Protein Science最新文献

{"title":"N-terminomics and proteomics analysis of Calpain-2 reveal key proteolytic processing of metabolic and cell adhesion proteins.","authors":"Anjali Kapilan, Mitchell Bulluss, Alexander R Ziegler, Mohamed Dabaja, Afshin Derakhshani, Anthonia Anowai, Victoria Armstrong, Rhiannon Campden, Daniel Young, Young Joo Sun, Nichollas E Scott, Laura E Edgington-Mitchell, Vinit B Mahajan, Antoine Dufour","doi":"10.1002/pro.70144","DOIUrl":"https://doi.org/10.1002/pro.70144","url":null,"abstract":"<p><p>Aberrant levels of the cysteine protease Calpain-2 have been linked to neurodegeneration, inflammation, and cancer, yet our understanding of this protease and its substrates remains limited. Systematic studies to identify Calpain-2 substrates have been largely confined to peptide libraries or in vitro studies, which fail to represent physiological cellular conditions and physiologically relevant substrates. To identify existing and novel Calpain-2 substrates, we used a genetic approach to knockout Calpain-2 in the THP-1 human monocyte-like cells, followed by proteomic and N-terminomic/TAILS mass spectrometry approaches to identify Calpain-2 substrates. We identified 51 substrates that may be cleaved directly by Calpain-2 or indirectly by downstream proteases. The direct cleavage of selected substrates by Calpain-2 was confirmed using in vitro assays. Finally, metabolomics analysis identified a role for Calpain-2 in the regulation of pyrimidine and glutathione metabolism. Our unbiased and quantitative mass spectrometry analytical pipeline provides new evidence on the physiological functions of Calpain-2 and its newly identified substrates in THP-1 cells.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70144"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12023407/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144018444","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":"Improving split-HaloTag through computational protein engineering.","authors":"Jonas Wilhelm, Lennart Nickel, Yin-Hsi Lin, Julien Hiblot, Kai Johnsson","doi":"10.1002/pro.70123","DOIUrl":"https://doi.org/10.1002/pro.70123","url":null,"abstract":"<p><p>Split-HaloTag can be used to transform transient molecular interactions into permanent marks through chemical labeling, thereby enabling the recording of transient physiological events in individual cells. However, applications of split-HaloTag-based recorders can be limited by slow labeling rates. To address this issue, we have engineered an improved version of cpHalo∆, the larger fragment of the split-HaloTag system. Using computational techniques, we identified stabilizing point mutations and designed a structured linker connecting the original N and C termini of the circular permutated protein, thereby significantly improving the thermostability and activity of cpHalo∆. These modifications decrease the time and substrate concentrations required for split-HaloTag-based assays and can expand their dynamic range and sensitivity.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70123"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006747/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144034153","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":"Mass balance approximation of unfolding boosts potential-based protein stability predictions.","authors":"Ivan Rossi, Guido Barducci, Tiziana Sanavia, Paola Turina, Emidio Capriotti, Piero Fariselli","doi":"10.1002/pro.70134","DOIUrl":"https://doi.org/10.1002/pro.70134","url":null,"abstract":"<p><p>Predicting protein stability changes upon single-point mutations is crucial in computational biology, with applications in drug design, enzyme engineering, and understanding disease mechanisms. While deep-learning approaches have emerged, many remain inaccessible for routine use. In contrast, potential-like methods, including deep-learning-based ones, are faster, user-friendly, and effective in estimating stability changes. However, most of them approximate Gibbs free-energy differences without accounting for the free-energy changes of the unfolded state, violating mass balance and potentially reducing accuracy. Here, we show that incorporating mass balance as a first approximation of the unfolded state significantly improves potential-like methods. While many machine-learning models implicitly or explicitly use mass balance, our findings suggest that a more accurate unfolded-state representation could further enhance stability change predictions.