Fluorescent guanine nucleotide analogs and G protein activation.
The N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Both MANT-GTP 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 nucleotide fluorescence was observed with excitation at 280 nm compared with 350 nm, due to energy transfer from tryptophan in G(o). G(o)-bound mGTP gamma S displays a blue-shift in its emission spectrum indicating a nonpolar environment for the G(o)-bound MANT. For the hydrolyzable analog, mGTP, the increase in fluorescence is followed by a decrease as it is hydrolyzed to mGDP. Unexpectedly, dissociation of mGDP was fast (t1/2 1.7 s) by comparison with GDP itself (t1/2 120 s). Binding of mGTP gamma S to G(o) was slow, but mastoparan increased the rate approximately 4-fold. For mGTP, mastoparan increased both the rate of binding 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 fully activate the G protein, even when bound. Second, mastoparan appears to increase the rate of the G protein conformational activation step, in addition to its known effect on GDP release.
Remmers
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AE
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
Posner
R
R
Neubig
R R
RR
D000818:Animals; D002417:Cattle; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000737:chemistry*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008956:Models, Chemical; D010455:Peptides; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014862:Wasp Venoms / Q000494:pharmacology; D062367:ortho-Aminobenzoates
D005456:Fluorescent Dyes; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms; D062367:ortho-Aminobenzoates; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C037500:mastoparan; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins