Changes of the Membrane Lipid Organization Characterized by Means of a New Cholesterol-Pyrene Probe

We synthesized 3β-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe self-association processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe (K): KDOPC ≫ KPOPC ≫ KDMPC > KDMPC/15 mol % Chol > KDMPC/30 mol % Chol. This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer (I1–I5) and excimer (IE) emission bands. In model membranes, I1/I3 and IE/I3 ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse δ-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse δ-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors

Unique Backbone-Water Interaction Detected in Sphingomyelin Bilayers with 1H/31P and 1H/13C HETCOR MAS NMR Spectroscopy

Two-dimensional 1H/31P dipolar heteronuclear correlation (HETCOR) magic-angle spinning nuclear magnetic resonance (NMR) is used to investigate the correlation of the lipid headgroup with various intra- and intermolecular proton environments. Cross-polarization NMR techniques involving 31P have not been previously pursued to a great extent in lipid bilayers due to the long 1H-31P distances and high degree of headgroup mobility that averages the dipolar coupling in the liquid crystalline phase. The results presented herein show that this approach is very promising and yields information not readily available with other experimental methods. Of particular interest is the detection of a unique lipid backbone-water intermolecular interaction in egg sphingomyelin (SM) that is not observed in lipids with glycerol backbones like phosphatidylcholines. This backbone-water interaction in SM is probed when a mixing period allowing magnetization exchange between different 1H environments via the nuclear Overhauser effect (NOE) is included in the NMR pulse sequence. The molecular information provided by these 1H/31P dipolar HETCOR experiments with NOE mixing differ from those previously obtained by conventional NOE spectroscopy and heteronuclear NOE spectroscopy NMR experiments. In addition, two-dimensional 1H/13C INEPT HETCOR experiments with NOE mixing support the 1H/31P dipolar HETCOR results and confirm the presence of a H2O environment that has nonvanishing dipolar interactions with the SM backbone