STRUCTURAL CONSEQUENCES OF ROS DAMAGE ON A MODEL SIGNAL TRANSDUCTION PROTEIN

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 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。

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.