Protein Science最新文献_第2页

The multifaceted role of XCL1 in health and disease. XCL1在健康和疾病中的多方面作用。

IF 4.5 3区生物学

Protein Science Pub Date : 2025-02-01 DOI: 10.1002/pro.70032

Muhammed Syed, Acacia F Dishman, Brian F Volkman, Tara L Walker

{"title":"The multifaceted role of XCL1 in health and disease.","authors":"Muhammed Syed, Acacia F Dishman, Brian F Volkman, Tara L Walker","doi":"10.1002/pro.70032","DOIUrl":"10.1002/pro.70032","url":null,"abstract":"The chemokine XC motif chemokine ligand 1 (XCL1) is an unusually specialized member of a conserved family of around 50 small, secreted proteins that are best known for their ability to stimulate the directional migration of cells. All chemokines adopt a very similar folded structure that binds a specific G protein-coupled receptor (GPCR), and most chemokines bind extracellular matrix glycosaminoglycans, often in a dimeric or oligomeric form. Owing in part to the lack of a disulfide bond that is conserved in all other chemokines, XCL1 interconverts between two distinct structures with distinct functions. One XCL1 fold resembles the structure of all other chemokines (chemokine fold), while the other does not (alternate fold). The chemokine fold of XCL1 displays high affinity for the GPCR XCR1, while the alternative fold binds GAGs and exhibits antimicrobial activity. Although the canonical role of XCL1 as a CD8+ dendritic cell chemoattractant was defined more than a decade ago, the misconception that XCL1 is a lymphocyte-specific chemoattractant still prevails in the recent literature. This review aims to highlight the structure-guided functions of XCL1 and reclarify its immunological role. In addition, the implications of this metamorphic chemokine in vaccine development and emerging functions in the nervous system will be explored.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70032"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751857/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143010477","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}

引用次数: 0

GDFold2: A fast and parallelizable protein folding environment with freely defined objective functions.

IF 4.5 3区生物学

Protein Science Pub Date : 2025-02-01 DOI: 10.1002/pro.70041

Tianyu Mi, Nan Xiao, Haipeng Gong

{"title":"GDFold2: A fast and parallelizable protein folding environment with freely defined objective functions.","authors":"Tianyu Mi, Nan Xiao, Haipeng Gong","doi":"10.1002/pro.70041","DOIUrl":"10.1002/pro.70041","url":null,"abstract":"An important step of mainstream protein structure prediction is to model the 3D protein structure based on the predicted 2D inter-residue geometric information. This folding step has been integrated into a unified neural network to allow end-to-end training in state-of-the-art methods like AlphaFold2, but is separately implemented using the Rosetta folding environment in some traditional methods like trRosetta. Despite the inferiority in prediction accuracy, the conventional approach allows for the sampling of various protein conformations compatible with the predicted geometric constraints, partially capturing the dynamic information. Here, we propose GDFold2, a novel protein folding environment, to address the limitations of Rosetta. On the one hand, GDFold2 is highly computationally efficient, capable of accomplishing multiple folding processes in parallel within the time scale of minutes for generic proteins. On the other hand, GDFold2 supports freely defined objective functions to fulfill diversified optimization requirements. Moreover, we propose a quality assessment (QA) model to provide reliable prediction on the quality of protein structures folded by GDFold2, thus substantially simplifying the selection of structural models. GDFold2 and the QA model could be combined to investigate the transition path between protein conformational states, and the online server is available at https://structpred.life.tsinghua.edu.cn/server_gdfold2.html.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70041"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11773392/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053470","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}

引用次数: 0

Investigation into the effect of phenylalanine gating on anaerobic haem breakdown using the energy landscape approach.

