Structure and dynamics of phospholipids in membranes elucidated by combined use of NMR and vibrational spectroscopies.

NMR is a sophisticated method for investigation of structure and dynamics of lipid and protein molecules in membranes. Vibrational spectroscopy is also powerful because of relatively high resolution and sensitivity, and easier access than NMR. A combined use of these spectroscopies could provide important insights into the membrane biophysics. A structural analysis of phosphatidylethanolamine (PE) bilayers in built-up films by infrared dichroism suggested that polar groups oriented parallel to the membrane surface. A Raman analysis of phosphatidylcholine (PC) revealed that the gauche conformation was preferred for the choline backbone not only in solid, but also in the gel and liquid-crystalline states. The polar group structure of DPPC bilayers in the liquid-crystalline state was determined by analyzing deuterium quadrupole splitting of the choline group and phosphorus chemical shift anisotropy of the phosphate group in combination with restriction of the gauche conformation of the choline group determined by Raman spectroscopy. This was an excellent complementarity of NMR and vibrational spectroscopies. The deuterium quadrupole splitting values mentioned above were found to change on addition of ions such as NaCl, CaCl, and LaCl, suggesting that a structural change takes place on ion binding and the polar group of PC works as an electric charge sensor of membranes. The ion-bound structure was determined by NMR using the restriction from Raman spectroscopy. The PN vector of phosphorylcholine group was inclined by 63° from the membrane surface, while the inclination was 18° in the ion-free form. The deuterium quadrupole splitting values and phosphorus powder patterns revealed that on mixing with phosphatidylglycerol (PG) or cardiolipin (CL), PC did not change its dynamic structure of the glycerol backbone, but PE did. The mixture of PE with PG or CL shared a new dynamic structure, suggesting their adaptive miscibility in the molecular level. PC was molecularly immiscible with any of PE, PG, and CL. The molecular miscibility would regulate not only interactions of proteins with mixed bilayers but also formation of asymmetric lipid membranes. Interactions of crown-ether (CE) modified artificial microbial peptides with phospholipid bilayers were investigated by NMR and FTIR. CE-modified 14-mers with one or two basic amino acid residues revealed position-specific selectivity for the suppression of calcein leakage from PC vesicles but did not for that from PG vesicles, suggesting that structures of the lipid polar groups play crucial roles in different responses of the vesicles to the positively charged peptides. Manipulation of the peptide-polar group interaction can be used for drug design.