Protein Science最新文献

{"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}

{"title":"Quantitative detection of amyloid fibrils using fluorescence resonance energy transfer between engineered yellow and cyan proteins.","authors":"Caitlyn Moustouka, George I Makhatadze","doi":"10.1002/pro.70094","DOIUrl":"10.1002/pro.70094","url":null,"abstract":"<p><p>Over 20 human diseases are caused by or associated with amyloid formation. Developing diagnostic tools to understand the process of amyloid fibril formation is essential for creating therapeutic agents to combat these widespread and growing health problems. Here, we capitalize on our recent striking discovery that green fluorescent protein (GFP), one of the most-used proteins in molecular and cell biology, has a high intrinsic binding affinity to various structural intermediates along the fibrillation pathway, independent of amyloid sequence. Using engineered GFP with the fluorescence properties of Aquamarine and mCitrine, we developed a fluorescence resonance energy transfer (FRET)-based sensor to quantitatively monitor amyloid fibrils. The proof-of-principle characterization was performed on a test system consisting of PAPf39 fibrils.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70094"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11915345/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657937","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":"Data-driven evaluation of suitable immunogens for improved antibody selection.","authors":"Katharina Waury, Hlin Kvartsberg, Henrik Zetterberg, Kaj Blennow, Charlotte E Teunissen, Sanne Abeln","doi":"10.1002/pro.70100","DOIUrl":"10.1002/pro.70100","url":null,"abstract":"<p><p>Antibodies are indispensable in laboratory and clinical applications due to their high specificity and affinity for protein antigens. However, selecting the right protein fragments as immunogens for antibody production remains challenging. Leveraging the Human Protein Atlas, this study systematically evaluates immunogen properties aiming to identify key factors that influence their suitability. Antibodies were classified as successful or unsuccessful based on standardized validation experiments, and the structural and functional properties of their immunogens were analyzed. Results indicated that longer immunogens often resulted in more successful but less specific antibodies. Shorter immunogens (50 residues or fewer) with disordered or unfolded regions at the N- or C-terminus and long coil stretches were more likely to generate successful antibodies. Conversely, immunogens with high beta sheet content, transmembrane regions, or disulfide bridges were associated with poorer antibody performance. Post-translational modification sites within immunogens appeared to mark beneficial regions for antibody generation. To support antibody selection, a novel R package, immunogenViewer, was developed, enabling researchers to easily apply these insights when immunogen sequences are disclosed. By providing a deeper understanding of immunogen suitability, this study promotes the development of more effective antibodies, ultimately addressing issues of reproducibility and reliability in antibody-based research. The findings are highly relevant to the research community, as end users often lack control over the immunogen selection process in antibody production. The R package is freely available as part of Bioconductor: https://bioconductor.org/packages/release/bioc/html/immunogenViewer.html.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70100"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11926642/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143674258","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":"Revisiting the structure of UBR box from human UBR6.","authors":"Bokyung Kim, Sohae Lee, Bong Heon Kim, Leehyeon Kim, Hyun Kyu Song","doi":"10.1002/pro.70092","DOIUrl":"10.1002/pro.70092","url":null,"abstract":"<p><p>Eukaryotic N-degron pathways are proteolytic systems with the ability to recognize specific N-terminal residues of substrate proteins, which are essential parts of their degradation signals. Domains, referred to as UBR boxes, of several E3 ubiquitin ligases can recognize basic N-terminal residues as N-degrons. UBR6 is among the seven mammalian UBR family proteins containing the UBR box domain. However, the recognition of basic type-1 N-degrons by UBR6 is still not well understood. The crystal structure of the UBR box from human UBR6 revealed zinc-mediated dimerization, a structural feature distinct from other monomeric UBR boxes. Furthermore, its folding pattern differed from that of the UBR fold, although the sequences aligned well with those of other UBR boxes. In this study, we re-determined the structure of the UBR box from human UBR6 to investigate whether the unusual domain-swapped dimer was structurally relevant. The newly determined UBR box of UBR6 at 1.5 Å resolution was a monomer with a classical UBR fold. Our structure was compared with previously reported structures of UBR boxes, and its structural features were further analyzed using N-degron binding assays.