Rapid kinetics of G protein subunit association: a rate-limiting conformational change?
G protein subunit association and dissociation are thought to play an important role in signal transduction. We measured alpha beta gamma heterocomplex formation using resonance energy transfer. Fluorescein-labelled alpha(F-alpha) emission was quenched approximately 10% on mixing with eosin-labelled beta gamma(E-beta gamma). Unlabelled beta gamma did not quench F-alpha fluorescence. Stopped-flow kinetics showed a t1/2 ranging from 2.5 s to 0.25 s for 50 nM to 1200 nM E-beta gamma. The rate saturated at high E-beta gamma concentrations consistent with a two-step mechanism. We report the first rapid-mix studies of G protein subunit association kinetics which suggest that alpha and beta gamma combine by a two-step process with a maximal rate of 4.1 +/- 0.4 s-1.
Neubig
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RR
Department of Pharmacology, University of Michigan, Ann Arbor 48109-0632.
Connolly
M P
MP
Remmers
A E
AE
D004735:Energy Transfer; D004801:Eosine Yellowish-(YS); D019793:Fluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015335:Molecular Probes; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D005456:Fluorescent Dyes; D015335:Molecular Probes; D019204:GTP-Binding Proteins; D004801:Eosine Yellowish-(YS); D019793:Fluorescein