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，主要是电子顺磁性 共振（EPR）光谱，以定义结构性变化 发生在运动蛋白中，肌球蛋白和动力素系列， 在其功能周期中。 肌球蛋白的轻型链域是 已知会在中风期间改变其方向，并将是 一条调查的重点。 我们将附上顺磁性 对肌球蛋白调节轻链（LC2）进行探测，并使用它们进行测量 肌球蛋白的轻链域（LC结构域）的方向 头在兔子骨骼肌纤维中以测量它的旋转 力产生期间的区域。 顺磁性标签也将是 在平滑肌中附着在肌球蛋白的轻链上。 我们将 探索ADP和LC2磷酸化在生理中的作用 平滑肌的反应，尤其是维护 闩锁状态。 我们将监视有关构象的变化 与吉姆·斯普迪奇（Jim Spudich）合作的肌球蛋白催化域 实验室。 顺磁性探针将连接到半胱氨酸 在“ Cys-Lite”肌球蛋白头（头部）中引入特定位置 从中所有天然反应性半胱氨酸都被去除）。 二 最初将调查区域，遍历50 kD的裂口 从肌动蛋白部位到核苷酸口袋的催化结构域， 位于催化之间的界面的转换器区域 域和LC域。 认为这两个地区都经历了 构象变化响应于核苷酸和/或的结合 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。 数据将确定什么 构象与核苷酸状态的构象相关，是否 发现特定核苷酸的多种构象，什么 充满活力的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 自旋标签以监测驱动蛋白的颈部区域的构象， 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 已经开发了一个“ Cys-Lite”动力素二聚体，并具有新的半胱氨酸 引入颈部区域。 人们认为这个地区是 允许两个驱动蛋白的头部与A同时相互作用 微管。 该假设将通过定义 使用单探针的光谱对该区域的构象 附着在引入该地区的半胱氨酸上，并通过测量 两个磁磁性探针附着在两个半胱氨酸上的距离。 我们正在使用计算机图形实验室设施来建造 并可视化我们的三维模型。