Michael R. Morrow

Chain-length dependence of lipid bilayer properties near the liquid crystal to gel phase transition

The temperature dependence of the mean orientational order parameter in the vicinity of the liquid crystal to gel phase transition is obtained from the first moment M1 of deuterium nuclear magnetic resonance spectra for bilayers of chain perdeuterated phosphatidylcholines with acyl chains of 12, 14, 16, and 18 carbons. The data clearly show an increasing temperature dependence of the orientational order parameter in the vicinity of the transition, with the effect becoming more pronounced with decreasing chain length. Assuming a linear relationship between the mean orientational order parameter and the extension of the acyl chain, estimates of the change in area of the membrane at the transition are shown to be consistent with those obtained from other measurements. It is shown that the transition may be modeled in terms of a Landau expansion of the free energy involving a small number of phenomenological parameters. From this it is shown that the behavior of these systems in the temperature range of interest is, in large part, controlled by the close proximity of a spinodal to the transition temperature.

The phase diagram of dimyristoyl phosphatidylcholine and chain-perdeuterated distearoyl phosphatidylcholine A deuterium NMR spectral difference study

Abstract The phase diagram for mixtures of the lipids dimyristoylphosphatidylcholine (DMPC) and chain perdeuterated distearoylphosphatidylcholine (DSPC-d70) is determined using 2H-NMR difference spectroscopy. It is found that the temperature of the solidus line increases monotonically with DSPC-d70 concentration and thus that there is no evidence for peritectic behavior in this mixture. This phase diagram is compared to others that have been determined for DMPC/DSPC mixtures and for mixtures of perdeuterated DMPC (DMPC-d54) with DSPC some of which do display peritectic behavior. The possibility that different isotopic compositions give rise to qualitatively different phase diagrams for this system is discussed

Smoothed acyl chain orientational order parameter profiles in dimyristoylphosphatidylcholine-distearoylphosphatidylcholine mixtures: a 2H-NMR study.

The accommodation of chain-length mismatch in liquid crystal phase bilayers was examined by using deuterium nuclear magnetic resonance to obtain smoothed orientational order parameter profiles for acyl chains of both components in binary lipid mixture bilayers. Mixtures of dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC) covering a range of compositions were prepared with either DSPC acyl chains or DMPC acyl chains perdeuterated. Orientational order parameters in the plateau regions of the smoothed profiles for both components were found to increase smoothly with increasing DSPC concentration. The orientational order parameters in the DSPC-smoothed profile were found to be slightly higher than corresponding values for DMPC over a wide range of bilayer composition. The shapes of the smoothed profiles for both components were found to be sensitive to bilayer composition. At low DSPC concentration, DSPC methylene deuterons near the bilayer center display a secondary plateau at low orientational order. At high DSPC concentration, the plateau of the DMPC-smoothed profile is stretched slightly. The concentration dependence of the smoothed profiles at low DSPC concentration appears to be consistent with a picture in which the last few segments of the DSPC chain cross the bilayer midplane, on average, but remain very disordered.

2H Solid-State Nuclear Magnetic Resonance Investigation of Whole Escherichia coli Interacting with Antimicrobial Peptide MSI-78

A key aspect of the activity of antimicrobial peptides (AMPs) is their interaction with membranes. Efforts to elucidate their detailed mechanisms have focused on applying biophysical methods, including nuclear magnetic resonance (NMR), to AMPs in model lipid systems. However, these highly simplified systems fail to capture many of the features of the much more complex cell envelopes with which AMPs interact in vivo. To address this issue, we have designed a procedure to incorporate high levels of (2)H NMR labels specifically into the cell membrane of Escherichia coli and used this approach to study the interactions between the AMP MSI-78 and the membranes of intact bacteria. The (2)H NMR spectra of these membrane-deuterated bacteria can be reproduced in the absence and presence of MSI-78. Because the (2)H NMR data provide a quantitative measure of lipid disorder, they directly report on the lipid bilayer disruption central to the function of AMPs, in the context of intact bacteria. Addition of MSI-78 to the bacteria leads to decreases in the order of the lipid acyl chains. The molar peptide:lipid ratios required to observe the effects of MSI-78 on acyl chain order are approximately 30 times greater than the ratios needed to observe effects in model lipid systems and approximately 100 times less than the ratios required to observe inhibition of cell growth in biological assays. The observations thus suggest that MSI-78 disrupts the bilayer even at sublethal AMP levels and that a large fraction of the peptide does not actually reach the inner membrane.

Effects of hydrostatic pressure on bilayer phase behavior and dynamics of dilauroylphosphatidylcholine

Deuterium nuclear magnetic resonance spectroscopy was used to study the thermotropic phase behavior of dilauroylphosphatidylcholine (DLPC) bilayers at pressures up to 221 MPa. Pressure was found to separate the liquid crystal to gel transition from the gel to ordered crystalline phase transition. The jump in chain order observed on cooling through the transition into the gel phase was found to be small and thus consistent with the trend in longer chain saturated diacyl phosphatidylcholines. On cooling, DLPC was observed to enter an unusual state above the transition into the gel phase. This unusual state displayed fluid-like conformational order but short transverse relaxation times. It was found to be much better pronounced and to span a broader temperature range at elevated pressure than at lower pressures. Transverse relaxation measurements of deuterons on the chain alpha-carbons revealed a substantial slowing of molecular motions within the temperature range of the unusual fluid phase. The observation of such a phase at high pressure appears to be consistent with recent reports of an unusual fluid phase, Lx, in DLPC at ambient pressure.

Studies on the interaction of human erythrocyte band 3 with membrane lipids using deuterium nuclear magnetic resonance and differential scanning calorimetry

Human erythrocyte band 3, reconstituted into large unilamellar phospholipid vesicles, has been used as a model system for studying the interactions between membrane lipids and large transmembrane glycoproteins. Both 2H-nuclear magnetic resonance (2H-NMR) and differential scanning calorimetric techniques have been used to probe dimyristoylphosphatidylcholine-band 3 interactions over the temperature range 4-32 degrees C. Analysis of 2H-NMR spectra allowed the assignment of liquid crystal, gel phase and two-phase regions for several protein/lipid mole fractions in the range (1-20) X 10(-4). Sample size was limited by the amount of available glycoprotein and this precluded exact determination of the phase boundaries for this system. The sharp discontinuity in the spectral first moment, M1, seen at the phase transition of the pure phospholipid is progressively diminished by addition of protein, and at the highest protein concentration the first moment varies smoothly between the two phases. For T greater than 26 degrees C or less than 16 degrees C, the moments are relatively insensitive to protein concentration, while between 20 and 26 degrees C the moments increase with protein concentration up to the boundary of the two-phase region. Beyond this boundary, they remain constant or decrease slightly with increasing amount of protein. A preliminary phase diagram for band 3 in this lipid system is presented, based on 2H-NMR data. Differential scanning calorimetry (DSC) showed that addition of glycoprotein dramatically alters the scan shape and tends to extend the coexistence of two phases to higher temperatures.

Perturbation of DPPC bilayers by high concentrations of pulmonary surfactant protein SP-B

Deuterium (2H) NMR has been used to observe perturbation of dipalmitoylphosphatidylcholine (DPPC) bilayers by the pulmonary surfactant protein B (SP-B) at concentrations up to 17% (w/w). Previous 2H NMR studies of DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3) bilayers containing up to 11% (w/w) SP-B and DPPC bilayers containing up to 11% (w/w) synthetic SP-B indicated a slight effect on bilayer chain order and a more substantial effect on motions that contribute to decay of quadrupole echoes obtained from bilayers of deuterated DPPC. This is consistent with the perturbation of headgroup-deuterated DPPC reported here for bilayers containing 6 and 9% (w/w) SP-B. For the higher concentrations of SP-B investigated in the present work, 2H NMR spectra of DPPC deuterated in both the headgroup and chain display a prominent narrow component consistent with fast, large amplitude reorientation of some labeled lipid. Similar spectral perturbations have been reported for bilayers in the presence of the antibiotic polypeptide nisin. The observation of large amplitude lipid reorientation at high SP-B concentration could indicate that SP-B can induce regions of high bilayer curvature and thus provides some insight into local interaction of SP-B with DPPC. Such local interactions may be relevant to the formation, in vitro and in vivo, of tubular myelin, a unique structure found in extracellular pulmonary surfactant, and to the delivery of surfactant material to films at the air–water interface.

