Reducing the NMR sample volume using a single organic liquid: increased sensitivity for mass-limited samples with standard NMR probes.

A simple inexpensive protocol for confining an aqueous sample to the active region of a standard NMR probe is examined for high-resolution NMR. The aqueous sample is sandwiched between an inert perfluorinated organic liquid that has been exploited in the design of micro-coil NMR probes. The procedure is demonstrated with 3mm NMR tubes at ambient and elevated temperatures but should be equally applicable to smaller diameter tubes. It is shown that confinement has minimal effects on line shape and provides at least a two fold increase in sensitivity over a conventional sample, for the same mass of solute.

Dynamic characterization of the water binding loop in the P-type cardiotoxin: implication for the role of the bound water molecule.

Recent studies of cobra P-type cardiotoxins (CTXs) have shown that the water-binding loop (loop II) plays a crucial role in toxin binding to biological membranes and in their cytotoxicity. To understand the role of bound water in the loop, the structure and dynamics of the major P-type CTX from Taiwan cobra, CTX A3, were determined by a comprehensive NMR analysis involving (1)H NOESY/ROESY, (13)C[1)H]NOE/T(1) relaxation, and (17)O triple-quantum filtered NMR. A single water molecule was found to be tightly hydrogen bonded to the NH of Met26 with a correlation time (5-7 ns) approaching the isotropic tumbling time (3.8-4.5 ns) of the CTX A3 molecule. Surprisingly, despite the relatively long residence time (ca. 5 ns to 100 micros), the bound water molecule of CTX A3 is located within a dynamic (order parameter S(2) approximately 0.7) and solvent accessible loop. Comparison among several P-type CTXs suggests that proline residues in the consensus sequence of MxAxPxVPV should play an important role in the formation of the water binding loop. It is proposed that the exchange rate of the bound water may play a role in regulating the lipid binding mode of amphiphilic CTX molecules near membrane surfaces.

An optimal strategy for recovering the deuterium (2H) quadrupolar interaction under magic-angle spinning NMR.

By exploiting the homology in the form of the truncated high-field homonuclear dipole-dipole and quadrupole coupling Hamiltonians, we have previously demonstrated that a simple adaptation of a rotor-synchronized pulse sequence (DRAMA) used for the recovery of dipole-dipole couplings can also be used to resurrect quadrupole couplings (QUADRAMA). In the canonical implementation of these recovery pulse sequences, the couplings are not significantly scaled down from their static sample values. While such minimal scaling is of course desirable in the recovery of typical homonuclear dipolar couplings (< or =2 kHz) and small quadrupole couplings, it is clearly not ideal for the recovery of the much larger quadrupole couplings (20-200 kHz) often encountered in solid-state 2H NMR. In such a case, some prior knowledge of the order of magnitude of the coupling is required to optimize the experimental conditions for QUADRAMA. In order to overcome this drawback, in this study, we have developed a general and optimized strategy for implementing the QUADRAMA technique which does not require any knowledge of the size of the coupling vQ. Experimental tests of the optimized protocol demonstrate that by judicious choices of a combination of scaling factors and recoupling times, 2H quadrupole couplings ranging over an order of magnitude from 3 to 42 kHz can be measured. Since this optimized protocol can reliably be used to recover couplings over a broad range, it expands the range of systems accessible to study by 2H NMR into a realm where static sample NMR and simple MAS NMR may fail.

Extraction of lipid membrane incorporated vitamin E by sucrose polyesters.

2H and 31P solid state NMR have been used to probe, at the molecular level, the interaction between structurally different sucrose polyesters and a phospholipid membrane into which alpha-tocopherol and specifically deuterated alpha-[5,7-(2)H(6)] tocopherol has been incorporated. Our results show that at high concentration (>or=10 mol%) sucrose octapalmitate (SOP) and sucrose hexapalmitate (SHxP) deplete bilayer-associated alpha-tocopherol in dipalmitoyl phosphatidalcholine (DPPC) multilamellar dispersions and preferentially sequester the alpha-tocopherol into a fluid sucrose polyesters (SPE) phase located proximal to the membrane surface. It is demonstrated that the ability of SPEs to function as a 'lipophilic sink' depends strongly on sucrose polyester concentration and degree of esterification.

Structures of heparin-derived disaccharide bound to cobra cardiotoxins: context-dependent conformational change of heparin upon binding to the rigid core of the three-fingered toxin.

