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Proteins are essential components of human cells and tissues, and they are commonly measured in clinical laboratories using immunoassays. However, these assays have certain limitations, such as non-specificity binding, insufficient selectivity, and interference of antibodies. More sensitive, accurate, and efficient technology is required to overcome these limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful analytical tool that provides high sensitivity and specificity, making it superior to traditional methods such as biochemical methods and immunoassays. While LC-MS/MS has been increasingly used for detecting small molecular analytes and steroid hormones in clinical practice recently, its application for protein or peptide analysis is still in its early stages. Established methods for quantifying proteins and peptides by LC-MS/MS are mainly focused on scientific research, and only a few proteins and peptides can be or have the potential to be detected and applied in clinical practice. Therefore, this article aims to review the clinical applications, advantages, and challenges of analyzing proteins and peptides using LC-MS/MS in clinical laboratories., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Solid-state nuclear magnetic resonance (NMR) is a potent tool for studying the structures and dynamics of insoluble proteins. It starts with signal assignment through multi-dimensional correlation experiments, where the aliphatic 13 Cα- 13 Cβ correlation is indispensable for identifying specific residues. However, developing efficient methods for achieving this correlation is a challenge in solid-state NMR. We present a simple band-selective zero-quantum (ZQ) recoupling method, named POST-C4 16 1 (PC4), which enhances 13 Cα- 13 C radio-frequency (RF) field and proton decoupling, exhibits high stability against RF variations, and achieves superior efficiency. Comparative tests on various samples, including the formyl-Met-Leu-Phe (fMLF) tripeptide, microcrystalline β1 immunoglobulin binding domain of protein G (GB1), and membrane protein of mechanosensitive channel of large conductance from Methanosarcina acetivorans (MaMscL), demonstrate that PC4 selectively enhances 13 C radio-frequency (RF) field and proton decoupling, exhibits high stability against RF variations, and achieves superior efficiency. Comparative tests on various samples, including the formyl-Met-Leu-Phe (fMLF) tripeptide, microcrystalline β1 immunoglobulin binding domain of protein G (GB1), and membrane protein of mechanosensitive channel of large conductance from Methanosarcina acetivorans (MaMscL), demonstrate that PC4 selectively enhances 13 Cα- 13 Cβ correlations by up to 50 % while suppressing unwanted correlations, as compared to the popular dipolar-assisted rotational resonance (DARR). It has addressed the long-standing need for selective 13 C- 13 C correlation methods. We anticipate that this simple but efficient PC4 method will have immediate applications in structural biology by solid-state NMR., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Quantum dots (QDs) are semiconductor nanocrystals (2-10 nm) with unique optical and electronic properties due to quantum confinement effects. They offer high photostability, narrow emission spectra, broad absorption spectrum, and high quantum yields, making them versatile in various applications. Due to their highly reactive surfaces, QDs can conjugate with biomolecules while being used, produced, or unintentionally released into the environment. This systematic review delves into intricate relationship between QDs and proteins, examining their interactions that influence their physicochemical properties, enzymatic activity, ligand binding affinity, and stability. The research utilized electronic databases like PubMed, WOS, and Proquest, along with manual reviews from 2013 to 2023 using relevant keywords, to identify suitable literature. After screening titles and abstracts, only articles meeting inclusion criteria were selected for full text readings. This systematic review of 395 articles identifies 125 articles meeting the inclusion criteria, categorized into five overarching themes, encompassing various mechanisms of QDs and proteins interactions, including adsorption to covalent binding, contingent on physicochemical properties of QDs. Through a meticulous analysis of existing literature, it unravels intricate nature of interaction, significant influence on nanomaterials and biological entities, and potential for synergistic applications harnessing both specific and nonspecific interactions across various fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The history of the topic of proteins at soft interfaces dates back to the 19 th century, and until the present day, it has continuously attracted great scientific interest. A multitude of experimental methods and theoretical approaches have been developed to serve the research progress in this large domain of colloid and interface science, including the area of soft colloids such as foams and emulsions. From classical methods like surface tension adsorption isotherms, surface pressure-area measurements for spread layers, and surface rheology probing the dynamics of adsorption, nowadays, advanced surface-sensitive techniques based on spectroscopy, microscopy, and the reflection of light, X-rays and neutrons at liquid/fluid interfaces offers important complementary sources of information. Apart from the fundamental characteristics of protein adsorption layers, i.e., surface tension and surface excess, the nanoscale structure of such layers and the interfacial protein conformations and morphologies are of pivotal importance for extending the depth of understanding on the topic. In this review article, we provide an extensive overview of the application of three methods, namely, ellipsometry, X-ray reflectometry and neutron reflectometry, for adsorption and structural studies on proteins at water/air and water/oil interfaces. The main attention is placed on the development of experimental approaches and on a discussion of the relevant achievements in terms of notable experimental results. We have attempted to cover the whole history of protein studies with these techniques, and thus, we believe the review should serve as a valuable reference to fuel ideas for a wide spectrum of researchers in different scientific fields where proteins at soft interface may be of relevance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The author is an Editorial Board Member for Advances in Colloid and Interface Science and was not involved in the editorial review or the decision to publish this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This review covers the analytical applications of protein partitioning in aqueous two-phase systems (ATPSs). We review the advancements in the analytical application of protein partitioning in ATPSs that have been achieved over the last two decades. Multiple examples of different applications, such as the quality control of recombinant proteins, analysis of protein misfolding, characterization of structural changes as small as a single-point mutation, conformational changes upon binding of different ligands, detection of protein-protein interactions, and analysis of structurally different isoforms of a protein are presented. The new approach to discovering new drugs for a known target (e.g., a receptor) is described when one or more previous drugs are already available with well-characterized biological efficacy profiles.

