Structural Mapping of Protein Complexes by Hydrogen/Deuterium Exchange

通过氢/氘交换绘制蛋白质复合物的结构图

• 批准号: 7267750
• 负责人: ALAN G MARSHALL
• 金额: $ 30.54万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7267750
• 关键词: Amides; Amino Acid Sequence; Area; Automation; Back; Biological; Capsid; Capsid Proteins; Cells; Charge; Chemicals; Complex; Data; Data Analyses; Data Collection; Deuterium; Digestion; Endopeptidases; Entropy; Enzymes; Future; Generations; HIV; HIV-1; Heterogeneity; High Pressure Liquid Chromatography; Holoenzymes; Human; Hydrogen; Imagery; Individual; Ions; Ligand Binding; Ligands; Link; Maps; Marshal; Mass Spectrum Analysis; Measurement; Measures; Mediating; Metal Ion Binding; Metalloproteins; Metals; Methods; Modification; Molecular Conformation; Molecular Weight; Monitor; Motor; NAD(P)+ transhydrogenase; Nuclear Magnetic Resonance; Pepsin A; Peptide Fragments; Peptide Hydrolases; Peptides; Pharmacologic Substance; Play; Polymerase; Post-Translational Protein Processing; Protein Conformation; Protein Fragment; Protein Interaction Mapping; Protein-Protein Interaction Map; Proteins; Proteolysis; Protons; RNA; Range; Reaction; Relative (related person); Reproducibility; Research Personnel; Research Project Grants; Resolution; Robot; Role; Sampling; Series; Site; Solutions; Solvents; Sorting - Cell Movement; Specificity; Structure; Supercritical Fluid Chromatography; Surface; System; Techniques; Time; Tuberculosis; Vertebral column; Viral; Virus; base; crosslink; data acquisition; day; glycosylation; human disease; improved; ionization; novel; novel strategies; programs; protein purification; protein structure; receptor; research study; response; tumor progression; viral RNA

DESCRIPTION (provided by applicant): The best techniques for identifying sites of contact in protein complexes are x-ray diffraction and nuclear magnetic resonance. However, those techniques may not be available for large complexes, due to limited sample amount, aggregation, insolubility, posttranslational modification heterogeneity, lack of suitable crystals, etc. Most alternative methods are based on exposing the complex to some sort of chemical perturbation (e.g., chemical reactivity toward general or specific agents, cross-linking, and the like). The contact surfaces may then be located as those sites that are linked or become protected against chemical reactivity on formation of the protein complex. The most generally applicable measure of solvent exposure is exchange of backbone amide hydrogens for deuteriums, because it is least dependent on the amino acid sequence. Major current difficulties for the H/D exchange method include: back-exchange of H for D during separation of proteolytic fragments for analysis; mass resolving power too low to separate and identify partially deuterated peptides by mass spectrometry; incomplete sequence coverage by pepsin cleavage (pepsin is usually used due to its activity at low pH after H/D exchange has been quenched); and the difficulty and duration of acquiring and interpreting the data. In this project, we shall combine several improvements: supercritical fluid chromatography to eliminate back-exchange; isotopic depletion and ultrahigh mass resolving power to simplify identification of peptides, a suite of enzymes of different proteolytic specificity to better span the sequence; and automation of data collection and data analysis. Relevance. It is becoming increasingly evident that protein complexes and assemblies play a key role in many human diseases: e.g., the protein "capsid" that protects RNA in the AIDS virus and in the biological "motors" that transport essential components into a cell or virus. A first step in understanding (and eventually controlling) those functions is to identify the sites of contact that hold proteins together. This project presents several new approaches for such "mapping", along with some suggested initial applications that could point to future targets with pharmaceutical applications. This project is multidisiplinary and pulls together several distinguished collaborators with focused research projects that will benefit directly from H/D exchange with high-resolution mass analysis.

描述（由申请人提供）：识别蛋白质复合物中接触位点的最佳技术是X射线衍射和核磁共振。但是，由于样本量有限，聚集，不溶性，翻译后修饰的异质性，缺乏合适的晶体等，这些技术可能无法用于大型复合物。大多数替代方法是基于将复合物暴露于某种化学扰动的络合物（例如，对某种化学反应性（例如，化学反应性）对普通或特定的特定或特定的交叉链接等）。然后可以将接触表面作为那些在形成蛋白质复合物形成时与化学反应性联系起来的位点。最普遍适用的溶剂暴露量度是将依丁膜的骨链酰胺氢换成，因为它最不依赖于氨基酸序列。 H/D交换方法的主要当前困难包括：H的h/d交换方法在分离蛋白水解片段进行分析时d的后交换；质量分离的能力太低而无法通过质谱法分离并鉴定部分剥离的肽；胃蛋白酶裂解不完整的序列覆盖率（胃蛋白酶通常是由于其在h/d交换后低pH值的活性而使用的）；以及获取和解释数据的困难和持续时间。在这个项目中，我们将结合几个改进：超临界流体色谱法以消除后交换；同位素耗竭和超高质量分辨能力简化肽的鉴定，肽是一组不同蛋白水解特异性的酶，以更好地跨越序列；以及数据收集和数据分析的自动化。关联。越来越明显的是，蛋白质复合物和组件在许多人类疾病中起着关键作用：例如，保护艾滋病病毒中的RNA以及将必需成分传输到细胞或病毒的生物“电动机”中的蛋白“ capsid”。理解（并最终控制）这些功能的第一步是识别将蛋白质结合在一起的接触位点。该项目提出了这种“映射”的几种新方法，以及一些建议的初始应用，这些应用可能指出了使用药品应用程序的未来目标。该项目是多学科的，并通过重点研究项目将几个杰出的合作者汇集在一起​​，这些项目将直接从H/D交流中受益，并具有高分辨率的质量分析。