Protein Science最新文献

{"title":"A synthetic heavy chain variable domain antibody library (VHL) provides highly functional antibodies with favorable developability.","authors":"Guiying Pang, Ruixue Wang, Hongxu Yang, Mengya Chai, Yanzhe Gao, Sisi Chen, Ting Mao, Luheng Du, Yujia Lan, Shu Li, Jiale Xu, Panpan Cui, Ruqing Cheng, Yuxin Huang, Xuncui Wang, Yi Yang","doi":"10.1002/pro.70090","DOIUrl":"10.1002/pro.70090","url":null,"abstract":"<p><p>Synthetic antibody libraries have been developed as an efficient source for the discovery of the heavy chain variable (VH) domain, which exhibits low immunogenicity, high tissue penetration, and diverse binding epitopes in therapeutic biopharmaceuticals. In this study, the human IGHV3-23*04 germline gene was chosen as the scaffold with a high expression level and favorable thermal stability. Amino acid diversity was introduced into the complementarity determining region 3 (CDR3) to exclude potential sequence liabilities. A library containing 2.6 × 10¹¹ independent clones was successfully constructed. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, interleukin-17A (IL17A), B-cell maturation antigen (BCMA), and G-protein coupled receptor family C group 5 member D (GPRC5D) were used as target antigens to screen and identify VHs. In each case, Thirty-one to fifty-five VHs were screened out. The VH-Fc antibodies showed superior affinities (as high as 4.6 nM) to the corresponding antigens but did not bind to antigen-irrelevant cell CHO-S. Furthermore, the anti-RBD and anti-IL17A VH-Fc antibodies showed strong functional activity in the receptor-blocking assays. The VH-Fc antibodies from the synthetic library exhibited favorable developability (thermal stability, colloidal stability, hydrophilicity, anti-aggregation ability, and no interaction with human IgGs). We demonstrated that high-affinity and highly functional VH domain antibodies were generated from the rationally designed library with desired physicochemical properties. This approach is generally universal to target any antigen and has significant potential to accelerate candidate selection.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70090"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11917115/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658394","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":"Tutorial on integrative spatiotemporal modeling by integrative modeling platform.","authors":"Andrew P Latham, Miha Rožič, Benjamin M Webb, Andrej Sali","doi":"10.1002/pro.70107","DOIUrl":"10.1002/pro.70107","url":null,"abstract":"<p><p>Cells function through dynamic interactions between macromolecules. Detailed characterization of the dynamics of large biomolecular systems is often not feasible by individual biophysical methods. In such cases, it may be possible to compute useful models by integrating multiple sources of information. We have previously developed an integrative method to model dynamic processes by computing biomolecular heterogeneity at fixed time points, then generating static integrative structural modes for each of these heterogeneity models, and finally connecting these static models to produce a scored trajectory model that depicts the process. Here, we demonstrate how to compute, score, and assess these integrative spatiotemporal models using our open-source Integrative Modeling Platform (IMP) program (https://integrativemodeling.org/).</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70107"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934212/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143700632","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":"PDBe tools for an in-depth analysis of small molecules in the Protein Data Bank.","authors":"Preeti Choudhary, Ibrahim Roshan Kunnakkattu, Sreenath Nair, Dare Kayode Lawal, Ivanna Pidruchna, Marcelo Querino Lima Afonso, Jennifer R Fleming, Sameer Velankar","doi":"10.1002/pro.70084","DOIUrl":"10.1002/pro.70084","url":null,"abstract":"<p><p>The Protein Data Bank (PDB) is the primary global repository for experimentally determined 3D structures of biological macromolecules and their complexes with ligands, proteins, and nucleic acids. PDB contains over 47,000 unique small molecules bound to the macromolecules. Despite the extensive data available, the complexity of small-molecule data in the PDB necessitates specialized tools for effective analysis and visualization. PDBe has developed a number of tools, including PDBe CCDUtils (https://github.com/PDBeurope/ccdutils) for accessing and enriching ligand data, PDBe Arpeggio (https://github.com/PDBeurope/arpeggio) for analyzing interactions between ligands and macromolecules, and PDBe RelLig (https://github.com/PDBeurope/rellig) for identifying the functional roles of ligands (such as reactants, cofactors, or drug-like molecules) within protein-ligand complexes. The enhanced ligand annotations and data generated by these tools are presented on the novel PDBe-KB ligand pages, offering a comprehensive overview of small molecules and providing valuable insights into their biological contexts (example page for Imatinib: https://pdbe.org/chem/sti). By improving the standardization of ligand identification, adding various annotations, and offering advanced visualization capabilities, these tools help researchers navigate the complexities of small molecules and their roles in biological systems, facilitating mechanistic understanding of biological functions. The ongoing enhancements to these resources are designed to support the scientific community in gaining valuable insights into ligands and their applications across various fields, including drug discovery, molecular biology, systems biology, structural biology, and pharmacology.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70084"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11917123/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657996","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":"SHARK-capture identifies functional motifs in intrinsically disordered protein regions.","authors":"Chi Fung Willis Chow, Swantje Lenz, Maxim Scheremetjew, Soumyadeep Ghosh, Doris Richter, Ceciel Jegers, Alexander von Appen, Simon Alberti, Agnes Toth-Petroczy","doi":"10.1002/pro.70091","DOIUrl":"10.1002/pro.70091","url":null,"abstract":"<p><p>Increasing insights into how sequence motifs in intrinsically disordered regions (IDRs) provide functions underscore the need for systematic motif detection. Contrary to structured regions where motifs can be readily identified from sequence alignments, the rapid evolution of IDRs limits the usage of alignment-based tools in reliably detecting motifs within. Here, we developed SHARK-capture, an alignment-free motif detection tool designed for difficult-to-align regions. SHARK-capture innovates on word-based methods by flexibly incorporating amino acid physicochemistry to assess motif similarity without requiring rigid definitions of equivalency groups. SHARK-capture offers consistently strong performance in a systematic benchmark, with superior residue-level performance. SHARK-capture identified known functional motifs across orthologs of the microtubule-associated zinc finger protein BuGZ. We also identified a short motif in the IDR of S. cerevisiae RNA helicase Ded1p, which we experimentally verified to be capable of promoting ATPase activity. Our improved performance allows us to systematically calculate 10,889 motifs for 2695 yeast IDRs and provide it as a resource. SHARK-capture offers the most precise tool yet for the systematic identification of conserved regions in IDRs and is freely available as a Python package (https://pypi.org/project/bio-shark/) and on https://git.mpi-cbg.de/tothpetroczylab/shark.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70091"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11917139/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658263","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":"Comparison of enriched charge variants from different anti-CD3 bispecific antibodies reveals differential susceptibility of each bispecific arm to post-translational modification.","authors":"Jennifer B Nguyen, Sophia Liu, Dylan A Howie, Zachary R Oberholtzer, Eric T Ong, Ramya Rao, Jethro E Prinston, Igor Dikiy, Jikang Wu, Zhijie Wu, Yimeng Zhao, Meinuo Li, Rosalynn Molden, Guido Molina, Kathleen Provoncha, Cristinel Sandu, Haibo Qiu, Ning Li, William Matousek, Michael P Rosconi, Erica A Pyles","doi":"10.1002/pro.70079","DOIUrl":"10.1002/pro.70079","url":null,"abstract":"<p><p>Charge heterogeneity is an important quality attribute of therapeutic antibodies, and a detailed understanding of charge heterogeneity arising from post-translational modifications (PTMs) is required by regulatory agencies during drug development. Among antibody therapeutics, the bispecific antibody with two distinct Fab domains targeting distinct antigens provides additional complexity to the charge profile. In this study, charge variant species were enriched from three bispecific antibodies (bsAbs) each containing one anti-CD3 binding arm designed with differential affinity to CD3. The charge heterogeneity corresponding to each anti-CD3 arm within each enriched fraction was evaluated using a domain-specific, digestion-assisted imaged capillary isoelectric focusing (icIEF) method known as DiCE. Through fractionation, we observed that the anti-CD3 arm of each bispecific antibody exhibited different distributions of acidic variants, even when the anti-CD3 arms were identical based on primary sequence. Reduced peptide mapping was performed on specific fractions to identify unique site-specific PTMs that were uncovered or enriched through fractionation. In each case, the bispecific arm that was most susceptible to PTMs exhibited a more basic isoelectric point. Conformational stability analysis of each bispecific antibody using differential scanning calorimetry suggested that the more basic Fab arm tended to be correlated with a lower melting temperature, although it is unclear the extent to which PTMs on the basic arm may contribute to reduced conformational stability. Overall, these results provide additional evidence that each of the two arms of a bispecific antibody may exhibit differential susceptibility to post-translational modification and that this susceptibility is likely correlated with subtle differences in overall bispecific antibody structure, which is influenced by electrostatic properties inherent to the primary sequence. Future studies to obtain high-resolution structures of full-length bispecific antibodies by crystallography or cryo-electron microscopy may help to elucidate the driving force for susceptibility to PTMs in bispecific antibodies.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70079"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11926628/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143674326","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":"Functional and structural characterization of Staphylococcus aureus N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) reveals a redox-sensitive acetyltransferase activity.","authors":"Jordan L Pederick, Akhil Kumar, Tara L Pukala, John B Bruning","doi":"10.1002/pro.70111","DOIUrl":"10.1002/pro.70111","url":null,"abstract":"<p><p>The bifunctional enzyme N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) is a promising antibiotic drug target, as it facilitates the biosynthesis of uridine 5'-diphospho-N-acetylglucosamine, an essential precursor of cell wall constituents. We identified that Staphylococcus aureus GlmU (SaGlmU), which was previously targeted for inhibitor development, possesses a dual-cysteine variation (C379/C404) within the acetyltransferase active site. Enzyme assays performed under reducing and non-reducing conditions revealed that the acetyltransferase activity of SaGlmU is redox-sensitive, displaying ~15-fold lower turnover and ~3-fold higher K_M value for the acetyl CoA substrate under non-reducing conditions. This sensitivity was absent in a C379A SaGlmU mutant. Analysis of SaGlmU by mass spectrometry, x-ray crystallography, and in silico modeling support that C379 and C404 act as a reversible, redox-sensitive switch by forming a disulfide under non-reducing conditions that impedes acetyl CoA recognition and turnover. Therefore, we recommend that future in vitro screening and characterization of SaGlmU inhibitors consider both reducing and non-reducing conditions.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70111"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11947611/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143721201","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":"One-pot dual protein labeling for simultaneous mechanical and fluorescent readouts in optical tweezers.","authors":"Laura-Marie Silbermann, Maximilian Fottner, Ronald van der Meulen, Nora Migdad, Kathrin Lang, Katarzyna Tych","doi":"10.1002/pro.70098","DOIUrl":"10.1002/pro.70098","url":null,"abstract":"<p><p>Optical tweezers are widely used in the study of biological macromolecules but are limited by their one-directional probing capability, potentially missing critical conformational changes. Combining fluorescence microscopy with optical tweezers, employing Förster resonance energy transfer (FRET) pairs, addresses this issue. When integrating fluorescence microscopy with optical tweezers, orthogonal protein conjugation methods are needed to enable simultaneous, site-specific attachment of fluorophores and DNA handles, commonly used to apply force to molecules of interest. In this study, we utilized commercially available reagents for dual site-specific labeling of the homodimeric heat shock protein 90 (Hsp90) using thiol-maleimide and inverse electron demand Diels-Alder cycloaddition (IEDDAC) bioorthogonal reactions. In a one-pot approach, Hsp90 modified with a cysteine mutation and the non-canonical amino acid cyclopropene-L-lysine (CpK) was labeled with the FRET pair maleimide-Atto 550 and maleimide-Atto 647N, alongside single-stranded methyltetrazine-modified DNA oligonucleotide. Optical tweezers experiments with this labeled Hsp90 construct revealed structural transitions consistent with previous studies, validating the approach. Fluorescence measurements confirmed the proximity of FRET pairs in the N-terminally closed state of Hsp90 in this experimental setup. This integrative method provides a powerful tool for probing complex protein conformational dynamics beyond the limitations of traditional optical tweezers.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70098"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11915586/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657900","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":"Templated trimerization of the phage L decoration protein on capsids.","authors":"Brianna M Woodbury, Rebecca L Newcomer, Makayla N Leroux, Andrei T Alexandrescu, Carolyn M Teschke","doi":"10.1002/pro.70089","DOIUrl":"10.1002/pro.70089","url":null,"abstract":"<p><p>The 134-residue phage L decoration protein (Dec) forms a capsid-stabilizing homotrimer that has an asymmetric tripod-like structure when bound to phage L capsids. The N-termini of the trimer subunits consist of spatially separated globular OB-fold domains that interact with the virions of phage L or the related phage P22. The C-termini of the trimer form a spike structure that accounts for nearly all the interactions that stabilize the trimer. A Dec mutant with the spike residues 99-134 deleted (Dec_1-98) was used to demonstrate that the globular OB-fold domain folds independently of the C-terminal