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 °C. Ensemble FRET measurements performed at 15–55 °C confirmed the cholesterol-induced stabilization of the dimer (at 25 °C, ΔΔ<i>G</i>_a = −9 kJ mol^–1 and ΔΔ<i>H</i>_a = −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 Δ<i>C</i>_<i>p</i>(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