Phase behavior and structural characteristics of hydrated bovine brain gangliosides

Hydrated bovin brain gangliosides have been studied by differential scanning calorimetry, X-ray diffraction, and polarized light microscopy. Over the hydration range 18–50 wt.% H₂O, mixed brain gangliosides exhibit a hexagonal mesophase structure, in which the ganglioside molecules form hexagonally packed rod-like structures. The apolar lipid chains radiate from the center of the rods, with the sugar groups on the cylinder surface in contact with water. At higher water contents, an isotropic micellar solution is formed.Over the hydration range 20–30 wt.% H₂O, two small thermal transitions with peak maxima at 30°C and 46°C are observed by differential scanning calorimetry. These transitions broaden and move apart in temperature as the hydration is increased to 50 wt.% H₂O. X-ray diffraction data indicate that this double transition is associated with a hydrocarbon chain rearrangement from a disordered state to another, possibly more disordered, state. Thus, the gangliosides, although membrane lipid components, have physical characteristics which are very different from those of the membrane phospholipids.     

A Raman spectroscopic study on the interaction of an ion-channel protein with a phospholipid in a model membrane system (gramicidin A/L-alpha-lysophosphatidylcholine)

The interaction of the ion channel polypeptide gramicidin A with the L-alpha-lysophosphatidylcholine micelles in a membrane state association (approximative molar ratio 1:9) was investigated by Raman spectroscopy. Studies were carried out over the spectral ranges of 700-1700 cm-1 and 2800-3100 cm-1 at 10 degrees C. The Raman spectrum of L-alpha-lysophosphatidylcholine micelles indicated a disordered structure of the lipid acyl chains by the high intensities of the gauche conformation vibrations. Changing from the micellar phase to the membrane state of association with gramicidin A, the intensities of all-trans stretching modes increased whereas the intensities of gauche conformation vibrations decreased, reflecting the emergence of ordered lipid chains. Hydrophobic interactions between the acyl chains and the polypeptide side chain residues were demonstrated. The absence of modifications in intensities of the very strong tryptophan vibrations in the complex spectrum indicated that, if the tryptophan-stacking interactions suggested by some authors exist, they are very weak ones.     

Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.     

Thermotropic phase properties of 1,2-di-O-tetradecyl-3-O-(3-O-methyl- beta-D-glucopyranosyl)-sn-glycerol.

The hydration properties and the phase structure of 1,2-di-O-tetradecyl-3-O(3-O-methyl-beta-D-glucopyranosyl)-sn-glycerol (3-O-Me-beta-D-GlcDAIG) in water have been studied via differential scanning calorimetry, 1H-NMR and 2H-NMR spectroscopy, and x-ray diffraction. Results indicate that this lipid forms a crystalline (Lc) phase up to temperatures of 60-70 degrees C, where a transition through a metastable reversed hexagonal (Hll) phase to a reversed micellar solution (L2) phase occurs. Experiments were carried out at water concentrations in a range from 0 to 35 wt%, which indicate that all phases are poorly hydrated, taking up < 5 mol water/mol lipid. The absence of a lamellar liquid crystalline (L alpha) phase and the low levels of hydration measured in the discernible phases suggest that the methylation of the saccharide moiety alters the hydrogen bonding properties of the headgroup in such a way that the 3-O-Me-beta-D-GlcDAIG headgroup cannot achieve the same level of hydration as the unmethylated form. Thus, in spite of the small increase in steric bulk resulting from methylation, there is an increase in the tendency of 3-O-Me-beta-D-GlcDAIG to form nonlamellar structures. A similar phase behavior has previously been observed for the Acholeplasma laidlawii A membrane lipid 1,2-diacyl-3-O-(6-O-acyl-alpha-D-glucopyranosyl)-sn-glycerol in water (Lindblom et al. 1993. J. Biol. Chem. 268:16198-16207). The phase behavior of the two lipids suggests that hydrophobic substitution of a hydroxyl group in the sugar ring of the glucopyranosylglycerols has a very strong effect on their physicochemical properties, i.e., headgroup hydration and the formation of different lipid aggregate structures.     

Intrinsic conformation of lipid A is responsible for agonistic and antagonistic activity.

