Characterization of Membrane Protein Interactions by Isothermal Titration Calorimetry

Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin alpha IIb beta 3 TM complex affinity, to study the consequences of helix-helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of alpha IIb beta 3 proceeded with a free energy change of -4.61 +/- 0.04 kcal/mol at bicelle conditions where the sampling of random helix-helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of >1 kcal/mol was obtained from helix-helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon alpha IIb beta 3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed alpha IIb beta 3 association and dissociation rates of 4500 +/- 100 s(-1) and 2.1 +/- 0.1 s(-1), respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins. (C) 2014 Elsevier Ltd. All rights reserved.