Structure, dynamics and function of spin labeled G proteins activated by dimerization studied by multi-frequency cw and pulsed EPR

GAD是通过二聚化活化的G蛋白的亚家族，其通过与已经针对相关Ras蛋白建立的机制不同的机制表现出不同的功能。尽管GAD蛋白类内的功能和结构的多样性，激活的共同模式表明其功能相似的机制原则。然而，有来自结构数据的不同的机制模型之间的不一致，使得它的问题是否GAD共享一个共同的激活机制。此外，关于G结构域二聚化如何与蛋白质功能偶联的知识仍然缺失。在这里，我们将研究GAD家族的两个“顺式”-二聚体形成成员，它们在生化和结构上都有很好的特征，即来自E. coli和C. tepidum Roco，人LRRK 2帕金森激酶的原核同源物。通过这种方式，我们的目标是研究G结构域的行为及其对蛋白质功能的影响。我们将用多频连续波和脉冲EPR波谱（9.5 - 95 GHz）研究这些蛋白质的定点自旋标记变体在GTdR循环期间的结构和构象变化，以揭示GAD功能的机制。

GADs are a subfamily of G proteins activated by dimerization, which, exhibit diverse functionality by mechanisms distinct from those that have been established for the related Ras proteins. Despite the functional and structural diversity within the GAD protein class, the common mode of activation suggests similar mechanistic principles for their function. However, there are inconsistencies between different mechanistic models derived from structural data, making it questionable whether GADs share a common mechanism for activation. Furthermore, knowledge of how G domain dimerization is coupled to protein function is still missing. Here we will examine two “cis”-dimer forming members, of the GAD family that have been quite well characterized biochemically and structurally, namely the tRNA modifying MnmE/GidA from E. coli and the Roc-COR tandem from C. tepidum Roco, a prokaryotic homologue of the human LRRK2 Parkinson kinase. In this way we aim to study the behaviour of the G domains and their influence on the function of the protein. The structure and conformational changes during the GTPase cycle of site-directed spin labeled variants of these proteins will be studied with multi-frequency cw and pulse EPR spectroscopy at (9.5 - 95 GHz) to unravel the mechanism of GAD function.