Lipopolysaccharides (LPS, endotoxin) represent a major virulence factor of Gram-negative bacteria, which can cause septic shock in mammals, including man. The lipid anchor of LPS to the bacterial outer membrane, lipid A, exhibits a peculiar chemical structure, harbours the 'endotoxic principle' of LPS and is also responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against endotoxically active LPS. By applying orientation measurements with attenuated total reflectance (ATR) infrared spectroscopy on hydrated lipid A samples, we show here that these different biological activities are directly correlated to the intrinsic conformation of lipid A. Bisphosphoryl-hexaacyl lipid A molecules with an asymmetric (4/2) distribution of the acyl chains linked to the diglucosamine backbone have a large tilt angle (> 45 degrees ) of the diglucosamine backbone with respect to the membrane surface, a conical molecular shape (larger cross-section of the hydrophobic than the hydrophilic moiety), and are endotoxically highly active. Monophosphoryl hexaacyl lipid A has a smaller tilt angle, and the conical shape is less expressed in favour of a more cylindrical shape. This correlates with decreasing endotoxic activity. Penta- and tetraacyl lipid A or hexaacyl lipid A with a symmetric acyl chain distribution (3/3) have a small tilt angle (< 25 degrees ) and a cylindrical shape and are endotoxically inactive, but may be antagonistic.     

Interaction of Zn2+ with phospholipid membranes

To characterize the specificity of zinc binding to phospholipid membranes in terms of headgroup structure, hydration and phase behavior we studied the zwitterionic lipid 1-palmitoyl-2-oleoyl-phosphatidylcholine as a function of hydration at 30 degreesC in the presence and absence of ZnCl2. Zinc forms a 2:1-1:1 complex with the lipid, and in particular with the negatively charged phosphate groups. Zn2(+)-bridges between neighboring lipid molecules stabilize the gel phase of the lipid relative to the liquid-crystalline state. Upon Zn2+ binding the C-O-P-O-C- backbone of the lipid headgroup changes from a gauche/gauche into the trans/trans conformation and it loses roughly 50% of the hydration shell. The ability of the Zn2(+)-bound phosphate groups to take up water is distinctly reduced, meaning that the headgroups have become less hydrophilic. The energetic cost (on the scale of Gibbs free energy) for completely dehydrating the lipid headgroups is decreased by approximately 10 kJ/mole in the presence of Zn2+. The interaction of phospholipid headgroups with Zn2+ is conveniently described by a hydrated zinc-phosphate complex the key energy contribution of which is more covalent than electrostatic in nature. Dehydration of phospholipid headgroups due to complexation with zinc cations is suggested to increase fusogenic potency of lipid membranes. Zinc appears to be one of the most potent divalent cation in inducing membrane fusion.     

Interaction of human apolipoprotein A-I with dipalmitoylphosphatidylcholine in vesicular and micellar complexes

The interactions of human apolipoprotein A-I (apo A-I) with dipalmitoylphosphatidylcholine (DPPC) in vesicular complexes at low protein concentrations and in micellar complexes at high protein concentrations are compared. The C-terminal segment of this protein, with a relative molecular weight (Mr) of about 11,000, is protected on trypsin treatment of apo A-I-vesicle complexes. A segment within the sequence from Leu-189 to Arg-215 of apo A-I penetrates the hydrophobic interior of the membrane, as found in a hydrophobic labeling experiment involving 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID). No appreciable stretch of apo A-I in micellar complexes was found to be protected from the tryptic digestion. This indicates that the interactions of apo A-I with lipids in the vesicular and micellar complexes are different. The binding equilibrium of apo A-I as to DPPC vesicles at low protein concentrations, as studied by hydrophobic labeling of the bilayer-penetrating segment, is reached within about 1 h, while the formation of micellar complexes at high protein concentrations takes about 24 h at 42 degrees C. Time-dependent labeling studies involving photoreactive phosphatidylcholine (PC) with high apo A-I concentrations suggested an initial interaction with the head group region of the bilayer followed by interaction with the tail ends of the acyl chains of the lipid. A possible mechanism for the micellization process is discussed.     