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70092"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11915344/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658220","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":"Fibrinogen-binding M-related proteins facilitate the recruitment of plasminogen by Streptococcus pyogenes.","authors":"Emma-Jayne Proctor, Hannah R Frost, Bhanu Mantri, Sandeep Satapathy, Gwenaëlle Botquin, Jody Gorman, David M P De Oliveira, Jason McArthur, Mark R Davies, Gökhan Tolun, Anne Botteaux, Pierre Smeesters, Martina Sanderson-Smith","doi":"10.1002/pro.70078","DOIUrl":"10.1002/pro.70078","url":null,"abstract":"<p><p>Group A Streptococcus (GAS) M-related proteins (Mrp) are dimeric α-helical coiled-coil cell-wall-attached proteins. During infection, Mrp recruit human fibrinogen (Fg) to the bacterial surface, enhancing phagocytosis resistance and promoting growth in human blood. However, Mrp exhibit a high degree of sequence diversity, clustering into four evolutionarily distinct groups. It is currently unknown whether this diversity affects the host-pathogen interactions mediated by Mrp. In this study, nine Mrp sequences from the four major evolutionary groups were selected to examine the effect of sequence diversity on protein-protein interactions with Fg. Negative staining transmission electron microscopy confirmed that Mrp are fibrillar proteins measuring between 45.4 and 47.3 nm in length, and mass photometry confirmed the ability of Mrp to form dimers. Surface plasmon resonance was used to evaluate the affinity of each Mrp for Fg. All Mrp studied bound to Fg via Fragment D (FgD) with nanomolar affinity. Previous studies have linked the acquisition of plasminogen (Plg) by GAS Fg-binding M proteins to tissue destruction and excessive stimulation of the human inflammatory response during infection. Our findings show that Mrp provide an alternative mechanism for Plg recruitment, as Plg binding by Mrp was significantly enhanced following pre-incubation with Fg. These data suggest that Mrp play an important role in GAS host-pathogen interactions. However, further studies are necessary to investigate the relevance of these findings in vivo.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70078"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11917135/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658397","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":"Lysine carbamoylation during urea denaturation remodels the energy landscape of human transthyretin dissociation linked to unfolding.","authors":"Marcus Jäger, David E Mortenson, Maziar S Ardejani, Gabriel M Kline, Maria T Dendle, Nicholas L Yan, Evan T Powers, Martin Gruebele, Jeffery W Kelly","doi":"10.1002/pro.70009","DOIUrl":"10.1002/pro.70009","url":null,"abstract":"<p><p>Chemical denaturants such as urea have become indispensable in modern protein science for measuring the energetics of protein folding and assembly. Denaturants bind to and preferentially stabilize denatured states, folding transition states, and folding intermediates over the native state, allowing experimental access to free energies of folding and insights into folding mechanisms. However, too little attention is paid to the established chemical instability of aqueous urea, that is, its decomposition into the reactive electrophile ammonium cyanate or isocyanic acid depending on the solution pH. Protein carbamoylation by cyanate/isocyanic acid can change the dissociation and/or unfolding free energy landscape of the protein under study with time. This problem is exemplified using the human blood protein transthyretin (TTR), a kinetically stable transporter of thyroid hormone and holo-retinol binding protein. The dissociation, misfolding, and aggregation of TTR are associated with a prominent human amyloid disease. We demonstrate that modification of TTR by cyanate reshapes the energy landscape of TTR tetramer dissociation and unfolding on multiple time scales. Like certain halide anions and the more chemically inert thiocyanate anion, cyanate binds weakly and non-covalently to the thyroid hormone binding interface in the TTR tetramer. The close proximity of the bound cyanate ion to the pK_a-perturbed lysine 15 ε-amino side chain nucleophile in the thyroid hormone binding sites of TTR favors carbamoylation of this nitrogen. Lysine 15 ε-amino carbamoylation substantially slows down TTR tetramer dissociation mediated by urea denaturation, thus introducing kinetic heterogeneity early in the unfolding reaction. Slower carbamoylation of the subpopulation of other, less pK_a-perturbed lysine ε-amino groups hastens tetramer unfolding, leading to non-exponential, sigmoidal unfolding trajectories. We thus demonstrate that lysine carbamoylation in urea solutions can strongly alter protein unfolding energetics and the mechanism of unfolding.