Philip W. Westerman

Lateral diffusion of cholesterol and dimyristoylphosphatidylcholine in a lipid bilayer measured by pulsed field gradient NMR spectroscopy.

The pulsed field gradient NMR method for measuring self-diffusion has been used for a direct determination of the lateral diffusion coefficient of cholesterol, fluorine labeled at the 6-position, for an oriented lamellar liquid-crystalline phase of dimyristoylphosphatidylcholine (DMPC)/cholesterol/water. It is found that the diffusion coefficients of DMPC and cholesterol are equal over a large temperature interval. The apparent energy of activation for the diffusion process (58 kJ/mol) is about the same as for a lamellar phase of DMPC/water, whereas the phospholipid lateral diffusion coefficient is approximately four times smaller in the presence of cholesterol.

The interaction of n-alkanols with lipid bilayer membranes: a 2H-NMR study

The interaction of eight n-alkanols with bilayers of dimyristoylphosphatidylcholine (DMPC) has been studied by deuterium nuclear magnetic resonance (2H-NMR). At comparable temperatures and concentrations of solute in the bilayer, order parameters measured at the 1-methylene segment of the n-alkanols show a maximum for n-dodecanol. For both n-dodecanol and n-tetradecanol, orientational ordering shows a maximum at the C-4 to C-7 methylene segments, with labels at both ends of the n-alkanol exhibiting reduced order. These observations are consistent with earlier findings for n-octanol and n-decanol. Unlike the longer chain n-alkanols, ordering in n-butanol decreases from the hydroxyl group end to the methyl group end of the molecule. Orientational ordering at nine inequivalent sites in DMPC, has also been measured as a function of temperature, for bilayers containing n-butanol, n-octanol, n-dodecanol and n-tetradecanol. At the 3R,S sites on the glycerol backbone, for comparable temperatures and solute concentrations, n-butanol produces a larger disordering than the other n-alkanols. This result probably reflects the greater fraction of time spent by the hydroxyl group of n-butanol in the vicinity of the lipid polar head group compared with the hydroxyl group in longer chain n-alkanols. It was found that n-octanol orders the acyl chains of DMPC, unlike n-butanol which disorders them, and the longer chain n-alkanols which have little effect. Within experimental error, the effect of n-dodecanol on order at all sites in DMPC is the same as n-tetradecanol. The influence of n-alkanols on DMPC ordering at twelve sites has been compared with that of cholesterol which is shown to interact with DMPC bilayers in a distinctly different manner from the n-alkanols.

Determination of Membrane Immersion Depth with O2: A High-Pressure 19F NMR Study

Oxygen is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior. At partial pressures (P(O2)) of 100 Atm or more, this concentration gradient is sufficient to induce paramagnetic effects that depend sensitively on membrane immersion depth. This effect is demonstrated for the fluorine nucleus by depth-dependent paramagnetic shifts and spin-lattice relaxation rates, using a fluorinated detergent, CF3(CF(2))(5)C(2)H(4)-O-maltose (TFOM), reconstituted into a lipid bilayer model membrane system. To interpret the spin-lattice relaxation rates (R) in terms of a precise immersion depth, two specifically fluorinated cholesterol species (6-fluorocholesterol and 25-fluorocholesterol), whose membrane immersion depths were independently estimated, were studied by (19)F NMR. The paramagnetic relaxation rates, R, of the cholesterol species were then used to parameterize a Gaussian profile that directly relates R to immersion depth z. This same Gaussian curve could then be used to determine the membrane immersion depth of all six fluorinated chain positions of TFOM and of two adjacent residues of specifically fluorinated analogs of the antibacterial peptide indolicidin. The potential of this method for determination of immersion depth and topology of membrane proteins is discussed.

A study of carbobenzoxy-D-phenylalanine-L-phenylalanine-glycine, an inhibitor of membrane fusion, in phospholipid bilayers with multinuclear magnetic resonance.

The anti-viral and membrane fusion inhibitor, carbobenzoxy-D-phenylalanine-L-phenylalanine-glycine (ZfFG), was studied in phospholipid bilayers, where earlier studies had indicated this peptide functioned. Multinuclear magnetic resonance (NMR) studies were performed with isotopically labeled peptide. A peptide labeled in the glycine carboxyl with 13C was synthesized, and the isotropic 13C-NMR chemical shift of that carbon was measured as a function of pH. A pKa of 3.6 for the carboxyl was determined from the peptide bound to a phosphatidylcholine bilayer. ZfFG inhibits the formation by sonication of highly curved, small unilamellar vesicles. Experiments as a function of pH revealed that this ability of ZfFG was governed by a pKa of 3.7. Therefore the protonation state of the carboxyl of ZfFG appeared to regulate the effectiveness of this anti-viral peptide at destabilizing highly curved phospholipid assemblies. Such destabilization had previously been discovered to be related to the mechanism of the anti-fusion and anti-viral activity of this peptide. The location of the carboxyl of ZfFG in the membrane was probed with paramagnetic relaxation enhancement of the 13C spin lattice relaxation of the carboxyl carbon in the glycine of ZfFG (enriched in 13C). Results suggested that this carboxyl is at or above the surface of the phospholipid bilayer. The dynamics of the molecule in the membrane were examined with 2H-NMR studies of ZfFG, deuterated in the alpha-carbon protons of the glycine. When ZfFG was bound to membranes of phosphatidylcholine, a sharp 2H-NMR spectral component was observed, consistent with a disordering of the glycine methylene segment of the peptide. When ZfFG was bound to N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE) bilayers at temperatures below 30 degrees C, a large quadrupole splitting was observed. These results suggest that ZfFG likely inhibits membrane fusion from the surface of the lipid bilayer, but not by forming a tight, stoichiometric complex with the phospholipids.

