A continuous, regenerative coupled GTPase assay for dynamin-related proteins

Dynamin-related proteins (DRPs) compose a diverse family of proteins that function, through GTPase stimulated self-assembly, to remodel cellular membranes. The molecular mechanism by which DRPs mediate membrane remodeling events and the specific role of their GTPase cycle is still not fully understood. Although DRPs are members of the GTPase superfamily, they possess unique kinetic properties. In particular, they have relatively low affinity for guanine nucleotides and, under conditions that favor self-assembly, they have high rates of GTP turnover. Established fixed time point assays used for the analysis of assembly stimulated GTPase activity are prone to inaccuracies due to substrate depletion and are also limited by lack of time resolution. We describe a simple, continuous, coupled GTP regenerating assay that tackles the limitations of the fixed time point assays and can be used for the kinetic analysis of DRP GTP hydrolysis under unassembled and assembled conditions.