Glycosaminoglycans (GAGs) have been suggested to be a potential target for cobra cardiotoxin (CTX) with high affinity and specificity via a cationic belt at the concave surface of the polypeptide. The interaction of GAGs, such as high-molecular weight heparin, with CTXs not only can induce aggregation of CTX molecules but also can enhance their penetration into membranes. The binding of short chain heparin, such as a heparin-derived disaccharide [DeltaUA2S(1-->4)-alpha-D-GlcNS6S], to CTX A3 from Taiwan cobra (Naja atra), however, will not induce aggregation and was, therefore, investigated by high-resolution (1)H NMR. A novel heparin binding site on the convex side of the CTX, near the rigid disulfide bond-tightened core region of Cys38, was identified due to the observation of intermolecular NOEs between the protein and carbohydrate. The derived carbohydrate conformation using complete relaxation and conformational exchange matrix analysis (CORCEMA) of NOEs indicated that the glycosidic linkage conformation and the ring conformation of the unsaturated uronic acid in the bound state depended significantly on the charge context of CTX molecules near the binding site. Specifically, comparative binding studies of several heparin disaccharide homologues with two CTX homologues (CTX Tgamma from Naja nigricollis and CTX A3) indicated that the electrostatic interaction of N-sulfate of glucosamine with NH(3)(+)zeta of Lys12 and of the 2-O-sulfate of the unsaturated uronic acid with NH(3)(+)zeta of Lys5 played an important role. These results also suggest a model on how the CTX-heparin interaction may regulate heparin-induced aggregation of the toxin via the second heparin binding site.

Membrane perturbing properties of sucrose polyesters.

Sucrose polyester (SPE), in the form of sucrose octaesters and sucrose hexaesters of palmitic (16:0), stearic (18:0), oleic (18:1cis), and linoleic (18:2cis) acids, have many uses. Applications include: a non-caloric fat substitute, detoxification agent, and oral contrast agent for human abdominal (MRI) magnetic resonance imaging. However, it has been shown that the ingestion of SPE was shown to generate a depletion of physiologically important lipidic vitamins and other lipophilic molecules. In order to better understand, at the molecular level, the type of interaction between SPE and lipid membrane, we have, first synthesized different type of labelled and non-labelled SPEs. Secondly, we have studied the effect of SPEs on multilamellar dispersions of dielaidoylphosphatidylethanolamine (DEPE) and dipalmitoylphosphocholine (DPPC) as a function of temperature, SPE composition and concentration. The effects of SPEs were studied by differential scanning calorimetry (DSC), X-ray diffraction, 2H and 31P NMR spectroscopy. At low concentration (< 1 mol%) all of the SPEs lowered the bilayer to the inverted hexagonal phase transition temperature of DEPE and induced the formation of a cubic phase in a composition dependent manner. At the same low concentration, SPEs in DPPC induce the formation of a non-bilayer phase as seen by 31P NMR. Order parameter measurements of DPPC-d62/SPE mixtures show that the SPE effect on the DPPC monolayer thickness is dependent on the SPE, concentration, chains length and saturation level. At higher concentration (> or = 10 mol%) SPE are very potent DEPE bilayer to HII phase transition promoters, although at that concentration the SPE have lost the ability to form cubic phases. SPEs have profound effects on the phase behaviour of model membrane systems, and may be important to consider when developing current and potential industrial and medical applications.

Solid state molecular motion in sucrose octapalmitate as studied by deuterium NMR spectroscopy.

Sucrose octapalmitate-d11, d24 and d248 have been synthesized. Using 2H NMR T1 and analyses of the temperature dependence of the lineshapes, a detailed description of the solid state molecular motional modes is presented. Activation energies for methyl and methylene group rotation in the fatty acyl chains have been determined. The sucrose moiety is found to be static on the solid state deuterium NMR timescale.

Discontinuous expansion linked to sector formation in Pseudomonas aeruginosa colonies.

Colonies of Pseudomonas aeruginosa exhibit sectors that were shown to be located at specific intervals within the colony. Maxima in the distribution of sectors were observed every 5 mm as measured from the center of the colony. These maxima correlated with changes in the expansion rates of colonies. The absolute average number of sectors per colony was higher for colonies grown at higher temperatures. These results increase our understanding of colony pattern formation.

Differential expression of nonagglutinating fimbriae and MR/P pili in swarming colonies of Proteus mirabilis.

