Structural Mapping of Protein Complexes by Hydrogen/Deuterium Exchange

描述（由申请人提供）：鉴定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。然而，由于样本量有限、聚集、不溶性、翻译后修饰不均一性、缺乏合适的晶体等原因，这些技术可能不适用于大型配合物。大多数替代方法是基于将络合物暴露于某种化学扰动（例如，对一般或特定试剂的化学反应性，交联等）。然后，接触面可以定位为那些在蛋白质复合物形成时被连接或被保护以防止化学反应的位点。溶剂暴露的最普遍适用的测量是主酰胺氢交换氘，因为它是最不依赖于氨基酸序列。目前H/D交换法的主要困难包括：在分离蛋白水解片段进行分析时H与D的反交换；质谱法分离和鉴定部分氘化肽的质量分辨能力太低；胃蛋白酶裂解不完全覆盖序列（通常使用胃蛋白酶，因为它在H/D交换被淬灭后在低pH下具有活性）；以及获取和解释数据的难度和时间。在这个项目中，我们将结合几个改进：超临界流体色谱法消除反交换；同位素耗竭和超高质量分辨率简化了多肽的鉴定，一套不同蛋白水解特异性的酶可以更好地跨越序列；以及数据收集和分析的自动化。的相关性。越来越明显的是，蛋白质复合物和组装在许多人类疾病中发挥关键作用：例如，在艾滋病病毒中保护RNA的蛋白质“衣壳”，以及在将基本成分运输到细胞或病毒中的生物“马达”中。了解（并最终控制）这些功能的第一步是确定将蛋白质结合在一起的接触位点。本项目提出了几种用于这种“绘图”的新方法，以及一些建议的初步应用，这些应用可能指向未来的制药应用目标。该项目是多学科的，汇集了几位杰出的合作者，他们专注于研究项目，将直接受益于高分辨率质量分析的H/D交换。

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.