Mapping the M2 Proton Channel by H/D-LC-MS

通过 H/D-LC-MS 绘制 M2 质子通道

• 批准号: 6643174
• 负责人: Patrick Robert Griffin
• 金额: $ 13.23万
• 依托单位: EXSAR CORPORATION
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-15 至 2003-11-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6643174
• 关键词: acidity /alkalinity; amantadine; binding sites; cell surface receptors; deuterium; hydrogen; liquid chromatography mass spectrometry; membrane proteins; method development; protein structure

DESCRIPTION (provided by applicant): We have developed high-throughput mass spectrometric methods for mapping the structure and the location of active sites of proteins. These methods rely on measuring changes in solvent-accessibility and stability in the presence and absence of a small or large molecule ligands, as determined from the exchange rates of solvent deuterons with amide NH and sidechain CH groups of the protein; collectively we term this hydrogen-deuterium-exchange LC-MS, or H/D-LC-MS. Although our methods are being extensively developed to assess the binding of drugs to water-soluble proteins, they have not yet been applied to membrane proteins. Here we propose to extend our technology to study a relatively simple, but medicinally important membrane protein, the proton channel from influenza A virus (M2). This M2 proton channel is essential to the infectivity of the virus, and is the target of the anti-influenza drugs, amantadine and rimantadine. We have three specific aims in this proposal: 1) Using M2 as a model, we will extend H/D-LCMS to characterize the structures and binding sites of membrane proteins. 2) The methods will then be used to map the solvent accessibility of amides and sidechains within the M2 proton channel. Specifically we will determine the extent to which we can map the structured water-soluble regions of the protein, the membrane-contacting regions, and the solvent-filled channel of the M2 protein. 3) We will determine the feasibility of using these methods to assess structural changes associated with ligand binding and alterations in pH; the M2 proton channel is activated at the low pH (near 5.5) where it functions. We therefore will examine whether our technology can be used to evaluate differences in its H/D exchange at pH 7 versus 5, in the presence and absence of amantadine. The results will have an impact on our knowledge of this important therapeutic target, and will have broad impact on the future analysis of membrane-protein structures. With methods in place, we will be able to define structure perturbations of M2 upon binding other amantidine-related ligands and new pharmacophores. One long-term goal will be to aid the discovery of new antiviral agents. In addition, the methods developed herein will fill the gaps in the ability to explore structures of membrane proteins. We hope to develop H/D exchange into platform for the study of important membrane-associated drug targets, such as G-protein coupled receptors (GPCRs).

描述（由申请人提供）：我们开发了高通量质谱方法，用于绘制蛋白质活性位点的结构和位置。这些方法依赖于测量存在和不存在小分子或大分子配体时溶剂可及性和稳定性的变化，这是根据溶剂氘核与蛋白质的酰胺 NH 和侧链 CH 基团的交换率确定的；我们统称为氢-氘交换 LC-MS，或 H/D-LC-MS。尽管我们的方法正在广泛开发以评估药物与水溶性蛋白质的结合，但尚未应用于膜蛋白。在这里，我们建议扩展我们的技术来研究一种相对简单但具有医学重要性的膜蛋白，即甲型流感病毒（M2）的质子通道。这种 M2 质子通道对于病毒的感染性至关重要，并且是抗流感药物金刚烷胺和金刚乙胺的靶标。我们在这个提案中有三​​个具体目标：1）使用M2作为模型，我们将扩展H/D-LCMS来表征膜蛋白的结构和结合位点。 2) 然后，这些方法将用于绘制 M2 质子通道内酰胺和侧链的溶剂可及性图。具体来说，我们将确定我们可以在多大程度上绘制蛋白质的结构化水溶性区域、膜接触区域和 M2 蛋白质的溶剂填充通道。 3) 我们将确定使用这些方法评估与配体结合和 pH 值变化相关的结构变化的可行性； M2 质子通道在其发挥作用的低 pH（接近 5.5）下被激活。因此，我们将检查我们的技术是否可用于评估在存在和不存在金刚烷胺的情况下，pH 7 与 5 时 H/D 交换的差异。结果将影响我们对这一重要治疗靶点的认识，并将对膜蛋白结构的未来分析产生广泛影响。有了适当的方法，我们将能够定义 M2 在结合其他金刚烷胺相关配体和新药效团时的结构扰动。一个长期目标是帮助发现新的抗病毒药物。此外，本文开发的方法将填补探索膜蛋白结构能力的空白。我们希望将H/D交换发展成为研究重要膜相关药物靶点的平台，例如G蛋白偶联受体（GPCR）。