One-Pot Reconstitution of GPCRs into Unilamellar Vesicles for Fluorescence-Based Phospholipid Scramblase Activity Assay.

Overview

Scramblases are membrane proteins that translocate phospholipids bidirectionally between the leaflets of a membrane bilayer. Examples of scramblases include Class A G protein-coupled receptors (GPCRs), members of the TMEM16 and DedA protein families, and protein insertases. To measure scramblase activity, the protein of interest is purified and reconstituted into large unilamellar vesicles that contain trace amounts of a fluorescent phospholipid reporter. The activity assay reports on the translocation of fluorescent lipids from the inner to the outer leaflet of the vesicles by using a membrane-impermeant reducing agent to eliminate the fluorescence of only those molecules that can reach the outer leaflet. Here we describe a one-pot reconstitution protocol whereby phospholipids, solubilized in the non-ionic detergent CHAPS, are combined with a purified GPCR to generate mixed micelles and then treated with detergent-adsorbing polystyrene beads (SM2 BioBeads) to yield proteoliposomes. The proteoliposomes are extruded to generate a more homogeneously sized, unilamellar population suitable for activity assays. We describe protocols for measuring the protein and phospholipid content of the vesicles and for assaying scramblase activity.