IF 4.5 3区生物学

Protein Science Pub Date : 2025-02-01 DOI: 10.1002/pro.5243

Alasdair D Keith, Elizabeth B Sawyer, Desmond C Y Choy, James L Cole, Cheng Shang, George S Biggs, Oskar James Klein, Paul D Brear, David J Wales, Paul D Barker

{"title":"Investigation into the effect of phenylalanine gating on anaerobic haem breakdown using the energy landscape approach.","authors":"Alasdair D Keith, Elizabeth B Sawyer, Desmond C Y Choy, James L Cole, Cheng Shang, George S Biggs, Oskar James Klein, Paul D Brear, David J Wales, Paul D Barker","doi":"10.1002/pro.5243","DOIUrl":"10.1002/pro.5243","url":null,"abstract":"We have recently demonstrated a novel anaerobic NADH-dependent haem breakdown reaction, which is carried out by a range of haemoproteins. The Yersinia enterocolitica protein, HemS, is the focus of further research presented in the current paper. Using conventional experimental methods, bioinformatics, and energy landscape theory (ELT), we provide new insight into the mechanism of the novel breakdown process. Of particular interest is the behavior of a double phenylalanine gate, which opens and closes according to the relative situations of haem and NADH within the protein pocket. This behavior suggests that the double phe-gate fulfills a regulatory role within the pocket, controlling the access of NADH to haem. Additionally, stopped-flow spectroscopy results provide kinetic comparisons between the wild-type and the selected mutants. We also present a fully resolved crystal structure for the F104AF199A HemS monomer, including its extensive loop, the first such structure to be completely resolved for HemS or any of its close homologues. The energy landscapes approach provided key information regarding the gating strategy employed by HemS, compensating for current limitations with conventional biophysical and molecular dynamics approaches. We propose that ELT become more widely used in the field, particularly in the investigation of the dynamics and interactions of weak-binding ligands, and for gating features, within protein cavities.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e5243"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11773379/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053473","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}

引用次数: 0

AFFIPred: AlphaFold2 structure-based Functional Impact Prediction of missense variations. 基于AlphaFold2结构的错义变异功能影响预测。

IF 4.5 3区生物学

Protein Science Pub Date : 2025-02-01 DOI: 10.1002/pro.70030

Mustafa S Pir, Emel Timucin

{"title":"AFFIPred: AlphaFold2 structure-based Functional Impact Prediction of missense variations.","authors":"Mustafa S Pir, Emel Timucin","doi":"10.1002/pro.70030","DOIUrl":"10.1002/pro.70030","url":null,"abstract":"Protein structure holds immense potential for pathogenicity prediction, albeit structure-based predictors are limited compared to the sequence-based counterparts due to the \"structure knowledge gap\" between large number of available protein sequences and relatively limited number of structures. Leveraging the highly accurate protein structures predicted by AlphaFold2 (AF2), we introduce AFFIPred, an ensemble machine learning classifier that combines sequence and AF2-based structural characteristics to predict missense variant pathogenicity. Based on the assessments on unseen datasets, AFFIPred reached a comparable level of performance with the state-of-the-art predictors such as AlphaMissense. We also showed that the recruitment of AF2 structures that are full-length and represent the unbound states ensures more precise SASA calculations compared to the recruitment of experimental structures. In line with the completeness of the AF2 structures, their use provide a more comprehensive view of the structural characteristics of the missense variation datasets by capturing all variants. AFFIPred maintains high-level accuracy without the limitations of PDB-based classifiers. AFFIPred has predicted over 210 million variations of the human proteome, which are accessible at https://affipred.timucinlab.com/.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70030"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751861/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143010354","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}

引用次数: 0

NumSimEX: A method using EXX hydrogen exchange mass spectrometry to map the energetics of protein folding landscapes.

IF 4.5 3区生物学

Protein Science Pub Date : 2025-02-01 DOI: 10.1002/pro.70045

Jasper A G Flint, Jacob Witten, Isabella Han, John Strahan, Jovan Damjanovic, Nevon Song, Tim Poterba, Alexis Jaramillo Cartagena, Angelika Hirsch, Tony Ni, Julie L Sohl, Amy S Wagaman, Sheila S Jaswal

