DETECTION AND QUANTITATION OF HETEROTRIMERIC G-PROTEINS BY FLUORESCENCE RESONANCE ENERGY-TRANSFER

N-Methyl-3'-O-anthranoyl (mant) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for detection of heterotr imeric guanine nucleotide binding proteins. The mant derivative of GTP gamma S, mGTP gamma S, is synthesized and purified by modification of a method initially described by Hiratsuka (1983, Biochim. Biophys. Ac ta 742, 496-508). The binding affinity of mGTP gamma S for G proteins G(i) and G(o) is comparable to that of GTP gamma S. The rate of bindin g is determined by the dissociation rate of the endogenously bound GDP . The large fluorescence increase observed upon mGTP gamma S binding t o G protein is due, in part, to resonance energy transfer from tryptop hans in the G protein to the mant guanine nucleotide. The magnitude of the fluorescence increase is dependent upon the concentration of G pr otein. Therefore, mGTP gamma S binding can be used to quantitate and l ocate G proteins during the protein purification process. This method is rapid compared to the [S-35]GTP gamma S binding assay in that (i) t he bound ligand does not need to be separated from the free ligand thu s avoiding vacuum filtration and (ii) the time required to measure flu orescence in each sample is less than that required for scintillation counting. In addition, the use of radioactivity can be avoided. Thus, the mGTP gamma S binding assay for the detection of G(i) and G(o) repr esents a rapid, reliable alternative to assays based on radio-labeled GTP gamma S binding or ADP-ribosylation with pertussis toxin. (C) 1998 Academic Press.