Protein Science最新文献

{"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":5.2,"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}

{"title":"Prokaryotic mechanosensitive channels mediate copper influx.","authors":"Yara Ghnamah, Caitlin D Palmer, Nurit Livnat-Levanon, Moti Grupper, Amy C Rosenzweig, Oded Lewinson","doi":"10.1002/pro.70205","DOIUrl":"10.1002/pro.70205","url":null,"abstract":"<p><p>Copper is an essential micronutrient in all kingdoms of life, requiring a meticulous balance between acquisition and toxic overload. While copper import in eukaryotes has been investigated extensively, few prokaryotic copper importers have been identified, leading to the notion that cytoplasmic copper uptake is unnecessary in prokaryotes. Here we report that mechanosensitive channels are key players in prokaryotic copper import. Deletion of the gene encoding the Escherichia coli small mechanosensitive channel, _EcMscS, leads to significantly reduced copper influx. Conversely, overexpression of _EcMscS leads to increased copper influx, elevated intracellular copper content, and renders cells hypersensitive to copper. Furthermore, specific channel blockers and competing permeating ions inhibit _EcMscS copper conductance, lowering intracellular copper accumulation and alleviating copper hypersensitivity. These findings extend beyond E. coli, as other prokaryotic small mechanosensitive channels of bacterial and archaeal origin also facilitate copper influx. Taken together, these results uncover a previously unknown moonlighting function for mechanosensitive channels as a pathway for prokaryotic copper uptake.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70205"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12198049/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144497939","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":"Loss-of-function coding variants in the Ras of complex proteins/GTPase domain of leucine rich repeat kinase 2.","authors":"Sarah Butterfield, Susanne Herbst, Patrick Alfryn Lewis","doi":"10.1002/pro.70190","DOIUrl":"10.1002/pro.70190","url":null,"abstract":"<p><p>The LRRK2 gene is a key contributor to the genetic risk of Parkinson's disease, and a priority drug target for the disorder. Leucine Rich Repeat Kinase 2, the protein product of LRRK2, is a multidomain enzyme implicated in a range of cellular processes-including endolysosomal trafficking and damage response. Based on the report that truncation and structural variants resulting in loss of LRRK2 protein are observed in human populations, genomic sequence repositories were queried for coding variants affecting key catalytic residues in LRRK2-resulting in the identification of three variants (K1347E, K1347R, and T1348P) predicted to ablate the capacity of LRRK2 to bind GTP. Biochemical and cellular characterization of these variants confirmed loss of GTP binding, as well as reduced or loss of kinase activity. These data demonstrate the presence of rare coding enzymatic loss-of-function variants in humans, with implications for our understanding of LRRK2 as a driver of disease and as a drug target.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70190"},"PeriodicalIF":5.2,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12183101/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144369172","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":"An iron-sulfur cluster as a new metal center in a flavodiiron protein.","authors":"Maria C Martins, Célia M Silveira, Miguel Teixeira, Filipe Folgosa","doi":"10.1002/pro.70204","DOIUrl":"10.1002/pro.70204","url":null,"abstract":"<p><p>Syntrophomonas wolfei contains two distinct multiple domain flavodiiron proteins (FDPs) of Classes H and E, presumably acting as oxygen reductases to protect this anaerobic bacterium from oxidative stress due to exposure to environments containing, even if only transiently, oxygen. The Class E FDP was previously predicted by us to have, besides the two core domains characteristic of this type of enzymes, an extra C-terminal domain putatively harboring an iron-sulfur center. This C-terminal domain is exclusive to this class of FDPs and has homology with a protein domain family \"Fer4_19\" which may contain a [3Fe-4S]^1+/0 or a [4Fe-4S]^2+/1+ cluster. In this work, we extensively characterized the enzyme from S. wolfei (wild type, site-directed mutants, and truncated iron-sulfur domain) and showed unequivocally, using EPR and Resonance Raman spectroscopies, that indeed it contains a [3Fe-4S]^1+/0 center, a novelty in the field of FDPs. Structure prediction using Alphafold2 indicated some similarities of the FeS domain to [3Fe-4S]^1+/0 containing ferredoxins. The identification of this new type of redox center associated with an FDP could represent the first step towards identifying a novel electron transfer chain within this protein family. Additionally, the spectroscopic characterization of the FMN from the flavodoxin-like domain suggests that the semiquinone form is the active reduced state of this flavin cofactor. Furthermore, the presence of a minor species possibly associated with the flavin moiety was identified, displaying a so far undescribed UV-visible spectrum.