Biochimica et biophysica acta. Proteins and proteomics最新文献

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Identification of the interfacial regions in misfolded transthyretin oligomers 识别折叠错误的转甲状腺素寡聚体中的界面区
IF 3.2 4区 生物学
Biochimica et biophysica acta. Proteins and proteomics Pub Date : 2024-05-23 DOI: 10.1016/j.bbapap.2024.141027
Anvesh K.R. Dasari , Matthew F. Coats , Abdullah B. Ali , Kwang Hun Lim
{"title":"Identification of the interfacial regions in misfolded transthyretin oligomers","authors":"Anvesh K.R. Dasari ,&nbsp;Matthew F. Coats ,&nbsp;Abdullah B. Ali ,&nbsp;Kwang Hun Lim","doi":"10.1016/j.bbapap.2024.141027","DOIUrl":"https://doi.org/10.1016/j.bbapap.2024.141027","url":null,"abstract":"<div><p>Misfolding and aggregation of transthyretin (TTR) is associated with numerous ATTR amyloidosis. TTR aggregates extracted from ATTR patients consist of not only full-length TTR, but also N-terminally truncated TTR fragments that can be produced by proteolytic cleavage, suggesting the presence of multiple misfolding pathways. Here, we report mechanistic studies of an early stage of TTR aggregation to probe the oligomerization process for the full-length as well as N-terminally truncated TTR. Our kinetic analyses using size exclusion chromatography revealed that amyloidogenic monomers dissociated from wild-type (WT) as well as pathogenic variants (V30M and L55P) form misfolded dimers, which self-assemble into oligomers, precursors of fibril formation. Dimeric interfaces in the full-length misfolded oligomers were investigated by examining the effect of single-point mutations on the two β-strands (F and H). The single-point mutations on the two β-strands (E92P on strand F and T119W on strand H) inhibited the dimerization of misfolded monomers, while the TTR variants can still form native dimers through the same F and H strands. These results suggest that the two strands are involved in intermolecular associations for both native and misfolded dimers, but detailed intermolecular interactions are different in the two forms of dimers. In the presence of a proteolytic enzyme, TTR aggregation is greatly accelerated. The two mutations on the two β-strands, however, inhibited TTR aggregation even in the presence of a proteolytic enzyme, trypsin. These results suggest that the two β-strands (F and H) play a critical role in aggregation of the N-terminally truncated TTR as well.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 5","pages":"Article 141027"},"PeriodicalIF":3.2,"publicationDate":"2024-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141097790","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Assessing the Martini 3 protein model: A review of its path and potential 评估马丁尼 3 号蛋白质模型:回顾其发展道路和潜力
IF 3.2 4区 生物学
Biochimica et biophysica acta. Proteins and proteomics Pub Date : 2024-04-25 DOI: 10.1016/j.bbapap.2024.141014
Luís Borges-Araújo , Gilberto P. Pereira , Mariana Valério , Paulo C.T. Souza
{"title":"Assessing the Martini 3 protein model: A review of its path and potential","authors":"Luís Borges-Araújo ,&nbsp;Gilberto P. Pereira ,&nbsp;Mariana Valério ,&nbsp;Paulo C.T. Souza","doi":"10.1016/j.bbapap.2024.141014","DOIUrl":"10.1016/j.bbapap.2024.141014","url":null,"abstract":"<div><p>Coarse-grained (CG) protein models have become indispensable tools for studying many biological protein details, from conformational dynamics to the organization of protein macro-complexes, and even the interaction of proteins with other molecules. The Martini force field is one of the most widely used CG models for bio-molecular simulations, partly because of the enormous success of its protein model. With the recent release of a new and improved version of the Martini force field – Martini 3 – a new iteration of its protein model was also made available. The Martini 3 protein force field is an evolution of its Martini 2 counterpart, aimed at improving many of the shortcomings that had been previously identified. In this mini-review, we first provide a general overview of the model and then focus on the successful advances made in the short time since its release, many of which would not have been possible before. Furthermore, we discuss reported limitations, potential directions for model improvement and comment on what the likely future development and application avenues are.