Rhodopsin reconstitution in nanodiscs.

All lipids used for nanodisc were purchased from Avanti and dissolved in the form of chloroform. 40% POPS or POPG were mixed with 60% POPC and the chloroform was dried under a gentle stream of N₂. The glass vials containing the mixed lipids were kept in a desiccator overnight to get rid of any residual chloroform. CHAPS was added to dissolve the lipids till it became a clear solution. Lipids, MSP1D1 and Rho were mixed at the ratio of 360: 6: 1 in the dark for 30 minutes. Biobeads were added to the mix and incubate overnight. The reconstituted Rho was loaded onto a Superdex 200 increase column in buffer containing 20 mM HEPES (pH 8.0) and 100 mM NaCl under the light. The peak fractions containing both empty nanodiscs or nanodiscs with rhodopsin were pooled and concentrated using a 100 kDa cutoff 50 ml centrifugal concentrator (Amicon^® Ultra). The amount of rhodopsin reconstituted in nanodiscs were determined using Coomassie staining with known amount of rhodopsin as standard.