title: Cholesterol-induced lipophobic interaction between transmembrane helices using ensemble and single-molecule fluorescence resonance energy transfer.
abstract: 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, ΔΔG(a) = -9 kJ mol(-1) and ΔΔHa = -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 ΔC(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.
authors: Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo-ku, Kyoto 606-8501, Japan.
authors: Yoshiaki
authors: Y
authors: Yano
authors: Kotaro
authors: K
authors: Kondo
authors: Ryota
authors: R
authors: Kitani
authors: Arisa
authors: A
authors: Yamamoto
authors: Katsumi
authors: K
authors: Matsuzaki
mesh_terms: D002784:Cholesterol / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D008565:Membrane Proteins / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D008565:Membrane Proteins; D002784:Cholesterol