Cholesterol containing model membranes studied by multinuclear solid state NMR spectroscopy

Abstract

Ternary phase diagrams have been shown to be very useful in understanding the phenomena of lipid rafts. Recently a comprehensive ternary phase diagram for DPPC-d₆₂/DOPC/Chol using static ²H-NMR has been reported [S. L. Veatch, O. Soubias, S. L. Keller, and K. Gawrisch Proceedings of the National Academy of Sciences of the United States of America, 2007, 104, 17650–17655]. However, it is often difficult to interpret these ternary phase diagrams as there are many possible ambiguities, in particular, the extent of the gel(s_o)–L_o–L_α(l_d) three phase region and the clear demarcation between the gel(s_o) and L_o phase. Here we examine the canonical model lipid raft system by a multinuclear approach using ²H static and ¹³C, ³¹P Magic Angle Spinning (MAS) NMR. By using deuterated DPPC (DPPC-d₆₂) it is possible to examine changes in the methylene chain order parameters of the saturated lipid whilst changing the ratio of saturated to unsaturated phospholipid. From these data it is possible to propose the existence at low temperatures (<20 °C) of a disrupted or disordered gel-type phase at ratios as high as 1:1:1 DOPC/DPPC/Chol, in equilibrium with fluid phases. The presence of a gel-type phase was also confirmed by ¹³C and ³¹P MAS NMR. This disrupted gel-type phase has different properties in terms of axial rotation than that of the liquid ordered phase (L_o). We have also shown that it is possible to observe different quadrupolar splittings for the inequivalent sn-1 and sn-2 methyl groups in a gel phase. We have concluded that this is not always indicative of a liquid-ordered lamellar L_o phase, as previously assumed. Above the melting point of the gel-type phase (∼20 °C), we observe a single fluid environment for both DPPC-d₆₂ and Chol-d₁ on the basis of the ²H-NMR spectra, which are an average of a fluid disordered (L_α) and an L_o phase. In this fluid regime, the order parameters for the 1:1:1 system DPPC-d₆₂/DOPC/Chol are close to that of the binary (1:1) DPPC-d₆₂:Chol system, indicating non-random distribution of the DPPC-d₆₂. Increasing the ratio of DOPC to DPPC, from 1:1:1 to 1.5:1:1 to 2:1:1 in the ternary system, causes an increase in chain length of the DPPC-d₆₂ in the fluid phase.