Protein Science最新文献

{"title":"ExploreTurns: A web tool for the exploration, analysis, and classification of beta turns and structured loops in proteins; application to beta-bulge and Schellman loops, Asx helix caps, beta hairpins, and other hydrogen-bonded motifs.","authors":"Nicholas E Newell","doi":"10.1002/pro.70046","DOIUrl":"10.1002/pro.70046","url":null,"abstract":"<p><p>The most common type of protein secondary structure after the alpha helix and beta sheet is the four-residue beta turn, which plays many key structural and functional roles. Existing tools for the study of beta turns operate in backbone dihedral-angle (Ramachandran) space, which presents challenges for the visualization, comparison and analysis of the wide range of turn conformations. In this work, a new turn-local coordinate system and structural alignment, together with a set of geometric descriptors for turn backbone shape, are incorporated into ExploreTurns, a web facility for the exploration, analysis, geometric tuning and retrieval of beta turns and their contexts which combines the advantages of Ramachandran- and Euclidean-space representations. Due to the prevalence of beta turns in proteins, this facility, supported by its interpreter for a new general nomenclature which classifies H-bonded loop motifs and beta hairpins, serves as an exploratory browser and analysis tool for most loop structure. The tool is applied to the detection of new H-bonded loops, including short and \"double\" Schellman loops, a large family of beta-bulge loops with a range of geometries and H-bond topologies, and other motifs. Other applications presented here include the mapping of sequence preferences in Asx helix N-caps and an investigation of the depth dependence of beta-turn geometry. ExploreTurns, available at www.betaturn.com, should prove useful in research, education, and applications such as protein design, in which an enhanced Euclidean-space picture of turn and motif structure and the ability to identify and tune structures suited to particular requirements may improve performance.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70046"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11836897/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449921","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":"Impact of local unfolding fluctuations on the evolution of regional sequence preferences in proteins.","authors":"Keila Voortman-Sheetz, James O Wrabl, Vincent J Hilser","doi":"10.1002/pro.70015","DOIUrl":"10.1002/pro.70015","url":null,"abstract":"<p><p>The number of distinct structural environments in the proteome (as observed in the Protein Data Bank) may belie an organizing framework, whereby evolution conserves the relative stability of different sequence segments, regardless of the specific structural details present in the final fold. If true, the question arises as to whether the energetic consequences of amino acid substitutions, and thus the frequencies of amino acids within each of these so-called thermodynamic environments, could depend less on what local structure that sequence segment may adopt in the final fold, and more on the local stability of that final structure relative to the unfolded state. To address this question, a previously described ensemble-based approach (the COREX algorithm) was used to define proteins in terms of thermodynamic environments, and the naturally occurring frequencies of amino acids within these environments were used to generate statistical energies (a type of knowledge-based potential). By comparing compatibility scores from the statistical energies with energies calculated using the Rosetta all-atom energy function, we assessed the information overlap between the two approaches. Results revealed a substantial correlation between the statistical scores and those obtained using Rosetta, directly demonstrating that a small number of thermodynamic environments are sufficient to capture the perceived multiplicity of different structural environments in proteins. More importantly, the agreement suggests that regional amino acid distributions within each protein in any proteome have been substantially driven by the evolutionary conservation of the regional differences in stabilities within protein families.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70015"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11837041/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449948","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":"Protein Data Bank Japan: Improved tools for sequence-oriented analysis of protein structures.","authors":"Gert-Jan Bekker, Chioko Nagao, Matsuyuki Shirota, Tsukasa Nakamura, Toshiaki Katayama, Daisuke Kihara, Kengo Kinoshita, Genji Kurisu","doi":"10.1002/pro.70052","DOIUrl":"10.1002/pro.70052","url":null,"abstract":"<p><p>Protein Data Bank Japan (PDBj) is the Asian hub of three-dimensional macromolecular structure data, and a founding member of the worldwide Protein Data Bank. We have accepted, processed, and distributed experimentally determined biological macromolecular structures for over two decades. Although we collaborate with RCSB PDB and BMRB in the United States, PDBe and EMDB in Europe and recently PDBc in China for our data-in activities, we have developed our own unique services and tools for searching, exploring, visualizing and analyzing protein structures. We have recently introduced a new UniProt-integrated portal to provide users with a quick overview of their target protein and shows a recommended structure with integrated data from various internal and external resources. The portal page helps users identify known genomic variations of their protein of interest and provide insights into how these modifications might impact the structure, stability and dynamics of the protein. Furthermore, the portal page also helps users to select the optimal structure to use for further analysis. We have also introduced another service to explore proteins using experimental and computational approaches, which enables experimental structural biologists to increase their insight to help them to more efficiently design their experimental studies. With these new additions, we have enhanced our service portfolio to benefit both experimental and computational structural biologists in their search to interpret protein structures, their dynamics and function.