Protein Science最新文献

{"title":"Navigating infection by pathogenic spirochetes: The host-bacteria interface at the atomic level.","authors":"Libor Hejduk, Norbert Müller, Adriana Rathner, Ján Štěrba, Shang-Cheng Hung, Chia-Lin Chyan, Ryan O M Rego, Martin Strnad","doi":"10.1002/pro.70185","DOIUrl":"10.1002/pro.70185","url":null,"abstract":"<p><p>Pathogenic spirochetes bind and interact with various host structures and molecules throughout the course of infection. By utilizing their outer surface molecules, spirochetes can effectively modulate their dissemination, interact with immune system regulators, and select specific destination niches within the host. The three-dimensional structures of multiple spirochetal surface proteins have been elucidated, providing insight into their modus operandi. This review focuses on the structural characteristics of these sticky molecules and their functional implications, highlighting how these features contribute to the pathogenicity of spirochetes and their ability to persist in the host and vector environments. Recognizing the structural motifs and ligands to which these important virulence determinants bind could open new avenues for developing strategies to block colonization by spirochetal pathogens.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70185"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12183334/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144369174","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 high throughput assay for measuring secreted protein based on a de novo fluorescent reporter reveals regulatory and structural insights in Salmonella type three secretion system.","authors":"Samuel Alexander Leach, Jordan Scott Summers, Edward Wen, Danielle Tullman-Ercek","doi":"10.1002/pro.70183","DOIUrl":"10.1002/pro.70183","url":null,"abstract":"<p><p>Intracellular protein production in bacteria is limited by the need for lysis and costly purification. A promising alternative is to engineer the host organism for protein secretion. While the Salmonella enterica serovar Typhimurium (Salmonella typhimurium) Type 3 Secretion System (T3SS) has been utilized for protein secretion, its study and eventual applicability for recombinant protein production are constrained by the lack of high-throughput assays to quantitatively measure secretion titer. Developing such assays is challenging, as proteins must remain unfolded for secretion, limiting the use of several common reporter proteins. In this work, we develop a high-throughput secretion assay using mini-fluorescence activating protein (mFAP). mFAP forms a chromophore only upon addition of an exogenous substrate, allowing secretion and subsequent fluorescence detection. We demonstrate mFAP secretion via the T3SS with an N-terminal secretion tag and show that the fluorescent signal in the secreted fraction is rapid and linear over three orders of magnitude. Using this assay, we screen S. typhimurium strains with secretion-enhancing mutations, identifying a constitutively active strain and revealing temporally controlled secretion dynamics. We also show that this assay may be applicable to other secretion systems, providing a universal tool for tracking heterologous protein secretion.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70183"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12198052/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144497891","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":"Unraveling antibody-induced mechanical stability of antigen: Insights from single-molecule studies.","authors":"Soham Chakraborty, Shivam Pandit, Krishnendu Sinha, Madhu Bhatt, Debojyoti Chowdhury, Suman Chakrabarty, Shubhasis Haldar","doi":"10.1002/pro.70201","DOIUrl":"10.1002/pro.70201","url":null,"abstract":"<p><p>Antigen-antibody interaction, as a prominent ligand-receptor reaction, plays a crucial role in immunological responses. Notably, antigens can contain multiple ligand binding sites that define their intermolecular interactions more intricately and thereby make them context-dependent. Here, we have investigated the binding-induced effect of the largest antibody isotype, IgM, on protein L mechanical stability using single-molecule magnetic tweezers. Our results showed that IgM elevates the protein L mechanical stability by increasing its unfolding time. Interestingly, we were able to resolve distinct IgM-bound states of protein L by characterizing their unfolding dwell time: while the IgM-unbound state has the lowest dwell time, it increases with the IgM concentration via binding to either one or both of its binding sites, reconciling the IgM-induced protein L mechanical stability. To delve into the plausible mechanism of such intricate phenomena, we performed steered a molecular dynamic simulation of protein L and determined its unfolding rupture force at those multiple IgM-bound states, their corresponding molecular insights, and interaction gymnastics through binding interfaces. Additionally, we unraveled the mechanical response of these binding interfaces to be different; and during dimer IgM complex formation, these binding interfaces synergistically increase the mechanical stability of the complex. This provides the underlying principles of IgM-induced protein L stability under mechanical constraints. Overall, this study provides an in-depth understanding of a generic mechanism of antibody-induced mechanical stability of antigenic substrate under physiological sheer stress.