Protein Science最新文献

{"title":"Insights into the low-temperature adaptation of an enzyme as studied through ancestral sequence reconstruction.","authors":"Shuang Cui, Subrata Dasgupta, Sota Yagi, Madoka Kimura, Ryutaro Furukawa, Shunsuke Tagami, Satoshi Akanuma","doi":"10.1002/pro.70071","DOIUrl":"10.1002/pro.70071","url":null,"abstract":"<p><p>For billions of years, enzymes have evolved in response to the changing environments in which their host organisms lived. Various lines of evidence suggest the earliest primitive organisms inhabited high-temperature environments and possessed enzymes adapted to such conditions. Consequently, extant mesophilic and psychrophilic enzymes are believed to have adapted to lower temperatures during the evolutionary process. Herein, we analyzed this low-temperature adaptation using ancestral sequence reconstruction. Previously, we generated the phylogenetic tree of 3-isopropylmalate dehydrogenases (IPMDHs) and reconstructed the sequence of the last bacterial common ancestor. The corresponding ancestral enzyme displayed high thermostability and catalytic activity at elevated temperatures but moderate activity at low temperatures (Furukawa et al., Sci. Rep., 2020;10:15493). Here, to identify amino acid residues that are responsible for the low-temperature adaptation, we reconstructed and characterized all 11 evolutionary intermediates that sequentially connect the last bacterial common ancestor with extant mesophilic IPMDH from Escherichia coli. A remarkable change in catalytic properties, from those suited for high reaction temperatures to those adapted for low temperatures, occurred between two consecutive evolutionary intermediates. Using a combination of sequence comparisons between ancestral proteins and site-directed mutagenesis analyses, three key amino acid substitutions were identified that enhance low-temperature catalytic activity. Intriguingly, amino acid substitutions that had the most significant impact on activity at low temperatures displayed no discernable effect on thermostability. However, these substitutions markedly reduced the activation energy for catalysis, thereby improving low-temperature activity. The results were further investigated by molecular dynamics simulations of the predicted structures of the ancestral enzymes. Our findings exemplify how ancestral sequence reconstruction can identify residues crucial for adaptation to low temperatures.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70071"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11836894/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449882","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":"Folding of a tandemly knotted protein: Evidence that a polypeptide chain can get out of deep kinetic traps.","authors":"Hongyu Zhang, Sophie E Jackson","doi":"10.1002/pro.70048","DOIUrl":"10.1002/pro.70048","url":null,"abstract":"<p><p>It is hard to imagine how proteins can thread and form knots in their polypeptide chains, but they do. These topologically complex structures have challenged the traditional protein folding views of simple funnel-shaped energy landscapes. Previous experimental studies on the folding mechanisms of deeply knotted proteins with a single trefoil knot have yielded evidence that this topology has a more complicated folding landscape than other simpler proteins. However, to date, there have been no attempts to study the folding of any protein in which multiple threading events are needed to create more than one knot within a single polypeptide chain. Here, we report the construction and characterization of an artificial tandemly knotted protein. We find compelling evidence that both domains of the protein form trefoil knots with similar structures and stabilities to the parent single trefoil-knotted protein. In addition, we show that this tandemly knotted protein has a complex folding pathway in which there are additional very slow folding phases that we propose correspond to the formation of the second knot within the system. We also find evidence that during folding this protein gets transiently trapped in deep kinetic traps, however, the majority of protein chains (>90%) manage to partially unfold and acquire the native tandem-knot topology. This work highlights the fact that Nature can tolerate more complex protein topologies than we thought, and despite considerable misfolding during folding, protein chains can find their way to the native state even in the absence of molecular chaperones.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70048"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11837048/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449940","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":"Torsional twist of the SARS-CoV and SARS-CoV-2 SUD-N and SUD-M domains.","authors":"Monica Rosas-Lemus, George Minasov, Joseph S Brunzelle, Taha Y Taha, Sofia Lemak, Shaohui Yin, Ludmilla Shuvalova, Julia Rosecrans, Kanika Khanna, H Steven Seifert, Alexei Savchenko, Peter J Stogios, Melanie Ott, Karla J F Satchell","doi":"10.1002/pro.70050","DOIUrl":"10.1002/pro.70050","url":null,"abstract":"<p><p>Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vesicle that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated but rather have significant rotational flexibility. Phylogenetic analysis supports that the potential to form the disulfide bond is common across betacoronavirus isolates from many bat species and civets, but also one or both of the cysteines that form the disulfide bond are absent across isolates from bats and pangolins. The absence of these cysteines does not impact viral replication or protein translation.