Techno-functional properties of protein isolates such as emulsification, foaming, and gelling serve as key indicators to determine their food applications. Conventional macro-volume techniques used to measure these techno-functional properties are usually time consuming, require large amounts of protein samples, and are impractical when diverse protein samples are handled at the early screening stage. To overcome these issues, we have developed scaled-down (miniaturized) assays to test techno-functional properties of protein samples. These assays are simple, efficient, and require <400 μl of protein solution. Specifically, the miniaturized emulsification and gelling assays require 25-fold less protein than conventional macro-volume techniques and the miniaturized foaming assay requires 100-fold less sample. The performance of these assays has been thoroughly validated using conventional techno-functional tests for each parameter. The protocols described herein offer high-throughput screening capabilities, accelerating the testing process for protein techno-functional properties and allowing for quick identification of samples of interest from diverse samples. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Miniaturized emulsification assay Alternate Protocol 1: Conventional macro-volume emulsification assay Basic Protocol 2: Miniaturized foaming assay Alternate Protocol 2: Conventional macro-volume foaming assay Basic Protocol 3: Miniaturized gelling assay Alternate Protocol 3: Conventional macro-volume gelling assay., (© 2024 Wiley Periodicals LLC.)

The food industry is undergoing a significant transformation with the advancement of 3D technology. Researchers in the field are increasingly interested in using protein and protein-polysaccharide composite materials for 3D printing applications. However, maintaining nutritional and sensory properties while guaranteeing printability of these materials is challenging. This review examines the commonly used protein and composite materials in food 3D printing and their roles in printing inks. This review also outlines the essential properties required for 3D printing, including extrudability, appropriate viscoelasticity, thixotropic properties, and gelation properties. Furthermore, it explores the wide range of potential applications for 3D printing technology in novel functional foods such as space food, dysphagia food, kid's food, meat analogue, and other specialized food products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

This review addresses the current trend of replacing petroleum-based polymers in food packaging with bio-based alternatives, specifically focusing on proteins and polysaccharides. While these biopolymers exhibit excellent film-forming properties and are abundant in nature, their individual use in packaging lacks ideal plastic-like characteristics, especially in terms of mechanical and barrier properties. A recent solution involves the formulation of biocomposites through the reinforcement of one biopolymer with another (e.g., protein with a polysaccharide), significantly enhancing the physical, mechanical, and barrier properties of packaging materials. The review concentrates on the integration of proteins and polysaccharides in biocomposite materials, emphasizing their potential applications in active and intelligent food packaging systems. It covers sources, manufacturing methods, interaction mechanisms, recent developments, perspectives, and opportunities. The exploration extends to practical implementations of these biocomposites in enhancing food quality, safety, and shelf life-a green technological approach contributing to the reduction of food waste and loss., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Elucidating protein conformational changes is essential because conformational changes are closely related to the functions of proteins. Cryo-electron microscopy (cryo-EM) experiment can be used to reconstruct protein conformational changes via a method that involves using the experimental data (two-dimensional protein images). In this study, a reconstruction method, referred to as the "four-dimensional imaging," was proposed. In our four-dimensional imaging technique, the protein conformational change was obtained using the two-dimensional protein images (the three-dimensional electron density maps used in previously proposed techniques were not used). The protein conformation for each two-dimensional protein image was obtained using our original protocol with molecular dynamics simulations. Using a manifold-learning technique and two-dimensional protein images, the protein conformations were arranged according to the conformational change of the protein. By arranging the protein conformations according to the arrangement of the protein images, four-dimensional imaging is constructed. A simulation for a cryo-EM experiment demonstrated the validity of our four-dimensional imaging technique., (© 2023 The Authors. Journal of Computational Chemistry published by Wiley Periodicals LLC.)

The mechanism of ammonia formation during the pyrolysis of proteins in biomass is currently unclear. To further investigate this issue, this study employed the AMS 2023.104 software to select proteins (actual proteins) as the model compounds and the amino acids contained within them (assembled amino acids) as the comparative models. ReaxFF molecular dynamics simulations were conducted to explore the nitrogen transformation and NH 3 generation mechanisms in three-phase products (char, tar, and gas) during protein pyrolysis. The research results revealed several key findings. Regardless of whether the model compounds are actual proteins or assembled amino acids, NH 3 is the primary nitrogen-containing product during pyrolysis. However, as the temperature rises to higher levels, such as 2000 K and 2500 K, the amount of NH 3 decreases significantly in the later stages of pyrolysis, indicating that it is being converted into other nitrogen-bearing species, such as HCN and N 2 . Simultaneously, we also observed significant differences between the pyrolysis processes of actual proteins and assembled amino acids. Notably, at 2000 K, the amount of NH 3 generated from the pyrolysis of assembled amino acids was twice that of actual proteins. This discrepancy mainly stems from the inherent structural differences between proteins and amino acids. In proteins, nitrogen is predominantly present in a network-like structure (NH-N), which shields it from direct external exposure, thus requiring more energy for nitrogen to participate in pyrolysis reactions, making it more difficult for NH 3 to form. Conversely, assembled amino acids can release NH 3 through a simpler deamination process, leading to a significant increase in NH 3 production during their pyrolysis.

Due to their excellent emulsification, biocompatibility, and biological activity, proteins are widely used as microcapsule wall materials for encapsulating drugs, natural bioactive substances, essential oils, probiotics, etc. In this review, we summarize the protein-based microcapsules, discussing the types of proteins utilized in microcapsule wall materials, the preparation process, and the main factors that influence their properties. Additionally, we conclude with examples of the vital role of protein-based microcapsules in advancing the food industry from primary processing to deep processing and their potential applications in the biomedical, chemical, and textile industries. However, the low stability and controllability of protein wall materials lead to degraded performance and quality of microcapsules. Protein complexes with polysaccharides or modifications to proteins are often used to improve the thermal instability, pH sensitivity, encapsulation efficiency and antioxidant capacity of microcapsules. In addition, factors such as wall material composition, wall material ratio, the ratio of core to wall material, pH, and preparation method all play critical roles in the preparation and performance of microcapsules. The application area and scope of protein-based microcapsules can be further expanded by optimizing the preparation process and studying the microcapsule release mechanism and control strategy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The present work describes an update to the protein covalent geometry and atomic radii parameters in the Xplor-NIH biomolecular structure determination package. In combination with an improved treatment of selected non-bonded interactions between atoms three bonds apart, such as those involving methyl hydrogens, and a previously developed term that affects the system's gyration volume, the new parameters are tested using structure calculations on 30 proteins with restraints derived from nuclear magnetic resonance data. Using modern structure validation criteria, including several formally adopted by the Protein Data Bank, and a clear measure of structural accuracy, the results show superior performance relative to previous Xplor-NIH implementations. Additionally, the Xplor-NIH structures compare favorably against originally determined NMR models., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA.)

