Structure and dynamics of the aliphatic cholesterol side chain in membranes as studied by ²H NMR spectroscopy and molecular dynamics simulation

Abstract

Cholesterol is an evolutionarily highly optimized molecule particularly known for its ability to condense the phospholipids in cellular membranes. Until recently, the accompanying increase in the chain order of the surrounding phospholipids was attributed to the planar and rigid tetracyclic ring structure of cholesterol. However, detailed investigations of cholesterol's aliphatic side chain demonstrated that this side chain is responsible for approximately half of the condensation effect. Therefore, we investigated the structure and dynamics of the aliphatic side chain of cholesterol using (2)H solid-state nuclear magnetic resonance (NMR) spectroscopy and microsecond timescale all-atom molecular dynamics (MD) simulations in four different model membranes: POPC, DPPC, PSM, and POPC/PSM (1 : 1 mol/mol) and at three different temperatures: 5 °C, 37 °C, and 50 °C. A cholesterol variant, in which 11 hydrogens of the aliphatic side chain were exchanged for deuterium, was used and the respective (2)H NMR spectra confirmed the axially asymmetric rotational diffusion of cholesterol in DPPC and PSM. Furthermore, NMR spectra indicated that some hydrogens showed an unexpected magnetic inequivalency. This finding was confirmed by all-atom molecular dynamics simulations and detailed analysis revealed that the hydrogens of the methylene groups at C22, C23, and C24 are magnetically inequivalent. This inequivalency is caused by steric clashes of the aliphatic side chain with the ring structure of cholesterol as well as the branched C21 methyl group. These excluded volume effects result in reduced conformational flexibility of the aliphatic side chain of cholesterol and explain its high order (order parameter of 0.78 for chain motions) and large contribution to the condensation effect. Additionally, the motional pattern of the side chain becomes highly anisotropic such that it shows larger fluctuations perpendicular to the ring plane of cholesterol with a biaxiality of the distribution of 0.046. Overall, our results shed light on the mechanism how the aliphatic side chain is able to contribute about half of the condensation effect of cholesterol.

Exploring the structure and stability of cholesterol dimer formation in multicomponent lipid bilayers

“…The simulation results agree well with experimental solid state NMR order parameters determined for 34% CHOL in POPC and CHOL tail NMR order parameters for 25% CHOL in POPC (Fig. a) . The sterol ring section of CHOL shows slightly more disorder in simulation than experiment.…”

“… NMR order parameters (S CH ) of CHOL C‐H bonds computed for (a) overall CHOL (b) CHOL dimers (D) and monomers (M) in four bilayer compositions. Experimentally measured NMR order parameters are shown for comparison (points) . Error bars on S CH values are too small to be visible.…”

Cholesterol-induced suppression of membrane elastic fluctuations at the atomistic level

“…Various biophysical methods have been used to study lipid-cholesterol interactions, including electron spin resonance (ESR) (Cheng et al, 2014; Delmelle et al, 1980; Hubbell and McConnell, 1971; Lai and Freed, 2014; Manukovsky et al, 2013; Semer and Gelerinter, 1979; Stepien et al, 2015; Vitiello et al, 2015; Williams et al, 2013), Raman (Lippert and Peticolas, 1971; Mendelsohn, 1972; Tantipolphan et al, 2006), Fourier transform infrared (FT-IR) (Umemura et al, 1980), fluorescence spectroscopy (Xu and London, 2000; Yasuda et al, 2015a), atomic force microscopy (AFM), multidimensional NMR spectroscopy (Holland and Alam, 2006; Leftin et al, 2013; Leftin et al, 2014a; Warschawski and Devaux, 2005), solid-state 2 H nuclear magnetic resonance (NMR) (Bartels et al, 2008; Brown, 1990; Bunge et al, 2008; Martinez et al, 2004; Martinez et al, 2002; Matsumori et al, 2012; Stockton et al, 1976; Vogel et al, 2016; Weisz et al, 1992; Yasuda et al, 2015a), and neutron diffraction methods (Armstrong et al, 2014; Toppozini et al, 2014). However, a thorough understanding of the physical basis for these observations in relation to the intricate lipid compositions of many biological membranes to some extent remains an enigma (Feigenson, 2015; McConnell, 2005; Meinhardt et al, 2013; Sodt et al, 2014; Stanich et al, 2013).…”

Enantiomers of phospholipids and cholesterol: A key to decipher lipid‐lipid interplay in membrane

“…Regarding the interaction between Cho and membrane lipids, the rigid cyclic core of Cho is always referred, while there are only few studies on the side chain. By examining the conformation by 2 H NMR, Vogel et al have been revealed that the side chain plays a major role in the lipid condensation effect of Cho . They have also shown that the two pairs of hydrogen atoms at the C23 and C24 methylene groups have different orientations relative to the molecular rotation axis.…”

“… 2 H NMR spectra showing a chiral environment of the Cho side chain, where each of the pro‐chiral methylene groups at 23‐d 2 ‐Cho (A) and 2‐d 2 ‐Cho (B) shows two pairs of 2 H signals with difference width under the lipid‐disordered phase of a ternary SSM‐DOPC‐Cho (1:1:1) system at 50 o C. Assignments of prochiral 2 H signals were made by previous studies . (Hanashima, S., Ibata, Y. and Murata, M. unpublished data)…”