Protein Science最新文献

{"title":"ShiftScan: A tool for rapid analysis of high-throughput differential scanning fluorimetry data and compound prioritization.","authors":"Samantha C Waterworth, Shilpa R Shenoy, Nirmala D Sharma, Chris Wolcott, Duncan E Donohue, Barry R O'Keefe, John A Beutler","doi":"10.1002/pro.70055","DOIUrl":"10.1002/pro.70055","url":null,"abstract":"<p><p>Differential scanning fluorimetry (DSF) can be an effective high-throughput screening assay in drug discovery for detecting protein-compound interactions that stabilize or destabilize macromolecules. Due to the magnitude and quality of the data produced by this biophysical assay, analyzing and prioritizing compounds from large-scale DSF data sets has proven challenging to the research community. Here, we present ShiftScan-a powerful, stand-alone tool designed for the rapid analysis of DSF data and compound prioritization based on thermal transition patterns. ShiftScan accurately and quickly predicts melting temperatures (Tm values) from both canonical and non-canonical transition patterns, efficiently filtering out spurious data to minimize false positives. We report on the use of this tool for data analysis of screens involving both pure compound and natural product fraction libraries and provide the software to the screening community to aid in the discovery of molecularly-targeted compounds. Instructions for installation and usage of ShiftScan can be found at our GitHub repository: https://github.com/samche42/ShiftScan.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70055"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11848206/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143483816","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":"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}

{"title":"Docking guidance with experimental ligand structural density improves docking pose prediction and virtual screening performance.","authors":"Althea T Hansel-Harris, Andreas F Tillack, Diogo Santos-Martins, Matthew Holcomb, Stefano Forli","doi":"10.1002/pro.70082","DOIUrl":"10.1002/pro.70082","url":null,"abstract":"<p><p>Recent advances in structural biology have led to the publication of a wealth of high-resolution x-ray crystallography (XRC) and cryo-EM macromolecule structures, including many complexes with small molecules of interest for drug design. While it is common to incorporate information from the atomic coordinates of these complexes into docking (e.g., pharmacophore models or scaffold hopping), there are limited methods to directly leverage the underlying density information. This is desirable because it does not rely on the determination of relevant coordinates, which may require expert intervention, but instead interprets all density as indicative of regions to which a ligand may be bound. To do so, we have developed CryoXKit, a tool to incorporate experimental densities from either cryo-EM or XRC as a biasing potential on heavy atoms during docking. Using this structural density guidance with AutoDock-GPU, we found significant improvements in re-docking and cross-docking, important pose prediction tasks, compared with the unmodified AutoDock4 force field. Failures in cross-docking tasks are additionally reflective of changes in the positioning of pharmacophores in the site, suggesting it is a fundamental limitation of transferring information between complexes. We additionally found, against a set of targets selected from the LIT-PCBA dataset, that rescoring of these improved poses leads to better discriminatory power in virtual screenings for selected targets. Overall, CryoXKit provides a user-friendly method for improving docking performance with experimental data while requiring no a priori pharmacophore definition and at virtually no computational expense. Map-modification code available at: https://github.com/forlilab/CryoXKit.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70082"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11854350/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143503771","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":"Nitro-fatty acids-mediated nitroalkylation modulates fine-tuning catalase antioxidant function during salinity stress in plants.","authors":"Mounira Chaki, Lorena Aranda-Caño, Juan C Begara-Morales, Beatriz Sánchez-Calvo, Francisco Javier López-Jaramillo, María N Padilla, Raquel Valderrama, José Rafael Pedrajas, Juan B Barroso","doi":"10.1002/pro.70076","DOIUrl":"10.1002/pro.70076","url":null,"abstract":"<p><p>Nitro-fatty acids (NO₂-FAs) are novel molecules resulting from the interaction of unsaturated fatty acids and nitric oxide (NO) or NO-related molecules. In plants, it has recently been described that NO₂-FAs trigger a powerful antioxidant and defense response against stressful situations, the induction of the heat-shock response (HSR), and they exert their signaling function mainly through a reversible post-translational modification called nitroalkylation. Catalase (CAT) is a key antioxidant enzyme for the control of the hydrogen peroxide (H₂O₂) levels generated by environmental oxidative stress. The data presented in this study provide novel information on the role of NO₂-FAs in modulating the antioxidant activity of catalase 2 (CAT2) during salinity stress in Arabidopsis thaliana. Initially, in vitro treatment with nitro-linolenic acid (NO₂-Ln) down-regulated Arabidopsis CAT2 activity, as a consequence of the nitroalkylation of His 156 and His 248, evolutionarily conserved residues with key functional implications for the quaternary structure and hence CAT2 activity. Any effect of NO₂-Ln on the heme group or S-nitrosylation of CAT2 was excluded. To further our knowledge of the regulatory mechanism of this antioxidant enzyme by nitroalkylation, the functional modulation of CAT by NO₂-FAs was analyzed in 5-day-old Arabidopsis cell suspension cultures subjected to salinity stress. In this situation, the oxidative stress generated caused the nitroalkylation of these residues to disappear through the cleavage of NO₂-Ln binding to CAT2, thus restoring CAT2 catalytic activity. Thus, during salinity stress, CAT2 enzymatic activity increased without changes in protein levels. These results highlight the amino acid targets that are susceptible to nitroalkylation and the modulatory effect of this post-translational modification on CAT2 enzymatic activity in vitro and in vivo. These findings underline the regulatory role of nitroalkylation in CAT2 functionality, which is strongly influenced by the redox state thus becoming a new key control mechanism of this antioxidant enzyme in abiotic stress cell response processes.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70076"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11862108/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143503775","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":"Human recombinant tyrosinase destabilization caused by the double mutation R217Q/R402Q.","authors":"Sarah Toay, Narin Sheri, Ian MacDonald, Yuri V Sergeev","doi":"10.1002/pro.70029","DOIUrl":"10.1002/pro.70029","url":null,"abstract":"<p><p>Oculocutaneous albinism is an autosomal recessive inherited disorder associated with mutations in the TYR gene. A single missense change in the tyrosinase (Tyr) could result in partial or complete loss of catalytic activity. The effect of two genetic mutations in the same Tyr as the molecule is less studied. Here, we study single mutation variants, R217Q, R402Q, and a double mutant variant, R217Q/R402Q, to establish a link between alterations at the level of the atomic model of the protein and the disease phenotype. Human recombinant intra-melanosomal Tyr domains of Tyr and three mutant variants were expressed in T. ni. Larvae were purified using the combination of IMAC and SEC, and diphenolase activities were measured. The Tyr homology model was equilibrated using 100 ns molecular dynamics and analyzed using computational methods. The purified R217Q and R217Q/R402Q variants show decreased catalytic activities compared to those of the Tyr and R402Q variants. The R217Q/R402Q variant has the lowest protein activity and is significantly reduced.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70029"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751874/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143010411","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":"α-N-Methyltransferase regiospecificity is mediated by proximal, redundant enzyme-substrate interactions.","authors":"Kathryn K Crone, Jason W Labonte, Mikael H Elias, Michael F Freeman","doi":"10.1002/pro.70021","DOIUrl":"10.1002/pro.70021","url":null,"abstract":"<p><p>N-Methylation of the peptide backbone confers pharmacologically beneficial characteristics to peptides that include greater membrane permeability and resistance to proteolytic degradation. The borosin family of ribosomally synthesized and post-translationally modified peptides offer a post-translational route to install amide backbone α-N-methylations. Previous work has elucidated the substrate scope and engineering potential of two examples of type I borosins, which feature autocatalytic precursors that encode N-methyltransferases that methylate their own C-termini in trans. We recently reported the first discrete N-methyltransferase and precursor peptide from Shewanella oneidensis MR-1, a minimally iterative, type IV borosin that allowed the first detailed kinetic analyses of borosin N-methyltransferases. Herein, we characterize the substrate scope and resilient regiospecificity of this discrete N-methyltransferase by comparison of relative rates and methylation patterns of over 40 precursor peptide variants along with structure analyses of nine enzyme-substrate complexes. Sequences critical to methylation are identified and demonstrated in assaying minimal peptide substrates and non-native peptide sequences for assessment of secondary structure requirements and engineering potential. This work grants understanding towards the mechanism of substrate recognition and iterative activity by discrete borosin N-methyltransferases.