium as compared to that under N2-treated conditions, while retaining selectivity to the target protein.

The specific recognition of protein surface elements is a fundamental challenge in the life sciences. New developments in this field will form the basis of advanced therapeutic approaches and lead to applications such as sensors, affinity tags, immobilization techniques, and protein-based materials. Synthetic supramolecular molecules and materials are creating new opportunities for protein recognition that are orthogonal to classical small molecule and protein-based approaches. As outlined here, their unique molecular features enable the recognition of amino acids, peptides, and even whole protein surfaces, which can be applied to the modulation and assembly of proteins. We believe that structural insights into these processes are of great value for the further development of this field and have therefore focused this Perspective on contributions that provide such structural data.

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. © 2017 Elsevier B.V.

A new anti-protein nonspecific adsorption polydopamine-based surface protein-imprinted magnetic microsphere was prepared by using surface imprinting and AGET-ATRP in this work. The highlights were as follows. Firstly, different from our previous work, we could guarantee that there was no anti-protein nonspecific adsorption MPC chain segment in the imprinted sites. This fact could eliminate the negative influence of MPC chain segment on imprinted sites and further ensure the high imprinting efficiency. Secondly, the length of MPC chain segment was optimized to weaken the influence of protein resistance effect on adsorption capacity on the premise of guaranteeing excellent anti-protein nonspecific adsorption. The results showed that the optimal dopamine layer thickness was 10 nm. The mass ratio of MPC and Fe3O4@SiO2@Pdop-MIPs-Br was determined as 12/1. Under such conditions, after the graft of MPC, the adsorption capacity of imprinted microspheres for BSA decreased only a little from 9.39 mg/g to 8.26 mg/g, whereas the imprinting factor increased markedly from 1.53 to 5.74, revealing the advantage of this work. Finally, the successful applications in protein recognition showed that the new strategy was expected to be an effective means for improving the detection sensitivity of molecularly imprinted biosensors.

Bulk imprinting of proteins was used combined with a grafting approach onto maghemite nanoparticles to develop a faster and simpler polymerization method for the synthesis of magnetic protein imprinted polymers with very high adsorption capacities and very strong affinity constants.

Self-folding deep cavitands with variably functionalized upper rims are able to selectively immobilize proteins at a biomimetic supported lipid bilayer surface. The immobilization process takes advantage of the dual-mode binding capabilities of the hosts, combining a defined binding pocket with upper rim charged/H-bonding groups. A variety of proteins can be selectively immobilized at the bilayer interface, either via complementary charge/H-bonding interactions, cavity-based molecular recognition, or a combination of both. The immobilization process can be used to bind unmodified native proteins, epitopes for bioadhesion, or proteins covalently modified with suitable RNMe3+ binding "handles" and charged groups that can either match or mismatch with the cavitand rim. The immobilization process can be monitored in real time using surface plasmon resonance (SPR) spectroscopy, and applied to the construction of cavitand:lipid arrays using the hosts and trehalose vitrified phospholipid vesicles. The selective, dual-mode protein recognition is maintained in the arrays, and can be visualized using SPR imaging.

Supramolecules such as calixarenes, cucurbiturils and foldamers are rapidly emerging as tools for protein assembly. This thesis builds on past successes with some of these scaffolds. Previously, sulfonato-calix[4]arene was shown to recognize and assemble the lysine-rich cytochrome c (cytc). Here, we tested calix[4]arenes with different upper or lower rims. A phosphonatomethyl derivative demonstrated enhanced selectivity for lysine whereas a lower rim oxomethylcarboxylate yielded a honeycomb network of cytc. A comparison of these structures highlighted how different substituents alter the recognition and the assembly-inducing behaviour. In related experiments a co-crystal structure revealed a surprising assembly of sulfonato-calix[8]arene and a cationic porphyrin. Inspired by the interactions of calixarenes with cytc, the sulfonato-calix[4]arene (n= 4, 6, 8) series was co-crystallized with another cationic protein – the small antifungal protein from Penicillium (PAF). It was an exciting and a rewarding project to solve the crystal structure of PAF for the first time. X-ray and solution-state studies enabled us to compare the influence of increasing calixarene size and charge on recognition and assembly. Another interesting project was to elucidate crystallographically the protein binding of a tether-free foldamer. The complex of a quinoline foldamer and cytc yielded a remarkable biohybrid assembly with chiral resolution of the foldamer helix handedness. NMR and CD experiments suggested differences in the solution state recognition processes. 2020-12-06

Like all outer membrane (OM) constituents, integral OM β-barrel proteins in Gram-negative bacteria are synthesized in the cytoplasm and trafficked to the OM, where they are locally assembled into the growing OM by the ubiquitous β-barrel assembly machine (Bam). While the identities and structures of all essential and accessory Bam components have been determined, the basic mechanism of Bam-assisted OM protein integration remains elusive. Here we review mechanistic analyses of OM β-barrel protein folding and Bam dynamics and summarize recent insights that inform a general model for OM protein recognition and assembly by the Bam complex.

