CONFORMATIONAL CHANGES IN MOLECULAR MOTORS

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， 发生在马达蛋白，肌球蛋白和驱动蛋白家族， 在其功能周期中。 肌球蛋白的轻链结构域是 已知在动力冲程期间改变其方向，并且将是 一条调查线的焦点。 我们将顺磁 肌球蛋白调节轻链（LC 2）的探针，并使用它们来测量 肌球蛋白轻链结构域（LC结构域）的方向 头部在兔骨骼肌纤维，以衡量旋转这一点 部队组建期间。 顺磁性标签也将是 附着在平滑肌肌球蛋白轻链上。 我们将 探讨ADP和LC 2磷酸化在细胞生理过程中的作用， 平滑肌的反应，特别是在维持 闩锁状态。 我们将监测构象的变化， 与Jim Spudich及其团队合作的肌球蛋白催化结构域 实验室 顺磁探针将附着在半胱氨酸上 引入到“cys-lite”肌球蛋白头部（头部）中的特定位置 其中所有天然反应性半胱氨酸已被除去）。 两 区域将进行初步调查，50 kD的裂缝，横贯 从肌动蛋白位点到核苷酸口袋的催化结构域，和 位于所述催化剂与所述催化剂之间的界面处的转化器区域 域和LC域。 这两个地区都被认为经历了 响应于核苷酸结合的构象变化和/或 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。 这些数据将决定 构象与哪些核苷酸状态有关， 对于特定的核苷酸，发现了多种构象， 能量的差异将不同的构象分开， 构象是通过与肌动蛋白结合而产生的。 我们将使用 自旋标记以监测驱动蛋白颈部区域的构象， 在与核苷酸和微管的相互作用中。 这个项目 将与罗恩维尔和他的实验室合作， 他们开发了一种“cys-lite”驱动蛋白二聚体， 导入颈部区域。 这个区域被认为是 允许驱动蛋白的两个头部同时与一个 微管 这一假设将通过定义 构象的这一地区使用单探针的光谱 连接到引入该区域的半胱氨酸，并通过测量 连接到两个半胱氨酸的两个顺磁性探针之间的距离。 我们正在使用计算机图形实验室的设施， 和可视化我们的三维模型。

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.