In vitro GEF and GAP assays.

Small GTPases act as tightly regulated molecular switches governing a large variety of critical cellular functions. Their activity is controlled by two different biochemical reactions, GDP/GTP exchange and GTP hydrolysis. These very slow reactions require catalysis in cells by two kinds of regulatory proteins. While the guanine nucleotide exchange factors (GEFs) activate small GTPases by stimulating the slow exchange of bound GDP for the cellularly abundant GTP, GTPase-activating proteins (GAPs) accelerate the slow intrinsic rate of GTP hydrolysis by several orders of magnitude, leading to inactivation. There are a number of methods that can be used to characterize the specificity and activity of such regulators, to understand the effect of binding on the protein structure, and, ultimately, to obtain insights into their biological functions. This unit describes (1) detailed protocols for the expression and the purification of small GTPases and the catalytic domains of GEFs and GAPs; (2) preparation of nucleotide-free and fluorescent nucleotide-bound small GTPases; and (3) methods for monitoring of the intrinsic and GEF-catalyzed nucleotide exchange as well as intrinsic and GAP-stimulated GTP hydrolysis.