The author's final peer reviewed version can be found by following the URI link. The Publisher's final version can be found by following the DOI link. Molecularly imprinted polymers (MIPs) have potential as alternatives to antibodies in the diagnosis and treatment of disease. However, atomistic level knowledge of the prepolymerization process is limited that would facilitate rational design of more efficient MIPs. Accordingly, we have investigated using computation and experiment the protein–monomer binding interactions that may influence the desired specificity. Myoglobin was used as the target protein and five different acrylamide-based monomers were considered. Protein binding sites were predicted using SiteMap and binding free energies of monomers at each site were calculated using MM-GBSA. Statistical thermodynamic analysis and study of atomistic interactions facilitated rationalization of monomer performance in MIP rebinding studies (% rebind; imprinting factors). CD spectroscopy was used to determine monomer effects on myoglobin secondary structure, with all monomers except the smallest monomer (acrylamide) causing significant changes. A complex interplay between different protein–monomer binding effects and MIP efficacy was observed. Validation of hypotheses for key binding features was achieved by rational selection of two different comonomer MIP combinations that produced experimental results in agreement with predictions. The comonomer studies revealed that uniform, noncompetitive binding of monomers around a target protein is favorable. This study represents a step toward future rational in silico design of MIPs for proteins.

ANTI-MODIFIED PROTEIN AUTOANTIBODIES IN RA DISPLAY IMPORTANT PEPTIDE CROSS-REACTIVITY BUT YET PROTEIN RECOGNITION SELECTIVITY

Smart gating membranes can regulate their permeability with high selectivity by controlling pore sizes based on the opening and closing behavior of their pores in response to external stimuli. This function arises from the volume change of stimuli-responsive polymers, which are immobilized inside the nano-sized pores. Among smart gating membranes, ion-recognition gating membranes are inspired by biomembranes that recognize ion signals; these artificial membranes can control their permeability in response to specific ions. Furthermore, biomolecule-recognition gating membranes, which work in protein recognition with high selectivity, have recently attracted attention because such membranes are of interest for biomedical applications, e.g., diagnostic devices. This chapter explains the design of and the principles underlying ion- and biomolecule-recognition smart gating membranes.

A facile method to prepare hollow molecularly imprinted polymers (HMIPs) for specific recognition of horseradish peroxidase (HRP) from biological samples was proposed in this paper. The HMIPs was prepared using silica nanoparticles as the sacrificial matrix and dopamine as functional monomer. The thickness of polydopamine shells can be easily modulated by tuning the mass ratio of silica matrix and dopamine. The polymerization conditions and recognition behaviors of the HMIPs were investigated systematically. The results suggested that the hollow structure endowed the HMIPs with fast adsorption kinetics of 25 min, high binding capacity of 172.1 mg/g, and reusability of no less than four adsorption-regeneration cycles without apparent deterioration. Meanwhile, excellent binding specificity towards HRP was presented in the selectivity studies. Moreover, enriching of HRP from human serum sample by the obtained HMIPs was conducted. The HMIPs displayed satisfactory binding specificity to HRP, in spite of the complex composition of the human serum.

Zinc is a biocompatible element that exists as the second most abundant transition metal ion and an indispensable trace element in the human body. Compared to traditional metal-organic complexes systems, d10 metal ZnII complexes not only exhibit a large Stokes shift and good photon stability but also possess strong emission and low cytotoxicity with a relatively small molecular weight. The use of ZnII complexes has emerged in the last decade as a versatile and convenient tool for numerous biological applications, including bioimaging, molecular and protein recognition, as well as photodynamic therapy. Herein, we review recent developments involving ZnII metal complexes applied as specific subcellular compartment imaging probes and their correlated utilizations.