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70009"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934213/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143701395","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":"High-throughput amino acid-level characterization of the interactions of plasminogen activator inhibitor-1 with variably divergent proteases.","authors":"Laura M Haynes, Matthew L Holding, Hannah L DiGiovanni, David Siemieniak, David Ginsburg","doi":"10.1002/pro.70088","DOIUrl":"10.1002/pro.70088","url":null,"abstract":"<p><p>While members of large paralogous protein families share structural features, their functional niches often diverge significantly. Serine protease inhibitors (SERPINs), whose members typically function as covalent inhibitors of serine proteases, are one such family. Plasminogen activator inhibitor-1 (PAI-1) is a prototypic SERPIN, which canonically inhibits tissue- and urokinase-type plasminogen activators (tPA and uPA) to regulate fibrinolysis. PAI-1 has been shown to also inhibit other serine proteases, including coagulation factor XIIa (FXIIa) and transmembrane serine protease 2 (TMPRSS2). The structural determinants of PAI-1 inhibitory function toward these non-canonical protease targets, and the biological significance of these functions, are unknown. We applied deep mutational scanning (DMS) to assess the effects of ~80% of all possible single-amino acid substitutions in PAI-1 on its ability to inhibit three putative serine protease targets (uPA, FXIIa, and TMPRSS2). Selection with each target protease generated a unique PAI-1 mutational landscape, with the determinants of protease specificity distributed throughout PAI-1's primary sequence. Next, we conducted a comparative analysis of extant orthologous sequences, demonstrating that key residues modulating PAI-1 inhibition of uPA and FXIIa, but not TMPRSS2, are maintained by purifying selection (also referred to as \"negative selection\"). PAI-1's activity toward FXIIa may reflect how protease evolutionary relationships predict SERPIN functional divergence, which we support via a cophylogenetic analysis of secreted SERPINs and their cognate serine proteases. This work provides insight into the functional diversification of SERPINs and lays the framework for extending these studies to other proteases and their regulators.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70088"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11917113/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143656882","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 naturally occurring standalone TrpB enzyme provides insights into allosteric communication within tryptophan synthase.","authors":"Thomas Kinateder, Lukas Drexler, Cristina Duran, Sílvia Osuna, Reinhard Sterner","doi":"10.1002/pro.70103","DOIUrl":"10.1002/pro.70103","url":null,"abstract":"<p><p>Allosteric regulation of catalytic activity is a widespread property of multi-enzyme complexes. The tryptophan synthase is a prototypical allosteric enzyme where the constituting α (TrpA) and β (TrpB) subunits mutually activate each other in a manner that is incompletely understood. Experimental and computational studies have shown that LBCA-TrpB from the last bacterial common ancestor contains six residues (Res₆) distal from the active site that allow for high stand-alone catalytic activity in the absence of a TrpA subunit. In the present study, a database search revealed that Res₆ is also present in the extant plTrpB from Pelodictyon luteolum. The plTrpB enzyme showed a high stand-alone activity and only a moderate activation by plTrpA. The replacement of LBCA-Res₆ in plTrpB with the consensus residues from a multiple sequence alignment yielded plTrpB-con, which showed a dramatically decreased stand-alone activity but was strongly stimulated by plTrpA. These findings suggest that the effect of these six key allosteric residues is largely independent of the protein context within a specific TrpB enzyme. Analysis of the conformational landscapes of plTrpB and plTrpB-con revealed that plTrpB in isolation displays efficient closure of both the active site and the communication (COMM) domain. In contrast, these catalytically competent states are destabilized in plTrpB-con but can be recovered by the addition of plTrpA. A correlation-based shortest path map (SPM) analysis reveals that the catalytically and allosterically relevant domains-specifically, the COMM domain in TrpB and loops 2 and 6 in TrpA-are tightly interconnected exclusively in plTrpA:plTrpB-con.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70103"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11917138/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658389","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}