PT  - JOURNAL ARTICLE
AU  - S Hoffenberg
AU  - T M Shannon
AU  - T P Noonan
AU  - S Liu
AU  - D S Daniel
AU  - J B Fishman
AU  - J B Rubins
AU  - H K Misra
AU  - G E Wright
AU  - B F Dickey
TI  - Specific and effective interaction of a guanine nucleotide analogue with small G proteins.
DP  - 1996 Jan 01
TA  - Molecular Pharmacology
PG  - 156--164
VI  - 49
IP  - 1
4099  - http://molpharm.aspetjournals.org/content/49/1/156.short
4100  - http://molpharm.aspetjournals.org/content/49/1/156.full
SO  - Mol Pharmacol1996 Jan 01; 49
AB  - G proteins are molecular switches that use a cycle of GTP binding and hydrolysis to regulate a wide variety of cellular biochemical processes. Because the functional state of these proteins is allosterically determined by bound guanine nucleotides, a nucleotide analogue with protein specificity might have pharmacological or biochemical value. The binding of [alpha-32P]GTP to four small G proteins immobilized on nitrocellulose was competed by a series of analogues with modifications at multiple sites. One analogue, N2-(p-n-butylphenyl)guanosine 5'-(beta,gamma-difluoromethylene)triphosphate, had a approximately 40-fold higher affinity for one small G protein than for two of the others. Systematic 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 guanosine-5'-(beta,gamma-difluoromethylene)triphosphate and guanosine-5'-O-(3-thiotriphosphate) than was rab5, and the Q79L GTPase-defective mutant of rab5 was 6-fold more discriminant than wild-type rab5. Guanosine-5'-(beta,gamma-difluoromethylene)triphosphate protected a 20-kDa fragment of rab5 from tryptic proteolysis with greater efficacy than guanosine-5'-O-(3-thiotriphosphate) or guanosine-5'-(beta,gamma-imido)triphosphate despite its lower affinity, and GMP stabilized a conformation indistinguishable from apo-rab5. These results identify a synthetic guanine nucleotide analogue with differential affinity for closely related G proteins, determine the atomic substitution in the analogue that confers specificity, demonstrate discrimination by the analogue between wild-type and a point-mutant G protein, and establish efficacy of the analogue in inducing conformational change of a target protein disproportionate to the affinity of the interaction.