Site-Directed Oxidative Modification of Muscle Protein Structural Dynamics

描述(申请人提供)：这项研究的长期目标是了解肌肉蛋白质中特定部位氧化的功能和结构后果，以阐明氧化应激影响人类健康和衰老的机制。本文主要研究了两种关键肌肉蛋白--钙调素(CaM)作为钙释放通道(Ryanodine Receptor，RyR)的调节者和肌球蛋白(Myosin)作为肌动蛋白依赖的力量生成器的蛋氨酸氧化作用。这项工作的基本原理很大程度上来自于前一个项目阶段，当时我们确定CaM和肌球蛋白中的蛋氨酸氧化是肌肉老化或氧化后功能下降和蛋白质结构变化的关键目标。在接下来的一段时间里，我们将重点研究位点特异性蛋氨酸氧化对这两种蛋白质的结构和功能的影响的基本问题。本项目采用定点突变有三个目的：(1)蛋氨酸突变将用于控制氧化敏感性，(2)将引入先前已确定的功能突变，以确定它们对其他位点氧化损伤易感性的影响，(3)半胱氨酸突变将用于将光谱探针连接到选定的位点，这些位点旨在检测CaM或肌球蛋白的功能重要结构变化或相互作用。因此，特定Met氧化的功能影响将直接与结构影响相关。将使用互补的光谱技术阵列--荧光共振能量转移(FRET)、瞬时磷光各向异性(TPA)、电子顺磁共振(EPR)和核磁共振(核磁共振)。核磁共振将使我们能够获得溶液中小蛋白(CaM)的高分辨率结构数据，而其他方法使我们能够获得补充核磁共振的远程约束，并检测这些蛋白质功能所在的大蛋白质复合体的结构变化。NIH资助的三个研究小组之间的富有成效的合作使这项工作的高潜在影响成为可能。这些小组通过联合出版物和初步数据表明，在实现上一个供资期间的目标和为新提案的所有目标确定可行性方面，它们的合作是有效的。该项目提供了独特和创新的方法组合，所有这些方法都集中在一个及时的目标上-解释特定的蛋氨酸氧化如何影响肌肉蛋白质的功能、结构和动力学。这一基本信息对于进一步了解蛋白质氧化的结构生物学是必要的。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the functional and structural consequences of site-specific oxidation in muscle proteins, in order to illuminate the mechanisms by which oxidative stress affects human health and aging. The present proposal focuses on the effect of methionine oxidation in two key muscle proteins - calmodulin (CaM), in its role as regulator of the calcium release channel (ryanodine receptor, RyR) and myosin, in its role as actin-dependent force generator. The rationale for this work comes largely from the previous project period, in which we identified methionine oxidations in CaM and myosin as critical targets of functional decline and protein structural changes in muscle that has been aged or oxidized. In the next period, we focus on fundamental questions about the effects of site-specific methionine oxidation on the structure and function of these two proteins. This project employs site-directed mutagenesis for three purposes: (1) Met mutagenesis will be used to control susceptibility to oxidation, (2) previously identified functional mutations will be introduced to determine their effect on susceptibility to oxidative damage at other sites, (3) Cys mutagenesis will be used to attach spectroscopic probes to selected sites that are designed to detect functionally important structural changes or interactions of CaM or myosin. Thus the functional impacts of specific Met oxidations will be correlated directly with structural impacts. A complementary array of spectroscopic techniques will be used - fluorescence resonance energy transfer (FRET), transient phosphorescence anisotropy (TPA), electron paramagnetic resonance (EPR), and nuclear magnetic resonance (NMR). NMR will allow us to obtain high-resolution structural data on small proteins (CaM) in solution, while the other methods allow us to obtain long-range distance constraints that complement NMR, and to detect structural changes in the large protein complexes in which these proteins function. The high potential impact of this work is made possible by a productive collaboration among three NIH- funded research groups. These groups have demonstrated, through joint publications and preliminary data, the effectiveness of their collaboration in achieving the aims of the previous funding period and establishing feasibility for all aims of the new proposal. This project offers a unique and innovative combination of approaches, all focused on a timely goal - to explain how specific Met oxidations affect muscle protein function, structure, and dynamics. This fundamental information is required for further progress in understanding the structural biology of protein oxidation.