Structural and dynamical changes of the bindin B18 peptide upon binding to lipid membranes. A solid-state NMR study

Structural and dynamical features of the B18 peptide from the sea urchin sperm binding protein were determined in the crystalline state and in zwitterionic lipid bilayers at a peptide:lipid molar ratio of 1:12 using solid-state NMR spectroscopy. The study was focused on three (13)C and (15)N uniformly labeled leucine residues, which were introduced into three different B18 peptides at positions evenly distributed along the B18 primary structure. Isotropic (13)C and (15)N chemical shift measurements showed that while B18 possesses a nonhelical and non-sheet-like structure in the crystalline state, the peptide adopts an oligomeric beta-sheet structure in the membrane in the presence of Zn(2+) ions at high peptide:lipid ratio. Torsion angle measurements for the three leucine sites supported these results, with phi torsion angles between -80 degrees and -90 degrees in the crystalline state and between -110 degrees and -120 degrees in the membrane-bound form. These phi torsion angles determined for membrane-bound B18 are consistent with a parallel beta-sheet secondary structure. Analysis of motionally averaged dipolar coupling measurements established an increase of the mobility in the leucine side chains upon binding to the membrane, whereas the backbone mobility remained essentially unchanged, except in the binding site of Zn(2+) ions. This difference in mobility was related to the H-bond network in the parallel beta-sheet structure, which involves the backbone and excludes the side chains of leucine residues. The parallel beta-sheet structure of B18 in the membrane in the presence of Zn(2+) appears to be an active state for the fusion of zwitterionic membranes in the presence of Zn(2+). A fluorescence fusion assay indicated that high B18 concentrations are required to induce fusion in these systems. Therefore, it was hypothesized that the oligomeric beta-sheet secondary structure revealed in the study represents an active state of the peptide in a membrane environment during fusion.     

Effect of cold shock on lipid A biosynthesis in Escherichia coli. Induction At 12 degrees C of an acyltransferase specific for palmitoleoyl-acyl carrier protein

Palmitoleate is not present in lipid A isolated from Escherichia coli grown at 30 degrees C or higher, but it comprises approximately 11% of the fatty acyl chains of lipid A in cells grown at 12 degrees C. The appearance of palmitoleate at 12 degrees C is accompanied by a decline in laurate from approximately 18% to approximately 5.5%. We now report that wild-type E. coli shifted from 30 degrees C to 12 degrees C acquire a novel palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase that acts on the key lipid A precursor Kdo2-lipid IVA. The palmitoleoyl transferase is induced more than 30-fold upon cold shock, as judged by assaying extracts of cells shifted to 12 degrees C. The induced activity is maximal after 2 h of cold shock, and then gradually declines but does not disappear. Strains harboring an insertion mutation in the lpxL(htrB) gene, which encodes the enzyme that normally transfers laurate from lauroyl-ACP to Kdo2-lipid IVA (Clementz, T., Bednarski, J. J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095-12102) are not defective in the cold-induced palmitoleoyl transferase. Recently, a gene displaying 54% identity and 73% similarity at the protein level to lpxL was found in the genome of E. coli. This lpxL homologue, designated lpxP, encodes the cold shock-induced palmitoleoyl transferase. Extracts of cells containing lpxP on the multicopy plasmid pSK57 exhibit a 10-fold increase in the specific activity of the cold-induced palmitoleoyl transferase compared with cells lacking the plasmid. The elevated specific activity of the palmitoleoyl transferase under conditions of cold shock is attributed to greatly increased levels of lpxP mRNA. The replacement of laurate with palmitoleate in lipid A may reflect the desirability of maintaining the optimal outer membrane fluidity at 12 degrees C.     

Phase behavior and structure of aqueous dispersions of sphingomyelin

The phase behavior of bovine brain sphingomyelin in water has been determined by polarizing light microscopy, differential scanning calorimetry, and X-ray diffraction. Lamellar phases, in which water is intercalated between sheets of lipid molecules arranged in the classical bilayer fashion, are present over much of the phase diagram. An order-disorder transition separates the high temperature, liquid crystalline, lamellar phase from a more ordered lamellar phase at low temperatures. The hydration characteristics of sphingomyelin are similar to the structurally related lecithin in that only limited amounts of water are incorporated above and below the transition. Above the transition at 47 degrees C, a maximum of 35% by weight of water can be incorporated between the lipid bilayers, the total thickness at maximum hydration being 60.2 A, the lipid thickness 38 A, and the surface area per lipid molecule at the interface 60 A(2). Water in excess of 35% by weight is present as a separate phase. Below the phase transition, at 25 degrees C a maximum of 42% by weight of water may be incorporated between the lipid bilayers. On increasing the hydration, the lamellar repeat distance increases from 63.5 A to a limiting value of 76 A. Within this hydration range the calculated lipid thickness decreases from 63.5 to 42.5 A, and the surface area per lipid molecule increases from 36.1 to 53.6 A(2). Although these changes may be accounted for by a structure in which the hexagonally packed ordered hydrocarbon chains tilt progressively with respect to the normal to the bilayer plane on increasing hydration, it is possible that changes in other more complex lamellar structures may be responsible for these variations in lipid thickness and surface area.     