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70052"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11837027/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143450135","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":"FASTIA: A rapid and accessible platform for protein variant interaction analysis demonstrated with a single-domain antibody.","authors":"Ryo Matsunaga, Kouhei Tsumoto","doi":"10.1002/pro.70065","DOIUrl":"10.1002/pro.70065","url":null,"abstract":"<p><p>Antibodies are critical tools in medicine and research, and their affinity for their target antigens is a key determinant of their efficacy. Traditional antibody affinity maturation and interaction analyses are often hampered by time-consuming steps such as cloning, expression, purification, and interaction assays. To address this, we have developed FASTIA (Fast Affinity Screening Technology for Interaction Analysis), a novel platform that integrates rapid gene fragment preparation, cell-free protein synthesis, and bio-layer interferometry with non-regenerative analysis. Using this approach, we can analyze the intermolecular interactions of over 20 variants over 2 days, requiring only the parent protein expression plasmid and basic equipment. We have demonstrated the ability of FASTIA to discriminate between single-domain antibody variants with different binding affinities using the anti-HEL VHH antibody D2-L29, and mapped the results to the crystal structure to identify key interaction sites. FASTIA provides results comparable to those obtained using traditional methods. Our system bypasses the need for genetic engineering facilities and can be easily adopted by laboratories, accelerating the protein engineering and optimization processes. In addition, FASTIA is applicable to other protein-protein interactions, making it a versatile tool for studying molecular recognition. FASTIA facilitates efficient affinity maturation, protein engineering, and analysis of protein-protein interactions. This provides a rapid and accessible route for improving antibodies and a broader understanding of protein interactions.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70065"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11843469/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143468938","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":"Taming interleukin-12: Engineering of bispecific antibody-based IL-12 mimetics with biased agonism capacities.","authors":"Britta Lipinski, Laura Unmuth, Paul Arras, Ron Endruszeit, Stefan Becker, Jonathan Mathias Rödel, Jürgen Scheller, Silke Pudewell, Doreen M Floss, Simon Krah, Julia Harwardt, Achim Doerner, Laura Helming, Chunxiao Xu, Andreas Menrad, Andreas Evers, Harald Kolmar, Desislava Elter, Lukas Pekar, Stefan Zielonka","doi":"10.1002/pro.70072","DOIUrl":"10.1002/pro.70072","url":null,"abstract":"<p><p>In this work, we have generated bispecific interleukin (IL)-12 surrogate agonists based on camelid-derived single-domain antibodies (sdAbs) targeting the IL-12 receptor (IL-12R) subunits IL-12Rβ1 and IL-12Rβ2. Following immunization and antibody display-based paratope isolation, respective sdAbs were combinatorially reformatted into a monovalent bispecific architecture by grafting resulting paratopes onto the hinge region of a heterodimeric Fc region. Functional characterization using NK-92 cells enabled the identification of multiple different sdAb-based bispecifics displaying divergent IL-12R agonism capacities as analyzed by STAT4 phosphorylation. Further investigations by harnessing peripheral blood mononuclear cells (PBMCs) from healthy donors revealed attenuated pSTAT4 activation compared to recombinant human (rh) wild-type IL-12 regarding both natural killer (NK)-cell and T-cell activation but robust IL-12R agonism on stimulated T cells. While several sdAb-based IL-12 mimetics were nearly inactive on NK cells as well as T cells obtained from PBMCs, they elicited significant STAT4 phosphorylation and interferon (IFN)-γ release on stimulated T cells as well as an IL-12-like transcriptional signature. Furthermore, we demonstrate that the activity of receptor agonism of generated bispecific IL-12 mimetics can also be biased towards stimulated T cells by changing the spatial orientation of the individual sdAbs within the molecular design architecture. Taken together, we present an alternative strategy to generate IL-12-like biologics with tailor-made characteristics.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70072"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11843475/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143468896","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":"Controlled by disorder: Phosphorylation modulates SRSF1 domain availability for spliceosome assembly.","authors":"Talia Fargason, Erin Powell, Naiduwadura Ivon Upekala De Silva, Trenton Paul, Zihan Zhang, Peter Prevelige, Jun Zhang","doi":"10.1002/pro.70070","DOIUrl":"10.1002/pro.70070","url":null,"abstract":"<p><p>Serine/arginine-rich splicing factor 1 (SRSF1) is key in the mRNA lifecycle including transcription, splicing, nonsense-mediated decay, and nuclear export. Consequently, its dysfunction is linked to cancers, viral evasion, and developmental disorders. The functionality of SRSF1 relies on its interactions with other proteins and RNA molecules. These processes are regulated by phosphorylation of its unstructured arginine/serine-rich tail (RS). Here, we characterize how phosphorylation affects SRSF1's protein and RNA interaction and phase separation. Using NMR paramagnetic relaxation enhancement and chemical shift perturbation, we find that when unphosphorylated, SRSF1's RS interacts with its first RNA-recognition motif (RRM1). Phosphorylation of RS decreases its interactions with the protein-binding site of RRM1 and increases its interactions with the RNA-binding site of RRM1. This change in SRSF1's intramolecular interactions increases the availability of protein-interacting sites on RRM1 and weakens RNA binding of SRSF1. Phosphorylation alters the phase separation of SRSF1 by diminishing the role of arginine in intermolecular interactions. These findings provide an unprecedented view of how SRSF1 influences the early-stage spliceosome assembly.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 3","pages":"e70070"},"PeriodicalIF":4.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11836896/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449916","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":"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":"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}