The diversity of the liquid ordered (Lo) phase of phosphatidylcholine/cholesterol membranes: a variable temperature multinuclear solid-state NMR and x-ray diffraction study

Abstract

To investigate the properties of a pure liquid ordered (Lo) phase in a model membrane system, a series of saturated phosphatidylcholines combined with cholesterol were examined by variable temperature multinuclear (1H, 2H, 13C, 31P) solid-state NMR spectroscopy and x-ray scattering. Compositions with cholesterol concentrations>or=40 mol %, well within the Lo phase region, are shown to exhibit changes in properties as a function of temperature and cholesterol content. The 2H-NMR data of both cholesterol and phospholipids were used to more accurately map the Lo phase boundary. It has been established that the gel-Lo phase coexistence extends to 60 mol % cholesterol and a modified phase diagram is presented. Combined 1H-, 2H-, 13C-NMR, and x-ray scattering data indicate that there are large changes within the Lo phase region, in particular, 1H-magic angle spinning NMR and wide-angle x-ray scattering were used to examine the in-plane intermolecular spacing, which approaches that of a fluid Lalpha phase at high temperature and high cholesterol concentrations. Although it is well known for cholesterol to broaden the gel-to-fluid transition temperature, we have observed, from the 13C magic angle spinning NMR data, that the glycerol region can still undergo a "melting", though this is broadened with increasing cholesterol content and changes with phospholipid chain length. Also from 2H-NMR order parameter data it was observed that the effect of temperature on chain length became smaller with increasing cholesterol content. Finally, from the cholesterol order parameter, it has been previously suggested that it is possible to determine the degree to which cholesterol associates with different phospholipids. However, we have found that by taking into account the relative temperature above the phase boundary this relationship may not be correct.

FIGURE 1

¹H MAS spectra of DPPC/40% Chol, DPPC/50% Chol, and DPPC/60% Chol between 10 and 50°C.

FIGURE 2

³¹P MAS spectra of DPPC/40% Chol, DPPC/50% Chol, and DPPC/60% Chol between 5 and 50°C.

FIGURE 3

¹³C MAS spectra of DPPC/40% Chol, DPPC/50% Chol, and DPPC/60% Chol between 5 and 60°C showing the glycerol and headgroup regions.

FIGURE 4

¹³C MAS spectra of DPPC/60% Chol between 5 and 60°C showing changes to chemical shift of the methylene region.

FIGURE 5

Graph of quadrupolar splitting versus relative temperature, T*, for DPPC/40% Chol-d₁, DPPC/50% Chol-d₁, and DPPC/60% Chol-d₁ between 5 and 55°C. T* is the temperature above the gel + L_o phase boundary.

FIGURE 6

²H spectra of DPPC-d₆₂/40% Chol, DPPC-d₆₂/50% Chol, and DPPC-d₆₂/60% Chol between 5 and 60°C.

FIGURE 7

Order profiles of DPPC-d₆₂/40% Chol, DPPC-d₆₂/50% Chol, and DPPC-d₆₂/60% Chol between 5 and 60°C.

FIGURE 8

Temperature dependence of the DPPC-d₆₂ chain length (sn-2 chain) for DPPC/40–60% Chol estimated from the order parameters.

FIGURE 9

In-plane spacing of DPPC/30–60% Chol as a function of temperature.

FIGURE 10

Quadrupolar splitting of DMPC/50% Chol-d₁ and DPPC/50% Chol-d₁ at the recorded temperature (left) and the temperature above the phase boundary (right).

FIGURE 11

Modified DPPC/Chol phase diagram adapted from Huang et al. (39).