Protein Science最新文献

{"title":"Nonlinear optical properties of photosensory core modules of monomeric and dimeric bacterial phytochromes.","authors":"Diana Galiakhmetova, Aleksandr Koviarov, Viktor Dremin, Tatjana Gric, Dmitrii Stoliarov, Andrei Gorodetsky, Marios Maimaris, Daria M Shcherbakova, Mikhail Baloban, Vladislav V Verkhusha, Sergei G Sokolovski, Edik Rafailov","doi":"10.1002/pro.70118","DOIUrl":"https://doi.org/10.1002/pro.70118","url":null,"abstract":"<p><p>Near-infrared (NIR) fluorescent proteins and optogenetic tools derived from bacterial phytochromes' photosensory core modules (PCMs) operate within the first (NIR-I) tissue transparency window under single-photon activation. Leveraging two-photon (2P) light in the second transparency window (NIR-II) for photoswitching bacterial phytochromes between Pr and Pfr absorption states offers significant advantages, including enhanced tissue penetration, spatial resolution, and signal-to-noise ratio. However, 2P photoconversion of bacterial phytochromes remains understudied. Here, we study the non-linear Pr to Pfr photoconversion's dependence on irradiation wavelength (1180-1360 nm) and energy fluence (41-339 mJ/cm²) for the PCM of DrBphP bacterial phytochrome. Our findings reveal substantially higher photoconversion efficiency for the engineered monomeric DrBphP-PCM (73%) compared to the natural dimeric DrBphP-PCM (57%). Molecular mechanical calculations, based on experimentally determined 2P absorption cross-section coefficients for the monomer (167 GM) and dimer (170 GM), further verify these results. We demonstrate both short- (SWE) and long-wavelength excitation (LWE) fluorescence of the Soret band using 405 and 810-890 nm laser sources, respectively. Under LWE, fluorescence emission (724 nm) exhibits saturation at a peak power density of 1.5 GW/cm². For SWE, we observe linear degradation of fluorescence for both DrBphP-PCMs, decreasing by 32% as the temperature rises from 19 to 38°C. Conversely, under LWE, the monomeric DrBphP-PCM's brightness increases up to 182% (at 37°C), surpassing the dimeric form's fluorescence rise by 39%. These findings establish the monomeric DrBphP-PCM as a promising template for developing NIR imaging and optogenetic probes operating under the determined optimal parameters for its 2P photoconversion and LWE fluorescence.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70118"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006755/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144021001","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":"The LptC transmembrane helix undergoes a rigid body movement upon LptB₂FG cavity collapse.","authors":"Nicholas P Cina, Candice S Klug","doi":"10.1002/pro.70133","DOIUrl":"https://doi.org/10.1002/pro.70133","url":null,"abstract":"<p><p>Lipopolysaccharide (LPS) is an essential component of the cellular envelope of Gram-negative bacteria and contributes to antibiotic resistance and pathogenesis. Proper localization of LPS at the outer membrane is facilitated via seven distinct LPS transport (Lpt) proteins that bridge the inner and outer membranes. Mature LPS diffuses into the membrane cavity of the inner membrane ABC transporter LptB₂FGC through a lateral gate formed by the LptF and LptG transmembrane (TM) helices. The TM helix of LptC intercalates within the LPS entry point and has been shown to regulate the ATPase activity of LptB₂FG and contribute to thermal stability. Determination of the LptB₂FGC open state structure revealed the location of the LptC TM helix within the membrane complex. However, in the closed state structure, the LptC TM helix is unresolved, suggesting the helix may be displaced from the lateral gate prior to or upon closure of the cavity. To determine the conformational states of the LptC TM helix in the open and closed LptB₂FGC conformations, we utilized site-directed spin labeling in combination with both continuous wave electron paramagnetic resonance (EPR) and double electron electron resonance (DEER) spectroscopies to investigate the LptC TM helix and linker region. These data indicate that the LptC TM helix undergoes a rigid body movement away from the central LptB₂FG cavity upon cavity closure. The findings presented here will support structure-based drug design optimization of recently discovered antibiotics that bind LptB₂FG and occlude the LptC TM helix from the lateral gate.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70133"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012751/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144053702","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":"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}