Analysis of molecular assemblies by flow cytometry: determinants of Gi1 and by binding

Noune A. Sarvazyan; Richard R. Neubig

doi:10.1117/12.307055

1 May 1998 Analysis of molecular assemblies by flow cytometry: determinants of G_i1 and by binding

Noune A. Sarvazyan, Richard R. Neubig

Proceedings Volume 3256, Advances in Optical Biophysics; (1998) https://doi.org/10.1117/12.307055
Event: BiOS '98 International Biomedical Optics Symposium, 1998, San Jose, CA, United States

Abstract

We report here a novel application of flow cytometry for the quantitative analysis of the high affinity interaction between membrane proteins both in detergent solutions and when reconstituted into lipid vesicles. The approach is further advanced to permit the analysis of binding to expressed protein complexes in native cell membranes. The G protein heterotrimer signal transduction function links the extracellularly activated transmembrane receptors and intracellular effectors. Upon activation, (alpha) and (beta) (gamma) subunits of G protein undergo a dissociation/association cycle on the cell membrane interface. The binding parameters of solubilized G protein (alpha) and (beta) (gamma) subunits have been defined but little is known quantitatively about their interactions in the membrane. Using a novel flow cytometry approach, the binding of low nanomolar concentrations of fluorescein-labeled G(alpha) _i1 (F- (alpha) ) to (beta) (gamma) both in detergent solution and in a lipid environment was quantitatively compared. Unlabeled (beta) $gama reconstituted in biotinylated phospholipid vesicles bound F-(alpha) tightly (K_d 6 - 12 nM) while the affinity for biotinylated-(beta) (gamma) in Lubrol was even higher (K_d of 2.9 nM). The application of this approach to proteins expressed in native cell membranes will advance our understanding of G protein function in context of receptor and effector interaction. More generally, this approach can be applied to study the interaction of any fluorescently labeled protein with a membrane protein which can be expressed in Sf9 plasma membranes.

Citation Download Citation

Noune A. Sarvazyan and Richard R. Neubig "Analysis of molecular assemblies by flow cytometry: determinants of G_i1 and by binding", Proc. SPIE 3256, Advances in Optical Biophysics, (1 May 1998); https://doi.org/10.1117/12.307055

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