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70201"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12168486/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302721","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":"Uncovering protein conformational dynamics within two-component viral biomolecular condensates.","authors":"Alice Colyer, Julia Acker, Alexander Borodavka, Antonio N Calabrese","doi":"10.1002/pro.70181","DOIUrl":"10.1002/pro.70181","url":null,"abstract":"<p><p>Biomolecular condensates selectively compartmentalize and organize biomolecules within the crowded cellular milieu and are instrumental in some disease mechanisms. Upon infection, many RNA viruses form biomolecular condensates that are often referred to as viral factories. The assembly mechanism of these viral factories remains poorly defined but involves transient, non-stoichiometric protein/RNA interactions, making their structural characterization challenging. Here, we sought to investigate the structural dynamics and intermolecular interactions of the key proteins responsible for condensate formation upon rotavirus infection, namely NSP2 (an RNA chaperone) and NSP5 (an intrinsically disordered protein [IDP]), using a combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS), native MS, and biophysical tools. Our data reveal key structural features of intrinsically disordered NSP5 that are vital for condensate assembly and highlight inter/intra-protein interactions involved in condensate assembly. Moreover, we demonstrate that within a condensate there are altered conformational dynamics within the C-terminal region of NSP2, which has previously been shown to play a role in regulating its RNA chaperoning activity, and in the disordered regions of NSP5. We propose that altered conformational dynamics in NSP2 and NSP5 are critical for regulation of RNA annealing within a biomolecular condensate and for condensate assembly/client recruitment, respectively. Combined, our data demonstrate that the unique environment within a biomolecular condensate can tune functionally important protein conformational dynamics, which may play a crucial role in the replication of rotaviruses.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70181"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12168090/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302720","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":"Biomolecular condensates-Prerequisites for anhydrobiosis?","authors":"Charles A Elder, Hannah M Skaggs, Lynnette M A Dirk, David F Grimm, Clinton J Belott, Willem F Wolkers, Harriëtte Oldenhof, Vladimir N Uversky, A Bruce Downie, Michael A Menze","doi":"10.1002/pro.70192","DOIUrl":"10.1002/pro.70192","url":null,"abstract":"<p><p>It is often underappreciated that despite water being a requirement for life on Earth, organisms belonging to all taxonomic kingdoms have developed mechanisms to survive desiccation. These organisms, referred to as anhydrobiotes, accumulate specific biomolecules during or before drying that facilitate the survival of desiccation stress. Compounds utilized by a wide variety of anhydrobiotes during desiccation include metabolites such as sugars and amino acids, as well as proteins with extensive intrinsically disordered regions. Intrinsically disordered proteins that are constitutively expressed or upregulated during the onset or in preparation for desiccation include late embryogenesis abundant proteins, tardigrade disordered proteins, hydrophilins, some small heat shock proteins, and prion-like proteins. Some of these proteins form biomolecular condensates in the cellular environment. We hypothesize that phase transitions driven by anhydrobiosis-related intrinsically disordered proteins play a substantial role in enabling anhydrobiosis by (1) contributing to the downregulation of metabolic and developmental processes, (2) selectively sequestering desiccation-sensitive molecules into a \"protective compartment\" during drying, (3) interfering with programmed cell death signaling pathways to confer optimal time for the cell to repair after rehydration, (4) resisting intracellular volume changes to aid in membrane stabilization during desiccation, and (5) changing the biophysical properties of water to reduce desiccation-induced cellular damage. Biochemical strategies in anhydrobiotes are certainly multifaceted and may differ among systems. Nevertheless, a better understanding of the relevance of phase transitions in anhydrobiosis may allow us to get one step closer to unraveling the enigmatic phenomenon of life without water.