Abstract This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of protein expression levels, and a fit of the binding curve determines the number of binding sites and the dissociation coefficient. We experimentally verified the technique, using myosin-1C-EGFP as a model system and fit its binding curve. Furthermore, we studied the protein-lipid interactions of the membrane binding domains from lactadherin and phospholipase C-δ1 to evaluate the feasibility of using competition binding experiments to identify specific lipid-protein interactions in living cells. Finally, we applied the technique to determine the lipid specificity, the number of binding sites, and the dissociation coefficient of membrane binding for the Gag matrix domain of human T-lymphotropic virus type 1, which provides insight into early assembly steps of the retrovirus.
Bibliographical noteFunding Information:
This research was supported by National Institutes of Health (grant Nos. R01 GM064589 and GM098550) and the National Science Foundation (grant No. PHY-0957728). E.M.S. acknowledges support by the National Institute of Allergy and Infection Diseases of the National Institutes of Health grant No. 5T32A1083196 (Minnesota Training Program in Virology) and the University of Minnesota Graduate School Doctoral Dissertation Fellowship.