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

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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.