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70021"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751858/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143010486","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":"Super-resolution imaging of proteins inside live mammalian cells with mLIVE-PAINT.","authors":"Haresh Bhaskar, Zoe Gidden, Gurvir Virdi, Dirk-Jan Kleinjan, Susan J Rosser, Sonia Gandhi, Lynne Regan, Mathew H Horrocks","doi":"10.1002/pro.70008","DOIUrl":"10.1002/pro.70008","url":null,"abstract":"<p><p>Super-resolution microscopy has revolutionized biological imaging, enabling the visualization of structures at the nanometer length scale. Its application in live cells, however, has remained challenging. To address this, we adapted LIVE-PAINT, an approach we established in yeast, for application in live mammalian cells. Using the 101A/101B coiled-coil peptide pair as a peptide-based targeting system, we successfully demonstrate the super-resolution imaging of two distinct proteins in mammalian cells, one localized in the nucleus, and the second in the cytoplasm. This study highlights the versatility of LIVE-PAINT, suggesting its potential for live-cell super-resolution imaging across a range of protein targets in mammalian cells. We name the mammalian cell version of our original method mLIVE-PAINT.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70008"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11761688/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143047703","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":"LEA_4 motifs function alone and in conjunction with synergistic cosolutes to protect a labile enzyme during desiccation.","authors":"Vincent Nicholson, Kenny Nguyen, Edith Gollub, Mary McCoy, Feng Yu, Alex S Holehouse, Shahar Sukenik, Thomas C Boothby","doi":"10.1002/pro.70028","DOIUrl":"10.1002/pro.70028","url":null,"abstract":"<p><p>Organisms from all kingdoms of life depend on Late Embryogenesis Abundant (LEA) proteins to survive desiccation. LEA proteins are divided into broad families distinguished by the presence of family-specific motif sequences. The LEA_4 family, characterized by 11-residue motifs, plays a crucial role in the desiccation tolerance of numerous species. However, the role of these motifs in the function of LEA_4 proteins is unclear, with some studies finding that they recapitulate the function of full-length LEA_4 proteins in vivo, and other studies finding the opposite result. In this study, we characterize the ability of LEA_4 motifs to protect a desiccation-sensitive enzyme, citrate synthase (CS), from loss of function during desiccation. We show here that LEA_4 motifs not only prevent the loss of function of CS during desiccation but also that they can do so more robustly via synergistically interactions with cosolutes. Our analysis further suggests that cosolutes induce synergy with LEA_4 motifs in a manner that correlates with transfer free energy. This research advances our understanding of LEA_4 proteins by demonstrating that during desiccation their motifs can protect specific clients to varying degrees and that their protective capacity is modulated by their chemical environment. Our findings extend beyond the realm of desiccation tolerance, offering insights into the interplay between IDPs and cosolutes. By investigating the function of LEA_4 motifs, we highlight broader strategies for understanding protein stability and function.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70028"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751883/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143010423","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":"Translation of the downstream ORF from bicistronic mRNAs by human cells: Impact of codon usage and splicing in the upstream ORF.","authors":"Philippe Paget-Bailly, Alexandre Helpiquet, Mathilde Decourcelle, Roxane Bories, Ignacio G Bravo","doi":"10.1002/pro.70036","DOIUrl":"10.1002/pro.70036","url":null,"abstract":"<p><p>Biochemistry textbooks describe eukaryotic mRNAs as monocistronic. However, increasing evidence reveals the widespread presence and translation of upstream open reading frames preceding the \"main\" ORF. DNA and RNA viruses infecting eukaryotes often produce polycistronic mRNAs and viruses have evolved multiple ways of manipulating the host's translation machinery. Here, we introduce an experimental model to study gene expression regulation from virus-like bicistronic mRNAs in human cells. The model consists of a short upstream ORF and a reporter downstream ORF encoding a fluorescent protein. We have engineered synonymous variants of the upstream ORF to explore large parameter space, including codon usage preferences, mRNA folding features, and splicing propensity. We show that human translation machinery can translate the downstream ORF from bicistronic mRNAs, albeit reporter protein levels are thousand times lower than those from the upstream ORF. Furthermore, synonymous recoding of the upstream ORF exclusively during elongation significantly influences its own translation efficiency, reveals cryptic splice signals, and modulates the probability of downstream ORF translation. Our results are consistent with a leaky scanning mechanism facilitating downstream ORF translation from bicistronic mRNAs in human cells, offering new insights into the role of upstream ORFs in translation regulation.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70036"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751868/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143010482","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}