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141014"},"PeriodicalIF":3.2,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140775132","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Chemical proteomics approaches for protein post-translational modification studies 用于蛋白质翻译后修饰研究的化学蛋白质组学方法
IF 3.2 4区 生物学
Biochimica et biophysica acta. Proteins and proteomics Pub Date : 2024-04-18 DOI: 10.1016/j.bbapap.2024.141017
Nan Zhang , Jinghua Wu , Qingfei Zheng
{"title":"Chemical proteomics approaches for protein post-translational modification studies","authors":"Nan Zhang ,&nbsp;Jinghua Wu ,&nbsp;Qingfei Zheng","doi":"10.1016/j.bbapap.2024.141017","DOIUrl":"https://doi.org/10.1016/j.bbapap.2024.141017","url":null,"abstract":"<div><p>The diversity and dynamics of proteins play essential roles in maintaining the basic constructions and functions of cells. The abundance of functional proteins is regulated by the transcription and translation processes, while the alternative splicing enables the same gene to generate distinct protein isoforms of different lengths. Beyond the transcriptional and translational regulations, post-translational modifications (PTMs) are able to further expand the diversity and functional scope of proteins. PTMs have been shown to make significant changes in the surface charges, structures, activation states, and interactome of proteins. Due to the functional complexity, highly dynamic nature, and low presence percentage, the study of protein PTMs remains challenging. Here we summarize and discuss the major chemical biology tools and chemical proteomics approaches to enrich and investigate the protein PTM of interest.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141017"},"PeriodicalIF":3.2,"publicationDate":"2024-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1570963924000244/pdfft?md5=074377f1e99350ee49a95930dfa4ebb8&pid=1-s2.0-S1570963924000244-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140631771","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Conformational and dynamic properties of the KH1 domain of FMRP and its fragile X syndrome linked G266E variant FMRP的KH1结构域及其与脆性X综合征相关的G266E变体的构象和动态特性
IF 3.2 4区 生物学
Biochimica et biophysica acta. Proteins and proteomics Pub Date : 2024-04-17 DOI: 10.1016/j.bbapap.2024.141019
Flavia Catalano , Daniele Santorelli , Alessandra Astegno , Filippo Favretto , Marco D'Abramo , Alessandra Del Giudice , Maria Laura De Sciscio , Francesca Troilo , Giorgio Giardina , Adele Di Matteo , Carlo Travaglini-Allocatelli
{"title":"Conformational and dynamic properties of the KH1 domain of FMRP and its fragile X syndrome linked G266E variant","authors":"Flavia Catalano ,&nbsp;Daniele Santorelli ,&nbsp;Alessandra Astegno ,&nbsp;Filippo Favretto ,&nbsp;Marco D'Abramo ,&nbsp;Alessandra Del Giudice ,&nbsp;Maria Laura De Sciscio ,&nbsp;Francesca Troilo ,&nbsp;Giorgio Giardina ,&nbsp;Adele Di Matteo ,&nbsp;Carlo Travaglini-Allocatelli","doi":"10.1016/j.bbapap.2024.141019","DOIUrl":"https://doi.org/10.1016/j.bbapap.2024.141019","url":null,"abstract":"<div><p>The Fragile X messenger ribonucleoprotein (FMRP) is a multi-domain protein involved in interactions with various macromolecules, including proteins and coding/non-coding RNAs. The three KH domains (KH0, KH1 and KH2) within FMRP are recognized for their roles in mRNA binding. In