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70192"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12168135/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302748","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":"Ancestral sequence reconstruction of the Mic60 Mitofilin domain reveals residues supporting respiration in yeast.","authors":"Friederike M C Benning, Tristan A Bell, Tran H Nguyen, Della Syau, Louise B Connell, Yi-Ting Liao, Matthew P Keating, Margaret Coughlin, Anja E H Nordstrom, Maria Ericsson, Corrie J B daCosta, Luke H Chao","doi":"10.1002/pro.70207","DOIUrl":"10.1002/pro.70207","url":null,"abstract":"<p><p>In eukaryotes, cellular respiration takes place in the cristae of mitochondria. The mitochondrial inner membrane protein Mic60, a core component of the mitochondrial contact site and cristae organizing system, is crucial for the organization and stabilization of crista junctions and its associated functions. While the C-terminal Mitofilin domain of Mic60 is necessary for cellular respiration, the sequence determinants for this function have remained unclear. Here, we used ancestral sequence reconstruction to generate Mitofilin ancestors up to and including the last opisthokont common ancestor (LOCA). We found that yeast-lineage derived Mitofilin ancestors as far back as the LOCA rescue respiration. By comparing Mitofilin ancestors, we identified four residues sufficient to explain the respiratory difference between yeast- and animal-derived Mitofilin ancestors. Our results provide a foundation for investigating the conservation of Mic60-mediated cristae junction interactions.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70207"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12183332/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144369168","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":"Mini-αA-crystallin protects a client lens protein from catastrophic aggregation due to heat stress.","authors":"Collin Sroge, Jaewon Suk, Jason Zhu, Maria Sophia Teresa Lee Padilla, Christian F Baca, Carter T Butts, Rachel W Martin","doi":"10.1002/pro.70199","DOIUrl":"10.1002/pro.70199","url":null,"abstract":"<p><p>The clarity and refractivity of the eye lens are mediated by the highly soluble crystallin proteins. Post-translational modifications impact solubility and stability of the structural and refractive βγ-crystallins, eventually leading to cataract. Such damaged proteins are kept in solution by the holdase chaperone α-crystallins, maintaining lens transparency over decades despite the absence of protein turnover. It was previously found that a short peptide from human αA-crystallin (mini-αA-crystallin [MAAC]) retains some chaperone activity, with hydrophobic interactions hypothesized to mediate chaperone-client interactions; MAAC has been hypothesized to have β-strand structure in solution, although its conformational ensemble under these conditions has not been well-characterized. Here, we employ a combination of nuclear magnetic resonance (NMR), circular dichroism spectroscopy, dynamic light scattering, and molecular dynamics simulations to examine the behavior of MAAC in dilute solution and in combination with human γS-crystallin. Structural ensembles of two alanine variants of MAAC (I4A and L6A) show that the variants lack well-defined secondary structure, but have a preference for a bent conformation with some self-interaction. A partial alanine scan indicates that several hydrophobic residues are important for peptide solubility, also modifying the peptide's conformational ensemble. Tests of wild-type MAAC chaperone activity on thermally stressed γS-crystallin show little interaction between MAAC and the client protein below its unfolding temperature. However, MAAC does inhibit large-scale aggregation at the γS-crystallin unfolding temperature. NMR measurements indicate only weak, transient interaction with the client protein during the intermediate aggregation phase, suggesting a sharp phase transition in the MAAC-client system.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70199"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12183329/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144369173","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 tool to dissect heterotypic determinants of homotypic protein phase behavior.","authors":"Hannah Kimbrough, Jacob Jensen, Caleb Weber, Tayla Miller, Lucinda E Maddera, Jillian F Blanck, Vignesh M P Babu, William B Redwine, Randal Halfmann","doi":"10.1002/pro.70194","DOIUrl":"10.1002/pro.70194","url":null,"abstract":"<p><p>Proteins commonly self-assemble to create liquid or solid condensates with diverse biological activities. The mechanisms of assembly are determined by each protein's sequence and cellular context. We previously developed distributed amphifluoric FRET (DAmFRET) to analyze sequence determinants of self-assembly in cells. Here, we extend the utility of DAmFRET by creating a nanobody (mEosNb) against the fluorescent protein mEos3 to physically tether other proteins to DAmFRET-enabled query proteins. This tool allows us to rapidly screen for effects on the phase behavior of query proteins by modulating the expression level and valence of mEosNb-fused modifier proteins. We use our system to identify thresholds of valence for liquid-liquid phase separation and to discriminate nucleation mechanisms of amyloid and other paracrystalline assemblies in cells. Our approach adds a new experimental dimension for interrogating the mechanisms of intracellular phase transitions.