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70204"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12183323/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144369167","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":"Investigating the unbinding mechanisms and kinetics of MmpL3 inhibitors: A computational study.","authors":"Likun Zhao, Xiuling Ma, Bo Liu, Xiaojun Yao, Huanxiang Liu, Qianqian Zhang","doi":"10.1002/pro.70163","DOIUrl":"10.1002/pro.70163","url":null,"abstract":"<p><p>Mycobacterial membrane protein Large 3 (MmpL3) is responsible for transporting trehalose monomycolates across the inner membrane for cell wall biosynthesis, a process driven by the proton motive force and essential for the survival of Mycobacterium tuberculosis. As a result, MmpL3 has become a promising target for anti-tuberculosis drugs. Although many inhibitors targeting MmpL3 have been discovered, their unbinding mechanisms and kinetics remain poorly understood. In this study, the τ-random acceleration molecular dynamics (τRAMD) and steered molecular dynamics (SMD) methods were employed to investigate the unbinding mechanisms and kinetics of four representative MmpL3 inhibitors: SQ109, AU1235, NITD349, and BM212. Analysis of 320 RAMD dissociation trajectories revealed considerable diversity in the dissociation pathways for these inhibitors, dissociating into intracellular, extracellular, or transmembrane regions. Notably, the H4H5H10 pathway, dissociating to the intracellular region, was the primary route. Also, τRAMD results demonstrated a strong correlation between the computed relative residence times and experimental data. Furthermore, SMD simulations along the H4H5H10 pathway indicated that SQ109, AU1235, and NITD349 disrupted hydrogen bonding with MmpL3 prior to dissociation. Meanwhile, inhibitor BM212 underwent conformational adjustments within the binding pocket. All these inhibitors must traverse the channel formed by Phe255 and Phe644 via the H4H5H10 pathway, necessitating the overcoming of significant energy barriers. Based on these findings, we suggest that enhancing inhibitor interactions with MmpL3, such as through hydrogen bonding or increasing inhibitor size to create larger physical barriers (e.g., interactions with Phe255 and Phe644), may prolong the inhibitors' residence times.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 6","pages":"e70163"},"PeriodicalIF":4.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12079479/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144078802","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 single buried cysteine acts as a hydrophobic stabilizer of a folding intermediate and transition state in the MATH domain of SPOP.","authors":"Livia Pagano, Awa Diop, Valeria Pennacchietti, Mariana Di Felice, Eduarda S Ventura, Julian Toso, Angelo Toto, Stefano Gianni","doi":"10.1002/pro.70138","DOIUrl":"10.1002/pro.70138","url":null,"abstract":"<p><p>Cysteine is a highly conserved amino acid with diverse roles in protein function. Whilst its role in the formation of disulfide bridges is well characterized, the contribution of isolated cysteines in protein folding is by and large unexplored. Here we investigate the impact of cysteine residues on the folding pathway of the MATH domain in the SPOP protein by comparing wild-type and serine mutants. Through kinetic analyses, we demonstrate that a buried cysteine residue stabilizes both an early folding intermediate and the main transition state. Most notably, such effects are disrupted upon substitution with serine but preserved with alanine. These findings suggest that, in certain structural contexts, cysteine behaves as a hydrophobic rather than a polar residue. Our results challenge the traditional classification of cysteine as a polar amino acid and highlight its unique contributions to protein folding, with implications for protein engineering and structural biology.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 6","pages":"e70138"},"PeriodicalIF":5.2,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12076002/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144029017","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}