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光谱，来定义在其功能周期中发生在马达蛋白（肌球蛋白和驱动蛋白家族）内的结构变化。这些数据将确定发生了什么结构变化，这些结构之间的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在动力冲程期间改变其方向，并且将是一条研究线的焦点。我们将顺磁性探针连接到肌球蛋白调节轻链（LC 2），并使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，以定义力产生过程中该区域的旋转。顺磁性标记也将附着在平滑肌中肌球蛋白的轻链上。我们将探讨ADP和LC 2磷酸化在平滑肌生理反应中的作用，特别是在维持闭锁状态中的作用。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。顺磁性探针将附着于引入“cys-lite”肌球蛋白头部（所有天然反应性半胱氨酸已被去除的头部）中特定位置的半胱氨酸。最初将研究两个区域，从肌动蛋白位点到核苷酸口袋横穿催化结构域的50 kD裂缝，以及位于催化结构域和LC结构域之间的界面处的转换器区域。这两个区域都被认为在核苷酸和/或肌动蛋白的结合反应中发生构象变化。这些区域的构象将在与肌动蛋白和核苷酸的相互作用过程中进行监测。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，不同构象之间的能量差异，以及与肌动蛋白结合产生的构象。我们将使用自旋标记监测的颈部区域的驱动蛋白的构象，在与核苷酸和微管的相互作用。该项目将与罗恩ale和他的实验室合作进行，他们开发了一种“cys-lite”驱动蛋白二聚体，在颈部区域引入了新的半胱氨酸。这一区域被设计成允许驱动蛋白的两个头部同时与微管相互作用。将通过使用单个探针的光谱定义该区域的构象来检验该假设。

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.