TOPOGRAPHICAL STUDIES OF SMOOTH AND NONMUSCLE MYOSINS

平滑肌肌球蛋白和非肌肉肌球蛋白的形貌研究

• 批准号: 6796889
• 负责人: Christine R Cremo
• 金额: $ 33.81万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6796889
• 关键词: adenosinetriphosphatase; binding sites; chemical kinetics; conformation; crosslink; enzyme activity; enzyme mechanism; enzyme structure; fluorescence spectrometry; fluorescent dye /probe; intermolecular interaction; isozymes; mass spectrometry; microfilaments; myosin light chain kinase; myosins; phosphorylation; photochemistry; physical model; protein folding; protein structure function; smooth muscle

Description:(Adapted from the application) Phosphorylation of smooth muscle myosin (SMM) and nonmuscle myosin II is required to activate cellular functions. A single serine in the regulatory light chain (RLC) of each of the two "head" domains of myosin is phosphorylated by a calcium-activated kinase. Current knowledge suggests that this regulatory effect is mediated through large conformational changes in myosin structure. Dr. Cremo's long-term goal is to determine the structural basis of the phosphorylation-dependent regulatory mechanism. Dr. Cremo hypothesizes that a specific interaction between the two heads is favored in the unphosphorylated "off" or down-regulated state. A prediction of the hypothesis is that the rod domain near the heads is altered by phosphorylation, thus assisting in breaking the interaction between the heads. Dr. Cremo now extends this hypothesis to the idea that the interaction between the two heads is not symmetrical, based upon recent findings from her laboratory. She presents a detailed "structural hypothesis" for how phosphorylation of the RLC controls the head-head interactions. She will also test the hypothesis proposed by others that interactions between the two catalytic domains, controlled by regulatory domain interactions, explain the down-regulated kinetics of the unphosphorylated state. These experiments should answer the question "How does phosphorylation of two residues at the head-tail junction alter the structure of two ATP binding sites 15 nm away?" The Specific Aims are: (1) Obtain secondary and tertiary structural data about the RLC of myosin and construct a 3-dimensional model to address the molecular mechanism of phosphorylation-dependent regulation. (2) Obtain initial quaternary structural data concerning the inter-head interactions between the two ELC (essential light chain), between the ELC and the heavy chain, and between the two heavy chains. (3) Determine which portions of one head must be present to down-regulate the partner head. (4) Obtain secondary and tertiary structural data about the rod region of myosin near the heads and construct a three-dimensional model to address the molecular mechanism of phosphorylation-dependent regulation.

描述：（从应用程序改编）平滑肌的磷酸化 需要肌球蛋白（SMM）和非肌肉肌球蛋白II激活细胞 功能。每个调节轻链（RLC）中的单个丝氨酸 肌球蛋白的两个“头”结构域被钙激活的激酶磷酸化。 当前的知识表明，这种调节效应是通过 肌球蛋白结构的大构象变化。 Cremo博士的长期目标是 确定磷酸化依赖性调节的结构基础 机制。 Cremo博士假设两个头之间的特定相互作用是 在未磷酸化的“ OFF”或下调状态中受到青睐。一个预测 假设是头部附近的杆域被改变 磷酸化，从而有助于打破头部之间的相互作用。 Cremo博士现在将这一假设扩展到了以下观点 根据她的最新发现，这两个头不是对称的 实验室。她提出了一个详细的“结构性假设” RLC的磷酸化控制头头相互作用。她也会 检验其他人提出的假设，即两者之间的相互作用 由调节域相互作用控制的催化域，解释 未磷酸化状态的下调动力学。这些实验应该 回答这个问题：“在头尾如何磷酸化两个残基的磷酸化 连接改变了15 nm远的两个ATP结合位点的结构吗？” 具体目的是： （1）获取有关肌球蛋白RLC和 构建一个三维模型来解决分子机制 磷酸化依赖性调节。 （2）获取有关头间的初始第四纪结构数据 两个ELC之间的相互作用（必需轻链），ELC和 重链，在两个重链之间。 （3）确定一个头必须存在的部分以下调 伴侣负责人。 （4）获取有关杆区域的二级和第三结构数据 头部附近的肌球蛋白并构建一个三维模型来解决 磷酸化依赖性调节的分子机制。