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The Diversity of the Liquid Ordered (L_o) Phase of Phosphatidylcholine/Cholesterol Membranes: A Variable Temperature Multinuclear Solid-State NMR and X-Ray Diffraction Study

Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom

Correspondence: Address reprint requests to Robert V. Law, E-mail: r.law{at}imperial.ac.uk; or John M. Seddon, E-mail: j.seddon{at}imperial.ac.uk.

To investigate the properties of a pure liquid ordered (L_o) 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 40 mol %, well within the L_o 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 L_o phase boundary. It has been established that the gel-L_o 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 L_o 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 L 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.

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