Poster Presentation 2014 International Biophysics Congress

Controlled reconstitution of membrane proteins by detergent extraction through cyclodextrin complexation (#607)

Martin Textor 1 , Carolyn Vargas 1 , Natalia Markova 2 , Sandro Keller 1
  1. Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany
  2. GE Healthcare, Uppsala, Sweden

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.

  1. Fiedler et al. 2010. Protein folding in membranes. Cell. Mol. Life Sci.. 67:1779–1798.
  2. Roosild et al. 2005. NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science. 307:1317–1321.