Effect of independent variations in fatty acid structure and chain length on lipid polar headgroup composition in Acholeplasma laidlawii B membranes: regulation of lamellar/nonlamellar phase propensity

We have studied the biosynthetic regulation of the membrane lipid polar headgroup distribution in Acholeplasma laidlawii B cells made fatty acid auxotrophic by growth in the presence of the biotin-binding agent avidin to test whether this organism has the ability to coherently regulate the lamellar/nonlamellar phase propensity of its membrane lipids. The addition of various single normal growth-supporting exogenous fatty acids to such cell cultures produces fatty acid-homogeneous cells in which the hydrocarbon chain length and structure of the fatty acyl chains of the membrane lipids can be independently varied. Moreover, in analyzing our results, we consider the fact that the individual membrane lipid classes of this organism can form either normal micellar, lamellar, or reversed cubic or hexagonal phases in isolation (Lewis, R. N. A. H., and McElhaney, R. N. (1995) Biochemistry 34, 13818-13824). When A. laidlawii cells are highly enriched in one of a homologous series of methyl isobranched, methyl anteisobranched, or omega-cyclohexyl fatty acids, neither the ratio of normal micellar/lamellar nor of inverted cubic or hexagonal/lamellar phase-forming lipids are coherently regulated, and in fact in the former case, the changes in lipid polar headgroup composition observed are generally in a direction opposite to that required to maintain the overall lamellar/nonlamellar phase preference of the total membrane lipids constant when hydrocarbon chain length is varied. Similarly, when lipid hydrocarbon structure is varied at a constant effective chain length, a similar lack of coherent regulation of membrane lipid polar headgroup distribution is also observed, although in this case a weak overall trend in the expected direction occurs. We also confirm our previous finding (Foht, P. J., Tran, Q. M., Lewis, R. N. A. H., and McElhaney, R. N. (1995) Biochemistry 34, 13811-13817) that the ratio of inverted phase-forming monoglucosyl diacylglycerol to the lamellar phase-forming glycolipid diglucosyl diacylglycerol, previously used to estimate membrane lipid phase preference in A. laidlawii A and B, is not by itself a reliable indicator of the overall lamellar/nonlamellar phase propensity of the total membrane lipids of these organisms. Our results indicate that A. laidlawii B lacks a coherent mechanism to biosynthetically regulate the polar headgroup distribution of its membrane lipids to maintain the micellar/lamellar/inverted phase propensity constant in the face of induced variations in either the chain length or the structure of its lipid hydrocarbon chains. Finally, we suggest that the lack of a coherent regulatory mechanism to regulate the overall phase-forming propensity of the total membrane lipids of this organism under these circumstances may result in part from its inability to optimize all of the biologically relevant physical properties of its membrane lipid bilayer simultaneously.     

Kinetic study of folding and misfolding of diacylglycerol kinase in model membranes

Despite the relevance of membrane protein misfolding to a number of common diseases, our understanding of the folding and misfolding of membrane proteins lags well behind soluble proteins. Here, the overall kinetics of membrane insertion and folding of the homotrimeric integral membrane protein diacylglycerol kinase (DAGK) is addressed. DAGK was purified into lipid/detergent-free urea and guanidinium solutions and subjected to general structural characterization. In urea, the enzyme was observed to be monomeric but maintained considerable tertiary structure. In guanidinium, it was also monomeric but exhibited much less tertiary structure. Aliquots of these DAGK stock solutions were diluted 200-fold into lipid vesicles or into detergent/lipid mixed micelles, and the rates and efficiencies of folding/insertion were monitored. Reactions were also carried out in which micellar DAGK solutions were diluted into vesicular solutions. Productive insertion of DAGK from denaturant solutions into mixed micelles occurred much more rapidly than into lipid vesicles, suggesting that bilayer transversal represents the rate-limiting step for DAGK assembly in vesicles. The efficiency of productive folding/insertion into vesicles was highest in reactions initiated with micellar DAGK stock solutions (where DAGK maintains a nativelike fold and oligomeric state) and lowest in reactions starting with guanidinium stocks (where DAGK is an unfolded monomer). Moreover, the final ratio of irreversibly misfolded DAGK to reversibly misfolded enzyme was highest following reactions initiated with guanidinium stock solutions and lowest when micellar stocks were used. Finally, it was also observed that very low concentrations of detergents were able to both enhance the bilayer insertion rate and suppress misfolding.     

