Determinants of gi1alpha and beta gamma binding. Measuring high affinity interactions in a lipid environment using flow cytometry.
G protein heterocomplex undergoes dissociation and association during its functional cycle. Quantitative measurements of alpha and betagamma subunit binding have been difficult due to a very high affinity. We used fluorescence flow cytometry to quantitate binding of fluorescein-labeled Gi1alpha (F-alpha) to picomolar concentrations of biotinylated G beta gamma. Association in Lubrol solution was rapid (kon = 0.7 x 10(6) M-1 s-1), and equilibrium binding revealed a Kd of 2.9 +/- 0.8 nM. The binding showed a complex dependence on magnesium concentration, but activation of F-alpha with either GDP/aluminum fluoride or guanosine 5'-O-(3-thiotriphosphate) completely prevented formation of the heterocomplex (Kd > 100 nM). The binding was also influenced by the detergent or lipid environment. Unlabeled betagamma reconstituted in biotinylated phospholipid vesicles (pure phosphatidylcholine or mixed brain lipids) bound F-alpha approximately 2-3-fold less tightly (Kd = 6-9 nM) than in Lubrol. In contrast, beta gamma in ionic detergents such as cholate and 3-[(cholamidopropyl)diethylammonio]-1-propanesulfonate exhibited substantially lower affinities for F-alpha. Dissociation of F-alpha from beta gamma reconstituted in lipid vesicles was observed upon addition of aluminum fluoride or excess unlabeled alpha subunit, indicating that myristoylated alpha subunit has only a weak interaction with lipids without the beta gamma subunit. The kinetics of aluminum fluoride-stimulated dissociation were slower than those of the alpha subunit conformational change detected by intrinsic fluorescence. These results quantitatively demonstrate G protein subunit dissociation upon activation and provide a simple but powerful new approach for studying high affinity protein/protein interactions in solution or in a lipid environment.
Sarvazyan
N A
NA
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-0632, USA.
Remmers
A E
AE
Neubig
R R
RR
D005434:Flow Cytometry; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008051:Lipid Bilayers; D011485:Protein Binding
D008051:Lipid Bilayers; D019204:GTP-Binding Proteins