Resonance energy transfer between guanine nucleotide binding protein subunits and membrane lipids.
Resonance energy transfer was used to estimate the distances of closest approach between fluorescent labels on G protein subunits (alpha, beta, and gamma) and the phospholipid bilayer surface. Fluorescein-labeled alpha, beta, and gamma subunits were the energy transfer donors and hexadecylaminoeosin (HAE) in phospholipid vesicles was the acceptor. Bovine brain G protein (alpha o, beta, and gamma subunits) were individually labeled with fluorescein as described in the accompanying paper [Kwon et al. (1993) Biochemistry (preceding paper in this issue)]. Fluorescein-labeled subunits were combined with the appropriate unlabeled G protein subunits and reconstituted into phospholipid vesicles. HAE quenched the fluorescein emission in a concentration-dependent manner which was dependent upon the G protein subunit labeled (gamma = beta > alpha o). From steady-state quenching data with hexadecylaminofluorescein (HAF) as a standard to determine the density of HAE in the phospholipid bilayer, the calculated distances between fluorescein-alpha o, -beta, and -gamma and HAE are 46 +/- 2, 38 +/- 3, and 37 +/- 2 A, respectively. Energy transfer was identified as the means of fluorescence quenching by two methods: (1) addition of 0.1% Lubrol reversed the quenching of fluorescein-labeled G protein subunits by HAE and (2) sensitized HAE emission was observed in the presence of fluorescein-labeled G protein subunits. These results are the first physical measurements of the distances between sites on G protein subunits and the lipid bilayer. These data demonstrate that the label in the beta and gamma subunits is closer to the lipid bilayer than that in the alpha subunit.
Remmers
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Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
Neubig
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D000818:Animals; D002417:Cattle; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000737:chemistry; D019793:Fluorescein; D005452:Fluoresceins; D019204:GTP-Binding Proteins / Q000737:chemistry*; D007454:Iodides; D008051:Lipid Bilayers; D008563:Membrane Lipids / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D007454:Iodides; D008051:Lipid Bilayers; D008563:Membrane Lipids; C080112:hexadecylaminoeosin; D019204:GTP-Binding Proteins; D004801:Eosine Yellowish-(YS); D019793:Fluorescein