CARDIAC MUSCLE--MOLECULAR MECHANISMS OF REGULATION

心肌--调节的分子机制

• 批准号: 2222437
• 负责人: JOHN Anthony PUTKEY
• 金额: $ 20.05万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-09 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2222437
• 关键词: calcium; carbon; conformation; electron spin resonance spectroscopy; electronic spectra; intermolecular interaction; magnesium; methionine; muscle contraction; mutant; myocardium; nuclear magnetic resonance spectroscopy; phosphorylation; protein engineering; protein sequence; protein structure function; site directed mutagenesis; stable isotope; synthetic peptide; troponin

Contraction in striated muscle is regulated through a cascade of protein-protein interactions initiated by the binding of Ca2+-to cardiac troponin C (cTnC), the regulatory subunit of the troponin complex. The regulatory properties of cardiac and fast skeletal muscle TnC are more dynamic than a simple on/off molecular switch, and may be modulated by a variety of factors including phosphorylation of other regulatory proteins and degree of myosin crossbridges. The critical role for cTnC in muscle contraction emphasizes a need to understand its molecular mechanism of action. The goal of this proposal is to study structure/function relationships in cTnC using a synergistic blend of NMR spectroscopy and molecular biology to elucidate Ca2+-dependent protein dynamics essential for triggering and modulating muscle contraction. The general strategy will be to use a bacterial expression system to mutate selected amino acids in cTnC and enrich the normal and mutated proteins with amino acids labeled with stable isotopes to permit easy detection by NMR. This will allow the analysis of the whole protein, and interactions between cTnC and other regulatory proteins. A detailed understanding of structure/function relationships in cTnC may provide insights into how muscle contraction may be artificially modified by designing reagents that specifically interfere with the normal function of cTnC. Moreover, molecular mechanisms of action identified for TnC may be applicable to other Ca2+-binding proteins with activator activity such as calmodulin. The first Specific Aim will be to systematically mutate Met and Cys residues and label the mutated protein with [methyl-13C]Met. Assigned Met residues will be used as markers for conformational changes. Mono-Cys derivatives of cTnC will be used for site-specific attachment of fluorescent and spin-label probes. For the second Specific Aim, NMR will be used to monitor conformational changes in labeled proteins. Protein dynamics will be followed as a function of Ca2+ and Mg2+ binding; association with TnI, TnT and TnI peptides; phosphorylation of TnI and TnI peptides; and pH. The third Specific Aim will be to make sequential assignments and define the partial solution structure of cTnC.

横纹肌肉的收缩通过级联的调节 Ca2+与心脏的结合引发的蛋白质 - 蛋白质相互作用 肌钙蛋白C（CTNC），肌钙蛋白复合物的调节亚基。 这 心脏和快速骨骼肌TNC的调节特性更多 动态性比简单的开/关分子开关，可以通过a调制 多种因素，包括其他调节蛋白的磷酸化 和肌球蛋白杂交的程度。 CTNC在肌肉中的关键作用 收缩强调需要了解其分子机制 行动。 该建议的目的是研究结构/功能 使用NMR光谱的协同混合物和CTNC的关系 分子生物学以阐明Ca2+依赖性蛋白质动力学必不可少的 用于触发和调节肌肉收缩。 一般策略 将使用细菌表达系统突变选定的氨基 CTNC中的酸并用氨基酸富集正常和突变的蛋白 用稳定的同位素标记，可通过NMR轻松检测。 这会 允许分析整个蛋白质，并在CTNC之间进行相互作用 和其他调节蛋白。 对 CTNC中的结构/功能关系可能会提供有关如何 肌肉收缩可以通过设计试剂进行人工修饰 特别干扰了CTNC的正常功能。 而且， 针对TNC鉴定的作用的分子机制可能适用于 其他Ca2+结合蛋白具有活性剂活性，例如钙调蛋白。 第一个具体目的是系统突变MET和CYS 残留物并用[甲基-13C] met将突变的蛋白标记。 分配 MET残留物将用作构象变化的标记。 CTNC的单-cys衍生物将用于特定于地点的附件 荧光和自旋标签探针。 对于第二个特定目标，NMR将 用于监测标记蛋白的构象变化。 蛋白质 动力学将作为Ca2+和Mg2+结合的函数。 与TNI，TNT和TNI肽的关联； TNI和 TNI肽；和ph。 第三个具体目的是进行顺序 分配并定义CTNC的部分溶液结构。