Bicelles stabilize a compact conformation of opsin with enhanced α-helical packing

Bicelles form a disc-like planar lipid bilayer surrounded by short-chain lipids at the peripheral rim. Therefore, they are well-suited to study purified transmembrane proteins in a more native-like environment. In this study, we investigated the physico-chemical properties of bicelles using transmission electron microscopy, dynamic light scattering, and fluorescence spectroscopy. The G protein-coupled receptor rhodopsin served as a prototypical membrane protein, which we reconstituted into bicelles having an average diameter of 11.6 ± 0.6 nm that increased to 14.9 ± 0.7 nm upon incorporation of rhodopsin. These results were confirmed by transmission electron microscopy and fluorescence spectroscopy. Comparing the molar concentration of bicelles and rhodopsin, we determined an average of 4 ± 1 bicelles per molecule of rhodopsin based on dynamic light scattering, and 6 ± 3 based on transmission electron microscopy data. Thus, only 14-25% of bicelles contained rhodopsin without evidence of aggregation. Infrared and circular dichroism spectroscopy measurements demonstrated that, in bicelles, rhodopsin forms a more packed structure compared to the detergent-solubilized condition, and exhibits enhanced α-helical packing. Moreover, the bicelle-reconstituted form exhibited increased thermal stability. When immobilized on sensor chip surfaces via concanavalin A anchoring, rhodopsin in bicelles showed at least a 10-fold lower binding efficiency to the G protein transducin than in detergent, although maintaining a 1:1 binding stoichiometry. These results indicate a monolamellar orientation of the bicelles on the sensor chip surface, exposing rhodopsin in native folding.