SPECIFIC AND EFFECTIVE INTERACTION OF A GUANINE-NUCLEOTIDE ANALOG WITH SMALL G-PROTEINS

G proteins are molecular switches that use a cycle of GTP binding and hydrolysis to regulate a wide variety of cellular biochemical processe s. Because the functional state of these proteins is allosterically de termined by bound guanine nucleotides, a nucleotide analogue with prot ein specificity might have pharmacological or biochemical value. The b inding of [alpha-P-32]GTP to four small G proteins immobilized on nitr ocellulose was competed by a series of analogues with modifications at multiple sites. One analogue, N-2-(p-n-butylphenyl)guanosine 5'-(beta ,gamma-difluoromethylene)triphosphate, had a similar to 40-fold higher affinity for one small G protein than for two of the others. Systemat ic analysis of each modification in the synthetic nucleotide revealed that specificity was conferred by the carbon substitution in the beta, gamma-phosphoanhydride bond. These observations were then extended to purified proteins of known sequence in solution by filtration binding studies with H-ras and rab5. Ras was 9-fold more discriminant between sine-5'-(beta,gamma-difluoromethylene)triphosphate and guanosine-5'-0- (3-thiotriphosphate) than was rab5, and the Q79L GTPase-defective muta nt of rab5 was 6-fold more discriminant than wild-type rab5. Guanosine 5'-(beta,gamma-difluoromethylene)triphosphate protected a 20-kDa frag ment of rab5 from tryptic proteolysis with greater efficacy than guano sine-5'-0-(3-thiotriphosphate) or guanosine-5'-(beta,gamma-imido)triph osphate despite its lower affinity, and GMP stabilized a conformation indistinguishable from apo-rab5. These results identify a synthetic gu anine nucleotide analogue with differential affinity for closely relat ed G proteins, determine the atomic substitution in the analogue that confers specificity, demonstrate discrimination by the analogue betwee n wild-type and a point-mutant G protein, and establish efficacy of th e analogue in inducing conformational change of a target protein dispr oportionate to the affinity of the interaction.