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，用于定义 存在于马达蛋白中，包括肌球蛋白和运动蛋白家族， 在它们的功能周期中。肌球蛋白的轻链结构域是 已知在动力冲程期间会改变其方向，并且将是 一条调查路线的重点。我们会把顺磁 肌球蛋白调控轻链(Lc2)的探针及其应用 肌球蛋白轻链结构域(LC结构域)的定位 头在兔骨骼肌纤维中测量旋转这一点 区域在部队生成过程中。顺磁性标签也将被 附着在平滑肌中肌球蛋白的轻链上。我们会 探讨ADP和LC2磷酸化在生理学中的作用 平滑肌的反应，特别是在维持 闩锁状态。我们将监测分子构象的变化 肌球蛋白的催化结构域与Jim Spudich和他的合作 实验室。顺磁探针将附着在半胱氨酸上 导入肌球蛋白头(头)中的特定位置 其中所有天然反应性半胱氨酸都已被去除)。二 将对区域进行初步调查，即横跨50kD的裂隙 从肌动蛋白位置到核苷酸口袋的催化结构域，以及 位于催化剂和催化剂之间的界面的转化器区域 域和LC域。这两个地区都被认为经历了 核苷酸和/或结合反应的构象变化 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。数据将决定 构象与哪种核苷酸状态有关，无论是 发现了特定核苷酸的多个构象，什么 能量差异将不同的构象分开，以及 构象是通过与肌动蛋白结合而产生的。我们将使用 自旋标记以监测激动素颈部区域的构象， 在与核苷酸和微管相互作用过程中。这个项目 将与罗恩·维尔和他的实验室合作进行， 他们开发了一种带有新半胱氨酸的“Cys-lite”激动素二聚体 进入颈部。这一地区被认为是向 允许Kinesin的两个头部同时与 微管。这一假设将通过定义 用单探针光谱研究这一区域的构象 与引入该地区的半胱氨酸结合，并通过测量 连接在两个半胱氨酸上的两个顺磁探针之间的距离。 我们正在使用计算机图形实验室的设施来建造 并将我们的三维模型可视化。

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.