Solid-phase fluorescence excitation-emission matrix (SPF-EEM) spectroscopy has potential for non-extractive, non-destructive, and non-contact analytical measurements of powder and solid-state samples, as well as front-face EEM spectroscopy for suspensions of high optical density. However, as there is no unified measurement method for SPF spectroscopy, comparing samples measured in different research fields is difficult. Therefore, this study designs a cell that can be created by a 3D printer and examines reproducibility on measuring fluorescent powders. The developed cell is applied to proteins (ovalbumin, BSA, gliadin, gluten, powdered collagen, casein), amino acids (tryptophan, tyrosine, and phenylalanine), soybean ingredients (daidzein, and genistein), and fluorescent chemicals (rhodamine B, fluorescein sodium salt, pyrene, and quinine sulfate dihydrate) and their spectra are compared with those in the solution states. When samples are refilled into the cell three times, the cell exhibits high reproducibility in terms of fluorescence peak wavelength and intensity. The solid proteins exhibit peaks attributed to the fluorescent amino acid residues, and broad peaks which are not detected for the proteins in the solution states. Powdered rhodamine B and fluorescein sodium salt do not exhibit fluorescence, possibly due to the inner-filter effect (IFE). Some non-colored molecules also exhibit loss of fluorescence or a remarkable difference between the solid and solution states, possibly due to the interaction of the fluorescent structure with the surrounding local environment, similar to the solvent effect, which is possibly affected by the molecular proximity, three-dimensional structure, and moisture absorption capacity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The stability of food emulsions is the basis for other properties. During their production and processing, emulsions tend to become unstable due to their thermodynamic instability, and it is usually necessary to add emulsifiers and proteins to stabilize emulsions. It becomes crucial to study the intrinsic mechanisms of emulsifiers and proteins and their joint stabilization of food emulsions. This paper summarizes the research on intrinsic mechanisms of food emulsions stabilized by emulsifiers and proteins in recent years. The destabilization and stabilization of emulsions are related to the added surfactants. The properties, type, and concentration of emulsifiers determine the stability of emulsions, and the emulsifiers can be classified into different types (e.g., ionic or nonionic, solid or liquid) according to their properties and sources. The physicochemical properties of proteins (e.g., spatial conformation, hydrophobicity) and the composition of proteins can also determine the stability of emulsions, and emulsions stabilized by emulsifiers and proteins together not only depend on these factors but also have a great relationship with the mutual combination and competition between the two. The instability and stability of emulsions are related to factors such as interfacial interaction forces, the rheological nature of the interface, and the added surfactant., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2024. Published by Elsevier B.V.)

Background: Autophagy is instrumental in various health conditions, including cancer, aging, and infections. Therefore, examining proteins and compounds associated with autophagy is paramount to understanding cellular biology and the origins of diseases, paving the way for potential therapeutic and disease prediction strategies. However, the complexity of autophagy, its intersection with other cellular pathways, and the challenges in monitoring autophagic activity make the experimental identification of these elements arduous., Methods: In this study, autophagy-related proteins and chemicals were catalogued on the basis of Human Autophagy-dedicated Database. These entities were mapped to their respective PubChem identifications (IDs) for chemicals and Ensembl IDs for proteins, yielding 563 chemicals and 779 proteins. A network comprising protein-protein, protein-chemical, and chemical-chemical interactions was probed employing the Random-Walk-with-Restart algorithm using the aforementioned proteins and chemicals as seed nodes to unearth additional autophagy-associated proteins and chemicals. Screening tests were performed to exclude proteins and chemicals with minimal autophagy associations., Results: A total of 88 inferred proteins and 50 inferred chemicals of high autophagy relevance were identified. Certain entities, such as the chemical prostaglandin E2 (PGE2), which is recognized for modulating cell death-induced inflammatory responses during pathogen invasion, and the protein G Protein Subunit Alpha I1 (GNAI1), implicated in ether lipid metabolism influencing a range of cellular processes including autophagy, were associated with autophagy., Conclusions: The discovery of novel autophagy-associated proteins and chemicals is of vital importance because it enhances the understanding of autophagy, provides potential therapeutic targets, and fosters the development of innovative therapeutic strategies and interventions., Competing Interests: The authors declare no conflict of interest. Given their role as Guest Editor or Editorial Board Members, Dr. Tao Huang and Dr. Yu-Dong Cai had no involvement in the peer review of this article and had no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Kavindra Kumar Kesari., (© 2024 The Author(s). Published by IMR Press.)

