Cholesterol-Induced Lipophobic Interaction between Transmembrane Helices Using Ensemble and Single-Molecule Fluorescence Resonance Energy Transfer

The solvent environment regulates the conformational dynamics and functions of solvated proteins. In cell membranes, cholesterol, a major eukaryotic lipid, can markedly modulate protein dynamics. To investigate the nonspecific effects of cholesterol on the dynamics and stability of helical membrane proteins, we monitored association-dissociation dynamics on the antiparallel dimer formation of two simple transmembrane helices (AALALAA)(3) with single-molecule fluorescence resonance energy transfer (FRET) using Cy3B- and Cy5-labeled helices in lipid vesicles (time resolution of 17 ms). The incorporation of 30 mol % cholesterol into phosphatidylcholine bilayers significantly stabilized the helix dimer with average lifetimes of 450-170 ms in 20-35 degrees C. Ensemble FRET measurements performed at 15-55 degrees C confirmed the cholesterol-induced stabilization of the dimer (at 25 degrees C, Delta Delta G(a) = -9 kJ mol(-1) and Delta Delta H-aa= -60 kJ mol(-1)), most of which originated from lipophobic interactions by reducing helix-lipid contacts and the lateral pressure in the hydrocarbon core region. The temperature dependence of the dissociation process (activation energy of 48 kJ) was explained by the Kramers-type frictional barrier in membranes without assuming an enthalpically unfavorable transition state. In addition to these observations, cholesterol-induced tilting of the helices, a positive Delta C-p(a), and slower dimer formation compared with the random collision rate were consistent with a hypothetical model in which cholesterol stabilizes the helix dimer into an hourglass shape to relieve the lateral pressure. Thus, the liposomal single-molecule approach highlighted the significance of the cholesterol-induced basal force for interhelical interactions, which will aid discussions of complex protein-membrane systems.