Protein Science最新文献

{"title":"Deep mutational scanning reveals a de novo disulfide bond and combinatorial mutations for engineering thermostable myoglobin.","authors":"Christoph Küng, Olena Protsenko, Rosario Vanella, Michael A Nash","doi":"10.1002/pro.70112","DOIUrl":"https://doi.org/10.1002/pro.70112","url":null,"abstract":"<p><p>Engineering protein stability is a critical challenge in biotechnology. Here, we used massively parallel deep mutational scanning (DMS) to comprehensively explore the mutational stability landscape of human myoglobin (hMb) and identify key mutations that enhance stability. Our DMS approach involved screening over 10,000 hMb variants by yeast surface display, single-cell sorting, and high-throughput DNA sequencing. We show how surface display levels serve as a proxy for thermostability of soluble hMb variants and report strong correlations between DMS-derived display levels and top-performing machine learning stability prediction algorithms. This approach led to the discovery of a variant with a de novo disulfide bond between residues R32C and C111, which increased thermostability by >12°C compared with wild-type hMb. By combining single stabilizing mutations with R32C, we engineered combinatorial variants that exhibited predominantly additive effects on stability with minimal epistasis. The most stable combinatorial variant exhibited a denaturation temperature exceeding 89°C, representing a >17°C improvement over wild-type hMb. Our findings demonstrate the capabilities in DMS-assisted combinatorial protein engineering to guide the discovery of thermostable variants and highlight the potential of massively parallel mutational analysis for the development of proteins for industrial and biomedical applications.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70112"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006728/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144009791","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"BioSpring: An elastic network framework for interactive exploration of macromolecular mechanics.","authors":"Benoist Laurent, André Lanrezac, Hubert Santuz, Nicolas Ferey, Olivier Delalande, Marc Baaden","doi":"10.1002/pro.70130","DOIUrl":"https://doi.org/10.1002/pro.70130","url":null,"abstract":"<p><p>BioSpring is an innovative tool for interactive molecular modeling and simulation, designed to explore the dynamics of biological structures in real time. Using an augmented elastic network model, the BioSpring framework enables researchers to intuitively examine complex biomolecules, and it combines real-time feedback with the user's experience. This capability makes it ideal for initial analysis of molecular systems, protein-protein and protein-DNA docking, protein mechanics, and protein-membrane interactions. The multi-resolution modeling approach combines accuracy and efficiency, supporting user-driven analysis of molecular interactions, conformational flexibility, and structural mechanics. This framework improves upon traditional methods in terms of robustness, accessibility, and ease of use, while requiring only modest computational resources and enabling a fast turnaround time to obtain initial results. It provides insights into molecular function and dynamics that advance the field of structural biology. Source code, executables, and examples for the BioSpring simulation engine are available at https://biospring.mol3d.tech.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70130"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006919/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144029046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"To be, or not to be cleaved: Directed evolution of a canonical serine protease inhibitor against active and inactive protease pair identifies binding loop residue critical for prevention of proteolytic cleavage.","authors":"Zsombor Köller, Bálint Zoltán Németh, Bence Kiss, Zoltán Attila Nagy, Gitta Schlosser, Csaba Magyar, Alexandra Demcsák, Miklós Sahin-Tóth, Gábor Pál","doi":"10.1002/pro.70146","DOIUrl":"https://doi.org/10.1002/pro.70146","url":null,"abstract":"<p><p>Canonical serine protease inhibitor proteins occupy the substrate-binding groove of their target enzyme via a surface loop. Unlike true substrates, inhibitors are cleaved by the target protease extremely slowly. Here, we applied an unbiased directed evolution approach to investigate which loop residues hamper proteolytic cleavage while maintaining high-affinity binding. As a protease inhibitor model system, we used human chymotrypsin C (CTRC) and Schistocerca gregaria protease inhibitor 2 (SGPI-2). We created an SGPI-2 library displayed on M13 phage by randomizing the binding loop amino acid positions, with the exception of the structurally indispensable Cys residues. We selected binding phage clones against active CTRC and the inactive mutant Ser195Ala. All CTRC-selected binders inhibited CTRC activity and also bound to the inactive Ser195Ala mutant, but the