{"title":"NumSimEX: A method using EXX hydrogen exchange mass spectrometry to map the energetics of protein folding landscapes.","authors":"Jasper A G Flint, Jacob Witten, Isabella Han, John Strahan, Jovan Damjanovic, Nevon Song, Tim Poterba, Alexis Jaramillo Cartagena, Angelika Hirsch, Tony Ni, Julie L Sohl, Amy S Wagaman, Sheila S Jaswal","doi":"10.1002/pro.70045","DOIUrl":"10.1002/pro.70045","url":null,"abstract":"Hydrogen exchange mass spectrometry (HXMS) is a powerful tool to understand protein folding pathways and energetics. However, HXMS experiments to date have used exchange conditions termed EX1 or EX2 which limit the information that can be gained compared to the more general EXX exchange regime. If EXX behavior could be understood and analyzed, a single HXMS timecourse on an intact protein could fully map its folding landscape without requiring denaturation. To address this challenge, we developed a numerical simulation method called NumSimEX that models EXX exchange for arbitrarily complex folding pathways. NumSimEx fits protein folding dynamics to experimental HXMS data by iteratively comparing the simulated and experimental timecourses, allowing for determination of both kinetic and thermodynamic protein folding parameters. After analytically verifying NumSimEX's accuracy, we demonstrated its power on HXMS data from beta-2 microglobulin (β2M), a protein involved in dialysis-related amyloidosis. In particular, using NumSimEX, we identified three-state kinetics that near-perfectly matched experimental observation. This proof-of-principle application of NumSimEX sets the stage for harnessing HXMS to expand our understanding of proteins currently excluded from traditional protein folding methods. NumSimEX is freely available at https://github.com/JaswalLab/NumSimEX_Public.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70045"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11761709/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143047692","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}

引用次数: 0

A functional helix shuffled variant of the B domain of Staphylococcus aureus. 金黄色葡萄球菌B结构域的一种功能性螺旋洗牌变体。

IF 4.5 3区生物学

Protein Science Pub Date : 2025-02-01 DOI: 10.1002/pro.70012

Hanna Bobolowski, Erik Fiedler, Ulrich Haupts, Hauke Lilie, Ulrich Weininger

{"title":"A functional helix shuffled variant of the B domain of Staphylococcus aureus.","authors":"Hanna Bobolowski, Erik Fiedler, Ulrich Haupts, Hauke Lilie, Ulrich Weininger","doi":"10.1002/pro.70012","DOIUrl":"10.1002/pro.70012","url":null,"abstract":"The B domain of protein A is a biotechnologically important three-helix bundle protein. It binds the Fc fragment of antibodies with helix 1/2 and the Fab region with helix 2/3. Here we designed a helix shuffled variant by changing the connectivity of the helices, in order to redesign the helix bundle, yielding altered helix-loop-helix properties. The new loops that generate the new connectivity were created in several protein libraries, and Fc binding variants were selected for a detailed biochemical characterization. We were able to create variants with Fc binding affinity at the same level as the wild type B but with significantly reduced thermal stability. The NMR structure proved that the overall three-dimensional structure was maintained not only in the helix shuffled variant but also points to some potential local differences to wild-type B, which could be the reason for the reduced thermal stability. Therefore, protein A is an example of an optimized structure being more important for stability than for function. Using the helix shuffled variant as a ligand on an affinity column facilitates a robust and straightforward purification of antibodies, but allows for a milder elution at less extreme pH. Therefore, the helix shuffled variant is a suitable ligand to purify more pH-sensitive antibodies.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70012"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751873/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143010352","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}

引用次数: 0

Cripto-1 acts as a molecular bridge linking nodal to ALK4 via distinct structural domains. Cripto-1通过不同的结构域作为连接nodal和ALK4的分子桥梁。

IF 4.5 3区生物学

Protein Science Pub Date : 2025-02-01 DOI: 10.1002/pro.70034

Kit-Yee Chu, Amberly N Crawford, Bradon S Krah, Vijayalakshmi Thamilselvan, Anjali Malik, Nina A Aitas, Erik Martinez-Hackert