Spin-labeled amphotericin B: synthesis, characterization, biological and spectroscopic properties

A biologically active spin-labeled derivative of amphotericin B has been synthesized by the nucleophilic addition of amphotericin B to 4-(2-iodoacetamido)-2,2',6,6'-tetramethylpiperadine-N-oxyl in dimethyl-sulphoxide at 40 degrees C. The derivative is a moderately water-soluble compound which displays the same biological activity of the parental compound against the sensitive organism Leishmania mexicana; also, the rates of proton-cation exchange induced by the two compounds in large unilamellar liposomes are indistinguishable. The ESR spectra of spin-labeled amphotericin B in lipid vesicles indicate a high degree of motion, very similar to that encountered for the compound in aqueous solutions at neutral pH and in deoxycholate micelles, and suggest that the structures formed by the antibiotic in membranes are composed by a small number of molecules. In contrast, the spectra of the labeled antibiotic in ethanol, diethyl ether and dimethylformamide indicate restricted motion and exchange interactions, probably resulting from the micellar aggregation induced in these media. Ascorbate at 10 mM is able to reduce completely the nitroxide group of the labeled antibiotic in lipid vesicles in less than 30 s, indicating that an asymmetric disposition of the antibiotic molecules across the membrane is capable of inducing its biological and ionophoric properties. Ni2+ and Cu2+ produce moderate exchange broadening of the ESR signal of spin-labeled amphotericin B in lipid vesicles; the comparison of this phenomenom with the exchange broadening produced by the same ions in the ESR spectrum of 2,2',6,6'-tetramethylpiperidine-N-oxyl in water solution suggests an specific Cu2+-amphotericin B interaction in membranes.     

The kinetics of formation and structure of the low-temperature phase of 1-stearoyl-lysophosphatidylcholine

A combination of differential scanning calorimetry (DSC) and X-ray diffraction have been used to study the kinetics of formation and the structure of the low-temperature phase of 1-stearoyl-lysophosphatidylcholine (18:0-lysoPC). For water contents greater than 40 weight %, DSC shows a sharp endothermic transition at 27 degrees C (delta H = 6.75 kcal/mol) corresponding to a low-temperature phase----micelle transition. This sharp transition is not reversible, but is regenerated in a time and temperature-dependent manner. For example, with incubation at 0 degrees C the maximum transition enthalpy (delta H = 6.75 kcal/mol) is generated in about 45 min after an initial slow nucleation process of approx. 20 min. The kinetics of formation of the low-temperature phase is accelerated at lower temperatures and may be related to the disruption of 18:0-lysoPC micelles by ice crystal formation. X-ray diffraction patterns of 18:0-lysoPC recorded at 10 degrees C over the hydration range 20-80% are characteristic of a lamellar gel phase with tilted hydrocarbon chains with the bilayer repeat distance increasing from 47.6 A at 20% hydration to a maximum of 59.4 A at 39% hydration. At this maximum hydration, approx. 19 molecules of water are bound per molecule of 18:0-lysoPC. Electron density profiles show a phosphate-phosphate distance of 30 A, indicating an interdigitated lamellar gel phase for 18:0-lysoPC at all hydration values. The angle of chain tilt is calculated to be between 20 and 30 degrees. For water contents greater than 40%, this interdigitated lamellar phase converts to the micellar phase at 27 degrees C in a kinetically fast process, while the reverse (micelle----interdigitated bilayer) transition is a kinetically slower process (see also Wu, W. and Huang, C. (1983) Biochemistry 22, 5068-5073).     

Disturb or Stabilize? A Molecular Dynamics Study of the Effects of Resorcinolic Lipids on Phospholipid Bilayers

Resorcinolic lipids, or resorcinols, are commonly found in plant membranes. They consist of a substituted benzene ring forming the hydrophilic lipid head, attached to an alkyl chain forming the hydrophobic tail. Experimental results show alternative effects of resorcinols on lipid membranes. Depending on whether they are added to lipid solutions before or after the formation of the liposomes, they either stabilize or destabilize these liposomes. Here we use atomistic molecular dynamics simulations to elucidate the molecular nature of this dual effect. Systems composed of either one of three resorcinol homologs, differing in the alkyl tail length, interacting with dimyristoylphosphatidylcholine lipid bilayers were studied. It is shown that resorcinols preincorporated into bilayers induce order within the lipid acyl chains, decrease the hydration of the lipid headgroups, and make the bilayers less permeable to water. In contrast, simulations in which the resorcinols are incorporated from the aqueous solution into a preformed phospholipid bilayer induce local disruption, leading to either transient pore formation or even complete rupture of the membrane. In line with the experimental data, our simulations thus demonstrate that resorcinols can either disturb or stabilize the membrane structure, and offer a detailed view of the underlying molecular mechanism.     