Ser195Ala-selected clones proved to be either inhibitors or substrates of active CTRC. Substrate-like behavior of SGPI-2 variants was associated with the absence of the P2 Thr, the residue next to the specificity determinant P1 amino acid. The selected SGPI-2 variants containing a P2 Thr bound strongly to CTRC even if the other loop residues deviated from the optimal inhibitory consensus sequence. In the absence of a P2 Thr, however, SGPI-2 variants became substrates unless all other loop residues were optimal for binding. Structural modeling confirmed that P2 Thr is important for organizing a stabilizing H-bond network. The observations indicate that binding loops of canonical serine protease inhibitors evolved amino acids not only to support tight binding to the target enzyme but also to inhibit proteolytic cleavage.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70146"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12038735/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144042158","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unlocking GPCR-ligand interactions: Measuring binding affinities with thermal shift assay.","authors":"Dmitrii Dashevskii, Aleksandra Luginina, Ivan Maslov, Marina Shevelyova, Polina Khorn, Daria Dmitrieva, Ivan Kapranov, Anatolii Belousov, Sergei Permyakov, Vadim Cherezov, Valentin Borshchevskiy, Alexey Mishin","doi":"10.1002/pro.70120","DOIUrl":"https://doi.org/10.1002/pro.70120","url":null,"abstract":"<p><p>G protein-coupled receptors (GPCRs) constitute the largest transmembrane protein superfamily, with over 800 representatives in the human genome. Recognized as pivotal targets in pharmacological research and drug discovery, these receptors play a crucial role in advancing therapeutics. Understanding the molecular mechanisms of receptor-ligand interactions is imperative for drug discovery applications. However, experimental procedures for measuring ligand binding are complicated by various factors, including the transmembrane nature of the receptors and the high cost associated with specialized instruments and consumables. Here we introduce an application of the thermal shift assay (TSA) to measuring ligand binding affinities for GPCRs. TSA is a cost-effective and user-friendly method that detects changes in protein stability induced by alterations in environmental conditions. Employing the human A_2A adenosine receptor as a representative GPCR, we determined binding constants for four orthosteric ligands and allosteric sodium using three mathematical models for TSA data approximation and analysis. Models were additionally validated by two antagonists of cysteinyl leukotriene GPCR (CysLT₁R), used as antiasthmatic drugs. Our results suggest that the TSA approach demonstrates a high degree of reproducibility and agreement with existing literature data, thereby affirming its suitability for investigating GPCR interactions with various types of ligands.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70120"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006757/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144014125","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization of synthetic human V_H affinity and solubility through in vitro affinity maturation and minimal camelization.","authors":"Kasandra Bélanger, Cunle Wu, Traian Sulea, Henk van Faassen, Deborah Callaghan, Annie Aubry, Marc Sasseville, Greg Hussack, Jamshid Tanha","doi":"10.1002/pro.70114","DOIUrl":"https://doi.org/10.1002/pro.70114","url":null,"abstract":"<p><p>An attractive feature of human V_Hs over camelid V_HHs as immunotherapeutics is their perceived lower risk of immunogenicity. While human V_Hs can readily be obtained from synthetic phage display libraries, they often suffer from low affinity and poor solubility compared to V_HHs derived from immune libraries. Using SARS-CoV-2 spike protein as a model antigen, we screened a synthetic human V_H phage display library and identified a diverse set of antigen-specific V_Hs. However, the V_Hs exhibited low affinity, and many had low solubility; that is, they were prone to aggregation. To explore the feasibility of improving the affinity, we subjected a representative V_H to in vitro affinity maturation. We created a yeast surface display library of V_H variants employing a site-saturated mutagenesis approach targeting complementarity-determining regions and selected against the target antigen. Next-generation sequencing of the selected variants, combined with structural modeling, identified a set of V_Hs as potentially improved candidates. Characterization of these candidates revealed several V_Hs with improved affinities of up to 100-fold (K_Ds as low as 3 nM) and potent neutralization capabilities; however, they still showed significant aggregation. By introducing as few as two camelid residues into the framework region 2 of a high-affinity V_H (a process referred to as camelization), we were able to completely solubilize the V_H without compromising its affinity and other important attributes, including thermostability and protein A binding. This study demonstrates the feasibility of generating high-affinity, -solubility, and -stability human V_Hs from synthetic libraries through a combination of in vitro affinity maturation and minimal camelization.