FLUORESCENT GUANINE-NUCLEOTIDE ANALOGS AND G-PROTEIN ACTIVATION

The N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are use ful environmentally sensitive fluorescent probes for studying G protei n mechanisms. Both MANT-GTF gamma S (mGTP gamma S) and MANT-GTP (mGTP) displayed a magnesium-dependent increase in fluorescence upon binding to bovine brain G(o). A much greater increase in MANT-guanine nucleot ide fluorescence was observed with excitation at 280 nm compared with 350 nm, due to energy transfer from tryptophan in G(o). G(o)- bound mG TP gamma S displays a blue-shift in its emission spectrum indicating a nonpolar environment for the G(o)- bound MANT For the hydrolyzable an alog, mGTP, the increase in fluorescence is followed by a decrease as it is hydrolyzed to mGDP. Unexpectedly, dissociation of mGDP was fast (t(1/2) 1.7 s) by comparison with GDP itself (t(1/2) 120 s). Binding o f mGTP gamma S to G(o) was slow, but mastoparan increased the rate app roximately 4-fold. For mGTP, mastoparan increased both the rate of bin ding and the peak fluorescence, even at saturating mGTP concentrations . Modeling the mGTP fluorescence kinetics in the presence and absence of mastoparan results in two novel conclusions. First, mGTP does not f ully activate the G protein, even when bound. Second, mastoparan appea rs to increase the rate of the G protein conformational activation ste p, in addition to its known effect on GDP release.