Use of Fluorescence Spectroscopy to Study the Regulation of Small G Proteins

The Ras-like low-molecular-weight guanosine triphosphate (GTP)-binding proteins form a superfamily whose members participate in a variety of biological pathways, including the regulation of cell growth and differentiation, vesicular transport, and cytoskeletal organization (1 ). In all cases, these GTP-binding proteins appear to act as molecular switches by cycling between an inactive guanosine diphosphate (GDP)-bound state and an active GTP-bound state. This cycle is tightly regulated by distinct proteins; in particular, the exchange of GDP to GTP is stimulated by guanine nucleotide exchange factors (GEFs), and the hydrolyses of GTP back to GDP is catalyzed by GTPase-activating proteins (GAPS) (2 ). In some cases, a third class of proteins participates in the regulation of the GTP-binding/GTPase cycle by inhibiting GDP dissociation (and thus have been designated the GDP-dissociation inhibitors or GDIs) and GTP hydrolysis and stimulating the dissociation of the GTP-binding protein from membranes (2 ,3 ).