Formation of Nanodiscs

The method used to induce the formation of lipid-bilayer nanodiscs was based on the protocol by Luthra et al.⁴⁹ 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was solubilized at a 50 mM concentration with the following method. 25.6 mg (32.6 μmol) of the lipid was dissolved in 2.5 mL of chloroform in a glass tube. The solvent was evaporated in an argon stream while the tube was turned at an angle. The lipid-coated glass tube was dried overnight in the desiccator. The lipid was again dissolved in 650 μL of a solution of 100 mM sodium cholate and 100 mM NaCl by repeated cycles of heating to 60 °C and sonicating for 3 min. The process was repeated until the solution was completely clear.

A 500 μL nanodisc formation solution was prepared from 70 μL of solubilized DOPC (7 mM final concentration) by adding MSP washing buffer 3 and purified protein to a final protein concentration of 50 μM (1:140 ratio of protein to lipid). This was incubated for 30 min on ice. After incubation, the mixture was transferred to a prehydrated dialysis cassette (10 kDa MWCO) and dialyzed against 200 mL of MSP washing buffer 3. The buffer was changed 2 times and the final dialysis step run overnight.