Structural and functional effects of oxidative modification of myosin

培训计划的主要目标是（a）为我提供肌肉生物化学，生理学和生物物理学方面的广泛培训，以完成肌肉蛋白质氧化的分子生物物理学博士论文，以及（B）为下一步沿着成为独立研究者的道路做好准备。托马斯实验室目前研究计划的长期目标是了解肌肉蛋白质氧化修饰的结构和功能基础，以确定由于衰老和退行性疾病导致肌肉力量下降的分子基础。本实验室以前的工作表明：（a）衰老导致肌肉力量下降，与萎缩无关，伴随着肌球蛋白的氧化修饰，肌动蛋白激活的ATP酶活性降低，（B）肌肉的过氧化物处理模拟衰老的功能效应，主要导致Met残基的修饰。我推测，肌球蛋白中的特定Met残基负责这些影响，这是由于肌球蛋白的催化结构域中的特定结构扰动。我将测试这一假设进行定点Met诱变的肌球蛋白在网骨藻细胞培养，然后进行功能和结构测试，以确定结构动力学和功能之间的相关性。主要的结构方法是定点自旋标记（SDSL），其次是电子顺磁共振（EPR）。我将追求三个目标。(1)蛋氨酸氧化对肌球蛋白催化结构域的功能影响。(2)肌球蛋白催化结构域的结构动力学。(3)蛋氨酸氧化对肌球蛋白催化结构域的影响。有相当多的科学证据表明，蛋白质氧化在与衰老相关的退行性疾病中起着重要作用，但关于氧化对特定蛋白质的生物化学和结构影响的信息很少。该项目旨在为肌肉力量随着氧化而下降提供位点特异性蛋白质结构解释，假设这在人类肌肉力量随着年龄的增长而丧失中起主要作用。对氧化机制的基本理解的这一进展对于在治疗与年龄相关的健康问题方面取得进展非常重要。利用定点突变技术和光谱学技术研究肌球蛋白氧化修饰的结构基础是一种新的方法。因此，除了为肌肉蛋白质氧化提供新的结构见解外，这项工作还可以作为未来研究的一般模型，重点关注氧化应激的特定结构后果。这个项目将建立在我过去在磁共振和衰老方面的经验的基础上，同时向我介绍一种新的生物系统（肌肉），其中氧化应激的机制被深入研究，以及一种新的光谱技术（EPR），它比NMR更敏感，可以在更多的生理条件下应用（例如，结合到肌动蛋白的肌球蛋白）。

The primary goals of the training program are (a) to provide me with extensive training in muscle biochemistry, physiology, and biophysics, toward completion of a PhD thesis in the molecular biophysics of muscle protein oxidation, and (b) to prepare me for the next step along the path to becoming an independent investigator. The long-term goal of the present research program in the Thomas lab is to understand the structural and functional basis of oxidative modification of muscle proteins, in order to determine the molecular basis of decline in muscle strength due to aging and degenerative diseases. Previous work in this lab has shown that (a) aging produces a decline in muscle force, independent of atrophy, accompanied by oxidative modification of myosin, decreasing actin-activated ATPase activity, and (b) peroxide treatment of muscle mimics the functional effects of aging and results primarily in modification of Met residues. I hypothesize that specific Met residues in myosin are responsible for these effects, which result from specific structural perturbations in the catalytic domain of myosin. I will test this hypothesis by performing site-directed Met mutagenesis on myosin in Dictyostelium cell culture, then performing functional and structural tests to determine the correlation between structural dynamics and function. The principal structural approach is site-directed spin labeling (SDSL), followed by electron paramagnetic resonance (EPR). I will pursue three aims. (1) Functional impact of methionine oxidation on myosin catalytic domain. (2) Structural dynamics of myosin catalytic domain. (3) Structural impact of methionine oxidation on the myosin catalytic domain. There is considerable scientific evidence that protein oxidation plays a major role in degenerative disorders associated with aging, but there is very little information about the biochemical and structural effects of oxidation on specific proteins. This project aims to provide a site-specific protein structural explanation for the decline in muscle strength with oxidation, which is hypothesized to play a major role in the loss of muscle strength with age in humans. This advance in fundamental understanding of oxidative mechanisms is important for making progress in treatment of age-related health problems. The approach of using site-directed mutagenesis and spectroscopy to probe the structural basis of oxidative modification in the muscle protein myosin is quite novel. Thus, in addition to providing new structural insight into muscle protein oxidation, this work can serve as a general model for future studies that focus on the specific structural consequences of oxidative stress. This project will build on my past experience in magnetic resonance and aging, while introducing me to a new biological system (muscle) for which the mechanisms of oxidative stress are intensely studied and a new spectroscopic technique (EPR) that is more sensitive than NMR and can be applied under more physiological conditions (e.g., myosin bound to actin).