Rab7: NMR and kinetics analysis of intact and C-terminal truncated constructs

Abstract

Rab proteins are a family of approximately 25kD ras-related GTPases which are associated with distinct intracellular membranes where they control vesicle traffic between intracellular compartments. The late-endosomal rab protein rab7(1-207), (lacking only the C-terminal lipids of the native molecule) and three C-terminal truncated constructs rab7(1-202), rab7(1-197) and rab7(1-182) were purified using an E. coli expression system. The C-terminal tail region of rab proteins is of special interest because it is thought to target rab proteins to particular intracellular membranes. A comparison of TOCSY-NMR spectra from intact rab7(1-207) and the tail-less construct rab7(1-182) suggested that much of the C-terminal tail is flexible in solution. The GTP hydrolysis, and GDP association and dissociation rates for all the truncated and intact constructs were similar, showing that the tail region of rab7(1-207) has little influence on the hydrolysis and exchange rates of the nucleotide.