An exponential increase in the number of publications that address artificial intelligence (AI) usage in life sciences has been noticed in recent years, while new modeling techniques are constantly being reported. The potential of these methods is vast-from understanding fundamental cellular processes to discovering new drugs and breakthrough therapies. Computational studies of protein-protein interactions, crucial for understanding the operation of biological systems, are no exception in this field. However, despite the rapid development of technology and the progress in developing new approaches, many aspects remain challenging to solve, such as predicting conformational changes in proteins, or more "trivial" issues as high-quality data in huge quantities.Therefore, this chapter focuses on a short introduction to various AI approaches to study protein-protein interactions, followed by a description of the most up-to-date algorithms and programs used for this purpose. Yet, given the considerable pace of development in this hot area of computational science, at the time you read this chapter, the development of the algorithms described, or the emergence of new (and better) ones should come as no surprise., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The protein delivery system is one of the innovative or novel drug delivery systems in the present era. Proteins play an indispensable role in our body and are mainly found in every part, like tissue and cells of our body. It also controls various functions, such as maintaining our tissue, transportation, muscle recovery, enzyme production and acting as an energy source for our body. Protein therapeutics have big future perspectives, and their use in the treatment of a wide range of serious diseases has transformed the delivery system in the pharmaceutical and biotechnology industries. The chief advantage of protein delivery is that it can be delivered directly to the systemic circulation. So far, parenteral routes, such as intravenous, intramuscular, and subcutaneous, are the most often used method of administering protein drugs. Alternative routes like buccal, oral, pulmonary, transdermal, nasal, and ocular routes have also shown a remarkable success rate. However, as with all other types of delivery, here, several challenges are posed due to the presence of various barriers, such as the enzymatic barrier, intestinal epithelial barrier, capillary endothelial barrier, and blood-brain barrier. There are several approaches that have been explored to overcome these barriers, such as chemical modification, enzymatic inhibitors, penetration enhancers, and mucoadhesive polymers. This review article discusses the protein, its functions, routes of administration, challenges, and strategies to achieve ultimate formulation goals. Recent advancements like the protein Pegylation method and Depofoam technology are another highlight of the article., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Proteins are the fundamental organic macromolecules in living systems that play a key role in a variety of biological functions including immunological detection, intracellular trafficking, and signal transduction. The docking of proteins has greatly advanced during recent decades and has become a crucial complement to experimental methods. Protein-protein docking is a helpful method for simulating protein complexes whose structures have not yet been solved experimentally. This chapter focuses on major search tactics along with various docking programs used in protein-protein docking algorithms, which include: direct search, exhaustive global search, local shape feature matching, randomized search, and broad category of post-docking approaches. As backbone flexibility predictions and interactions in high-resolution protein-protein docking remain important issues in the overall optimization context, we have put forward several methods and solutions used to handle backbone flexibility. In addition, various docking methods that are utilized for flexible backbone docking, including ATTRACT, FlexDock, FLIPDock, HADDOCK, RosettaDock, FiberDock, etc., along with their scoring functions, algorithms, advantages, and limitations are discussed. Moreover, what progress in search technology is expected, including not only the creation of new search algorithms but also the enhancement of existing ones, has been debated. As conformational flexibility is one of the most crucial factors affecting docking success, more work should be put into evaluating the conformational flexibility upon binding for a particular case in addition to developing new algorithms to replace the rigid body docking and scoring approach., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Proteins and peptides are highly desirable as therapeutic agents, being highly potent and specific. However, there are myriad challenges with processing them into patient-friendly formulations: they are often unstable and have a tendency to aggregate or degrade upon storage. As a result, the vast majority of protein actives are delivered parenterally as solutions, which has a number of disadvantages in terms of cost, accessibility, and patient experience. Much work has been undertaken to develop new delivery systems for biologics, but to date this has led to relatively few products on the market. In this chapter, we review the challenges faced when developing biologic formulations, discuss the technologies that have been explored to try to overcome these, and consider the different delivery routes that can be applied. We further present an overview of the currently marketed products and assess the likely direction of travel in the next decade., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Protein-protein docking is considered one of the most important techniques supporting experimental proteomics. Recent developments in the field of computer science helped to improve this computational technique so that it better handles the complexity of protein nature. Sampling algorithms are responsible for the generation of numerous protein-protein ensembles. Unfortunately, a primary docking output comprises a set of both near-native poses and decoys. Application of the efficient scoring function helps to differentiate poses with the most favorable properties from those that are very unlikely to represent a natural state of the complex. This chapter explains the importance of sampling and scoring in the process of protein-protein docking. Moreover, it summarizes advances in the field., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bioconjugation techniques have emerged as powerful tools for enhancing the stability and targeting efficiency of protein and peptide therapeutics. This review provides a comprehensive analysis of the various bioconjugation strategies employed in the field. The introduction highlights the significance of bioconjugation techniques in addressing stability and targeting challenges associated with protein and peptide-based drugs. Chemical and enzymatic bioconjugation methods are discussed, along with crosslinking strategies for covalent attachment and site-specific conjugation approaches. The role of bioconjugation in improving stability profiles is explored, showcasing case studies that demonstrate successful stability enhancement. Furthermore, bioconjugation techniques for ligand attachment and targeting are presented, accompanied by examples of targeted protein and peptide therapeutics. The review also covers bioconjugation approaches for prolonging circulation and controlled release, focusing on strategies to extend half-life, reduce clearance, and design-controlled release systems. Analytical characterization techniques for bioconjugates, including the evaluation of conjugation efficiency, stability, and assessment of biological activity and targeting efficiency, are thoroughly examined. In vivo considerations and clinical applications of bioconjugated protein and peptide therapeutics, including pharmacokinetic and pharmacodynamic considerations, as well as preclinical and clinical developments, are discussed. Finally, the review concludes with an overview of future perspectives, emphasizing the potential for novel conjugation methods and advanced targeting strategies to further enhance the stability and targeting efficiency of protein and peptide therapeutics., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Despite the development of methods for the experimental determination of protein structures, the dissonance between the number of known sequences and their solved structures is still enormous. This is particularly evident in protein-protein complexes. To fill this gap, diverse technologies have been developed to study protein-protein interactions (PPIs) in a cellular context including a range of biological and computational methods. The latter derive from techniques originally published and applied almost half a century ago and are based on interdisciplinary knowledge from the nexus of the fields of biology, chemistry, and physics about protein sequences, structures, and their folding. Protein-protein docking, the main protagonist of this chapter, is routinely treated as an integral part of protein research. Herein, we describe the basic foundations of the whole process in general terms, but step by step from protein representations through docking methods and evaluation of complexes to their final validation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Electrochemical methods can be used not only for the sensitive analysis of proteins but also for deeper research into their structure, transport functions (transfer of electrons and protons), and sensing their interactions with soft and solid surfaces. Last but not least, electrochemical tools are useful for investigating the effect of an electric field on protein structure, the direct application of electrochemical methods for controlling protein function, or the micromanipulation of supramolecular protein structures. There are many experimental arrangements (modalities), from the classic configuration that works with an electrochemical cell to miniaturized electrochemical sensors and microchip platforms. The support of computational chemistry methods which appropriately complement the interpretation framework of experimental results is also important. This text describes recent directions in electrochemical methods for the determination of proteins and briefly summarizes available methodologies for the selective labeling of proteins using redox-active probes. Attention is also paid to the theoretical aspects of electron transport and the effect of an external electric field on the structure of selected proteins. Instead of providing a comprehensive overview, we aim to highlight areas of interest that have not been summarized recently, but, at the same time, represent current trends in the field., (© 2023. The Author(s).)

