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。本提案的重点是蛋氨酸氧化的影响，在两个关键的肌肉蛋白-钙调蛋白（CaM），在其作为钙释放通道（ryanodine受体，RyR）和肌球蛋白，在其作为肌动蛋白依赖性力发生器的作用的调节器的作用。这项工作的基本原理主要来自于前一个项目期间，在该项目中，我们确定了钙调素和肌球蛋白中的蛋氨酸氧化是肌肉功能下降和蛋白质结构变化的关键目标，这些肌肉已经老化或氧化。在接下来的时间里，我们专注于这两种蛋白质的结构和功能的影响，位点特异性蛋氨酸氧化的基本问题。该项目采用定点诱变有三个目的：（1）Met诱变将用于控制对氧化的敏感性，（2）将引入先前鉴定的功能突变以确定它们对在其它位点对氧化损伤的敏感性的影响，（三）半胱氨酸诱变将被用来连接光谱探针选择的网站，旨在检测功能上重要的结构变化或相互作用钙调蛋白或肌球蛋白。因此，特定Met氧化的功能影响将与结构影响直接相关。将使用光谱技术的互补阵列-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（NMR）。NMR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NMR的长距离限制，并检测这些蛋白质功能的大蛋白质复合物中的结构变化。 这项工作的高潜在影响是由美国国立卫生研究院资助的三个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，证明了它们在实现上一个供资期的目标和确定新提案所有目标的可行性方面的合作成效。该项目提供了一种独特而创新的方法组合，所有这些方法都集中在一个及时的目标上-解释特定的Met氧化如何影响肌肉蛋白质功能，结构和动力学。这些基本信息是理解蛋白质氧化的结构生物学的进一步进展所必需的。

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.