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70050"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11837046/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143450198","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":"Conformational flexibility associated with remote residues regulates the kinetic properties of glutamate dehydrogenase.","authors":"Barsa Kanchan Jyotshna Godsora, Parijat Das, Prasoon Kumar Mishra, Anjali Sairaman, Sandip Kaledhonkar, Narayan S Punekar, Prasenjit Bhaumik","doi":"10.1002/pro.70038","DOIUrl":"10.1002/pro.70038","url":null,"abstract":"<p><p>Glutamate dehydrogenase (GDH) is a pivotal metabolic enzyme in all living organisms, and some of the GDHs exhibit substrate-dependent homotropic cooperativity. However, the mode of allosteric communication during the homotropic effect in GDHs remains poorly understood. In this study, we examined two homologous GDHs, Aspergillus niger GDH (AnGDH) and Aspergillus terreus GDH (AtGDH), with differing substrate utilization kinetics to uncover the factors driving their distinct behavior. We report the crystal structures and first-ever cryo-EM structures of apo- AtGDH and AnGDH that captured arrays of conformational ensembles. A wider mouth opening (~ 21 Å) is observed for the cooperative AnGDH as compared to the non-cooperative AtGDH (~17 Å) in their apo states. A network of interactions related to the substitutions in Domain II influence structural flexibility in these GDHs. Remarkably, we have identified a distant substitution (R246 to S) in Domain II, as a part of this network, which can impact the mouth opening and converts non-cooperative AtGDH into a cooperative enzyme. Our study demonstrates that remote residues can influence structural and kinetic properties in homologous GDHs.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70038"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11843732/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143468919","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 accelerated molecular dynamics study for investigating protein pathways using the bond-boost hyperdynamics method.","authors":"Soon Woo Park, Moon-Ki Choi, Byung Ho Lee, Sangjae Seo, Woo Kyun Kim, Moon Ki Kim","doi":"10.1002/pro.70073","DOIUrl":"10.1002/pro.70073","url":null,"abstract":"<p><p>Molecular dynamics (MD) simulation is an important tool for understanding protein dynamics and the thermodynamic properties of proteins. However, due to the high computational cost of MD simulations, it is still challenging to explore a wide conformational space. To solve this problem, a variety of accelerated MD (aMD) schemes have been proposed over the past few decades. The bond-boost method (BBM) is one of such aMD schemes, which expedites escape events from energy basins by adding a bias potential based on changes in bond length. In this paper, we present a new methodology based on the BBM for accelerating the conformational transition of proteins. In our modified BBM, the bias potential is constructed using the dihedral angle and hydrogen bond, which are more suitable variables to monitor the conformational change in proteins. Additionally, we have developed an efficient algorithm compatible with the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) package. The method is validated with the conformational change of ribose binding protein and adenylate kinase by comparing the conventional and accelerated MD simulation results. Based on the aMD results, the characteristics of the proteins are investigated by monitoring the conformational transition pathways. Moreover, the free energy landscape calculated using umbrella sampling confirms all the states identified by the aMD simulation are the free energy minima, and the system makes transitions following the path indicated by the free energy landscape. Our efficient approach is expected to play a key role in investigating transition pathways in a wide range of protein simulations.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70073"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11854359/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143503768","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 the molecular grammar and the structural transitions underlying the fibrillation of a viral fibrillogenic domain.","authors":"Frank Gondelaud, Julien Leval, Lisha Arora, Anuja Walimbe, Christophe Bignon, Denis Ptchelkine, Stefania Brocca, Samrat Mukhopadyay, Sonia Longhi","doi":"10.1002/pro.70068","DOIUrl":"10.1002/pro.70068","url":null,"abstract":"<p><p>Hendra virus (HeV) is a biosafety level 4 human pathogen belonging to the Henipavirus genus within the Paramyxoviridae family. In HeV, the phosphoprotein-encoding gene also drives the synthesis of the V and W proteins that are two major players in the host innate immune response evasion. These three proteins share a common intrinsically disordered N-terminal domain (NTD) and have distinct C-terminal domains. We recently reported the ability of a short region (i.e., PNT3), located within the shared NTD, to form fibrils. We subsequently identified a PNT3 motif (EYYY) critically involved in fibrillation and deciphered the contribution of each tyrosine to the process. Herein, we combined mutational studies with various biochemical and biophysical approaches to further investigate the molecular mechanisms underlying PNT3 fibrillation. The results show that (i) lysine residues play a critical role in driving fibrillation, (ii) hydrophobic residues affect the nucleation step, and (iii) charge distribution strongly affects the fibrillation propensities. Vibrational Raman spectroscopy data further validated the role of lysine residues in promoting fibrillation and enabled documenting the formation of cross-β amyloid structures. Altogether, these results illuminate the molecular mechanisms involved in fibril formation and pave the way towards the rational design of inhibitors.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70068"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11845978/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143477089","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":"Interfacing bacterial microcompartment shell proteins with genetically encoded condensates.","authors":"Michele Costantino, Eric J Young, Abesh Banerjee, Cheryl A Kerfeld, Giovanna Ghirlanda","doi":"10.1002/pro.70061","DOIUrl":"10.1002/pro.70061","url":null,"abstract":"<p><p>Condensates formed by liquid-liquid phase separation are promising candidates for the development of synthetic cells and organelles. Here, we show that bacterial microcompartment shell proteins from Haliangium ochraceum (BMC-H) assemble into coatings on the surfaces of protein condensates formed by tandem RGG-RGG domains, an engineered construct derived from the intrinsically disordered region of the RNA helicase LAF-1. WT BMC-H proteins formed higher-order assemblies within RGG-RGG droplets; however, engineered