CONFORMATIONAL CHANGES IN MOLECULAR MOTORS

分子马达的构象变化

• 批准号: 6338665
• 负责人: Roger A Cooke
• 金额: $ 20.03万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6338665
• 关键词: bioenergetics; cell motility; conformation; cytoskeletal proteins; electron spin resonance spectroscopy; fluorescent dye /probe; kinesin; laboratory rabbit; microtubules; muscle strength; myofibrils; myosin light chain kinase; myosins; nucleotides; protein binding; protein structure function; site directed mutagenesis; smooth muscle; striated muscles

Description (taken from the application): We will use spectroscopic techniques, mainly electron paramagnetic resonance spectroscopy, to define the structural changes that occur within the motor proteins, both myosin and kinesin families, during their functional cycles. The data will determine what structural changes occur, what energetic differences separate these structu4res and how these structures fit into the kinetic cycle. 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 of the myosin head in rabbit skeletal muscle fibers to define 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 ex0plore 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 ale and his laboratory, who have developed a "cys-lite" kinesin dimer, with new cysteines introduced into the neck region. This region is th0ught to unf9old 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.

描述（摘自申请）：我们将使用光谱技术，主要是电子顺磁共振光谱，来定义运动蛋白（肌球蛋白和驱动蛋白家族）在其功能周期期间发生的结构变化。这些数据将确定发生哪些结构变化、这些结构之间有哪些能量差异以及这些结构如何适应动力学循环。已知肌球蛋白的轻链结构域会在动力冲程期间改变其方向，并且将成为一项研究的焦点。我们将顺磁探针连接到肌球蛋白调节轻链 (LC2) 上，并用它们测量兔子骨骼肌纤维中肌球蛋白头轻链结构域的方向，以定义该区域在力产生过程中的旋转。顺磁性标签也会附着在平滑肌中肌球蛋白的轻链上。我们将探讨 ADP 和 LC2 磷酸化在平滑肌生理反应中的作用，特别是在维持闩锁状态中的作用。我们将与 Jim Spudich 及其实验室合作监测肌球蛋白催化结构域构象的变化。顺磁探针将附着到引入“cys-lite”肌球蛋白头（所有天然反应性半胱氨酸已被去除的头）中特定位置的半胱氨酸上。最初将研究两个区域，即从肌动蛋白位点穿过催化结构域到核苷酸口袋的 50 kD 裂口，以及位于催化结构域和 LC 结构域之间界面的转换器区域。据认为，这两个区域都会响应核苷酸和/或肌动蛋白的结合而发生构象变化。在与肌动蛋白和核苷酸相互作用期间将监测这些区域的构象。这些数据将确定哪些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，什么能量差异区分不同的构象，以及通过与肌动蛋白结合产生什么构象。我们将使用自旋标记来监测驱动蛋白颈部区域在与核苷酸和微管相互作用过程中的构象。该项目将与 Ron ale 及其实验室合作进行，他们开发了一种“cys-lite”驱动蛋白二聚体，并将新的半胱氨酸引入颈部区域。该区域被认为可以让驱动蛋白的两个头部同时与微管相互作用。该假设将通过使用单个探针的光谱定义该区域的构象来检验。