the context of Fragile X syndrome (FXS), over-and-above CGG triplet repeats expansion, three specific point mutations have been identified, each affecting one of the three KH domains (<sup>R138Q</sup>KH0, <sup>G266E</sup>KH1, and <sup>I304N</sup>KH2) resulting in the expression of non-functional FMRP. This study aims to elucidate the molecular mechanism underlying the loss of function associated with the <sup>G266E</sup>KH1 pathological variant. We investigate the conformational and dynamic properties of the isolated KH1 domain and the two KH1 site-directed mutants <sup>G266E</sup>KH1 and <sup>G266A</sup>KH1. Employing a combined <em>in vitro</em> and <em>in silico</em> approach, we reveal that the <sup>G266E</sup>KH1 variant lacks the characteristic features of a folded domain. This observation provides an explanation for functional impairment observed in FMRP carrying the G266E mutation within the KH1 domain, as it renders the domain unable to fold properly. Molecular Dynamics simulations suggest a pivotal role for residue 266 in regulating the structural stability of the KH domains, primarily through stabilizing the α-helices of the domain. Overall, these findings enhance our comprehension of the molecular basis for the dysfunction associated with the <sup>G266E</sup>KH1 variant in FMRP.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141019"},"PeriodicalIF":3.2,"publicationDate":"2024-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140645123","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The molecular interplay between human and bacterial amyloids: Implications in neurodegenerative diseases 人类和细菌淀粉样蛋白之间的分子相互作用:对神经退行性疾病的影响
IF 3.2 4区 生物学
Biochimica et biophysica acta. Proteins and proteomics Pub Date : 2024-04-17 DOI: 10.1016/j.bbapap.2024.141018
Neha Jain
{"title":"The molecular interplay between human and bacterial amyloids: Implications in neurodegenerative diseases","authors":"Neha Jain","doi":"10.1016/j.bbapap.2024.141018","DOIUrl":"https://doi.org/10.1016/j.bbapap.2024.141018","url":null,"abstract":"<div><p>Neurodegenerative disorders such as Parkinson's (PD) and Alzheimer's diseases (AD) are linked with the assembly and accumulation of proteins into structured scaffold called amyloids. These diseases pose significant challenges due to their complex and multifaceted nature. While the primary focus has been on endogenous amyloids, recent evidence suggests that bacterial amyloids may contribute to the development and exacerbation of such disorders. The gut-brain axis is emerging as a communication pathway between bacterial and human amyloids. This review delves into the novel role and potential mechanism of bacterial amyloids in modulating human amyloid formation and the progression of AD and PD.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141018"},"PeriodicalIF":3.2,"publicationDate":"2024-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140631772","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) bifunctional enzyme from Candidatus Liberibacer asiaticus Asiaticus 自由杆菌中的 AICAR 转化酶/IMP 环水解酶(ATIC)双功能酶的特性分析
IF 3.2 4区 生物学
Biochimica et biophysica acta. Proteins and proteomics Pub Date : 2024-04-12 DOI: 10.1016/j.bbapap.2024.141015
Sapna Lonare , Surabhi Rode , Preeti Verma, Shalja Verma, Harry Kaur, Md Shahid Alam, Padma Wangmo, Pravindra Kumar, Partha Roy, Ashwani Kumar Sharma