The expression of nonagglutinating fimbriae (NAF) and mannose-resistant/Proteus-like (MR/P) pili in swarming colonies of Proteus mirabilis was investigated. Elongated swarmer cells do not express pili, and the relative number of bacteria expressing NAF during swarming and early consolidation phases was very low (<5%). Relative expression of NAF in a terrace increased to approximately 30% at 48 h. We also determined the expression of NAF and MR/P pili in two phenotypically distinguishable regions of each terrace. The expression of both NAF and MR/P pili was always higher in the region closer (proximal) to the middle of the colony than in the distal region of the terrace. The relative numbers of bacteria expressing NAF or MR/P pili in the proximal region were between 39.1 and 63% and between 5.9 and 7.7%, respectively. In the distal region, expression levels were between 20.8 and 27.3% and between 3.7 and 5. 6%, respectively. A time course experiment testing NAF expression in both the proximal and distal regions of a terrace indicated that NAF expression in the proximal regions was always higher than in the distal regions and increased to a plateau 40 to 50 h after the start of the swarming phase for any given terrace. These results indicate that expression of NAF or MR/P pili in swarming colonies of P. mirabilis is highly organized, spatially and temporally. The significance of this controlled differentiation remains to be uncovered.

Phosphatidyl choline-mediated inhibition of Streptococcus pneumoniae adherence to type II pneumocytes in vitro.

Nearly 80% of the adherence of several strains of Streptococcus pneumoniae to A549 lung cells was inhibited by dimyristoylphosphatidylcholine (DMPC), as well as by the following mixtures of lipids: DMPC/globoside, DMPC/asialo GM-1 and DMPC/asialo GM-1/globoside liposomes. Control phosphatidylserine liposomes were ineffective at inhibiting bacterial adherence demonstrating the specificity of the interaction between bacteria and liposomes. FITC-labelled bacteria were shown to adhere directly to silica beads coated with DMPC. The proportion of S. pneumoniae bacteria binding to DMPC-coated beads did not exceed 20% of the bacterial population as shown by the binding isotherm. This clearly demonstrates that only a fraction of the bacterial population (a subpopulation) was capable of binding to the beads. The specificity of bacterial binding to DMPC was further demonstrated by surface plasmon resonance. By this latter technique, the affinity between DMPC and bacteria was shown to be high and substantially non-reversible. Finally, we established that in order to be efficient at inhibiting bacterial binding to A549 cells the average liposome diameter must be greater than approximately 200 nm suggesting that a multivalent attachment of the bacterium to a liposome is required for high affinity binding.

The nonrandom binding distribution of Streptococcus pneumoniae to type II pneumocytes in culture is dependent on the relative distribution of cells among the phases of the cell cycle.

The adherence of Streptococcus pneumoniae to epithelial (A549) lung cells was studied and the bacterial binding distribution was found to be nonrandom (non-Gaussian). Analysis of the dependency of bacterial binding on the cell cycle of A549 cells revealed that approximately 1.8 times more bacteria bind to G2 cells than to G0-G1 phase cells. Furthermore, bacterial binding curves exhibited a plateau of binding to G2 cells at a normalized bacteria to cell ratio approximately 1.8 times larger than that at which the plateau of binding to G0-G1 cell was observed. Since G2 cells are on average 1.4-1.8 times larger than G0-G1 cells, the results indicate that bacterial binding is proportional to cell size and not to the preferential binding (higher affinity) of bacteria to A549 cells in the G2 phase. Finally, the non-Gaussian distribution of bacterial binding could be mathematically modeled by a linear combination of three Gaussian distributions each representing bacterial binding to cells in a particular phase of the cell cycle (G0-G1, S, and G2-M). Because the Gaussian function contains a term that takes into account the relative number of cells in each of the phases, this last result implies that the overall (non-Gaussian) binding distribution (and hence the median of bacterial binding) can be highly sensitive to the relative proportion of cells in the various phases of the cell cycle.

Lateral diffusion of the total polar lipids from Thermoplasma acidophilum in multilamellar liposomes.

