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, ΔΔGa = −9 kJ mol–1 and ΔΔHa = −60 kJ mol–1), most of which originated from “lipophobic” interactions by reducing helix–lipid contacts and the l...