Protein-protein interactions are ubiquitous, essential to almost all known biological processes, and offer attractive opportunities for therapeutic intervention. Linear peptide drugs, however, can be applied therapeutically as protein recognition motifs only to a limited extent because of their poor permeability, decreased receptor selectivity, and proteolytic stability. A major strategy in peptide chemistry is directed toward chemical modification and macrocyclization in order to limit a peptide's conformational possibilities, to increase its chemical and enzymatic stability, to prolong the time of action, and to increase activity and selectivity toward the receptor.

The strong affinity of biological receptors for their targets has been studied for many years. Noncovalent interactions between these natural recognition elements and their ligands form the basis for a broad range of biosensor applications. Although these sensing platforms are usually appreciably sensitive and selective, certain drawbacks are associated with biological receptors under nonphysiological conditions in terms of temperature, pH, or ionic strength. Therefore, there are considerable efforts to mimic such molecular interactions with robust, synthetic receptors. Molecular imprinting is the best-known technique to obtain antibody mimics by synthesizing a polymer matrix in the presence of a template species, such as molecules or larger aggregates. Extraction of the template results in sterically and functionally adapted binding cavities in or on a porous matrix. Although in principle possible, the detection of larger bioparticles such as proteins, microorganisms, or cells remains challenging when using the classical MIP concept. To tackle inherent difficulties, extending the concept of molecular imprinting toward surface imprinting is a promising approach: Here, binding cavities are formed directly on the surface of a cross linked polymer layer, thus facilitating the removal of the templates. This article reviews the main surface-imprinting techniques and focuses on the implementation of surface-imprinted polymers (SIPS) into various biomimetic sensors and related applications. In addition, we provide an outlook on emerging research on surface imprinting and the development of biomimetic tools for diagnostic purposes.

Molecularly imprinted polymers (MIPs) for the recognition of proteins are expected to possess high affinity through the establishment of multiple interactions between the polymer matrix and the large number of functional groups of the target. However, while highly affine recognition sites need building blocks rich in complementary functionalities to their target, such units are likely to generate high levels of non-specific binding. This paradox, that nature solved by evolution for biological receptors, needs to be addressed by the implementation of new concepts in molecular imprinting of proteins. Additionally, the structural variability, large size and incompatibility with a range of monomers made the development of protein MIPs to take a slow start. While the majority of MIP preparation methods are variants of chemical polymerization, the polymerization of electroactive functional monomers emerged as a particularly advantageous approach for chemical sensing application. Electropolymerization can be performed from aqueous solutions to preserve the natural conformation of the protein templates, with high spatial resolution and electrochemical control of the polymerization process. This review compiles the latest results, identifying major trends and providing an outlook on the perspectives of electrosynthesised protein-imprinted MIPs for chemical sensing.

Molecularly imprinted polymer brush layers and gel layers with both a lectin (ConA) and an antibody-IgG as biomolecular ligands for a target protein were formed on surface plasmon resonance (SPR) sensor chips via surface-initiated atom transfer radical polymerization (SIATRP) without and with a crosslinker, respectively. While the IgG-imprinted brush layers chip had almost the same affinity constant for target IgG as the nonimprinted brush layer chip, the affinity constant of the IgG-imprinted gel layer chip was approximately twice than that of the nonimprinted gel layer chip. These indicate that chemical crosslinks are very important factor to create distinct molecular recognition sites by molecular imprinting. Thus, biomolecular imprinting that uses biomolecular ligands and crosslinkers enables us to design polymer layer chips with distinct molecular recognition sites with a strong affinity for a target biomolecule. The molecularly imprinted gel layers chips with lectin and antibody ligands are promising candidates for fabricating SPR sensor systems to monitor target biomolecules such as proteins.

Carbohydrate-protein interactions underpin wide-ranging aspects of biology. However, such interactions remain relatively unexplored in pharmaceutical and biotechnological applications, in part due to the challenges associated with their structural characterisation, both experimentally and computationally. Knowledge-based approaches have shown great success in the prediction of drug-protein and protein-protein interactions, although have not been comprehensively investigated for carbohydrate-protein interactions. In this work, carbohydrate-protein complexes from the Protein Data Bank were comprehensively obtained and analysed to identify patterns in how carbohydrate-protein interactions are mediated.

