Structural impact of methionine oxidation in muscle proteins

蛋氨酸氧化对肌肉蛋白的结构影响

基本信息

批准号：
8955526
负责人：
Jennifer Claire Klein
金额：
$ 32.92万
依托单位：
UNIVERSITY OF WISCONSIN LA CROSSE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-15 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8955526
关键词：
Actomyosin Affect Aging Aging-Related Process Amino Acids Back Biochemical Biological Biological Aging Biological Markers C-terminal Calcium Calmodulin Collaborations Complement Complex Computer Simulation Cysteine Data Degenerative Disorder Effectiveness Electron Spin Resonance Spectroscopy Elements Event Fluorescence Resonance Energy Transfer Functional disorder Future Generations Goals Heart failure Human In Vitro Institution Joints Link Methionine Methods Modification Molecular Motor Muscle Muscle Contraction Muscle Proteins Muscle Weakness Muscle function Mutagenesis Myocardium Myosin ATPase Nuclear Magnetic Resonance Oxidants Oxidative Stress Pattern Predisposition Production Protein Engineering Proteins Publications Pump Regulation Research Research Personnel Resolution Ryanodine Receptor Calcium Release Channel Signal Transduction Signaling Protein Site Site-Directed Mutagenesis Solutions Spectrum Analysis Staging Structural Models Structural Protein Structure Techniques Work Zebrafish age effect age related aged base biological adaptation to stress computerized tools design functional decline genetic regulatory protein in vivo innovation molecular dynamics muscle aging muscle degeneration oxidation prevent protein complex protein function protein structure public health relevance stem structural biology

项目摘要

DESCRIPTION (provided by applicant): Our goal is to explain in molecular detail how age-related muscle degeneration is related to the oxidation of muscle proteins by biological oxidants, from atomic-level changes in protein structure, to changes in protein regulatory interactions, and finally, to biological aging on the organismal level. The general hypothesis is that site-specific oxidative modifications of the amino acids methionine (Met) and cysteine (Cys) trigger changes in muscle protein function by altering protein structural dynamics. This advance in fundamental understanding of oxidative mechanisms is important for making progress in treating a broad range of human degenerative diseases, not just muscle. Aim 1 involves the proteins directly involved in muscle contraction, myosin and actin, and focuses on the key element of force generation: actomyosin interaction. Aim 2 involves key proteins involved in muscle regulation, calmodulin (CaM) and the ryanodine receptor complex (RyR), and focuses on the CaM/RyR structural interaction that modulates calcium release. The rationale for this work comes, in part, from the investigator's previous work, in which Met oxidations in myosin and CaM were identified as critical targets of functional decline and protein structural changes in muscle that has been aged or oxidized. We focus on fundamental questions about the effects of site-specific oxidation on the structure and function of CaM and myosin. This project employs site-directed mutagenesis for two purposes: (1) Met and Cys mutagenesis will be used to control susceptibility to oxidative modifications, and (2) Cys mutagenesis will be used to attach spectroscopic probes to selected sites that are designed to detect functionally critical structural changes or interactions of CaM or myosin. Thus the functional impacts of specific oxidations, including aging biomarkers, will be correlated directly with structural impacts. A complementary array of spectroscopic techniques will be used - fluorescence resonance energy transfer (FRET), electron paramagnetic resonance (EPR), and nuclear magnetic resonance (NMR). NMR will allow us to obtain high-resolution structure and dynamics 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 productive collaboration between investigators at small, primarily undergraduate institutions, and larger research-driven institutions. These groups have demonstrated, through joint publications and preliminary data, the effectiveness of their collaboration. This project offers a unique and innovative combination of approaches, all focused on a timely goal - to explain how specific oxidative modifications affect muscle protein function, structure, and dynamics. This fundamental information is required for further progress in understanding the structural biology of protein oxidation, and therefore the molecular basis of aging and age-related muscle dysfunction.

描述（由申请人提供）：我们的目标是从分子细节上解释与年龄相关的肌肉退化如何与生物氧化剂对肌肉蛋白的氧化相关，从蛋白质结构的原子水平变化到蛋白质调节相互作用的变化，以及最后，在有机体水平上进行生物衰老。一般假设是，氨基酸蛋氨酸（Met）和半胱氨酸（Cys）的位点特异性氧化修饰通过改变肌肉蛋白质功能而引发变化。对氧化机制的基本理解的这一进展对于治疗广泛的人类退行性疾病（而不仅仅是肌肉）方面取得进展非常重要。目标 1 涉及直接参与肌肉收缩的蛋白质、肌球蛋白和肌动蛋白，并重点关注力产生的关键要素：肌动球蛋白相互作用。目标 2 涉及参与肌肉调节的关键蛋白质、钙调蛋白 (CaM) 和兰尼碱受体复合物 (RyR)，并重点关注 CaM/RyR 结构相互作用。这项工作的基本原理部分来自于研究人员之前的工作，其中肌球蛋白和 CaM 中的 Met 氧化被确定为老化或氧化的肌肉中功能衰退和蛋白质结构变化的关键目标。我们重点研究有关位点特异性氧化对 CaM 和肌球蛋白的结构和功能的影响的基本问题。该项目采用定点诱变有两个目的：(1) Met 和 Cys 诱变。将用于控制对氧化修饰的敏感性，并且（2）Cys诱变将用于将光谱探针附着到旨在检测功能关键结构的选定位点因此，特定氧化的功能影响（包括衰老生物标志物）将与结构影响直接相关，将使用一系列互补的光谱技术 - 荧光共振能量转移（FRET）、电子顺磁共振（ EPR）和核磁共振（NMR）将使我们能够获得溶液中小蛋白质（CaM）的高分辨率结构和动力学数据，而其他方法使我们能够获得补充的长程距离约束。核磁共振，并检测这些蛋白质在其中发挥作用的大型蛋白质复合物的结构变化，这项工作的巨大潜在影响是通过小型（主要是本科机构）和大型研究驱动机构的研究人员之间的富有成效的合作而实现的。通过联合出版物和初步数据证明了他们合作的有效性，该项目提供了独特且创新的方法组合，所有这些方法都集中于一个及时的目标 - 解释特定的氧化修饰如何影响肌肉蛋白质的功能、结构和动力学。为了进一步理解结构生物学，需要基础信息蛋白质氧化，因此是衰老和与年龄相关的肌肉功能障碍的分子基础。