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（由适用提供）：我们的目标是用分子详细说明与年龄相关的肌肉变性与生物氧化物对肌肉蛋白的氧化有关，从蛋白质结构的原子水平变化到蛋白质调节性相互作用的变化，最后，以及最终的生物衰变。一般假设是氨基酸方法（Met）和半胱氨酸（CYS）的位点特异性氧化修饰通过改变蛋白质结构动力学来触发肌肉蛋白功能的变化。对氧化机制的基本理解的这种进步对于在治疗广泛的人类退化性疾病方面，不仅仅是肌肉而言，取得进展至关重要。 AIM 1涉及直接参与肌肉收缩，肌球蛋白和肌动蛋白的蛋白质，并专注于产生力的关键要素：肌动蛋白相互作用。 AIM 2涉及参与肌肉调节的关键蛋白，钙调蛋白（CAM）和Ryanodine接收器配合物（RYR），并着重于调节钙释放的CAM/RYR结构相互作用。这项工作的基本原理是从研究者的先前工作中得出的，其中肌球蛋白和CAM中的氧化被确定为功能下降和蛋白质结构变化的关键靶标，而肌肉已经老化或氧化。我们专注于有关位点特异性氧化对CAM和肌球蛋白结构和功能的影响的基本问题。该项目员工出于两个目的而定向诱变：（1）MET和CYS诱变将用于控制对氧化修饰的敏感性，并且（2）CYS诱变将用于将光谱问题附加到旨在检测功能上关键结构性结构性结构性结构性结构的位点上 CAM或肌球蛋白的变化或相互作用。包括衰老生物标志物在内的特定氧化的功能影响将与结构影响直接相关。将使用一系列光谱技术 - 荧光共振能量转移（FRET），电子顺磁共振（EPR）和核磁共振（NMR）。 NMR将使我们能够在溶液中获得有关小蛋白（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NMR的远程距离约束，并检测这些蛋白质功能的大蛋白质复合物中的结构变化。小型本科机构的研究人员与更大的研究驱动机构之间的研究人员之间的产品合作使这项工作的高潜在影响成为可能。这些小组通过共同出版物和初步数据证明了其协作的有效性。该项目提供了一种独特而创新的方法组合，都集中在及时的目标上 - 解释特定的氧化修饰如何影响肌肉蛋白质功能，结构和动力学。在理解蛋白质氧化的结构生物学以及衰老和与年龄相关的肌肉功能障碍的分子基础上，需要进一步进展，需要这种基本信息。