{"title":"Cripto-1 acts as a molecular bridge linking nodal to ALK4 via distinct structural domains.","authors":"Kit-Yee Chu, Amberly N Crawford, Bradon S Krah, Vijayalakshmi Thamilselvan, Anjali Malik, Nina A Aitas, Erik Martinez-Hackert","doi":"10.1002/pro.70034","DOIUrl":"10.1002/pro.70034","url":null,"abstract":"The TGF-β family ligand Nodal is an essential regulator of embryonic development, orchestrating key processes such as germ layer specification and body axis formation through activation of SMAD2/3-mediated signaling. Significantly, this activation requires the co-receptor Cripto-1. However, despite their essential roles in embryogenesis, the molecular mechanism through which Cripto-1 enables Nodal to activate the SMAD2/3 pathway has remained elusive. Intriguingly, Cripto-1 also has been shown to antagonize other TGF-β family ligands, raising questions about its diverse functions. To clarify how Cripto-1 modulates TGF-β signaling, we integrated AlphaFold3 modeling, surface plasmon resonance (SPR)-based protein-protein interaction analysis, domain-specific anti-Cripto-1 antibodies, and functional studies in NTERA-2 cells. In contrast to canonical TGF-β signaling, where ligands bridge type I and type II receptors for activation, Nodal, bound to the type II receptor, utilizes Cripto-1 to recruit the type I receptor ALK4, forming a unique ternary complex for SMAD2/3 activation. Our molecular characterization of Cripto-1-mediated Nodal signaling clarifies the unique role of this enigmatic co-receptor and advances our understanding of signaling regulation within the TGF-β family. These insights have potential implications for both developmental biology and cancer research.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70034"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751877/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143010403","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}

引用次数: 0

Chemical and temporal manipulation of early steps in protein assembly tunes the structure and intermolecular interactions of protein-based materials.

IF 4.5 3区生物学

Protein Science Pub Date : 2025-02-01 DOI: 10.1002/pro.70000

Valeria Italia, Amanda Jons, Bhavika Kaparthi, Britt Faulk, Marco Maccarini, Paolo Bertoncello, Ken Meissner, Donald K Martin, Sarah E Bondos

{"title":"Chemical and temporal manipulation of early steps in protein assembly tunes the structure and intermolecular interactions of protein-based materials.","authors":"Valeria Italia, Amanda Jons, Bhavika Kaparthi, Britt Faulk, Marco Maccarini, Paolo Bertoncello, Ken Meissner, Donald K Martin, Sarah E Bondos","doi":"10.1002/pro.70000","DOIUrl":"https://doi.org/10.1002/pro.70000","url":null,"abstract":"The Drosophila intrinsically disordered protein Ultrabithorax (Ubx) undergoes a series of phase transitions, beginning with noncovalent interactions between apparently randomly organized monomers, and evolving over time to form increasingly ordered coacervates. This assembly process ends when specific dityrosine covalent bonds lock the monomers in place, forming macroscale materials. Inspired by this hierarchical, multistep assembly process, we analyzed the impact of protein concentration, assembly time, and subphase composition on the early, noncovalent stages of Ubx assembly, which are extremely sensitive to their environment. We discovered that in low salt buffers, we can generate a new type of Ubx material from early coacervates using 5-fold less protein, and 100-fold less assembly time. Comparison of the new materials with standard Ubx fibers also revealed differences in the extent of wrinkling on the fiber surface. A new image analysis technique based on autocorrelation of scanning electron microscopy (SEM) images was developed to quantify these structural differences. These differences extend to the molecular level: new materials form more dityrosine covalent cross-links per monomer, but without requiring the specific tyrosine residues necessary for crosslinking previously established materials. We conclude that varying the assembly conditions represents a facile and inexpensive process for creating new materials. Most new biopolymers are created by changing the composition of the monomers or the method used to drive assembly. In contrast, in this study we used the same monomers and assembly approach, but altered the assembly time and chemical environment to create a new material with unique properties.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70000"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751906/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143060563","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}

引用次数: 0

Allosteric modulation of NF1 GAP: Differential distributions of catalytically competent populations in loss-of-function and gain-of-function mutants. NF1 GAP的变构调节：功能丧失和功能获得突变体中具有催化能力的群体的差异分布。

