STRUCTURAL CONSEQUENCES OF ROS DAMAGE ON A MODEL SIGNAL TRANSDUCTION PROTEIN

ROS 损伤对模型信号转导蛋白的结构影响

• 批准号: 8168864
• 负责人: JOSHUA S SHARP
• 金额: $ 3.37万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-10 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168864
• 关键词: Chemicals; Circular Dichroism Spectroscopy; Computer Retrieval of Information on Scientific Projects Database; Dose; Event; Funding; Grant; Institution; Kinetics; Maps; Methionine; Modeling; Pathway interactions; Proteins; Radiation; Reaction; Research; Research Personnel; Resolution; Resources; Series; Signal Transduction; Site-Directed Mutagenesis; Source; Staging; Sulfoxide; Surface; Techniques; Testing; Time; United States National Institutes of Health; Water; Work; oxidation; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed a technique dubbed Dose-Dependent Protein Oxidative Surface Mapping (DD-POSuM), wherein we oxidize a protein by continuous gamma radiolysis of water for different amounts of time under pseudo-first order reaction conditions. We then generate a series of single point kinetic approximations for the rate constants of oxidation for each oxidation target. Radiation dose-dependent changes in the rate constant of oxidation are indicative of oxidation-induced conformational changes, and allow us to examine these conformational changes with some structural resolution. Previous work using DD-POSuM has indicated that oxidation of Spo0F, a single domain response regulator involved in Stage 0 of the sporulation response pathway, undergoes a specific unfolding event upon oxidation; however, the apparent kinetics observed do not fit very well with the two-state model suggested by circular dichroism spectroscopy. We have some evidence that this conformational change is triggered by oxidation of Met81 to the sulfoxide. In order to test this hypothesis, we are utilizing chemical oxidation of methionine as well as site-directed mutagenesis.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们开发了一种称为剂量依赖性蛋白质氧化表面图谱 (DD-POSuM) 的技术，其中我们通过在伪一级反应条件下连续伽马射线分解水不同的时间来氧化蛋白质。 然后，我们为每个氧化目标的氧化速率常数生成一系列单点动力学近似。 氧化速率常数的辐射剂量依赖性变化表明了氧化诱导的构象变化，并且使我们能够通过某些结构分辨率来检查这些构象变化。 先前使用 DD-POSuM 的工作表明，Spo0F（参与孢子形成反应途径第 0 阶段的单域反应调节剂）的氧化在氧化后经历特定的去折叠事件；然而，观察到的表观动力学与圆二色光谱所建议的双态模型不太吻合。 我们有一些证据表明这种构象变化是由 Met81 氧化成亚砜引发的。 为了检验这一假设，我们利用蛋氨酸的化学氧化以及定点诱变。