Cholesterol Stiffening of Lipid Membranes

Abstract

Biomembrane order, dynamics, and other essential physicochemical parameters are controlled by cholesterol, a major component of mammalian cell membranes. Although cholesterol is well known to exhibit a condensing effect on fluid lipid membranes, the extent of stiffening that occurs with different degrees of lipid acyl chain unsaturation remains an enigma. In this review, we show that cholesterol locally increases the bending rigidity of both unsaturated and saturated lipid membranes, suggesting there may be a length-scale dependence of the bending modulus. We review our published data that address the origin of the mechanical effects of cholesterol on unsaturated and polyunsaturated lipid membranes and their role in biomembrane functions. Through a combination of solid-state deuterium NMR spectroscopy and neutron spin-echo spectroscopy, we show that changes in molecular packing cause the universal effects of cholesterol on the membrane bending rigidity. Our findings have broad implications for the role of cholesterol in lipid-protein interactions as well as raft-like mixtures, drug delivery applications, and the effects of antimicrobial peptides on lipid membranes.

Keywords: Area per lipid; Cholesterol; Membrane elasticity; Neutron spin-echo; Rafts; Solid-state NMR spectroscopy.

Fig. 1

Energy landscapes and mobilities of phospholipids in membranes entail characteristic timescales. a Fluctuations of ¹³C–¹H or C–²H bonds are given by Ω_ID Euler angles for internal segmental frame (I) with respect to the membrane director axis (D). Phospholipid molecular motions entail anisotropic reorientation of the molecule-fixed frame (M) with respect to the membrane director axis (D) as described by Ω_MD Euler angles. The liquid-crystalline bilayer lends itself to propagation of thermally excited, quasi-periodic fluctuations due to motion of the local membrane normal (N) relative to the average director axis (D) characterized by Ω_ND Euler angles. Examples are shown respectively for glycerophospholipids, b POPC, c DOPC, and d DMPC where the lipid headgroups are highlighted in brown; e glycolipids where R denotes the rest of the lipid molecule; f cholesterol which is the sterol component of animal biomembranes; and g lanosterol which is a precursor in the sterol biosynthesis pathway

Fig. 2

Excitations of a fluid lipid bilayer are described within the continuum elastic approximation. a Planar bilayer, b splay, c twist, and d bend deformations, together with axial rotations about the local director

Fig. 3

Solid-state ²H NMR spectroscopy of membrane lipids provides both lineshape data and relaxation times yielding information about structure and dynamics. a Inversion recovery of ²H nuclear magnetization for random DMPC-d₅₄/cholesterol (1:1) multilamellar dispersion in the liquid-ordered phase at T = 44 °C showing partially relaxed ²H NMR spectra. b Deconvolved (de-Paked) ²H NMR spectra corresponding to bilayer normal parallel to magnetic field (θ = 0°). The sample contained 20 mM Tris buffer at pH 7.3 (50 wt% H₂O). Data were acquired at 76.8 MHz using a phase-cycled, inversion-recovery quadrupolar-echo pulse sequence, π − t₁ − (π /2)_x − τ − (π/2)_y − t₂ (acquire), where t₁ is a variable delay ranging from 5 ms (bottom) to 3 s (top). Adapted from Ref. (Martinez et al. 2002b)

Fig. 4

Functional dependence of relaxation times and order parameters from solid-state ²H NMR spectroscopy reveal dynamical bending rigidity of lipid membranes. Data are for unoriented lipid dispersions at 55.4 MHz and various temperatures (T ≥ T_m + 6 °C): a DLPC-d₄₆, b DMPC-d₅₄, c DPPC-d₆₂, and d DSPC-d₇₀, with acyl lengths of n = 12, 14, 16, and 18 carbons, respectively. Square-law functional relation of spin–lattice relaxation rates R_1Z and order parameters |S_CD| along the acyl chains (index i) for a homologous series of PCs in the L_α phase characterizes the influence of the acyl length (bilayer thickness). Adapted from Ref. (Brown et al. 2001)

Fig. 5

Cholesterol reduces the conformational degrees of freedom for lipid acyl chain segments yielding closer molecular packing. a DMPC-d₅₄ in the liquid-disordered (l_d) phase, and b–d DMPC-d₅₄ containing various mole fractions of cholesterol (Chol) in the liquid-ordered (l_o) phase. Powder type spectra (brown) of randomly oriented multilamellar dispersions were numerically inverted (de-Paked) to yield subspectra corresponding to the θ = 0° orientation (green). Note that a distribution of residual quadrupolar couplings (RQCs) corresponds to the various C²H₂ and C²H₃ groups with a progressive increase due to cholesterol. Adapted from Ref. (Martinez et al. 2004)

Fig. 6

Square-law relations of NMR relaxation times and order parameters indicate bilayer stiffening by cholesterol and lanosterol. a Dependence of spin–lattice relaxation rates $R_{\text{1Z}}^{(i)}$ on squared order parameters $|S_{\text{CD}}^{(i)}|$ for resolved ²H NMR splittings of DMPC-d₅₄. Data were obtained at T = 44 °C and at 76.8 MHz (11.8 T). b Bending rigidity values calculated for DMPC-d₅₄/cholesterol (DMPC-d₅₄/Chol) and DMPC-d₅₄/lanosterol (DMPC-d₅₄/Lan) systems. Adapted from Ref. (Martinez et al. 2004)

Fig. 7

Results from solid-state ²H NMR spectroscopy shows cholesterol increases acyl chain ordering and bending stiffness of lipid membranes due to closer molecular packing in the bilayer. a Segmental order parameter versus acyl position for POPC-d₃₁ probe lipid in DOPC/cholesterol membranes with different mol% cholesterol at T = 25 °C. b Dependence of spin–lattice relaxation rate $R_{\text{1Z}}^{(i)}$ on squared order parameters $|S_{\text{CD}}^{(i)}|$ indicating a decrease in square-law slopes due to bilayer stiffening by cholesterol. c Structural parameters, i.e., steric thickness ($D_{\text{B}}^{\prime }$) and bilayer hydrocarbon thickness (2D_C), obtained from ²H NMR lineshapes using the first-order mean-torque model. Adapted from Ref. (Chakraborty et al. 2020)

Fig. 8

Neutron spin-echo (NSE) spectroscopy and solid-state ²H NMR relaxometry show nearly identical increases in bending modulus of unsaturated lipid membranes with cholesterol mole fraction. a Electron density (ED) profiles along the membrane normal (z-axis) obtained from SAXS data for DOPC/cholesterol membranes, such that z = 0 denotes the center of the membrane. b NSE-measured intermediate scattering functions on 50-nm vesicles of protiated DOPC membranes with 20 mol% cholesterol. Error bars represent ± 1 standard deviation. The lines are fits to the data using a stretched exponential function. c Relative bending rigidity moduli (κ/κ₀) calculated from nanoscale bending fluctuations sampled by NSE spectroscopy, solid-state ²H NMR relaxometry, and analysis of MD simulations. Adapted from Ref. (Chakraborty et al. 2020)