residues. However, Dec_1-98 was unable to bind phage P22 virions, indicating the C-terminal spike is essential for stable capsid interaction. The full-length Dec trimer is disassembled into monomers by acidification to pH <2. These monomers retain the folded globular OB-fold domain structure, but the spike is unfolded. Increasing the pH of the Dec monomer solution to pH 6 allowed for slow trimer formation in vitro over the course of days. The infectious cycle of phage L is only around an hour, thereby implying Dec trimer assembly in vivo is templated by the phage capsid. The thermodynamic hypothesis holds that protein folding is determined by the amino acid sequence. Dec serves as an unusual example of an oligomeric folding step that is kinetically accelerated by a viral capsid template. The capsid templating mechanism could satisfy the flexibility needed for Dec to adapt to the unusual quasi-symmetric binding site on the mature phage L capsid.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70089"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11917118/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658317","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":"A suite of pre-assembled, pET28b-based Golden Gate vectors for efficient protein engineering and expression.","authors":"Deepika Gaur, Matthew L Wohlever","doi":"10.1002/pro.70106","DOIUrl":"10.1002/pro.70106","url":null,"abstract":"<p><p>Expression and purification of recombinant proteins in Escherichia coli is a bedrock technique in biochemistry and molecular biology. Expression optimization requires testing different combinations of solubility tags, affinity purification techniques, and site-specific proteases. This optimization is laborious and time-consuming as these features are spread across different vector series and require different cloning strategies with varying efficiencies. Modular cloning kits based on the Golden Gate system exist, but they are not optimized for protein biochemistry and are overly complicated for many applications, such as undergraduate research or simple screening of protein purification features. An ideal solution is for a single gene synthesis or PCR product to be compatible with a large series of pre-assembled Golden Gate vectors containing a broad array of purification features at either the N or C terminus. To our knowledge, no such system exists. To fulfill this unmet need, we Golden Gate domesticated the pET28b vector and developed a suite of 21 vectors with different combinations of purification tags, solubility domains, visualization/labeling tags, and protease sites. We also developed a vector series with nine different N-terminal tags and no C-terminal cloning scar. The system is modular, allowing users to easily customize the vectors with their preferred combinations of features. To allow for easy visual screening of cloned vectors, we optimized constitutive expression of the fluorescent protein mScarlet3 in the reverse strand, resulting in a red to white color change upon successful cloning. Testing with the model protein sfGFP shows the ease of visual screening, high efficiency of cloning, and robust protein expression. These vectors provide versatile, high-throughput solutions for protein engineering and functional studies in E. coli.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70106"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934214/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143701388","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":"Kingfisher: An open-sourced web-based platform for the analysis of hydrogen exchange mass spectrometry data.","authors":"Nolan K McLaughlin, Juan P Rincon Pabon, Samantha Gies, Reza Dastvan, Michael L Gross","doi":"10.1002/pro.70096","DOIUrl":"10.1002/pro.70096","url":null,"abstract":"<p><p>Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is now a critical tool in molecular biology and structural proteomics. It is routinely used to probe protein and conformational dynamics through a well-established experiment where amide hydrogens exchange with deuterium atoms in a buffer containing D₂O. Although there have been numerous advances in the field, data analysis still poses challenges mainly due to the need for manual curation of the data and the lack of standardized statistics and accessible software. In response, we developed Kingfisher, an open-source, user-friendly, web-based solution that facilitates downstream analysis using well-established statistics and provides advanced high-resolution representations of the HDX results. Kingfisher is able to read data directly as exported from common software packages and usually takes less than a minute to run the analysis, without the need to download the raw code or install any software. We foresee Kingfisher as a valuable tool for both newcomers and experts in the field of Hydrogen Exchange Mass Spectrometry. Kingfisher is available to all users as an interactive web application at https://kingfisher.wustl.edu/.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70096"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11915630/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657065","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}