31P NMR lineshapes of multilamellar liposomes composed mostly of a bilayer-spanning tetraether lipid are consistent with rapid axially symmetric motion about the bilayer normal. The residual chemical shift anisotropy of 36 ppm is comparable to that seen for diacylphosphatidylglycerol systems and suggests comparable headgroup motion. The lateral diffusion rates for Thermoplasma acidophilum total polar lipids in mutilamellar liposomes was measured by two dimensional exchange NMR as a function of temperature. At 55 degrees C, near the growth temperature, the rate of lateral diffusion, DL, is comparable to that of diester phospholipids in the Lalpha liquid crystalline phase, having a value of 2 x 10(-8) cm2/s. DL decreases with temperature reaching a value of 8-6 x 10(-9) cm2/s at 30 degrees C. The activation energy Ea for lateral diffusion is estimated to be 10 kcal/mol (approximately 42 kJ/mol). The lateral diffusion rates indicate that the tetraether liposomes have a membrane viscosity at 30 degrees C which is considerably higher than that of diester phospholipids in the liquid crystalline phase.

NMR and molecular dynamics studies of the conformational epitope of the type III group B Streptococcus capsular polysaccharide and derivatives.

The conformational epitope of the type III group B Streptococcus capsular polysaccharide (GBSP III) exhibits unique properties which can be ascribed to the presence of sialic acid in its structure and the requirement for an extended binding site. By means of NMR and molecular dynamics studies on GBSP III and its fragments, the extended epitope of GBSP III was further defined. The influence of sialic acid on the conformational properties of GBSP III was examined by performing conformational analysis on desialylated GBSP III, which is identical to the polysaccharide of Streptococcus pneumoniae type 14, and also on oxidized and reduced GBSP III. Conformational changes were gauged by 1H and 13C chemical shift analysis, NOE, 1D selective TOCSY-NOESY experiments, J(HH) and J(CH) variations, and NOE of OH resonances. Changes in mobility were examined by 13C T1 and T2 measurements. Unrestrained molecular dynamics simulations with explicit water using the AMBER force field and the GLYCAM parameter set were used to assess static and dynamic conformational models, simulate the observable NMR parameters and calculate helical parameters. GBSP III was found to be capable of forming extended helices. Hence, the length dependence of the conformational epitope could be explained by its location on extended helices within the random coil structure of GBSP III. The interaction of sialic acid with the backbone of the PS was also found to be important in defining the conformational epitope of GBSP III.

Acyl chain length effects related to glycosphingolipid crypticity in phospholipid membranes: probed by 2H-NMR.

Wideline 2H-NMR was used to consider the relationships amongst glycosphingolipid and phospholipid fatty acid chain length and glycosphingolipid receptor function, in a system classically associated with crypticity. Galactosyl ceramide (GalCer), having 18- or 24-carbon fatty acid, was deuterium labelled at the conformationally-restricted fatty acid alpha-carbon (C-2). 2H-NMR spectra of N-[2,2-2H2]stearoyl and N-[2,2-2H2]lignoceroyl GalCer (GalCer with 18-vs. 24-carbon selectively deuterated fatty acid) were then compared over a range of temperatures in phosphatidylcholine/cholesterol membranes in which the host phospholipid had dimyristoyl, dipalmitoyl, or distearoyl fatty acid composition. Findings were evaluated in the light of known sensitivity of antibody interaction with GalCer to temperature and to both glycolipid fatty acid chain length and host matrix fatty acid chain length. Under the conditions of experimentation, spectra were not obtainable for glycolipids having rigid body motions that were slow on the NMR timescale (10(-4)-10(-5) s)-i.e.. motions typical of non-fluid (gel phase) membranes. The systems, DPPC/cholesterol and DSPC/cholesterol, in which the original observation was made of increased antibody binding to GalCer with long fatty acid, proved to be characterised by receptor motions that were in this slow timescale for both 18:0 and 24:0 GalCer at 22-24 degrees C. Under conditions for which spectra could be obtained, those for GalCer with [2,2-2H2]lignoceroyl (24-carbon alpha-deuterated) fatty acid were qualitatively similar to those of its 18-carbon analogue in all (fluid) membranes examined. However, spectral splittings differed quantitatively between deuterated 18:0 and 24:0 GalCer at a given temperature, dependent upon host matrix. These differences were most marked at lower temperatures and in the longer chain (more ordered) matrices, DPPC/cholesterol and DSPC/cholesterol. This suggests that maximum effects of glycolipid chain length on glycolipid receptor function may be expected to occur in spatially and motionally constrained lipid environments. There was little effect of temperature on spectral splittings seen for a given sample containing deuterated 18:0 GalCer. The small differences seen could be adequately accounted for by relatively minor alterations in glycolipid order and backbone conformation. In contrast, 24:0 GalCer in DPPC/cholesterol and DSPC/cholesterol displayed significant variation in its spectral splittings as the temperature was reduced; and these proved to be the source of the quantitative differences between 18:0 and 24:0 GalCer referred to above.(ABSTRACT TRUNCATED AT 400 WORDS)

Glycosphingolipid backbone conformation and behavior in cholesterol-containing phospholipid bilayers.

2H NMR spectroscopy was used to consider correspondence between existing single-crystal X-ray data for glycosphingolipids and their ceramide backbone conformation in fluid phospholipid membranes. A monoglycosylated sphingolipid, glucosylceramide (GlcCer), which represents the core structure of many important glycosphingolipids, was derived by partial synthesis through replacement of all native fatty acids with the 18-carbon species, stearic acid, deuterated at C2. N-[2,2-2H2]stearoyl-GlcCer was used to probe glycosphingolipid orientation and motion at low concentration in "fluid" phospholipid bilayers composed of dimyristoylphosphatidylcholine (DMPC), with and without physiological amounts of cholesterol. Spectral analysis, aided by stereoselective monodeuteration of the GlcCer fatty acid at C2, demonstrated that glycosphingolipid average acyl chain backbone conformation in fluid phospholipid membranes, with or without cholesterol, is likely closely related to that predicted from single crystal X-ray studies [Pascher, I. (1976) Biochim. Biophys. Acta 455, 433-451; Pascher, I., & Sundell, S. (1977) Chem. Phys. Lipids 20, 175-191]. To test the generality of this observation, specific comparisons were made involving galactosylceramide (GalCer) and globoside. GalCer provided a glycolipid differing only in monosaccharide stereochemistry (galactose vs glucose). Globoside permitted isolation of the effect of headgroup size, since it is derived from GlcCer via extension of the carbohydrate portion by the oligosaccharide, GalNAc beta 1-->3Gal alpha 1-->4Gal attached in beta 1-->4 linkage to the Glc residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of aminoglycoside antibiotics with phospholipids. A deuterium nuclear magnetic resonance study.

The effect of several aminoglycoside (AG) antibiotics on aqueous multilamellar dispersions of mixtures of phosphatidylinositol (PI) and deuterated phosphatidylcholine (PC) has been studied by deuterium (2H) NMR. Isepamicin and amikacin gave rise to no significant changes in 2H-NMR lineshape relative to that of the lipid mixture without antibiotic. Both kanamycin A and B, which have a greater affinity for PI than the other two antibiotics examined in this study, induced temperature-dependent changes in 2H-NMR lineshapes and associated spectral moments. The results are consistent with an antibiotic-induced lateral phase separation giving rise to PC-enriched domains free of drug and PI-AG domains. These effects are correlated with the inhibitory potency of aminoglycosides towards PC degradation.

Influence of the membrane surface on glycolipid conformation and dynamics. An interpretation of NMR results using conformational energy calculations.

Glycolipids constitute an important class of biomolecules that are involved in biomolecular recognition. The importance of carbohydrate head group conformation in such processes is well recognized. Glycolipids typically occur as minor components of the complex heterogeneous matrix of a biological membrane. As a result, the membrane surface may not only influence head group conformation but also serves as a spatial frame in which the glycolipid is oriented and recognized. In this study, conformational energy calculations have been used to assess the conformational space available to the glucose head group of 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL) in a liquid-crystalline membrane matrix. 2H NMR quadrupolar splittings are calculated and compared with those observed experimentally. This study demonstrates the importance of including surface interactions when considering the conformational space accessible to cell surface carbohydrates. The empirical approach taken here provides considerable insight at the molecular level, and offers the possibility of exploring even more complex systems.

Molecular motions and dynamics of a diunsaturated acyl chain in a lipid bilayer: implications for the role of polyunsaturation in biological membranes.