IF 4.5 3区生物学

Protein Science Pub Date : 2025-02-01 DOI: 10.1002/pro.70042

Liang Xu, Hyunbum Jang, Ruth Nussinov

{"title":"Allosteric modulation of NF1 GAP: Differential distributions of catalytically competent populations in loss-of-function and gain-of-function mutants.","authors":"Liang Xu, Hyunbum Jang, Ruth Nussinov","doi":"10.1002/pro.70042","DOIUrl":"10.1002/pro.70042","url":null,"abstract":"Neurofibromin (NF1), a Ras GTPase-activating protein (GAP), catalyzes Ras-mediated GTP hydrolysis and thereby negatively regulates the Ras/MAPK pathway. NF1 mutations can cause neurofibromatosis type 1 manifesting tumors, and neurodevelopmental disorders. Exactly how the missense mutations in the GAP-related domain of NF1 (NF1GRD) allosterically impact NF1 GAP to promote these distinct pathologies is unclear. Especially tantalizing is the question of how same-domain, same-residue NF1GRD variants exhibit distinct clinical phenotypes. Guided by clinical data, we take up this dilemma. We sampled the conformational ensembles of NF1GRD in complex with GTP-bound K-Ras4B by performing molecular dynamics simulations. Our results show that mutations in NF1GRD retain the active conformation of K-Ras4B but with biased propensities of the catalytically competent populations of K-Ras4B-NF1GRD complex. In agreement with clinical depiction and experimental tagging, compared to the wild type, NF1GRD E1356A and E1356V mutants effectively act through loss-of-function and gain-of-function mechanisms, leading to neurofibromatosis and developmental disorders, respectively. Allosteric modulation of NF1GRD GAP activity through biasing the conformational ensembles in the different states is further demonstrated by the diminished GAP activity by NF1GRD isoform 2, further manifesting propensities of conformational ensembles as powerful predictors of protein function. Taken together, our work identifies a NF1GRD hotspot that could allosterically tune GAP function, suggests targeting Ras oncogenic mutations by restoring NF1 catalytic activity, and offers a molecular mechanism for NF1 phenotypes determined by their distinct conformational propensities.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70042"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751910/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143010372","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}

引用次数: 0

A novel method for expressing and purifying large quantities of functional and stable human voltage-gated proton channel (hH_v1).

IF 4.5 3区生物学

Protein Science Pub Date : 2025-02-01 DOI: 10.1002/pro.70017

Emerson M Carmona, D Marien Cortes, Luis G Cuello

{"title":"A novel method for expressing and purifying large quantities of functional and stable human voltage-gated proton channel (hHv1).","authors":"Emerson M Carmona, D Marien Cortes, Luis G Cuello","doi":"10.1002/pro.70017","DOIUrl":"10.1002/pro.70017","url":null,"abstract":"Purifying membrane proteins has been the limiting step for studying their structure and function. The challenges of the process include the low expression levels in heterologous systems and the requirement for their biochemical stabilization in solution. The human voltage-gated proton channel (hHv1) is a good example of that: the published protocols to express and purify hHv1 produce low protein quantities at high costs, which is an issue for systematically characterizing its structure and function. Based on a pipeline approach, we developed a novel method to produce large quantities of properly folded and fully functional hHv1. We found that using the correct Escherichia coli strain in an autoinduction medium at low temperatures maximized protein expression. Furthermore, solubilization screenings showed that the detergent Anzergent 3-12 was a better alternative than Fos-choline-12 to purify hHv1, considerably reducing the costs. Buffers with high ionic strength increased the protein extracted during detergent solubilization and the stability of hHv1 during downstream processing. Finally, a further improvement was achieved when an enterokinase cutting site was inserted at the N-terminus of the protein. Our novel method produces properly folded and fully functional hHv1, increasing the protein yield by 100 times and reducing the cost by 96% while improving the protein stability compared to the previously published protocols. Our work will accelerate studies on hHv1 and its possible future therapeutic use, while serving as an example for developing purification methodologies for other challenging membrane proteins.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 2","pages":"e70017"},"PeriodicalIF":4.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11761714/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143047670","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}

引用次数: 0