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.

GXXXG‐Mediated Parallel and Antiparallel Dimerization of Transmembrane Helices and Its Inhibition by Cholesterol: Single‐Pair FRET and 2D IR Studies

“…As a proof-of-concept, antiparallel and parallel host helix dimers were prepared in palmitoyl oleoyl phosphocholine (POPC)/cholesterol (7/3) vesicles, and observed with total internal reflection fluorescence microscopy ( Figure 1b). As expected, most of the bright spots including a donor and acceptor pair exhibited FRET fluctuation due to transient associations in antiparallel topology (86%, n = 28), [15] whereas no FRET was observed for parallel topology (0%, n = 28) in the time resolution (17 msec).…”

“…Approximate energetic contributions of the GXXXG motif are summarized in Table 1, obtained from sp-FRET, ensemble FRET, or self-quenching (Supporting Information, Figure S5) experiments. We have observed that the single-molecule and ensemble measurements give the same association energy values, [15] although the introduction of Cys and linker residues for the topology control can slightly alter the ΔG a (~3 kJ mol −1 ) ( Table 1). In POPC, the motif contributed to ΔΔG a of −17 and −15 kJ mol −1 (in doubled mole fraction units) for parallel and antiparallel associations, respectively, comparable with the effect of a Gly mutation on glycophorin A dimerization.…”

“…[14] Recently, single-pair fluorescence resonance energy transfer (sp-FRET) experiments in liposomes revealed that cholesterol stabilizes an hourglass-shaped antiparallel dimer by 'lipophobic' forces. [15] Using this transmembrane helix as a host, we investigated the effect of the guest GXXXG motif at the center of the sequence (AALALAA-AGLALGA-AALALAA).…”

“…The host helices form cholesterol-induced hourglass-shaped dimers with a crossing angle of 50-60° to release the higher lateral pressure by cholesterol in the hydrocarbon core by the penetration of water between helices. [15] On the other hand, the GXXXG dimers have small crossing angles (~10°) (Figure 3a) therefore should be mechanically unstable in the presence of cholesterol. Furthermore, introduction of Gly residues enhance flexibility of the main chain, thus cannot release the cholesterol-originated lateral pressure even by forming hourglass-shaped dimers.…”

Be straight to better coordinate two -- Head groups and extended acyl chains of raft-like bilayer lipids can localize in groove between dimerized transmembrane helical peptides and assist sequence-nonspecific stabilization of peptide dimers by cholesterol

“…Sequence-nonspecific effects could be important to set the basal activities of receptor proteins, thereby adjusting the tone of cellular activities such as intensity of inflammation [3]. Yano et al [5] showed that homodimer of a helical peptide with a sequence of (AALALAA) 3 is stabilized in a 7:3 POPC/cholesterol bilayer compared to the POPC bilayer.…”