The nature and dynamics of the motions of a diunsaturated fatty acyl chain in a lipid bilayer were examined using a comprehensive simulation program for 2H NMR line shapes developed by Wittebort et al. [Wittebort, R. J., Olejniczak, E. T., & Griffin, R. G. (1987) J. Chem. Phys. 36, 5411-5420]. A motional model in which the isolinoleoyl chain (18:2 delta 6,9) adopts two conformations consistent with the low energy structures proposed for 1,4-pentadiene [Applegate, K. R., & Glomset, J. A. (1986) J. Lipid Res. 27, 658-680], but undergoes a rapid jump between these states, is sufficient to account for the experimentally observed quadrupolar couplings, the 2H-2H and 1H-2H dipolar couplings, the longitudinal relaxation times, and the changes in the average conformation of the chain that occur with a variation in temperature. The jump motion originates via rotations about the C7-C8 and the C8-C9 carbon bonds and leads to the low order parameters assigned to the C8 methylene segment (0.18) and the C9-C10 double bond (0.11). In contrast, the C6-C7 double bond, which is not involved in the two-site jump, characterized by a relatively large order parameter (0.56). Fatty acyl chains containing three or more double bonds likely cannot undergo the same jump motion and consequently will be highly ordered structures. Correlation times for diffusion of the molecular long axis of the diunsaturated acyl chain about the bilayer normal (approximately 10(-10) s) and for the local jump motion (approximately 10(-10) s) were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycosphingolipids: 2H NMR study of the influence of ceramide fatty acid characteristics on the carbohydrate headgroup in phospholipid bilayers.

Galactosylceramides bearing a variety of different pure fatty acid chains were 2H labeled in the carbohydrate headgroup at C6 of the terminal galactose residue, for study by 2H NMR. Fatty acids investigated included the 24-carbon saturated lignoceric acid, 18-carbon saturated stearic acid, cis-9,10-unsaturated oleic acid, and D- and L-stereoisomers of alpha-hydroxystearic acid. Headgroup-deuterated glycolipids were incorporated at 10 mol % into unsonicated bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine, and 2H NMR spectra were recorded at 65 and 40 degrees C. Under these experimental conditions, the membranes studied were primarily in the liquid-crystalline phase. At a given temperature, spectra for deuterated galactosylceramides dispersed in the fluid phase were remarkably similar, regardless of the nature of the fatty acid attached to the glycolipid sphingosine backbone. In each case, the spectrum consisted of a superposition of two quadrupolar powder patterns of approximately equal intensity. The spectra may be interpreted as arising from equal populations of two stereoisomers (pro-R and pro-S) of the deuterated galactose hydroxymethyl function, which is undergoing rapid (greater than 10(6) s-1) interconversion among the possible rotamers about the C5-C6 bond of the sugar ring. Within experimental error, the only fatty-acid-induced spectral difference detected among these glycosphingolipids deuterated in the carbohydrate headgroup was in the species with alpha-hydroxy-substituted fatty acids. At 65 degrees C, N-(D-alpha-hydroxy)stearoyl- and N-(L-alpha-hydroxy)stearoylgalactosylceramide gave rise to the same quadrupole splittings, but these differed marginally from the splittings observed for the other glycolipids studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of fatty acid alpha-hydroxylation on glycosphingolipid properties in phosphatidylcholine bilayers.

The role of glycosphingolipid fatty acid alpha-hydroxylation as a modulator of glycolipid organization and dynamics was considered by 2H-NMR in bilayer membranes. For these experiments, galactosylceramides were prepared in which the natural fatty acid mixture was replaced with perdeuterated 18-carbon hydroxylated or non-hydroxylated stearic acid. The L-stereoisomer of N-(alpha-OH-stearoyl-d34)galactosylceramide and its naturally-occurring D-alpha-OH analogue, were isolated for independent study. Bilayers were formed using 10 mol% galactosylceramide in a shorter chain phospholipid, dimyristoylphosphatidylcholine, in an attempt to reproduce several features of glycolipid-phospholipid interactions typical of cell membranes. Spectra of deuterated galactosylceramide in gel phase phospholipid membranes indicated that alpha-hydroxylation led to greater motional freedom and/or conformational disorder, with no measurable difference between D- and L-alpha-OH fatty acid derivatives. In fluid phosphatidylcholine bilayers the effects were modest. Glycolipid fatty acid hydroxylation led to broadening of the range of order parameters associated with methylene groups near the membrane surface (frequently referred to as the 'plateau region') - this effect being more marked for the naturally-occurring (D) stereoisomer. The degree of overall molecular order sensed by the glycolipid fatty acid chain in a fluid host matrix was minimally affected by alpha-hydroxylation; although the plateau region of the D isomer was slightly more ordered than that of the L isomer and the non-hydroxylated species. These results suggest that a significant aspect of the alpha-hydroxy group effect on glycosphingolipid behaviour in bilayer membranes with low glycolipid content was interference with glycolipid packing amongst host phospholipids in the upper portion of the acyl chains. For the D stereoisomer, there was some evidence that the hydroxy group led to strengthening of interlipid interaction near the membrane surface.