{"title":"Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) bifunctional enzyme from Candidatus Liberibacer asiaticus","authors":"Sapna Lonare ,&nbsp;Surabhi Rode ,&nbsp;Preeti Verma,&nbsp;Shalja Verma,&nbsp;Harry Kaur,&nbsp;Md Shahid Alam,&nbsp;Padma Wangmo,&nbsp;Pravindra Kumar,&nbsp;Partha Roy,&nbsp;Ashwani Kumar Sharma","doi":"10.1016/j.bbapap.2024.141015","DOIUrl":"https://doi.org/10.1016/j.bbapap.2024.141015","url":null,"abstract":"<div><p>The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from <em>Candidatus</em> Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (<em>K</em><sub>m</sub>, 146.6 μM and <em>V</em><sub>max</sub>, 0.95 μmol/min/mg) and AICAR (<em>K</em><sub>m</sub>, 34.81 μM and <em>V</em><sub>max</sub>, 0.56 μmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (<em>K</em><sub>m</sub>, 1.81 μM and <em>V</em><sub>max</sub>, 2.87 μmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 μM and 34.2 μM, respectively) compared to AICAR (Kd, 83.4 μM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing <em>C</em>Las and its unique cell protective, wound-healing properties for future biotechnological applications.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141015"},"PeriodicalIF":3.2,"publicationDate":"2024-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140618733","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The apo-acyl coenzyme A binding protein of Leishmania major forms a unique ‘AXXA’ motif mediated dimer 大利什曼原虫的载脂蛋白辅酶 A 结合蛋白形成独特的 "AXXA "基团介导的二聚体
IF 3.2 4区 生物学
Biochimica et biophysica acta. Proteins and proteomics Pub Date : 2024-04-12 DOI: 10.1016/j.bbapap.2024.141016
Shalini Verma , Rohit Singh Dangi , Manoj Kumar Rajak, Ravi Kant Pal, Monica Sundd
{"title":"The apo-acyl coenzyme A binding protein of Leishmania major forms a unique ‘AXXA’ motif mediated dimer","authors":"Shalini Verma ,&nbsp;Rohit Singh Dangi ,&nbsp;Manoj Kumar Rajak,&nbsp;Ravi Kant Pal,&nbsp;Monica Sundd","doi":"10.1016/j.bbapap.2024.141016","DOIUrl":"https://doi.org/10.1016/j.bbapap.2024.141016","url":null,"abstract":"<div><p>Acyl-Coenzyme A binding domain containing proteins (ACBDs) are ubiquitous in nearly all eukaryotes. They can exist as a free protein, or a domain of a large, multidomain, multifunctional protein. Besides modularity, ACBDs also display multiplicity. The same organism may have multiple ACBDs, differing in sequence and organization. By virtue of this diversity, ACBDs perform functions ranging from transport, synthesis, trafficking, signal transduction, transcription, and gene regulation. In plants and some microorganisms, these ACBDs are designated ACBPs (acyl-CoA binding proteins). The simplest ACBD/ACBP is a small, ∼10 kDa, soluble protein, comprising the acyl-CoA binding (ACB) domain. Most of these small ACBDs exist as monomers, while a few show a tendency to oligomerize. In sync with those studies, we report the crystal structure of two ACBDs from <em>Leishmania major,</em> named ACBP<sub>103,</sub> and ACBP<sub>96</sub> based on the number of residues present. Interestingly, ACBP<sub>103</sub> crystallized as a monomer and a dimer under different crystallization conditions. Careful examination of the dimer disclosed an exposed ‘AXXA’ motif in the helix I of the two ACBP<sub>103</sub> monomers, aligned in a head-to-tail arrangement in the dimer. Glutaraldehyde cross-linking studies confirm that apo-ACBP<sub>103</sub> can self-associate in solution. Isothermal titration calorimetry studies further show that ACBP<sub>103</sub> can bind ligands ranging from C<sub>8</sub> – to C<sub>20</sub>-CoA, and the data could be best fit to a ‘two sets of sites’/sequential binding site model. Taken together, our studies show that <em>Leishmania major</em> ACBP<sub>103</sub> can self-associate in the apo-form through a unique dimerization motif, an interaction that may play an important role in its function.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141016"},"PeriodicalIF":3.2,"publicationDate":"2024-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140557883","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
A role of methionines in the functioning of oxidatively modified fibrinogen 蛋氨酸在氧化修饰纤维蛋白原功能中的作用
IF 3.2 4区 生物学
Biochimica et biophysica acta. Proteins and proteomics Pub Date : 2024-04-04 DOI: 10.1016/j.bbapap.2024.141013