Solution nuclear magnetic resonance spectroscopy provides unique opportunities to study the structure and dynamics of biomolecules in aqueous environments. While spin relaxation methods are well recognized for their ability to probe timescales of motion, residual dipolar couplings (RDCs) provide access to amplitudes and directions of motion, characteristics that are important to the function of these molecules. Although observed in the 1960s, the acquisition and computational analysis of RDCs has gained significant momentum in recent years, and particularly applications to motion in proteins have become more numerous. This trend may well continue as RDCs can easily leverage structures produced by new computational methods (e.g., AlphaFold) to produce functional descriptions. In this report, we provide examples and a summary of the ways that RDCs have been used to confirm the existence of internal dynamics, characterize the type of dynamics, and recover atomic-scale structural ensembles that define the full range of conformational sampling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Published by Elsevier Ltd.)

Protein unfolding is a ubiquitous process responsible for the loss of protein functionality (denaturation), which, in turn, can be accompanied by the death of cells and organisms. The nature of enthalpy-entropy compensation (EEC) in the kinetics of protein unfolding is a subject of debate. In order to investigate the nature of EEC, the "completely loose" transition state (TS) model has been applied to calculate the Arrhenius parameters for the unfolding of polyglycine dimers as a model process. The calculated Arrhenius parameters increase with increasing dimer length and demonstrate enthalpy-entropy compensation. It is shown that EEC results from the linear correlations of enthalpy and entropy of activation with dimer length, which are derived directly from the properties of the transition state. It is shown that EEC in solvated (hydrated, etc.) proteins is a direct consequence of EEC in proteins themselves. The suggested model allows us also to reproduce and explain "exotic" very high values of the pre-exponential factor measured for the proteins unfolding, which are drastically higher than those known for unimolecular reactions of organic molecules. A similar approach can be applied to analyzing the nature of EEC phenomena observed in other areas of chemistry.

Carrier-mediated intracellular protein delivery holds tremendous application potential in biology and medicine. The ideal carrier should be well-controlled and cost-effective and able to facilitate robust delivery of diverse types of proteins into the target cells, thus ensuring efficacy in different application scenarios. Here, we describe a modular chemistry approach for generating a small-molecule amphiphile molecular library based on the Ugi four-component reaction under one-pot and mild conditions. Then, two different types of amphiphiles with the dimeric or trimeric architecture were obtained for intracellular protein delivery through in vitro screening test. Depending on the precise adjustment of the hydrophobic tails of amphiphiles, the optimized trimeric amphiphile (TA) exhibited more superior protein loading performance and a higher efficiency of delivering proteins into cells through the endocytosis pathway and subsequent endosomal escape. Furthermore, we demonstrated that the TA could be a universal delivery carrier capable of transporting broad-spectrum proteins, especially for the hard-to-deliver native antibodies, into the cytosol. Overall, we describe a robust amphiphile platform with a well-defined and cost-effective design to improve the cytosolic protein delivery capacity, exhibiting great promise for developing intracellular protein-based therapeutics.

Increasing protein demands directly require additional resources to those presently and recurrently available. Emerging green technologies have witnessed an escalating interest in "Cavitation Processing" (CP) to ensure a non-invasive, non-ionizing and non-polluting extraction. The main intent of this review is to present an integrated summary of cavitation extraction methods specifically applied to food protein sources. Along with a comparative assessment carried out for each type of cavitation model, protein extraction yield and implications on the extracted protein's structural and functional properties. The basic principle of cavitation is due to the pressure shift in the liquid flow within milliseconds. Hence, cavitation emerges similar to boiling; however, unlike boiling (temperature change), cavitation occurs due to pressure change. Characterization and classification of sample type is also a prime candidate when considering the applications of cavitation models in food processing. Generally, acoustic and hydrodynamic cavitation is applied in food applications including extraction, brewing, microbial cell disruption, dairy processing, emulsification, fermentation, waste processing, crystallisation, mass transfer and production of bioactive peptides. Micro structural studies indicate that shear stress causes disintegration of hydrogen bonds and Van der Waals interactions result in the unfolding of the protein's secondary and/or tertiary structures. A change in the structure is not targeted but rather holistic and affects the physicochemical, functional, and nutritional properties. Cavitation assisted extraction of protein is typically studied at a laboratory scale. This highlights limitations against the application at an industrial scale to obtain potential commercial gains., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Protein structure, function, and evolution depend on local and collective epistatic interactions between amino acids. A powerful approach to defining these interactions is to construct models of couplings between amino acids that reproduce the empirical statistics (frequencies and correlations) observed in sequences comprising a protein family. The top couplings are then interpreted. Here, we show that as currently implemented, this inference unequally represents epistatic interactions, a problem that fundamentally arises from limited sampling of sequences in the context of distinct scales at which epistasis occurs in proteins. We show that these issues explain the ability of current approaches to predict tertiary contacts between amino acids and the inability to obviously expose larger networks of functionally relevant, collectively evolving residues called sectors. This work provides a necessary foundation for more deeply understanding and improving evolution-based models of proteins., Competing Interests: Declaration of interests R.R. is a founder and shareholder of Evozyne Inc. and a member of its corporate board. R.R. is also a member of the scientific advisory board of this journal., (Copyright © 2022. Published by Elsevier Inc.)