Synthesis of methyl ω-deuterated tetradecanoate and hexadecanoate

Abstract Methyl ω-deuterated tetradecanoates have been prepared in high purity by two synthetic routes from methyl hydrogen tetradecanedioate. One method utilizes the selective reducing properties of sodium borodeuteride and sodium cyanoborodeuteride towards, acid chloride, ester, tosyloxy, and iodo groups to introduce the deuterium label at only the carboxyl group of methyl hydrogen tetradecanedioate. The second procedure utilizes a coupling reaction between an organic halide and lithium di-(trideuteriomethyl) cuprate (I). Corresponding ω-deuterated derivatives of methyl hexadecanoate may be prepared by the same methods from methyl hydrogen hexadecanedioate. The two methods should be generally applicable in the synthesis of ω-deuterated alkanoic acids.

Disposition of chloroform in phosphatidylcholine membranes: a 2H- and 31P-NMR study

Abstract The interaction of chloroform with bilayers of dimyristoylphosphatidylcholine (DMPC) has been studied by deuterium and phosphorus-31 nuclear magnetic resonance (NMR). Orientational order has been measured as a function of temperature at many sites in DMPC, water and chloroform for aqueous multilamellar dispersions of the lipid. At equivalent temperatures above the main phase transition temperature for a molar ratio of DMPC to chloroform of approximately 10 to 1, disordering at several sites in the head group of DMPC is observed, unlike the acyl chains where no disordering is observed. With higher concentrations of chloroform (DMPC/chloroform ≈ 4:1) greater disordering occurs at the same head group sites and is accompanied by disordering of the aryl chains. The pattern of solvent-induced changes in DMPC is similar to that produced by benzyl alcohol and n -alkanols. With 2 H-labelled chloroform, the 2 H-NMR spectra show two components, one isotropic and the other ordered ( Δν ≈ 1.5 kHz) arising from solute intercalated in the bilayer. In DMPC/water systems at low hydration the ordering of the 2 H 2 O in the L α phase is little affected by chloroform at comparable temperatures whereas at temperatures below the main phase temperature a large disordering of the water is observed. A model of the mode of interaction between chloroform and DMPC is proposed, in which the chloroform is localized principally in an ordered environment in the vicinity of the choline head group at lower temperatures and solute concentrations. Increasing either of these parameters favors the penetration of the chloroform into the center of the bilayer.

Synthesis of esters of tetradecanoic acid deuterated at the penultimate carbon: Some general procedures for the synthesis of selectively deuterated fatty acids

Abstract Methyl tetradecanoate, deuterated in the penultimate position has been prepared by three synthetic routes. One method in which an oxoester is an intermediate, utilizes the selective reducing properties of sodium borodeuteride and sodium cyanoborodeuteride towards oxo, tosyloxy and ester groups to introduce the deuterium label at only the oxo carbon of the oxoester. The second procedure, introduces a single deuteriumat at the oxo carbon of an oxoester, in a selective deoxygenation step which reduces an intermediate tosylhydrazone with sodium cyanoborodeuteride in acidic dimethylformamide-sulfolane. The third pathway, couples lithium dimethylcuprate (I) to the tosylate derivative of a ω-deuterio-ω-hydroxy methylester, prepared from the half-ester of a dicarboxylic acid. Utilizing suitable modifications, these three methods should be generally applicable in the synthesis of any saturated straight-chain alkanoate, selectively deuterated in the penultimate ( ω − 1) carbon, or at any other carbon in the hydrocarbon chain.

Physicochemical characterization of liquid crystalline phases in model bile and lipid digestive mixtures : A 2H NMR study

Abstract Phase properties of aqueous dispersions of model bile mixtures containing cholesterol, lecithin and bile salts and model mixed lipid digestive mixtures containing cholesterol, fatty acid, monoglyceride, lecithin and bile salts have been characterized by 2H NMR. We have chemically incorporated a deuteriomethyl (CD3) group into one or two lipid components and utilized differences in motionally-averaged quadrupole splittings (Δv) and CD3 chemical shifts, to determine directly by peak-area integration, for a number of compositions on the phase diagram, the distribution of a 2H-labeled component between micellar, lamellar and solid phases, as well as the chemical compositions of these phases. Our data show that increasing cholesterol stabilizes lamellar phases whereas bile salts disrupt them and favor micelle formation.

On the stability of the ripple phase in the DPPC/PLPC/water ternary system

Abstract The effect of incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine (PLPC) on the structure of the Pβ′ ripple mesophase in aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) has been studied by differential scanning calorimetry (DSC) and scanning dilatometry (SD). For samples containing 34 wt. % 2H2O and 0–15 wt. % PLPC, a pretransition was observed by DSC. The pretransition disappears at 15 wt. % PLPC. The behavior of thermodynamic functions at the pretransition and main transition gives new insights on the structural changes produced by PLPC on bilayers of DPPC.

A Thermodynamic and NMR Investigation of 1-Lysopalmitoyllecithin / 1,2-Dipalmitoylphosphatidylethanolamine/Water System

Abstract The effect of incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine (PLPC) on the bilayer structures occurring in aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) has been studied by phosphorus 31 nuclear magnetic resonance and calorimetric methods. The polymorphism of the system as a function of PLPC concentration has been defined. Experimental data allowed us to determine phase boundaries. Changes of molecular packing of the PLPC molecules in DPPE bilayers in the phase diagram are invoked to explain the experimental findings.