Controlled Reconstitution of Integral Membrane Proteins by Detergent Extraction through Cyclodextrin Complexation

Abstract

The reconstitution of purified membrane proteins from a detergent-solubilized state into lipid bilayer membranes is a prerequisite for many in vitro studies on membrane channels and transporters. Among the diverse methods used 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 selectively and at defined stoichiometries, which would, in principle, allow for a tight control of the reconstitution process and facilitate the rational optimization of experimental protocols. However, no systematic, quantitative studies on the complex interactions among cyclodextrins, detergents, and lipids have been reported to date. Thus, we adopted a microcalorimetric approach to thermodynamically characterize the complexation of a homologous series of alkyl maltoside detergents by various substituted β-cyclodextrins.The binding affinity increased with alkyl chain length, as reflected by a Gibbs free energy increment of about 3 kJ/mol per methylene group. In contrast with many other complexation reactions involving cyclodextrins, detergent binding did not reveal enthalpy−entropy compensation. Instead, the increase in affinity with chain length resulted from both a more favorable entropy term and a less unfavorable enthalpy change. These correlations can be ascribed to enhanced conformational flexibility and decreased repulsion between cyclodextrin and the detergent headgroup, respectively, as the alkyl chain becomes longer. The thermodynamic data thus obtained were used to optimize the efficiency of detergent extraction from mixed micelles to form well-defined, unilamellar, and uniformly sized bilayer vesicles, and a quantitative model was established to simulate and analyze this phase transition. Finally, we combined the results from experiment and theory to develop new protocols for the online monitoring of the reconstitution process to aid the functional reconstitution of membrane proteins such as ion and water channels.