title: Calorimetric Quantification of Cyclodextrin-Mediated Detergent Extraction for Membrane-Protein Reconstitution.
abstract: For many in vitro studies, purified membrane proteins need to be reconstituted from detergent micelles into lipid bilayers to regain their native structures and functions. Stoichiometric complexation of detergent by cyclodextrin provides a tightly controllable strategy for detergent extraction. Here, we describe a practical approach making use of isothermal titration calorimetry to obtain a complete set of thermodynamic parameters that allows for quantitative prediction of the transition from micelles to bilayer membranes during reconstitution. These parameters include the dissociation constant of the cyclodextrin/detergent inclusion complex, the critical micellar concentration of the detergent, and the phase boundaries of the lipid/detergent phase diagram. The underlying theoretical framework involves linked equilibria among all pseudophases, as described previously (Textor, Vargas, & Keller, 2015). This chapter focuses on practical aspects of the approach and discusses caveats and calorimetry-specific details of data analysis. With the entire parameter set at hand, exploration of different reconstitution trajectories within the lipid/detergent phase diagram is possible. Together with the straightforward control over the rate of detergent extraction offered by cyclodextrin complexation, this opens the possibility of systematically tuning and optimizing the reconstitution process of membrane proteins. Provided some particular precautions are taken, the approach can be adapted to many other combinations of proteins, lipids, detergents, and cyclodextrins.
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Textor
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany. Electronic address: mail@sandrokeller.com.
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
mesh_terms: D002151:Calorimetry / Q000379:methods*; D003505:Cyclodextrins / Q000737:chemistry; D003902:Detergents / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D003505:Cyclodextrins; D003902:Detergents; D008565:Membrane Proteins
keywords: Critical micellar concentration; Demicellization; Inclusion complex; Isothermal titration calorimetry; Micelles; Mistic; Phase diagram; Reconstitution; Solubilization; Vesicles