Ancestral sequence reconstruction (ASR) is a powerful tool to study the evolution of proteins and thus gain deep insight into the relationships among protein sequence, structure, and function. A major barrier to its broad use is the complexity of the task: it requires multiple software packages, complex file manipulations, and expert phylogenetic knowledge. Here we introduce topiary, a software pipeline that aims to overcome this barrier. To use topiary, users prepare a spreadsheet with a handful of sequences. Topiary then: (1) Infers the taxonomic scope for the ASR study and finds relevant sequences by BLAST; (2) Does taxonomically informed sequence quality control and redundancy reduction; (3) Constructs a multiple sequence alignment; (4) Generates a maximum-likelihood gene tree; (5) Reconciles the gene tree to the species tree; (6) Reconstructs ancestral amino acid sequences; and (7) Determines branch supports. The pipeline returns annotated evolutionary trees, spreadsheets with sequences, and graphical summaries of ancestor quality. This is achieved by integrating modern phylogenetics software (Muscle5, RAxML-NG, GeneRax, and PastML) with online databases (NCBI and the Open Tree of Life). In this paper, we introduce non-expert readers to the steps required for ASR, describe the specific design choices made in topiary, provide a detailed protocol for users, and then validate the pipeline using datasets from a broad collection of protein families. Topiary is freely available for download: https://github.com/harmslab/topiary., (© 2022 The Protein Society.)

The demand for creation of protein diversity and regulation of protein function through native protein modification and post-translational modification has ignited the development of selective chemical modification methods for peptides and proteins. Chemical bioconjugation offers selective functionalization providing bioconjugates with desired properties and functions for diverse applications in chemical biology, medicine, and biomaterials. The amino group existing at the lysine residue and N-terminus of peptides and proteins has been extensively studied in bioconjugation because of its good nucleophilicity and high surface exposure. Herein, we review the development of chemical methods for modification of the amino groups on lysine residue and N-terminus featuring excellent selectivity, mild reaction conditions, short reaction time, high conversion, biocompatibility, and preservation of protein integrity. This review is organized based on the chemoselectivity and site-selectivity of the chemical bioconjugation reagents to the amino acid residues aiming to provide guidance for the selection of appropriate bioconjugation methods.

Protein-ligand binding affinity prediction is an important task in structural bioinformatics for drug discovery and design. Although various scoring functions (SFs) have been proposed, it remains challenging to accurately evaluate the binding affinity of a protein-ligand complex with the known bound structure because of the potential preference of scoring system. In recent years, deep learning (DL) techniques have been applied to SFs without sophisticated feature engineering. Nevertheless, existing methods cannot model the differential contribution of atoms in various regions of proteins, and the relationship between atom properties and intermolecular distance is also not fully explored. We propose a novel empirical graph neural network for accurate protein-ligand binding affinity prediction (EGNA). Graphs of protein, ligand and their interactions are constructed based on different regions of each bound complex. Proteins and ligands are effectively represented by graph convolutional layers, enabling the EGNA to capture interaction patterns precisely by simulating empirical SFs. The contributions of different factors on binding affinity can thus be transparently investigated. EGNA is compared with the state-of-the-art machine learning-based SFs on two widely used benchmark data sets. The results demonstrate the superiority of EGNA and its good generalization capability., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Long noncoding RNA (lncRNA) is a kind of noncoding RNA with a length of more than 200 nucleotide units. Numerous research studies have proven that although lncRNAs cannot be directly translated into proteins, lncRNAs still play an important role in human growth processes by interacting with proteins. Since traditional biological experiments often require a lot of time and material costs to explore potential lncRNA-protein interactions (LPI), several computational models have been proposed for this task. In this study, we introduce a novel deep learning method known as combined graph auto-encoders (LPICGAE) to predict potential human LPIs. First, we apply a variational graph auto-encoder to learn the low dimensional representations from the high-dimensional features of lncRNAs and proteins. Then the graph auto-encoder is used to reconstruct the adjacency matrix for inferring potential interactions between lncRNAs and proteins. Finally, we minimize the loss of the two processes alternately to gain the final predicted interaction matrix. The result in 5-fold cross-validation experiments illustrates that our method achieves an average area under receiver operating characteristic curve of 0.974 and an average accuracy of 0.985, which is better than those of existing six state-of-the-art computational methods. We believe that LPICGAE can help researchers to gain more potential relationships between lncRNAs and proteins effectively., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Proteins and peptides possess considerable potential in treating solid tumors because of their unique properties. At present, there are over 100 peptide-based formulations on the market. Today, peptides and proteins are in more demand due to their selective nature and high target-binding efficiency. Targeting solid tumors with compounds of molecular weight less than 10 kDa are much more desirable because they undergo excessive penetration in view of the fact that they are small sized. The solid tumors have thick tissues and possess excessive interstitial fluid pressure, because of which high molecular compounds cannot enter. The properties of proteins and peptides induce low toxic effects and lessen the major side effects caused by chemical-based drugs. However, their delivery is quite challenging as most proteins and peptides stop functioning therapeutically when following a parenteral route of administration. This paper elaborates on the importance of new age formulations of peptides and proteins followed by their recently documented advancements that increase their stability and delay their metabolism, which helps to target solid tumors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Solution-state nuclear magnetic resonance (NMR) spectroscopy can be used to monitor protein-carbohydrate interactions. Two-dimensional 1 H- 15 N heteronuclear single quantum coherence (HSQC)-based techniques described in this chapter can be used quickly and effectively to screen a set of possible carbohydrate-binding partners, to quantify the dissociation constant (K d ) of any identified interactions, and to the map the carbohydrate-binding site on the structure of a protein. Here, we describe the titration of a family 32 carbohydrate-binding module from Clostridium perfringens (CpCBM32) with the monosaccharide N-acetylgalactosamine (GalNAc), in which we calculate the apparent dissociation of the interaction and map the GalNAc binding site onto the structure of CpCBM32. This approach can be applied to other CBM- and protein-ligand systems., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13 C, 18 O, or 15 N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labelled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labelled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labelling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, or even metagenomes and metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labelled microorganisms. Analysis of labelled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allowed using labelled substrates at environmentally relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter, we provide protocols for obtaining labelled DNA, RNA, and proteins that can be used for downstream omics-based analyses., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