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70134"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12023412/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144042829","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":"Molecular mechanisms of RaTG13 and SARS-CoV-2 RBD bound to Rhinolophus affinis bat ACE2.","authors":"Chenghai Wang, Min Li, Xiaoyan Nan, Yang Deng, Shilong Fan, Jun Lan","doi":"10.1002/pro.70117","DOIUrl":"https://doi.org/10.1002/pro.70117","url":null,"abstract":"<p><p>The discovery of the RaTG13 coronavirus in Rhinolophus affinis bats in 2013, sharing 96.3% genome homology with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suggested bats as the origin of SARS-CoV-2. Although both human angiotensin-converting enzyme 2 (hACE2) and Rhinolophus affinis bat ACE2 (bACE2-Ra, seven polymorphic variants named 01-07) are known to serve as entry receptors for these coronaviruses, the binding mechanism of RaTG13 receptor binding domain (RBD) bound to bACE2-Ra remains poorly understood. Here, we found that RaTG13 RBD bound to bACE2-Ra-07 with a weaker affinity (2.42 μM) compared with SARS-CoV-2 RBD (372 nM). Additional glycosylation at residue N370 of RaTG13 had little influence on bACE2-Ra-07 binding by RaTG13 RBD. Crystal structures of the SARS-CoV-2 and RaTG13 N370Q RBD bound to bACE2-Ra-07 were solved. Interface analysis and surface plasmon resonance (SPR) assay indicated that residue substitutions at 493, 498, 501, and 505 may play a more important role in the cross-species recognition of bACE2-Ra-07 by the SARS-CoV-2 RBD. Besides, the N370Q mutation enhanced the binding affinity between the RBD of pangolin coronavirus isolated from Guangxi (PCoV-GX) and the bACE2-Ra-07 receptor by over 10-fold. Furthermore, the recently prevalent SARS-CoV-2 variant RBDs extensively retained the interaction with the bACE2-Ra-07 receptor. Our findings give new lights on the cross-species evolution of SARS-CoV-2 and prompt the urgency to monitor the circulation of coronaviruses in bats to better prevent future spillover.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70117"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012733/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144046044","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":"Immobilization and enhancement of a heterodimeric fluorescence biosensor in fibrous protein biomaterials.","authors":"Rebecca M Booth, Amanda Jons, Xue Gong, Shounak Banerjee, Britt Faulk, Hays Rye, Christopher Bystroff, Sarah E Bondos","doi":"10.1002/pro.70119","DOIUrl":"https://doi.org/10.1002/pro.70119","url":null,"abstract":"<p><p>Leave-one-out green fluorescent proteins (LOO_GFPs) have a reduced quantum yield relative to the parent protein and form fluorescent oligomers in the unbound state. Immobilizing LOO_GFPs in materials composed of the Drosophila protein Ultrabithorax (Ubx) via gene fusion increased the fluorescent signal, significantly stabilized the biosensor, and prevented oligomerization into fluorescent aggregates, which has the potential to elevate the sensor's noise well above the signal. Interactions between LOO_GFP and Ubx hampered analyte rebinding. By optimizing the concentrations of LOO_GFP, salt, and detergent in the assay, the signal to noise ratio for the biosensor increased fourfold. These modified fibers represent the first incorporation of a protein complementation assay into protein-based materials, as well as the first incorporation, via gene fusion, of a heterodimeric functional protein into materials composed of a different self-assembling protein. This study highlights the advantages and identifies potential pitfalls associated with protein immobilization in materials.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70119"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012991/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144028844","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":"PackPPI: An integrated framework for protein-protein complex side-chain packing and ΔΔG prediction based on diffusion model.","authors":"Jingkai Zhang, Yuanyan Xiong","doi":"10.1002/pro.70110","DOIUrl":"https://doi.org/10.1002/pro.70110","url":null,"abstract":"<p><p>Deep learning methods have played an increasingly pivotal role in advancing side-chain packing and mutation effect prediction (ΔΔG) for protein complexes. Although these two tasks are inherently closely related, they are typically treated separately in practice. Furthermore, the lack of effective post-processing in most approaches results in sub-optimal refinement of generated conformations, limiting the plausibility of the predicted conformations. In this study, we introduce an integrated framework, PackPPI, which employs a diffusion model and a proximal optimization algorithm to improve side-chain prediction for protein complexes while using learned representations to predict ΔΔG. The results demonstrate that PackPPI achieved the lowest atom RMSD (0.9822) on the CASP15 dataset. The proximal optimization algorithm effectively reduces spatial clashes between side-chain atoms while maintaining a low-energy landscape. Furthermore, PackPPI achieves state-of-the-art performance in predicting binding affinity changes induced by multi-point mutations on the SKEMPI v2.0 dataset. These findings underscore the potential of PackPPI as a robust and versatile computational tool for protein design and engineering. The implementation of PackPPI is available at https://github.com/Jackz915/PackPPI.