Optimized conditions for GTP loading of Ras

Ras and the small GTPase group are essential for myriad cellular processes and cycle between GDP- and GTP-loaded states to allow stringent control of downstream signaling pathways. Biochemical studies of small GTPases can therefore require a specific nucleotide-bound state. Small GTPases possess basal intrinsic activity to process GTP into GDP plus inorganic phosphate; therefore, in vitro exchange of GDP for GTP is necessary for assays on GTP-loaded states. Here, we assess the methodology of in vitro nucleotide exchange for soluble H-Ras. We begin by describing a protocol to quantify the nucleotide-bound content of H-Ras using anion exchange chromatography and use this protocol to investigate optimal strategies for loading Ras with GTP by assessing the effects of time, temperature, H-Ras concentration, magnesium, excess nucleotide, and isoform identity. Together, we achieve a maximum of approximately 80% GTP-loading efficiency. We continue by considering storage of GTP-loaded H-Ras and present optimal conditions to minimize intrinsic GTP hydrolysis. Finally, we conclude by investigating the nucleotide composition of recombinantly expressed H-Ras encompassing cancer mutations at residues Gly12, Gly13, and Gln61 and find each to increase bound GTP compared with wildtype H-Ras. We therefore describe a methodology to quantitatively analyze the nucleotide content of small GTPases and their mutants and demonstrate conditions to achieve efficient GTP loading of Ras.