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70114"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012759/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144050592","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Direct comparison of the structural dynamics between spontaneous and ligand-induced folding of staphylococcal nuclease.","authors":"Yujiro Mori, Takuya Mizukami, Issei Suzuki, Shingo Fukazawa, Kosuke Miki, Heinrich Roder, Kosuke Maki","doi":"10.1002/pro.70135","DOIUrl":"https://doi.org/10.1002/pro.70135","url":null,"abstract":"<p><p>Despite numerous studies focusing on the folding mechanism of globular proteins as well as ligand-induced folding of intrinsically disordered proteins (IDPs), a unified framework for understanding both types of folding mechanisms has remained elusive. To explore the similarities and differences in the structural dynamics of spontaneous versus ligand-dependent folding, we investigated the folding dynamics of staphylococcal nuclease (SNase) in the presence and absence of the substrate analog adenosine 3',5'-diphosphate (prAp). We employed equilibrium and kinetic measurements, using fluorescence and NMR spectroscopy, to study the folding of SNase coupled with the binding of prAp as a function of ligand and urea concentrations, including conditions favoring either conformational selection (CS; folding before binding) or induced fit (IF; binding before folding) scenarios. Our findings revealed that during ligand-induced folding under IF conditions, the N-terminal β-barrel domain is formed first, followed by the α-helical domain. In contrast, under CS conditions, the α-helical domain forms before the β-barrel domain. Additionally, the dynamics of ligand-induced folding mirrors the sequence of events encountered along the minor of the two parallel pathways governing the spontaneous folding process. Therefore, some of the apparent mechanistic differences between spontaneous versus ligand-induced folding can be attributed to the fact that interactions with a nucleotide ligand result in a shift in flux from the major to the minor folding pathway.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70135"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12032610/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144021354","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrated structural model of the palladin-actin complex using XL-MS, docking, NMR, and SAXS.","authors":"Rachel Sargent, David H Liu, Rahul Yadav, Drew Glennenmeier, Colby Bradford, Noely Urbina, Moriah R Beck","doi":"10.1002/pro.70122","DOIUrl":"https://doi.org/10.1002/pro.70122","url":null,"abstract":"<p><p>Palladin is an actin-binding protein that accelerates actin polymerization and is linked to the metastasis of several types of cancer. Previously, three lysine residues in an immunoglobulin-like domain of palladin have been identified as essential for actin binding. However, it is still unknown where palladin binds to F-actin. Evidence that palladin binds to the sides of actin filaments to facilitate branching is supported by our previous study showing that palladin was able to compensate for Arp2/3 in the formation of Listeria actin comet tails. Here, we used chemical crosslinking to covalently link palladin and F-actin residues based on spatial proximity. Samples were then enzymatically digested, separated by liquid chromatography, and analyzed by tandem mass spectrometry. Peptides containing the crosslinks and specific residues involved were then identified for input to the HADDOCK docking server to model the most likely binding conformation. Small-angle x-ray scattering was used to provide further insight into palladin flexibility and the binding interface, and NMR spectra identified potential interactions between palladin's Ig domains. Our final structural model of the F-actin:palladin complex revealed how palladin interacts with and stabilizes F-actin at the interface between two actin monomers. Three actin residues that were identified in this study also appear commonly in the actin-binding interface with other proteins such as myotilin, myosin, and tropomodulin. An accurate structural representation of the complex between palladin and actin extends our understanding of palladin's role in promoting cancer metastasis through the regulation of actin dynamics.