With an estimate of hundred thousands of protein molecules per cell and the number of metabolites several orders of magnitude higher, protein-metabolite interactions are omnipresent. In vitro analyses are one of the main pillars on the way to establish a solid understanding of how these interactions contribute to maintaining cellular homeostasis. A repertoire of biophysical techniques is available by which protein-metabolite interactions can be quantitatively characterized in terms of affinity, specificity, and kinetics in a broad variety of solution environments. Several of those provide information on local or global conformational changes of the protein partner in response to ligand binding. This review chapter gives an overview of the state-of-the-art biophysical toolbox for the study of protein-metabolite interactions. It briefly introduces basic principles, highlights recent examples from the literature, and pinpoints promising future directions., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The roles of small molecules in every aspect of life have been gaining increased recognition. Many are known to exert their effect by binding proteins-but a comprehensive overview of protein-metabolite interactions (PMIs) is missing. Recently we devised a non-targeted method for detecting PMIs using size-exclusion chromatography followed by proteomic and metabolomic analysis: PROMIS. Under test this method was able to identify known PMIs such as enzyme-cofactor complexes as well as novel ones., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein-protein interaction mapping has gained immense importance in understanding protein functions in diverse biological pathways. There are various in vivo and in vitro techniques associated with the protein-protein interaction studies but generally, the focus is confined to understanding the protein interaction in the nucleus of the cell, and thus it limits the availability to explore protein interactions that are happening in the cytoplasm of the cell. Since posttranslational modification is a crucial step in signaling pathways and cellular protein interactions harnessing the cytoplasmic protein and evaluating the interaction in the cytoplasm, this protocol will provide more information about studying these types of protein interactions. Cytotrap is a type of yeast-two-hybrid system that differs in its ability to anchor along the membrane, thus directing the protein of interest to anchor along the membrane through the myristoylation signaling unit. The vector containing the target protein contains the myristoylation unit, called the prey, and the bait unit contains the protein of interest as a fusion with the hSos protein. In an event of interaction between the target and the protein of interest, the hSos protein unit will be localized to the membrane and the GDP/GTP exchange unit will trigger the activation of the Ras pathway that leads to the survival of the temperature-sensitive yeast strain at a higher temperature., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Dried reconstituted vesicle (DRV) liposomes are formulated under mild conditions. The method has the capability to entrap substantially higher amounts of hydrophilic solutes, compared to other passive-loading liposome preparation methods. These characteristics make this liposome type ideal for entrapment of labile substances, such as peptides, proteins, or DNA's (or other nucleotides or oligonucleotides), or in general biopharmaceuticals and sensitive drugs. In this chapter, all possible types of DRV liposomes (in respect to the encapsulated molecule characteristics and/or their applications in therapeutics) are introduced, and preparation methodologies (for each type) are described in detail., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The characterization of protein-protein interactions (PPI) often provides functional information about a target protein. Yeast-two-hybrid (Y2H) and luminescence/fluorescence-based detections, therefore, have been widely utilized for assessing PPI. In addition, a co-immunoprecipitation (co-IP) method has also been adopted with transient protein expression in Nicotiana benthamiana (N. benthamiana) infiltrated with Agrobacterium tumefaciens. Herein, we describe a co-IP procedure in which structural maintenance of chromosome 1 (SMC1), identified from a Y2H screening, was verified as an interacting partner for microchidia 1 (MORC1), a protein well known for its function in plant immunity and epigenetics. SMC1 and MORC1 were transiently expressed in N. benthamiana when infiltrated by Agrobacterium with the respective genes. From this approach, we identified a region of SMC1 responsible for interacting with MORC1. The co-IP method, of which outputs are mainly from immunoblot analysis, provided information about target protein expression as well, which is often useful for troubleshooting. Using this feature, we showcased a PPI confirmation from our SMC1-MORC1 study in which a full-length SMC1 protein was not detectable, and, therefore, a subsequent truncated mutant analysis had to be employed for PPI verification., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Camellia bee pollen protein isolates were extracted by cell wall disruption using ultrasonication, freeze-thawing, enzymatic hydrolysis, and their combinations. The effects of these methods on microstructure of cell wall, protein release, protein yield, physiochemical properties and structure of proteins were investigated. As compared with physical treatments (ultrasonication, freeze-thawing and their combination), the enzymatic hydrolysis significantly improved the yield of proteins, because it not only promoted the release of proteins from the inside of pollen, but also released proteins in pollen wall. The proteins extracted by enzymatic hydrolysis method also exhibited better solubility, emulsifying and gelation properties due to the partial hydrolysis of proteins by protease. In addition, when ultrasound was combined with freeze-thawing or enzymatic hydrolysis, it could further improve the yield of proteins and the functional properties of proteins, which was mainly related to the changes of protein structure induced by cavitation effect of ultrasound., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Mapping protein-protein interactions is crucial to understand protein function. Recent advances in proximity-dependent biotinylation (BioID) coupled to mass spectrometry (MS) allow the characterization of protein complexes in diverse plant models. Here, we describe the use of BioID in hairy root cultures of tomato and provide detailed information on how to analyze the data obtained by MS., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The nonrandom distribution of alleles of common genomic variants produces haplotypes, which are fundamental in medical and population genetic studies. Consequently, protein-coding genes with different co-occurring sets of alleles can encode different amino acid sequences: protein haplotypes. These protein haplotypes are present in biological samples and detectable by mass spectrometry, but they are not accounted for in proteomic searches. Consequently, the impact of haplotypic variation on the results of proteomic searches and the discoverability of peptides specific to haplotypes remain unknown., Findings: Here, we study how common genetic haplotypes influence the proteomic search space and investigate the possibility to match peptides containing multiple amino acid substitutions to a publicly available data set of mass spectra. We found that for 12.42% of the discoverable amino acid substitutions encoded by common haplotypes, 2 or more substitutions may co-occur in the same peptide after tryptic digestion of the protein haplotypes. We identified 352 spectra that matched to such multivariant peptides, and out of the 4,582 amino acid substitutions identified, 6.37% were covered by multivariant peptides. However, the evaluation of the reliability of these matches remains challenging, suggesting that refined error rate estimation procedures are needed for such complex proteomic searches., Conclusions: As these procedures become available and the ability to analyze protein haplotypes increases, we anticipate that proteomics will provide new information on the consequences of common variation, across tissues and time., (© The Author(s) 2023. Published by Oxford University Press GigaScience.)

