Detection and Quantitation of Heterotrimeric G Proteins by Fluorescence Resonance Energy Transfer

Abstract

N-Methyl-3′-O-anthranoyl (mant) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for detection of heterotrimeric guanine nucleotide binding proteins. The mant derivative of GTPγS, mGTPγS, is synthesized and purified by modification of a method initially described by Hiratsuka (1983,Biochim. Biophys. Acta742, 496–508). The binding affinity of mGTPγS for G proteins Giand Gois comparable to that of GTPγS. The rate of binding is determined by the dissociation rate of the endogenously bound GDP. The large fluorescence increase observed upon mGTPγS binding to G protein is due, in part, to resonance energy transfer from tryptophans in the G protein to the mant guanine nucleotide. The magnitude of the fluorescence increase is dependent upon the concentration of G protein. Therefore, mGTPγS binding can be used to quantitate and locate G proteins during the protein purification process. This method is rapid compared to the [35S]GTPγS binding assay in that (i) the bound ligand does not need to be separated from the free ligand thus avoiding vacuum filtration and (ii) the time required to measure fluorescence in each sample is less than that required for scintillation counting. In addition, the use of radioactivity can be avoided. Thus, the mGTPγS binding assay for the detection of Giand Gorepresents a rapid, reliable alternative to assays based on radiolabeled GTPγS binding or ADP-ribosylation with pertussis toxin.