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: 8361801
• 负责人: JOSHUA S SHARP
• 金额: $ 3.54万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361801
• 关键词: Chemicals; Circular Dichroism Spectroscopy; Dose; Event; Funding; Grant; Kinetics; Maps; Methionine; Modeling; National Center for Research Resources; Pathway interactions; Principal Investigator; Proteins; Radiation; Reaction; Research; Research Infrastructure; Resolution; Resources; Series; Signal Transduction; Site-Directed Mutagenesis; Source; Staging; Sulfoxide; Surface; Techniques; Testing; Time; United States National Institutes of Health; Water; Work; cost; oxidation; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们已经开发了一种称为剂量依赖性蛋白质氧化表面映射（DD-POSuM）的技术，其中我们通过在伪一级反应条件下连续γ辐解水不同的时间来氧化蛋白质。 然后，我们生成一系列的单点动力学近似为每个氧化目标的氧化速率常数。 氧化速率常数的辐射剂量依赖性变化指示氧化诱导的构象变化，并允许我们检查这些构象变化与一些结构分辨率。 以前的工作使用DD-POSuM已经表明，Spo 0 F，一个单域响应调节剂参与孢子形成反应途径的第0阶段，氧化后经历了一个特定的展开事件;然而，观察到的表观动力学不太符合圆二色光谱所建议的两态模型。 我们有一些证据表明，这种构象变化是由氧化Met 81的亚砜。 为了检验这一假设，我们正在利用蛋氨酸的化学氧化以及定点诱变。