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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们开发了一种被称为剂量依赖的蛋白质氧化表面映射(DD-POSuM)的技术，在这种技术中，我们通过在准一级反应条件下将水连续伽马辐解不同时间来氧化蛋白质。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。氧化速率常数随辐射剂量的变化预示着氧化诱导的构象变化，并使我们能够以一定的结构分辨率来研究这些构象变化。以前使用DD-POSuM的工作表明，Spo0F是参与孢子形成反应途径0阶段的单域反应调节因子，在氧化时经历了特定的展开事件；然而，观察到的表观动力学不太符合圆二色谱提出的两态模型。我们有一些证据表明，这种构象变化是由金属81氧化成亚硫醚引发的。为了验证这一假设，我们利用蛋氨酸的化学氧化以及定点突变。

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.