Functional Reconstitution of Membrane Proteins Monitored by Isothermal Titration Calorimetry

Abstract

Membrane protein reconstitution denotes the transfer of a purified (but usually inactive) membrane protein from detergent micelles into lipid bilayers. The aim is to restore the native protein fold and function in a well-defined membrane environment. The reconstitution yield critically depends on a wide range of parameters, including temperature, pH, ionic strength, as well as the type and concentration of detergent, lipid, protein, and additives. Moreover, it is of paramount importance to initiate the reconstitution process from a suitable lipid-to-detergent ratio. Unfortunately, however, assessing the success of a reconstitution experiment has thus far been limited to a trial-and-error approach, which has substantially slowed progress in the field.To address this problem, we have established high-sensitivity isothermal titration calorimetry (ITC) as a powerful tool for monitoring the reconstitution of membrane proteins into lipid vesicles. Using ITC, the complex changes in the physical state of a protein/lipid/detergent mixture during reconstitution can be followed in a non-invasive and fully automated manner. Here we exemplify this approach for the prokaryotic potassium channel KcsA, which we first purified in detergent micelles and then reconstituted into stable proteoliposomes at very high protein densities. Electrophysiological experiments performed in planar lipid membranes confirmed that KcsA regained its functional activity upon reconstitution.