The reconstitution of purified membrane proteins from a detergent-solubilised state into lipid bilayer membranes is a prerequisite for many in vitro studies on membrane channels and transporters [1]. In comparison with other, well-established methods for the removal of detergent from ternary protein/lipid/detergent mixtures, detergent complexation with cyclodextrins offers a number of unique advantages. In particular, cyclodextrins sequester detergents at defined stoichiometries, which allows for a tight control of the reconstitution process and facilitates the rational optimization of experimental protocols.
We have established a quantitative model to describe the phase transition during cyclodextrin-mediated detergent extraction from mixed micelles, which is monitored by isothermal titration calorimetry. Proof-of-principle reconstitution of the α-helical membrane protein Mistic from B. subtilis [2] demonstrates the formation of well-defined, unilamellar, and uniformly sized proteoliposomes, as verified by dynamic light scattering and circular dichroism spectroscopy. The model facilitates adaption of the microcalorimetric approach to different detergent/lipid combinations, and the option of using various substituted cyclodextrin derivatives offers great flexibility with regard to detergent/cyclodextrin complex properties.