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70122"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006749/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144041979","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Computational studies on the functional and structural impact of pathogenic mutations in enzymes.","authors":"Upeksha C Dissanayake, Arkanil Roy, Yazdan Maghsoud, Sarthi Polara, Tanay Debnath, G Andrés Cisneros","doi":"10.1002/pro.70081","DOIUrl":"10.1002/pro.70081","url":null,"abstract":"<p><p>Enzymes are critical biological catalysts involved in maintaining the intricate balance of metabolic processes within living organisms. Mutations in enzymes can result in disruptions to their functionality that may lead to a range of diseases. This review focuses on computational studies that investigate the effects of disease-associated mutations in various enzymes. Through molecular dynamics simulations, multiscale calculations, and machine learning approaches, computational studies provide detailed insights into how mutations impact enzyme structure, dynamics, and catalytic activity. This review emphasizes the increasing impact of computational simulations in understanding molecular mechanisms behind enzyme (dis)function by highlighting the application of key computational methodologies to selected enzyme examples, aiding in the prediction of mutation effects and the development of therapeutic strategies.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70081"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11926659/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143674253","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dynamic networks connect the USP14 active site region with the proteasome interaction surface.","authors":"Johannes Salomonsson, Linda Sjöstrand, Arvid Eskilson, Dean Derbyshire, Pádraig D'Arcy, Maria Sunnerhagen, Alexandra Ahlner","doi":"10.1002/pro.70077","DOIUrl":"10.1002/pro.70077","url":null,"abstract":"<p><p>Ubiquitin-specific protease 14 (USP14) is a member of the USP family responsible for the catalytic removal of ubiquitin (Ub) from proteins directed to the proteasome, implicated in the pathogenesis of neurodegeneration and cancer. Crystallography and cryo-EM analysis have identified loop regions crucial for the deubiquitinase activity of USP14, specifically those involved in Ub and proteasome binding. However, the structural changes in USP14 upon ligand binding to these regions are minimal, indicating significant yet uncharacterized dynamic contributions to its function. In this study, through structural and dynamical NMR experiments and functional evaluation, we demonstrate that small mutations designed to impact Ub binding and catalytic activity without disturbing the USP structure display both local and long-range effects. The affected residues connect the catalytic site and the Ub binding region with the proteasome interaction surface through a network of loops, which show varied dynamics on the ps-ms time scale. Collectively, our findings experimentally reveal different aspects of dynamic connections within USP14, suggesting the presence of allosteric networks that link enzyme activity with regulatory function. The identification of coupled clusters of possible allostery participants in the free USP domain provides new insights into the dynamic regulation of USP14, with potential implications for understanding its role in cellular processes.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70077"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11912437/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143650295","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deciphering opening mechanisms of 14-3-3 proteins.","authors":"Exequiel E Barrera, Rostislav Skrabana, Diego M Bustos","doi":"10.1002/pro.70108","DOIUrl":"10.1002/pro.70108","url":null,"abstract":"<p><p>The 14-3-3 proteins are a highly conserved family of regulatory molecules that play crucial roles in various cellular processes. They are known for their ability to bind to phosphorylated serine and threonine residues on target proteins, which allows them to modulate their activity, localization, and stability. In mammals, there are seven known paralogs of 14-3-3 proteins, designated as β, ε, ζ, η, σ, τ, and γ. Each paralog has distinct biological functions and tissue distributions, which allow a diverse range of regulatory roles in cellular processes. The conformational plasticity of 14-3-3s regulates their interaction with protein partners but has not yet been thoroughly characterized. We investigated this topic by classical molecular dynamics simulations and observed how the γ, ε, and ζ paralogs exhibit different opening rates. A PCA analysis identified the main modes of these opening-conformational variations. Using correlation-based tools and simulations with single amino acid substitutions, we have recognized how the amphipathic 14-3-3 groove opening is triggered by a distally located aliphatic-π interaction. The identified residues form a partially conserved small cavity between helices H6, H7, and H8, representing a potential paralog-specific drug site.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70108"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934215/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143701393","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}