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70194"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12168132/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302744","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":"ConsAMPHemo: A computational framework for predicting hemolysis of antimicrobial peptides based on machine learning approaches.","authors":"Peilin Xie, Lantian Yao, Jiahui Guan, Chia-Ru Chung, Zhihao Zhao, Feiyu Long, Zhenglong Sun, Tzong-Yi Lee, Ying-Chih Chiang","doi":"10.1002/pro.70087","DOIUrl":"10.1002/pro.70087","url":null,"abstract":"<p><p>Many antimicrobial peptides (AMPs) function by disrupting the cell membranes of microbes. While this ability is crucial for their efficacy, it also raises questions about their safety. Specifically, the membrane-disrupting ability could lead to hemolysis. Traditionally, the hemolytic activity of AMPs is evaluated through experiments. To reduce the cost of assessing the safety of an AMP as a drug, we introduce ConsAMPHemo, a two-stage framework based on deep learning. ConsAMPHemo performs conventional binary classification of the hemolytic activities of AMPs and predicts their hemolysis concentrations through regression. Our model demonstrates excellent classification performance, achieving an accuracy of 99.54%, 82.57%, and 88.04% on three distinct datasets, respectively. Regarding regression prediction, the model achieves a Pearson correlation coefficient of 0.809. Additionally, we identify the correlation between features and hemolytic activity. The insights gained from this work shed light on the underlying physics of the hemolytic nature of an AMP. Therefore, our study contributes to the development of safer AMPs through cost-effective hemolytic activity prediction and by revealing the design principles for AMPs with low hemolytic toxicity. The codes and datasets of ConsAMPHemo are available at https://github.com/Cpillar/ConsAMPHemo.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70087"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12168091/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302750","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 model predicts function of Rho-GTPase binding for plexin receptor GAP activity on Rap1b via dynamic allosteric changes.","authors":"Nisha Bhattarai, Lindsay Morrison, Alexandre F Gomes, Paul Savage, Amita R Sahoo, Matthias Buck","doi":"10.1002/pro.70196","DOIUrl":"10.1002/pro.70196","url":null,"abstract":"<p><p>Plexin-semaphorin signaling regulates key processes such as cell migration, neuronal development, angiogenesis, and immune responses. Plexins stand out because they can directly bind with both Rho- and Ras-family small GTPases through their intracellular domains when these GTPases are in their active, GTP-bound states. This binding occurs via intracellular regions, which include a Rho-GTPase binding domain and a GTPase-activating protein (GAP) segment. Studies have shown that Rho and Ras GTPases play vital roles in plexin signaling and activation. However, the structural dynamics of plexins and GTPases and how these conformational changes affect interactions when plexin is bound with both Ras and Rho-GTPases or bound to only one specific GTPase have remained unclear. In this study, we conducted molecular dynamics simulations on six distinct plexin-GTPase bound systems to investigate the differences in conformations and dynamics between plexin-B1 and three GTPases: Rap1b, Rnd1, and Rac1. Our analysis revealed that dynamics with Rac1 are more altered compared to Rnd1, depending on whether plexin's GAP domain is bound or unbound to Rap1b. In addition, we further investigated alterations in network centralities and compared the network dynamics of the plexin-GTPase complexes, focusing on the differences when plexin is bound to both Ras (Rap1b) and Rho-GTPases (Rnd1/Rac1) versus when it is bound to only one GTPase. Our study revealed that Rnd1 exhibits stronger and more stable interactions with plexin-B1 in the absence of Rap1b, while Rac1 shows fewer and less stable connections in comparison. These computational models have features that broadly agree with experimental results from hydrogen-deuterium exchange detected by mass spectrometry. Such insights provide a better understanding of the molecular mechanisms underlying plexin-GTPase interactions and the complexities of signaling mechanisms involving GTPases in general.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70196"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12183121/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144369169","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}