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70110"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012842/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144064180","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":"Crowding-induced stabilization and destabilization in a single protein.","authors":"Jordyn M Markle, Tarynn D Neal, Hania S Kantzer, Gary J Pielak","doi":"10.1002/pro.70126","DOIUrl":"https://doi.org/10.1002/pro.70126","url":null,"abstract":"<p><p>The protein concentration in cells can reach 300 g/L. These crowded conditions affect protein stability. Classic crowding theories predict entropically driven stabilization, which occurs via steric repulsion, but growing evidence shows a role for non-covalent chemical interactions. To aid our understanding of physiologically relevant crowding, we used NMR-detected ¹H-²H exchange to examine a simple, semi-reductionist system: protein self-crowding at the residue level using the widely studied model globular protein, GB1 (the B1 domain streptococcal protein G) at concentrations up to its solubility limit, 100 g/L. The surprising result is that self-crowding stabilizes some residues but destabilizes others, contradicting predictions. Two other observations are also contradictory. First, temperature-dependence data show that stabilization can arise enthalpically, not just entropically. Second, concentration-dependence data show destabilization often increases with increasing concentration. These results show a key role for chemical interactions. More specifically, self-crowding increases the free energy required to expose those residues that are only exposed upon complete unfolding, and stabilization of these globally unfolding residues increases with GB1 concentration, a result we attribute to repulsive chemical interactions between GB1 molecules. On the other hand, residues exposed upon local unfolding tend to be destabilized, with destabilization increasing with concentration, a result we attribute to attractive chemical interactions between GB1 molecules.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70126"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012844/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144041962","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":"Fine-tuning the yeast GAL10 promoter and growth conditions for efficient recombinant membrane protein production and purification.","authors":"Rebecca Moussa, François Gellé, Sandrine Masscheleyn, Alexandre Pozza, Christel Le Bon, Karine Moncoq, Françoise Bonneté, Bruno Miroux","doi":"10.1002/pro.70125","DOIUrl":"https://doi.org/10.1002/pro.70125","url":null,"abstract":"<p><p>One of the most common issues in producing membrane proteins in heterologous expression systems is the low yield of purified protein. The solubilization efficiency of the recombinant membrane protein from biological membranes is often the limiting step. Here, we study the effects of titration of the GAL10-CYC promoter of Saccharomyces cerevisiae, induction time, and culture media, on the rat mitochondrial uncoupling protein (UCP1) production and solubilization levels. We found that a maximum threshold of solubilized UCP1 (70%) is reached at 0.003% galactose concentration, independently of time, temperature, and detergent-to-protein ratio during solubilization. Supplementation with 0.1% amino acids of the S-lactate medium at induction resumes cell growth and recombinant protein production. The purified UCP1 protein (0.2 mg/L) is homogenous in DDM detergent and active after reconstitution in proteoliposomes. To extend the impact of our findings, we applied the same promoter titration to produce the GFP-AT7B human transporter and found an optimal galactose concentration of 0.0015%. The protein data bank analysis revealed that these galactose concentrations are 300 times lower than usual. We propose a novel strategy for the recombinant production of membrane proteins in the yeast S. cerevisiae, which unlocks the use of this inexpensive eukaryotic host for membrane protein production.