BMC-H variants fused to RGG truncations formed coatings on droplet surfaces. These intrinsically disordered tags controlled the interaction with the condensed phase based on their length and sequence, and one of the designs, BMC-H-T2, assembled preferentially on the surface of the droplet and prevented droplet coalescence. The formation of the coatings is dependent on the pH and protein concentration; once formed, the coatings are stable and do not exchange with the dilute phase. Coated droplets could sequester and concentrate folded proteins, including TEV protease, with selectivity similar to uncoated droplets. Addition of TEV protease to coated droplets resulted in the digestion of RGG-RGG to RGG and a decrease in droplet diameter, but not in the dissolution of the coatings. BMC shell protein-coated protein condensates are entirely encodable and provide a way to control the properties of liquid-liquid phase-separated compartments in the context of synthetic biology.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70061"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11837282/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449969","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":"PEG-mCherry interactions beyond classical macromolecular crowding.","authors":"Liam Haas-Neill, Khalil Joron, Eitan Lerner, Sarah Rauscher","doi":"10.1002/pro.5235","DOIUrl":"10.1002/pro.5235","url":null,"abstract":"<p><p>The dense cellular environment influences bio-macromolecular structure, dynamics, interactions, and function. Despite advancements in understanding protein-crowder interactions, predicting their precise effects on protein structure and function remains challenging. Here, we elucidate the effects of PEG-induced crowding on the fluorescent protein mCherry using molecular dynamics simulations and fluorescence-based experiments. We identify and characterize specific PEG-induced structural and dynamical changes in mCherry. Importantly, we find interactions in which PEG molecules wrap around specific surface-exposed residues in a binding mode previously observed in protein crystal structures. Fluorescence correlation spectroscopy experiments capture PEG-induced changes, including aggregation, suggesting a potential role for the specific PEG-mCherry interactions identified in simulations. Additionally, mCherry fluorescence lifetimes are influenced by PEG and not by the bulkier crowder dextran or by another linear polymer, polyvinyl alcohol, highlighting the importance of crowder-protein soft interactions. This work augments our understanding of macromolecular crowding effects on protein structure and dynamics.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e5235"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11836898/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Functional characterization of two AA10 lytic polysaccharide monooxygenases from Cellulomonas gelida.","authors":"Rosaliina Turunen, Tina R Tuveng, Zarah Forsberg, Valerie C Schiml, Vincent G H Eijsink, Magnus Ø Arntzen","doi":"10.1002/pro.70060","DOIUrl":"10.1002/pro.70060","url":null,"abstract":"<p><p>Lytic polysaccharide monooxygenases (LPMOs) are redox enzymes targeting the crystalline region of recalcitrant polysaccharides such as cellulose and chitin. Functional characterization of two LPMOs from the cellulose-degrading soil bacterium Cellulomonas gelida, CgLPMO10A and CgLPMO10B, showed expected activities on cellulose but also revealed novel features of AA10 LPMOs. While clustering together with strictly C1-oxidizing and strictly cellulose-active AA10 LPMOs, CgLPMO10A exhibits activity on both cellulose and chitin, oxidizing the C1 carbon of both substrates. This combination of substrate and oxidative specificity has not been previously observed for family 10 LPMOs and may be due to a conspicuous divergence in two hydrophobic residues on the substrate-binding surface. CgLPMO10B oxidizes cellulose at both the C1 and C4 positions and is also active on chitin, in line with predictions based on phylogeny. Interestingly, while coming from the same organism and both acting on cellulose, the two enzymes have markedly different redox properties with CgLPMO10B displaying the lowest redox potential and the highest oxidase activity observed for an AA10 LPMO so far. These results provide insight into the LPMO machinery of C. gelida and expand the known catalytic repertoire of bacterial LPMOs.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70060"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11837042/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449944","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":"Understanding dry proteins and their protection with solid-state hydrogen-deuterium exchange.","authors":"Julia A Brom, Gary J Pielak","doi":"10.1002/pro.70075","DOIUrl":"10.1002/pro.70075","url":null,"abstract":"<p><p>Protein-based drugs are among our most powerful therapeutics, but their manufacture, storage, and distribution are hindered by solution instability and the expense of the necessary refrigeration. Formulating proteins as dry products, which is an almost entirely empirical endeavor, can ameliorate the problem, but recovery of an acceptable product upon resuspension is not always possible. Additional knowledge about dry protein structure and protection is necessary to make dry formulation both more rational and effective. While most biophysical and biochemical techniques necessitate solvated protein, solid-state hydrogen-deuterium exchange enables the study of dry proteins. Fourier-transform infrared spectroscopy, mass spectrometry, and liquid-observed vapor exchange nuclear magnetic resonance have all been used to measure isotopic exchange. These methods report on secondary structure, peptide, and residue level exposure, respectively. Recent studies using solid-state hydrogen-deuterium exchange provide insight into the mechanisms of dry protein protection and uncover stabilizing and destabilizing interactions, bringing us closer to rational formulation of these lifesaving products.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70075"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11854353/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143503777","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}