CONFORMATIONAL CHANGES IN MOLECULAR MOTORS

描述(摘自申请)：我们将使用光谱技术，主要是电子顺磁共振波谱，来定义在马达蛋白中发生的结构变化，包括肌球蛋白和运动蛋白家族，在它们的功能周期中。这些数据将决定发生了什么结构变化，是什么能量差异将这些结构分开，以及这些结构如何适应动力学周期。肌球蛋白的轻链结构域已知会在力量卒中时改变其方向，这将是一系列研究的重点。我们将顺磁探针连接到肌球蛋白调节轻链(LC2)上，并用它们来测量兔骨骼肌纤维中肌球蛋白头部轻链结构域的方向，以确定该区域在力产生过程中的旋转。顺磁性标记也将被贴在平滑肌中肌球蛋白的轻链上。我们将阐述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方面的作用。我们将与Jim Spudich和他的实验室合作，监测肌球蛋白催化结构域的构象变化。顺磁探针将被连接到半胱氨酸上，这些半胱氨酸被引入“Cys-lite”肌球蛋白头(所有天然反应性半胱氨酸都已从其上移除的头)中的特定位置。最初将研究两个区域，50kD的裂隙横跨催化结构域从肌动蛋白位置到核苷酸口袋，以及位于催化结构域和LC结构域之间的转换区。这两个区域都被认为在核苷酸和/或肌动蛋白结合后发生构象变化。在与肌动蛋白和核苷酸相互作用的过程中，将监测这些区域的构象。这些数据将决定什么构象与哪种核苷酸状态相关，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，以及通过与肌动蛋白结合产生什么构象。在与核苷酸和微管相互作用过程中，我们将使用自旋标记来监测动蛋白颈部区域的构象。该项目将与罗恩·阿莱和他的实验室合作进行，他们已经开发出一种“Cys-lite”激动素二聚体，并将新的半胱氨酸引入颈部。这一区域很难使两个运动蛋白头部同时与微管相互作用。这一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。

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.