Molecular dynamics simulations and neutron reflectivity as an effective approach to characterize biological membranes and related macromolecular assemblies.

In combination with other spectroscopy, microscopy, and scattering techniques, neutron reflectivity is a powerful tool to characterize biological systems. Specular reflection of neutrons provides structural information at the nanometer and subnanometer length scales, probing the composition and organization of layered materials. Currently, analysis of neutron reflectivity data involves several simplifying assumptions about the structure of the sample under study, affecting the extraction and interpretation of information from the experimental data. Computer simulations can be used as a source of structural and dynamic data with atomic resolution. We present a novel tool to compare the structural properties determined by neutron reflectivity experiments with those obtained from molecular simulations. This tool allows benchmarking the ability of molecular dynamics simulations to reproduce experimental data, but it also promotes unbiased interpretation of experimentally determined quantities. Two application examples are presented to illustrate the capabilities of the new tool. The first example is the generation of reflectivity profiles for a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer from molecular dynamics simulations using data from both atomistic and coarse-grained models, and comparison with experimentally measured data. The second example is the calculation of lipid volume changes with temperature and composition from all atoms simulations of single and mixed 1,2-di-palmitoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) bilayers.

Spectroscopic and permeation studies of phospholipid bilayers supported by a soft hydrogel scaffold.

Polarized attenuated total reflection infrared (ATR-IR) spectroscopy, fluorescence microscopy, and fluorescence spectroscopy were used to characterize a lipid coating composed of 70 mol % 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 30 mol % cholesterol, supported on a spherical hydrogel scaffold. The fluorescence microscopy images show an association between the lipid coating and the hydrogel scaffold. Fluorescence permeability measurements revealed that the phospholipid coating acts as a permeability barrier, exhibiting characteristics of a lamellar bilayer coating structure. Variable evanescent wave penetration depth ATR-IR spectroscopy studies validated the determination of quantitative molecular orientation information for a lipid coating supported on a spherical scaffold. These polarized ATR-IR studies measured an average DMPC acyl chain tilt angle of ∼21-25°, with respect to the surface normal.

Evaluation of the effect of fluorination on the property of monofluorinated dimyristoylphosphatidylcholines.

The synthesis of three monofluorinated dimyristoylphosphatidylcholines (F-DMPC's), with the fluorine atom located at the extremities of the acyl chain in position 2 of the glycerol (sn-2), is described. The synthetic strategy relies on the coupling of 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (14:0 lyso-PC) and three different fluorinated fatty acids. FTIR results suggest that the presence of the fluorine atom does not significantly perturb the lipid phase transition temperature and conformational order even though a small increase in the phase transition temperature is observed for the 14F derivative. Overall, comparison with previously reported F-DMPC's where the fluorine atom is located in the middle or close from either side supports the fact that monofluorination of the acyl chain in sn-2 brings minimal perturbation to the lipid bilayer. F-DMPC's could therefore potentially be used as NMR probes for the investigation at the molecular level of the interaction between drugs or peptides and lipid membranes and for the study of membrane topology.

Synthesis and properties of monofluorinated dimyristoylphosphatidylcholine derivatives: potential fluorinated probes for the study of membrane topology.

The synthesis of three monofluorinated dimyristoylphosphatidylcholine derivatives (F-DMPC), with the fluorine atom located on the acyl chain in position 2 of the glycerol (sn-2) is described. The synthetic strategy relies on the coupling of 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PC) and three different fluorinated fatty acids. The latter were obtained from two different and complementary synthetic routes. Preliminary FTIR studies suggest that the presence of the fluorine atom does not significantly perturb the lipid conformational order and phase transition temperature and that these monofluorinated PC derivatives could be used as probes for the study of membrane topology, i.e. the location of drugs, peptides or proteins in membranes.

Use of 31PNMR spectroscopy to follow the time course of phosphatidylcholine chemical synthesis.

31PNMR spectroscopy is shown to be useful for studying the chemical synthesis of phosphatidylcholine from phosphatidic acid and choline. Sharp, well-resolved resonances were obtained by chelating multivalent cations, thereby enabling quantitation of reactants, products, and intermediates. The syntheses of several types of phosphatidylcholines were monitored by 31PNMR spectroscopy, including perdeuterated and headgroup spin-labeled molecules. For perdeuterated phosphatidylcholines, this analysis led to reaction conditions giving much better conversion to product than conditions previously observed. In addition, a polyphosphate side-product was identified in reactions which do not produce phosphatidylcholine, implying either a polyphosphate intermediate in the reaction mechanism, or else a competing side reaction.

Improved method for the synthesis of phosphatidylcholines.

An improved method for the synthesis of phosphatidylcholines from phosphatidic acid and choline is described. The technique utilizes the tetraphenylborate salt of choline together with the condensing agent 2,4,6-triisopropylbenzenesulfonyl chloride. The yields in the reaction are consistently in the range 70-75%.

Direct evidence of incorporation of 12-O-[20-2H1]tetradecanoylphorbol-13-acetate into artificial membranes as determined by deuterium magnetic resonance.

In order to elucidate the manner of interaction of 12-O-tetradecanoylphorbol-13-acetate (TPA), 2H-NMR spectra of [20-2H1]TPA and 31P-NMR spectra were recorded in the presence of multibilayers of dimyristoylphosphatidylcholine (DMPC). Observation of several pairs of quadrupole splittings directly proves the TPA molecules are intercalated into the multibilayers. Further, reduction of the 31P chemical shift anisotropy of DMPC multibilayers by TPA is more pronounced than that of phorbol 12,13-diacetate (PDA) whose activity of tumor promotion is very weak.