L.V. Yurina , A.D. Vasilyeva , E.S. Gavrilina , V.S. Ivanov , S.I. Obydennyi , I.A. Chabin , M.I. Indeykina , A.S. Kononikhin , E.N. Nikolaev , M.A. Rosenfeld
{"title":"A role of methionines in the functioning of oxidatively modified fibrinogen","authors":"L.V. Yurina ,&nbsp;A.D. Vasilyeva ,&nbsp;E.S. Gavrilina ,&nbsp;V.S. Ivanov ,&nbsp;S.I. Obydennyi ,&nbsp;I.A. Chabin ,&nbsp;M.I. Indeykina ,&nbsp;A.S. Kononikhin ,&nbsp;E.N. Nikolaev ,&nbsp;M.A. Rosenfeld","doi":"10.1016/j.bbapap.2024.141013","DOIUrl":"https://doi.org/10.1016/j.bbapap.2024.141013","url":null,"abstract":"<div><p>Posttranslational modifications in fibrinogen resulting from induced oxidation or oxidative stress in the organism can have deleterious influence on optimal functioning of fibrinogen, causing a disturbance in assembly and properties of fibrin. The protective mechanism supporting the ability of fibrinogen to function in ROS-generating environment remains completely unexplored. The effects of very low and moderately low HOCl/<sup>−</sup>OCl concentrations on fibrinogen oxidative modifications, the fibrin network structure as well as the kinetics of both fibrinogen-to-fibrin conversion and fibrin hydrolysis have been explored in the current study. As opposed to 25 Μm, HOCl/<sup>−</sup>OCl, 10 μM HOCl/<sup>−</sup>OCl did not affect the functional activity of fibrinogen. It is shown for the first time that a number of Met residues, AαMet476, AαMet517, AαMet584, BβMet367, γMet264, and γMet94, identified in 10 μM HOCl/–OCl fibrinogen by the HPLC-MS/MS method, operate as ROS scavengers, performing an important antioxidant function. In turn, this indicates that the fibrinogen structure is adapted to the detrimental action of ROS. The results obtained in our study provide evidence for a protective mechanism responsible for maintaining the structure and functioning of fibrinogen molecules in the bloodstream under conditions of mild and moderate oxidative stress.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141013"},"PeriodicalIF":3.2,"publicationDate":"2024-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140539713","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Epitope mapping of SARS-CoV-2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls 通过羟基自由基蛋白足迹法绘制 SARS-CoV-2 RBD 的表位图揭示了纳入阴性抗体对照的重要性。
IF 3.2 4区 生物学
Biochimica et biophysica acta. Proteins and proteomics Pub Date : 2024-03-17 DOI: 10.1016/j.bbapap.2024.141011
Daniel Nyberg Larsen , Jakub Zbigniew Kaczmarek , Yaseelan Palarasah , Jonas Heilskov Graversen , Peter Højrup
{"title":"Epitope mapping of SARS-CoV-2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls","authors":"Daniel Nyberg Larsen ,&nbsp;Jakub Zbigniew Kaczmarek ,&nbsp;Yaseelan Palarasah ,&nbsp;Jonas Heilskov Graversen ,&nbsp;Peter Højrup","doi":"10.1016/j.bbapap.2024.141011","DOIUrl":"10.1016/j.bbapap.2024.141011","url":null,"abstract":"<div><p>Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogen‑deuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups.</p><p>This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis.</p><p>Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD.</p><p>The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141011"},"PeriodicalIF":3.2,"publicationDate":"2024-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1570963924000189/pdfft?md5=e1464e1e200d56909b302bd9a8c71d90&pid=1-s2.0-S1570963924000189-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140157531","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Active roles of lytic polysaccharide monooxygenases in human pathogenicity 裂解多糖单氧化酶在人类致病性中的积极作用。
IF 3.2 4区 生物学
Biochimica et biophysica acta. Proteins and proteomics Pub Date : 2024-03-16 DOI: 10.1016/j.bbapap.2024.141012
Daniel Kracher , Tina Lanzmaier , Leonor Vieira Carneiro
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