Protein Science最新文献

{"title":"AlphaFold2's training set powers its predictions of some fold-switched conformations.","authors":"Joseph W Schafer, Lauren L Porter","doi":"10.1002/pro.70105","DOIUrl":"10.1002/pro.70105","url":null,"abstract":"<p><p>AlphaFold2 (AF2), a deep-learning-based model that predicts protein structures from their amino acid sequences, has recently been used to predict multiple protein conformations. In some cases, AF2 has successfully predicted both dominant and alternative conformations of fold-switching proteins, which remodel their secondary and/or tertiary structures in response to cellular stimuli. Whether AF2 has learned enough protein folding principles to reliably predict alternative conformations outside of its training set is unclear. Previous work suggests that AF2 predicted these alternative conformations by memorizing them during training. Here, we use CFold-an implementation of the AF2 network trained on a more limited subset of experimentally determined protein structures-to directly test how well the AF2 architecture predicts alternative conformations of fold switchers outside of its training set. We tested CFold on eight fold switchers from six protein families. These proteins-whose secondary structures switch between α-helix and β-sheet and/or whose hydrogen bonding networks are reconfigured dramatically-had not been tested previously, and only one of their alternative conformations was in CFold's training set. Successful CFold predictions would indicate that the AF2 architecture can predict disparate alternative conformations of fold-switched conformations outside of its training set, while unsuccessful predictions would suggest that AF2 predictions of these alternative conformations likely arise from association with structures learned during training. Despite sampling 1300-4300 structures/protein with various sequence sampling techniques, CFold predicted only one alternative structure outside of its training set accurately and with high confidence while also generating experimentally inconsistent structures with higher confidence. Though these results indicate that AF2's current success in predicting alternative conformations of fold switchers stems largely from its training data, results from a sequence pruning technique suggest developments that could lead to a more reliable generative model in the future.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70105"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934219/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143701391","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 suite of pre-assembled, pET28b-based Golden Gate vectors for efficient protein engineering and expression.","authors":"Deepika Gaur, Matthew L Wohlever","doi":"10.1002/pro.70106","DOIUrl":"10.1002/pro.70106","url":null,"abstract":"<p><p>Expression and purification of recombinant proteins in Escherichia coli is a bedrock technique in biochemistry and molecular biology. Expression optimization requires testing different combinations of solubility tags, affinity purification techniques, and site-specific proteases. This optimization is laborious and time-consuming as these features are spread across different vector series and require different cloning strategies with varying efficiencies. Modular cloning kits based on the Golden Gate system exist, but they are not optimized for protein biochemistry and are overly complicated for many applications, such as undergraduate research or simple screening of protein purification features. An ideal solution is for a single gene synthesis or PCR product to be compatible with a large series of pre-assembled Golden Gate vectors containing a broad array of purification features at either the N or C terminus. To our knowledge, no such system exists. To fulfill this unmet need, we Golden Gate domesticated the pET28b vector and developed a suite of 21 vectors with different combinations of purification tags, solubility domains, visualization/labeling tags, and protease sites. We also developed a vector series with nine different N-terminal tags and no C-terminal cloning scar. The system is modular, allowing users to easily customize the vectors with their preferred combinations of features. To allow for easy visual screening of cloned vectors, we optimized constitutive expression of the fluorescent protein mScarlet3 in the reverse strand, resulting in a red to white color change upon successful cloning. Testing with the model protein sfGFP shows the ease of visual screening, high efficiency of cloning, and robust protein expression. These vectors provide versatile, high-throughput solutions for protein engineering and functional studies in E. coli.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70106"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934214/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143701388","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":"Lysine carbamoylation during urea denaturation remodels the energy landscape of human transthyretin dissociation linked to unfolding.","authors":"Marcus Jäger, David E Mortenson, Maziar S Ardejani, Gabriel M Kline, Maria T Dendle, Nicholas L Yan, Evan T Powers, Martin Gruebele, Jeffery W Kelly","doi":"10.1002/pro.70009","DOIUrl":"10.1002/pro.70009","url":null,"abstract":"<p><p>Chemical denaturants such as urea have become indispensable in modern protein science for measuring the energetics of protein folding and assembly. Denaturants bind to and preferentially stabilize denatured states, folding transition states, and folding intermediates over the native state, allowing experimental access to free energies of folding and insights into folding mechanisms. However, too little attention is paid to the established chemical instability of aqueous urea, that is, its decomposition into the reactive electrophile ammonium cyanate or isocyanic acid depending on the solution pH. Protein carbamoylation by cyanate/isocyanic acid can change the dissociation and/or unfolding free energy landscape of the protein under study with time. This problem is exemplified using the human blood protein transthyretin (TTR), a kinetically stable transporter of thyroid hormone and holo-retinol binding protein. The dissociation, misfolding, and aggregation of TTR are associated with a prominent human amyloid disease. We demonstrate that modification of TTR by cyanate reshapes the energy landscape of TTR tetramer dissociation and unfolding on multiple time scales. Like certain halide anions and the more chemically inert thiocyanate anion, cyanate binds weakly and non-covalently to the thyroid hormone binding interface in the TTR tetramer. The close proximity of the bound cyanate ion to the pK_a-perturbed lysine 15 ε-amino side chain nucleophile in the thyroid hormone binding sites of TTR favors carbamoylation of this nitrogen. Lysine 15 ε-amino carbamoylation substantially slows down TTR tetramer dissociation mediated by urea denaturation, thus introducing kinetic heterogeneity early in the unfolding reaction. Slower carbamoylation of the subpopulation of other, less pK_a-perturbed lysine ε-amino groups hastens tetramer unfolding, leading to non-exponential, sigmoidal unfolding trajectories. We thus demonstrate that lysine carbamoylation in urea solutions can strongly alter protein unfolding energetics and the mechanism of unfolding.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70009"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934213/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143701395","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":"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}