Glycosphingolipid phase behaviour in unsaturated phosphatidylcholine bilayers : a 2H-NMR study

2H-NMR was employed to consider the arrangement of a glycosphingolipid, N-(lignoceroyl-d47)galactosylceramide, in bilayers of the mono-unsaturated phospholipid, 1-stearoyl-2-oleoylphosphatidylcholine. The deuterated glycolipid prepared by partial synthesis was incorporated at concentrations ranging from 5 mol% to 53 mol% into unsonicated liposomes, and its spectra were recorded from +76 degrees C to -10 degrees C. First spectral moments were plotted as a function of temperature for each sample composition and, along with inspection of the spectra, were employed to infer a phase diagram describing glycolipid behaviour in the unsaturated phospholipid host matrix. It was possible to refine the result using 2H-NMR difference spectroscopy. The phase diagram obtained was indicative of peritectic behaviour. At glycolipid concentrations exceeding about 20 mol% there was considerable tendency to glycolipid phase separation--as indicated by coexistence of fluid phospholipid-enriched and gel phase glycolipid-enriched domains over a wide range of temperatures, and by coexistence of distinct ordered phase domains at lower temperature. In contrast, at lower glycolipid concentrations reflective of many biological membranes, the lipid components were miscible in both the liquid crystal and gel phases, with only a narrow temperature range of fluid and ordered phase coexistence. For the fluid phase at low glycolipid concentrations, spectra of the deuterated glycolipid 24-carbon fatty acid suggest that orientational order is low for a number of methylene groups near the methyl end of the chain.

Hydrostatic pressure-induced conformational changes in phosphatidylcholine headgroups: a 2H NMR study

The effects of pressure and temperature on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine headgroup conformations were examined using deuterium nuclear magnetic resonance. Isothermal compression was found to produce a decrease in the choline alpha deuteron quadrupole splitting and increases in the choline beta and gamma deuteron quadrupole splittings. A similar counterdirectional change, seen in the presence of positive surface charge, has been attributed to tilting of the headgroup away from the bilayer surface in response to the torque exerted on the phosphocholine dipole by positive surface charges. The direction of the change in headgroup deuteron quadrupole splitting is consistent with the pressure-induced reduction in area per lipid in the liquid crystalline phase, which can be inferred from the ordering of phospholipid acyl chains under comparable conditions. The temperature dependences of the headgroup deuteron quadrupole splittings were also examined. It was found that at elevated pressure, the alpha splitting was insensitive to temperature, whereas the beta and gamma splittings decreased. The response of the beta deuteron splitting to temperature was found to be weaker at elevated pressure than at ambient pressure.

Glycosphingolipid headgroup orientation in fluid phospholipid/cholesterol membranes: similarity for a range of glycolipid fatty acids.

Galactosyl ceramide (GalCer) was labeled for nuclear magnetic resonance (NMR) spectroscopy by replacement of a hydrogen atom at C6 of the galactose residue with deuterium. Wideline 2H NMR of [d1]GalCer permitted consideration of a mechanism traditionally entertained for cell surface recognition site modulation: that the nature of the fatty acid attached to the sphingosine backbone of glycosphingolipids (GSLs) importantly influences carbohydrate headgroup orientation. Comparison was made among various glycolipid fatty acids by altering hydroxylation, saturation, and chain length. Studies were carried out in unsonicated bilayer membranes mimicking several important characteristics of cell plasma membranes: fluidity, low GSL content, predominant [sn-2]monounsaturated phosphatidylcholine (PC) (1-palmitoyl-2-oleoyl PC), and the presence of cholesterol. Spectroscopy was performed on samples over a range of temperatures, which included the physiological. 2H NMR spectra of [d1]GalCer having 18-carbon saturated fatty acid (stearic acid), cis-9-unsaturated fatty acid (oleic acid), D- and L-stereoisomers of alpha-OH stearic acid, or 24-carbon saturated fatty acid (lignoceric acid) were importantly similar. This argues that for GSLs dispersed as minor components in fluid membranes, variation of the glycolipid fatty acid does not provide as much potential for direct conformational modulation of the carbohydrate portion as has sometimes been assumed. However, there was some evidence of motional differences among the species studied. The 2H NMR spectra that were obtained proved to be more complex than was anticipated. Their features could be approximated by assuming a combination of axially symmetric and axially asymmetric glycolipid motions. Presuming the appropriateness of such a analysis, at a magnetic field of 3.54 T (23.215 MHz), the experimental spectra suggested predominantly asymmetric motional contributions. At the higher field of 11.7 T (76.7 MHz, equivalent to a proton frequency of 500 MHz), spectra indicated dominance by axially symmetric rotational modes. There was also evidence of some bilayer orientation in the stronger magnetic field. The unusual observation of spectral differences between the two magnetic field strengths may involve a diamagnetic response to high field on the part of some liposome physical characteristics.