PARTIAL G-PROTEIN ACTIVATION BY FLUORESCENT GUANINE-NUCLEOTIDE ANALOGS - EVIDENCE FOR A TRIPHOSPHATE-BOUND BUT INACTIVE STATE

N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein me chanisms. Previously, we showed that MANT fluorescence intensity when bound to G protein was related to the degree of G protein activation w here MANT-guanosine-5'-O-(3-thio-triphosphate) (mGTP gamma S) had the highest fluorescence followed by mGTP and mGDP, respectively (Remmers, A. E., Posner, R., and Neubig, R. R. (1994) J. Biol. Chem. 269, 13771 -13778). To directly examine G protein conformations with nucleotide t riphosphates bound, we synthesized several nonhydrolyzable MANT-labele d guanine nucleotides. The relative maximal fluorescence levels observ ed upon binding to recombinant myristoylated G(o alpha) (myrG(o alpha) ) and myrG(i alpha 1) were: mGTP gamma S > MANT-5'-guanylyl-imidodipho sphate > MANT-guanylyl-(beta,gamma-methylene)-diphosphonate > MANT-gua nosine 5'-O-2(thio)diphosphate. Using protection against tryptic diges tion as a measure of the activated conformation, the ability of the MA NT guanine nucleotides to maximally activate myrG(o alpha) correlated with maximal fluorescence. Biphasic dissociation kinetics were observe d for all of the RIANT guanine nucleotides. The data were consistent w ith the following model. [GRAPHICS] where G protein activation (G-GXP ) is determined by a conformational equilibrium between two triphospha te bound states as well as by the balance between binding and hydrolys is of the nucleotide triphosphate. Compared with myrG(i alpha 1), maxi mal mGTP fluorescence was only 2-fold higher for the myrG(i alpha 1) Q 204L mutant, a mutant with greatly reduced GTPase activity, and only 2 4% that of mGTP gamma S, indicating that partial activation by mGTP wa s not just due to hydrolysis of mGTP. These results extend our previou s conclusion that GTP analogs do not fully activate G protein.