Antibody–peptide/protein interactions are instrumental for many processes in the pharmaceutical and biotechnology industries and as tools for biomedical and biochemical research. The recent development of molecularly imprinted polymer nanoparticles displaying antibody-like recognition of peptides and proteins offers the possibility for substituting antibodies with these robust materials for applications where the structural integrity and function of antibodies is compromised by temperature, pH, solvent, etc. The background to the development of this class of antibody-mimicking material and the state-of-the-art in their synthesis and application is presented in this review.

The structural flexibility of RNA allows it to exist in several shapes and sizes. Thus, RNA is functionally diverse and is known to be involved in processes such as catalysis, ligand binding, and most importantly, protein recognition. RNA can adopt different structures, which can often dictate its functionality. When RNA binds onto protein to form a ribonucleoprotein complex (RNP), multiple interactions and conformational changes occur with the RNA and protein. However, there is the question of whether there is a specific pattern for these changes to occur upon recognition. In particular when RNP complexity increases with the addition of multiple proteins/RNA, it becomes difficult to structurally characterize the overall changes using the current structural determination techniques. Hence, there is a need to use a combination of biochemical, structural and computational modeling to achieve a better understanding of the processes that RNPs are involved. Nevertheless, there are well-characterized systems that are evolutionarily conserved [such as the signal recognition particle (SRP)] that give us important information on the structural changes of RNA and protein upon complex formation.

Considerable progress has been made characterizing the structures and interactions of leukocyte cell-surface molecules. We have focused on small T-cell expressed adhesion molecules and costimulatory receptors belonging to the Immunoglobulin Superfamily (IgSF), which comprises the largest group of proteins present on leukocyte surfaces. Our work has helped reveal how weak, specific protein recognition is achieved at cell surfaces. Studies of CD2 identified a new mode of protein recognition dominated by electrostatic contacts at interfaces with very poor shape complementarity. Work on costimulatory proteins revealed a second, more conventional mode of protein recognition at the cell surface, wherein much more hydrophobic, better-matched surfaces interact over smaller areas. The rigid-body nature of the interactions of costimulatory molecules placed helpful constraints on possibilities for how downstream signaling is initiated following ligand engagement by receptors. Here we review these findings and suggest a structure-based explanation for the remarkable evolutionary success of IgSF signaling proteins.

The egg coat, an extracellular matrix made up of glycoprotein filaments, plays a key role in animal fertilization by acting as a gatekeeper for sperm. Egg coat components polymerize using a common zona pellucida (ZP) "domain" module that consists of two related immunoglobulin-like domains, called ZP-N and ZP-C. The ZP module has also been recognized in a large number of other secreted proteins with different biological functions, whose mutations are linked to severe human diseases. During the last decade, tremendous progress has been made toward understanding the atomic architecture of the ZP module and the structural basis of its polymerization. Moreover, sperm-binding regions at the N-terminus of mollusk and mammalian egg coat subunits were found to consist of domain repeats that also adopt a ZP-N fold. This discovery revealed an unexpected link between invertebrate and vertebrate fertilization and led to the first structure of an egg coat-sperm protein recognition complex. In this review we summarize these exciting findings, discuss their functional implications, and outline future challenges that must be addressed in order to develop a comprehensive view of this family of biomedically important extracellular molecules.

The objective of this study is to evaluate the structure and protein recognition properties of hybrid four-way junctions (4WJs) composed of DNA and peptide nucleic acid (PNA) strands. We compare a classic immobile DNA junction, J1, vs. six PNA-DNA junctions, including a number with blunt DNA ends and multiple PNA strands. Circular dichroism (CD) analysis reveals that hybrid 4WJs are composed of helices that possess structures intermediate between A- and B-form DNA, the apparent level of A-form structure correlates with the PNA content. The structure of hybrids that contain one PNA strand is sensitive to Mg(+2). For these constructs, the apparent B-form structure and conformational stability (Tm) increase in high Mg(+2). The blunt-ended junction, b4WJ-PNA3, possesses the highest B-form CD signals and Tm (40.1 °C) values vs. all hybrids and J1. Protein recognition studies are carried out using the recombinant DNA-binding protein, HMGB1b. HMGB1b binds the blunt ended single-PNA hybrids, b4WJ-PNA1 and b4WJ-PNA3, with high affinity. HMGB1b binds the multi-PNA hybrids, 4WJ-PNA1,3 and b4WJ-PNA1,3, but does not form stable protein-nucleic acid complexes. Protein interactions with hybrid 4WJs are influenced by the ratio of A- to B-form helices: hybrids with helices composed of higher levels of B-form structure preferentially associate with HMGB1b.