The study of protein structure, dynamics and function by NMR spectroscopy commonly requires samples that have been enriched ('labelled') with the stable isotopes 13C and/or 15N. The standard approach is to uniformly label a protein with one or both of these nuclei such that all C and/or N sites are in principle 'NMR-visible'. NMR spectra of uniformly labelled proteins can be highly complicated and suffer from signal overlap. Moreover, as molecular size increases the linewidths of NMR signals broaden, which decreases sensitivity and causes further spectral congestion. Both effects can limit the type and quality of information available from NMR data. Problems associated with signal overlap and signal broadening can often be alleviated though the use of alternative, non-uniform isotopic labelling patterns. Specific isotopic labelling 'turns on' signals at selected sites while the rest of the protein is NMR-invisible. Conversely, specific isotopic unlabelling (also called 'reverse' labelling) 'turns off' selected signals while the rest of the protein remains NMR-visible. Both approaches can simplify NMR spectra, improve sensitivity, facilitate resonance assignment and permit a range of different NMR strategies when combined with other labelling tools and NMR experiments. Here, we review methods for producing proteins with enrichment of stable NMR-visible isotopes, with particular focus on residue-specific labelling and reverse labelling using Escherichia coli expression systems. We also explore how these approaches can aid NMR studies of proteins., (© 2022 The Author(s).)

Backbone-dependent rotamer libraries are commonly used to assign the side chain dihedral angles of amino acids when modeling protein structures. Most rotamer libraries are created by curating protein crystal structure data and using various methods to extrapolate the existing data to cover all possible backbone conformations. However, these rotamer libraries may not be suitable for modeling the structures of cyclic peptides and other constrained peptides because these molecules frequently sample backbone conformations rarely seen in the crystal structures of linear proteins. To provide backbone-dependent side chain information beyond the α-helix, β-sheet, and PPII regions, we used explicit-solvent metadynamics simulations of model dipeptides to create a new rotamer library that has high coverage in the (ϕ, ψ) space. Furthermore, this approach can be applied to build high-coverage rotamer libraries for noncanonical amino acids. The resulting Metadynamics of Dipeptides for Rotamer Distribution (MEDFORD) rotamer library predicts the side chain conformations of high-resolution protein crystal structures with similar accuracy (~80%) to a state-of-the-art rotamer library. Our ability to test the accuracy of MEDFORD at predicting the side chain dihedral angles of amino acids in noncanonical backbone conformation is restricted by the limited structural data available for cyclic peptides. For the cyclic peptide data that are currently available, MEDFORD and the state-of-the-art rotamer library perform comparably. However, the two rotamer libraries indeed make different rotamer predictions in noncanonical (ϕ, ψ) regions. For noncanonical amino acids, the MEDFORD rotamer library predicts the χ 1 values with approximately 75% accuracy., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Characterization of the hydrated state of a protein is crucial for understanding its structural stability and function. In the present study, we have investigated the 3D hydration structure of the protein BPTI (bovine pancreatic trypsin inhibitor) by molecular dynamics (MD) and the integral equation method in the three-dimensional reference interaction site model (3D-RISM) approach. Both methods have found a well-defined hydration layer around the protein and revealed the localization of BPTI buried water molecules corresponding to the X-ray crystallography data. Moreover, under 3D-RISM calculations, the obtained positions of waters bound firmly to the BPTI sites are in reasonable agreement with the experimental results mentioned above for the BPTI crystal form. The analysis of the 3D hydration structure (thickness of hydration shell and hydration numbers) was performed for the entire protein and its polar and non-polar parts using various cut-off distances taken from the literature as well as by a straightforward procedure proposed here for determining the thickness of the hydration layer. Using the thickness of the hydration shell from this procedure allows for calculating the total hydration number of biomolecules properly under both methods. Following this approach, we have obtained the thickness of the BPTI hydration layer of 3.6 Å with 369 water molecules in the case of MD simulation and 3.9 Å with 333 water molecules in the case of the 3D-RISM approach. The above procedure was also applied for a more detailed description of the BPTI hydration structure near the polar charged and uncharged radicals as well as non-polar radicals. The results presented for the BPTI as an example bring new knowledge to the understanding of protein hydration.

Surface-enhanced Raman spectroscopy (SERS) is powerful for structural characterization of biomolecules under physiological condition. Owing to its high sensitivity and selectivity, SERS is useful for probing intrinsic structural information of proteins and is attracting increasing attention in biophysics, bioanalytical chemistry, and biomedicine. This review starts with a brief introduction of SERS theories and SERS methodology of protein structural characterization. SERS-active materials, related synthetic approaches, and strategies for protein-material assemblies are outlined and discussed, followed by detailed discussion of SERS spectroscopy of proteins with and without cofactors. Recent applications and advances of protein SERS in biomarker detection, cell analysis, and pathogen discrimination are then highlighted, and the spectral reproducibility and limitations are critically discussed. The review ends with a conclusion and a discussion of current challenges and perspectives of promising directions.

The direct correlation between proteoforms and biological phenotype necessitates the exploration of mass spectrometry (MS)-based methods more suitable for proteoform detection and characterization. Here, we couple nano-hydrophobic interaction chromatography (nano-HIC) to ultraviolet photodissociation MS (UVPD-MS) for separation and characterization of intact proteins and proteoforms. High linearity, sensitivity, and sequence coverage are obtained with this method for a variety of proteins. Investigation of collisional cross sections of intact proteins during nano-HIC indicates semifolded conformations in low charge states, enabling a different dimension of separation in comparison to traditional, fully denaturing reversed-phase separations. This method is demonstrated for a mixture of intact proteins from Escherichia coli ribosomes; high sequence coverage is obtained for a variety of modified and unmodified proteoforms.

In 1987, Susi & Byler published a groundbreaking paper for the determination of the secondary structure of proteins. Notably, they determined the characteristic signature of the β-strand in the infrared spectrum. As a result, Fourier-transform infrared spectroscopy became a general method to determine protein structure., (Copyright © 2022 Elsevier Inc. All rights reserved.)