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 heterotrimeric 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. Acta 742, 496-508). The binding affinity of mGTP gamma S for G proteins Gi and G(o) is comparable to that of GTP gamma S. The rate of binding is determined by the dissociation rate of the endogenously bound GDP. The large fluorescence increase observed upon mGTP gamma S 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, mGTP gamma S binding can be used to quantitate and locate G proteins during the protein purification process. This method is rapid compared to the [35S]GTP gamma S 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 mGTP gamma S binding assay for the detection of Gi and G(o) represents a rapid, reliable alternative to assays based on radiolabeled GTP gamma S binding or ADP-ribosylation with pertussis toxin.
Remmers
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Department of Pharmacology, University of Michigan, Ann Arbor 48109-0632, USA.
D001704:Biopolymers; D005456:Fluorescent Dyes / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000737:chemistry; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000737:chemistry / Q000302:isolation & purification
D001704:Biopolymers; D005456:Fluorescent Dyes; C111797:N-methyl-3'-O-anthranoyl guanosine 5'-O-(3-thiotriphosphate); D062367:ortho-Aminobenzoates; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins