CONFORMATIONAL CHANGES IN MOLECULAR MOTORS

分子马达的构象变化

• 批准号: 6220223
• 负责人: Roger A Cooke
• 金额: $ 0.79万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6220223
• 关键词: biological products; biomedical resource

We use spectroscopic techniques, mainly electron paramagnetic resonance (EPR) spectroscopy, to define the structural changes that occur within the motor proteins, both myosin and kinesin families, during their functional cycles. The light chain domain of myosin is known to alter its orientation during the power stroke, and will be a focus of one line of investigation. We will attach paramagnetic probes to myosin regulatory light chains (LC2) and use them to measure the orientation of the light chain domain (LC domain) of the myosin head in rabbit skeletal muscle fibers to measure the rotation of this region during force generation. Paramagnetic labels will also be attached to the light chains of myosin in smooth muscle. We will explore the roles of ADP and LC2 phosphorylation in the physiological responses of smooth muscle, and in particular in the maintenance of the latch state. We will monitor changes in the conformation of the catalytic domain of myosin in collaboration with Jim Spudich and his laboratory. Paramagnetic probes will be attached to cysteines introduced into specific locations in "cys-lite" myosin heads (heads from which all native reactive cysteines have been removed). Two regions will be initially investigated, the 50 kD cleft that traverses the catalytic domain from the actin site to the nucleotide pocket, and the converter region which lies at the interface between the catalytic domain and the LC domain. Both regions are thought to undergo conformational changes in response to binding of nucleotides and/or actin. The conformation of these regions will be monitored during interaction with actin and nucleotides. The data will determine what conformations are associated with which nucleotide states, whether multiple conformations are found for specific nucleotides, what energetic differences separate the different conformations, and what conformations are produced by binding to actin. We will use spin-labels to monitor the conformation of the neck region of kinesin, during interaction with nucleotides and microtubules. This project will be carried out in collaboration with Ron Vale and his laboratory, who have developed a "cys-lite" kinesin dimer, with new cysteines introduced into the neck region. This region is thought to unfold to allow both heads of kinesin to interact simultaneously with a microtubule. This hypothesis will be tested by defining the conformation of this region using the spectra of single probes attached to cysteines introduced into the region, and by measuring the distance between two paramagnetic probes attached to two cysteines. We are using the Computer Graphics Laboratory facilities for building and visualizing our three-dimensional models.

我们使用光谱技术，主要是电子顺磁 共振（EPR）光谱，以确定结构变化 发生在运动蛋白（肌球蛋白和驱动蛋白家族）中， 在它们的功能周期中。 肌球蛋白的轻链结构域是 已知会在动力冲程期间改变其方向，并且将是 一条线调查的重点。 我们将附加顺磁 探测肌球蛋白调节轻链 (LC2) 并用它们来测量 肌球蛋白轻链结构域（LC 结构域）的方向 兔子骨骼肌纤维中的头部来测量其旋转 力生成期间的区域。 顺磁标签也将 附着在平滑肌中肌球蛋白的轻链上。 我们将 探讨 ADP 和 LC2 磷酸化在生理学中的作用 平滑肌的反应，特别是在维持 锁存状态。 我们将监测构象的变化 与 Jim Spudich 及其同事合作研究肌球蛋白的催化结构域 实验室。 顺磁性探针将附着在半胱氨酸上 引入“cys-lite”肌球蛋白头的特定位置（头 其中所有天然反应性半胱氨酸已被去除）。 二 将首先研究区域，穿过的 50 kD 裂缝 从肌动蛋白位点到核苷酸口袋的催化结构域，以及 转换器区域位于催化器之间的界面处 域和LC域。 这两个地区都被认为正在经历 响应于核苷酸和/或结合的构象变化 肌动蛋白。 这些区域的构象将在 与肌动蛋白和核苷酸的相互作用。 数据将决定什么 构象与哪些核苷酸状态相关，是否 发现特定核苷酸的多种构象，什么 能量差异将不同的构象分开，什么 构象是通过与肌动蛋白结合产生的。 我们将使用 自旋标签监测驱动蛋白颈部构象， 在与核苷酸和微管相互作用期间。 这个项目 将与 Ron Vale 及其实验室合作进行， 他们开发了一种带有新半胱氨酸的“cys-lite”驱动蛋白二聚体 引入颈部区域。 该区域被认为将展开 允许驱动蛋白的两个头同时与 微管。 该假设将通过定义 使用单个探针的光谱确定该区域的构象 附着在引入该区域的半胱氨酸上，并通过测量 连接到两个半胱氨酸的两个顺磁探针之间的距离。 我们正在使用计算机图形实验室设施来构建 并将我们的三维模型可视化。