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70125"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012841/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144008683","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":"Intramolecular epistasis correlates with divergence of specificity in promiscuous and bifunctional NSAR/OSBS enzymes.","authors":"Dat P Truong, Roopa Dharmatti, Dylan Suriadinata, Jamison Huddleston, Rebecca Skouby, Gladys Owusu Addo, Mingzhao Zhu, Anjana Delpe Acharige, Reethu Sankari Bayana, Cristian Davila, Susan C Fults, Frank M Raushel, Kenneth G Hull, Daniel Romo, Margaret E Glasner","doi":"10.1002/pro.70113","DOIUrl":"https://doi.org/10.1002/pro.70113","url":null,"abstract":"<p><p>Understanding the functions and evolution of specificity-determining residues is essential for improving strategies to predict and design enzyme functions. Whether the function of an amino acid residue is retained during evolution depends on intramolecular epistasis, which occurs when the same residue contributes to different phenotypes in different genetic backgrounds. This study examines the relationship between epistasis and functional divergence by investigating a conserved specificity determinant in five homologs from the N-succinylamino acid racemase (NSAR)/o-succinylbenzoate synthase (OSBS) subfamily. NSAR activity originated as a promiscuous (non-biological) activity of an ancestral OSBS. Some extant NSAR/OSBS subfamily enzymes still have OSBS activity as a biological function and NSAR as a promiscuous activity, while some use both OSBS and NSAR activities as biological functions. Others use only NSAR activity as a biological function but can still catalyze the OSBS reaction as a promiscuous activity. Previously, we determined that the conserved residue R266 in Amycolatopsis sp. T-1-60 NSAR contributes to NSAR specificity by enabling K263 to act as a general acid/base catalyst. Here, we show that mutating R266 decreased relative specificity for NSAR activity in four of five NSAR/OSBS subfamily enzymes, as predicted. However, other phenotypes exhibited epistasis related to the pleiotropy of R266, including the proton exchange rate between the catalytic lysines and the substrate, the impact on OSBS activity, and thermostability. The strength of epistasis was associated with functional and evolutionary divergence of NSAR/OSBS enzymes. These results illustrate the benefits of comparing multiple homologs for understanding mechanisms of enzyme specificity.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70113"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006748/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144009029","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":"Expanding the bioanalytical application of β-hydroxybutyrate binding proteins through characterization of their metabolite interactions and site-directed mutagenesis.","authors":"Bryant J Kane, Kyle V Murphy, Koji Sode","doi":"10.1002/pro.70129","DOIUrl":"https://doi.org/10.1002/pro.70129","url":null,"abstract":"<p><p>β-hydroxybutyrate binding proteins (BHBBPs) are a newly identified group of periplasmic solute-binding proteins (SBPs) that interact with β-hydroxybutyrate (BHB), a key physiological metabolite. In this study, we systematically characterized the interaction properties of both previously reported and newly identified BHBBPs, including \"NovoS\" and \"EDC10\" from Gram-negative bacteria. Following recombinant production, we assessed the specificity and affinity of these proteins against a library of 23 different metabolites using a label-free derivative of differential scanning fluorimetry (nanoDSF). Positive interactions were further evaluated for their binding affinity via tryptophan fluorescence spectroscopy, which confirmed D/L-BHB as the preferred ligand for all proteins, with slight enantioselectivity. BHBBPs also exhibited binding to other compounds such as acetoacetate, D/L-α-hydroxybutyrate, L-lactate, and pyruvate, albeit with reduced affinity. These findings expand the classification of BHBBPs, suggesting that similar proteins and associated transporters may be widespread in prokaryotes involved in the carbon cycle of polyhydroxybutyrate. Guided by the crystal structure of the homologous BMA2936 protein, we introduced targeted point mutations in conserved polar residues of the BHBBPs EDC24 and NovoS. It was determined through this experimental pipeline that their affinity towards BHB was reduced by a factor between 25 and 750, shifting their binding constants towards the millimolar range. Collectively, the affinities of both wild-type and mutant proteins span 4 orders of magnitude, from nanomolar to millimolar recognition of BHB. Leveraging the versatility of SBP-based biosensing, these receptors and their wide affinity range could facilitate the development of effective bioanalytical tools for BHB detection in diverse physiological environments.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70129"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012846/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144051067","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}