Site-Directed Oxidative Modification of Muscle Protein Structural Dynamics

描述（由申请人提供）：本研究的长期目标是了解肌肉蛋白中位点特异性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。目前的建议侧重于蛋氨酸氧化对两个关键肌肉蛋白的影响-钙调蛋白（CaM），其作为钙释放通道的调节剂（ryanodine受体，RyR）和肌球蛋白，其作为动作蛋白依赖的力发生器的作用。这项工作的基本原理主要来自于之前的项目时期，在该项目期间，我们确定了CaM和肌球蛋白中的蛋氨酸氧化是衰老或氧化肌肉功能下降和蛋白质结构变化的关键靶点。在下一阶段，我们将重点研究位点特异性蛋氨酸氧化对这两种蛋白质结构和功能的影响。该项目采用位点定向诱变有三个目的：(1)Met诱变将用于控制氧化易感性，(2)将引入先前确定的功能突变，以确定它们对其他位点氧化损伤易感性的影响，(3)Cys诱变将用于将光谱探针附着在选定的位点上，这些位点旨在检测功能重要的结构变化或CaM或肌球蛋白的相互作用。因此，特定Met氧化的功能影响将与结构影响直接相关。将使用互补的光谱技术阵列-荧光共振能量转移（FRET），瞬态磷光各向异性（TPA），电子顺磁共振（EPR）和核磁共振（NMR）。核磁共振将使我们能够获得溶液中小蛋白质（CaM）的高分辨率结构数据，而其他方法使我们能够获得远距离距离约束，以补充核磁共振，并检测这些蛋白质功能的大蛋白质复合物的结构变化。这项工作的高潜在影响是由三个NIH资助的研究小组之间的富有成效的合作所实现的。这些小组通过联合出版物和初步数据表明，它们的合作在实现上一个筹资期的目标和确定新提议的所有目标的可行性方面是有效的。这个项目提供了一个独特的和创新的方法组合，都集中在一个及时的目标-解释特定的Met氧化如何影响肌肉蛋白质的功能，结构和动力学。这些基本信息对于进一步了解蛋白质氧化的结构生物学是必需的。

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. PUBLIC HEALTH RELEVANCE: This project's goal is to understand how muscle proteins become damaged by oxidation, the addition of oxygen atoms, which occurs in the processes of aging and degenerative disease. Protein engineering will be done to prevent, mimic, or reverse the oxidation process. This fundamental information will inform future efforts to prevent or reverse the effects of aging and degenerative diseases, such as heart failure and muscle weakness.