CHEMISTRY OF MUSCLE PROTEINS

肌肉蛋白质的化学性质

• 批准号: 6171453
• 负责人: SUSAN LOWEY
• 金额: $ 43.84万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-01-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6171453
• 关键词: adenosine diphosphate; adenosinetriphosphatase; bioenergetics; chemical kinetics; conformation; cryoscopy; enzyme activity; fluorescence polarization; fluorescence spectrometry; fluorescent dye /probe; intermolecular interaction; muscle contraction; muscle proteins; muscle tension; mutant; myofibrils; myosins; phosphorylation; protein engineering; protein isoforms; protein sequence; protein structure function; site directed mutagenesis; smooth muscle; striated muscles

DESCRIPTION: Current theories of the force generating step in the contractile cycle involve a reorientation of the myosin head relative to the actin filament during sliding. The inability to detect any large conformational changes in the globular, catalytic domain of myosin has focused interest on the neck region, which consists of a long alpha-helical heavy chain segment that is stabilized by the regulatory (RCL) and essential ELC light chains. It has been proposed that small structural changes in the motor domain serve to rotate this light chain binding domain. This hypothesis will be tested by: (1) Using an in vitro motility assay to measure the working stroke and unitary force of single myosin molecules from which the RCL or ELC has been removed by biochemical methods, or from which the LC-binding site has been deleted by recombinant techniques. The mechanical properties of single-headed myosin will be compared to the two-headed species. (2) Light chain interactions within a head and between heads will be determined by labelling mutant RLCs and ELCs with fluorescent probes. Changes in distance between fluorescent LCs bound to myosin will be measured by resonance energy transfer, and changes in orientation of the LC-binding region in fibers will be measured by transient fluorescence polarization. (3) The effect of light and heavy chain isoforms on force and movement will be determined by (a) analyzing the interaction of a labelled N-terminal region of ELC with actin by fluorescence spectroscopy and cro-electron microscopy, and (b) mutating variable regions in myosin isoforms to identify sequences in the head that are responsible for coupling ATP hydrolysis to movement.

描述：当前关于力产生步骤的理论 收缩周期涉及肌球蛋白头相对于肌球蛋白的重新定向 滑动时的肌动蛋白丝。 无法检测到任何大的 肌球蛋白球状催化结构域的构象变化 重点关注颈部区域，该区域由长α螺旋组成 由监管 (RCL) 和必需的稳定的重链片段 ELC轻链。 有人建议，在结构上进行小的调整 运动结构域用于旋转该轻链结合结构域。 这 假设将通过以下方式进行检验：（1）使用体外运动测定 测量单个肌球蛋白分子的工作冲程和统一力 其中 RCL 或 ELC 已通过生化方法去除，或其中 LC 结合位点已通过重组技术删除。 这 单头肌球蛋白的机械性能将与 双头物种。 (2) 头部内和头部之间的轻链相互作用 头将通过用荧光标记突变 RLC 和 ELC 来确定 探针。 与肌球蛋白结合的荧光 LC 之间的距离变化将是 通过共振能量转移和方向变化来测量 纤维中的 LC 结合区域将通过瞬态荧光进行测量 极化。 (3)轻链和重链异构体对力和力的影响 运动将通过（a）分析标记的相互作用来确定 通过荧光光谱分析 ELC 的 N 末端区域和肌动蛋白 微电子显微镜，以及 (b) 肌球蛋白可变区的突变 同工型来识别头部中负责耦合的序列 ATP水解进行运动。