Properties of ganglioside GM1 in phosphatidylcholine bilayer membranes.

Gangliosides have been shown to function as cell surface receptors, as well as participating in cell growth, differentiation, and transformation. In spite of their multiple biological functions, relatively little is known about their structure and physical properties in membrane systems. The thermotropic and structural properties of ganglioside GM1 alone and in a binary system with 1,2-dipalmitoyl phosphatidylcholine (DPPC) have been investigated by differential scanning calorimetry (DSC) and x-ray diffraction. By DSC hydrated GM1 undergoes a broad endothermic transition TM = 26 degrees C (delta H = 1.7 kcal/mol GM1). X-ray diffraction below (-2 degrees C) and above (51 degrees C) this transition indicates a micellar structure with changes occurring only in the wide angle region of the diffraction pattern (relatively sharp reflection at 1/4.12 A-1 at -2 degrees C; more diffuse reflection at 1/4.41 A-1 at 51 degrees C). In hydrated binary mixtures with DPPC, incorporation of GM1 (0-30 mol%; zone 1) decreases the enthalpy of the DPPC pretransition at low molar compositions while increasing the TM of both the pre- and main transitions (limiting values, 39 and 44 degrees C, respectively). X-ray diffraction studies indicate the presence of a single bilayer gel phase in zone 1 that can undergo chain melting to an L alpha bilayer phase. A detailed hydration study of GM1 (5.7 mol %)/DPPC indicated a conversion of the DPPC bilayer gel phase to an infinite swelling system in zone 1 due to the presence of the negatively charged sialic acid moiety of GM1. At 30-61 mol % GM1 (zone 2), two calorimetric transitions are observed at 44 and 47 degrees C, suggesting the presence of two phases. The lower transition reflects the bilayer gel --> L alpha transition (zone 1), whereas the upper transition appears to be a consequence of the formation of a nonbilayer, micellar or hexagonal phase, although the structure of this phase has not been defined by x-ray diffraction. At > 61 mol % GM1 (zone 3) the calorimetric and phase behavior is dominated by the micelle-forming properties of GM1; the presence of mixed GM1/DPPC micellar phases is predicted.     

An electron-spin-resonance spin-label study of the interaction of purified Mojave toxin with synaptosomal membranes from rat brain

The structural properties of isolated purified rat brain synaptosomal membranes, both in the presence and absence of purified active toxin of the Mojave snake Crotalus scutulatus scutulatus, were studied by spin-label electron spin resonance techniques. The spectra from eight different positional isomers of nitroxide-labelled stearic acids, a rigid steroid androstanol, and a spin-labelled phosphatidylcholine intercalated into the synaptosomal membranes, were obtained as a function of temperature from 4-40 degrees C. The flexibility gradient (from spin-label order parameters) and polarity profile (from isotropic splitting factors) across the synaptosomal membranes, was characteristic for lipid bilayers. The nitroxide spin-labelled steroid, androstanol, intercalated into the synaptosomal membrane, revealed the abrupt onset of rapid cooperative rotation about the long axis of the molecule at 12 degrees C showing that the lipid molecules are rotating rapidly around their long axes at physiological temperatures. The presence of the Mojave toxin affected the synaptosomal membrane in a complex manner, depending upon the temperature and the position of the nitroxide label on the alkyl chain of the stearic acid probe. Mojave toxin exerted little effect on the flexibility gradient of the synaptosomal membrane at 20 degrees C, a temperature at which the acyl chain labels detected a structural change in the membranes. At temperatures lower than 20 degrees C, the Mojave toxin produced a change in the flexibility gradient of the synaptosomal membrane which indicated an increased disordering in the upper region of the membrane and a concomitant increased ordering of the acyl chains in the deeper regions of the membrane. At temperatures higher than 20 degrees C, the order profile of the synaptosomal membrane was shifted by the presence of the Mojave toxin in a manner which indicated that the outer parts of the membrane were more rigid and the inner regions more fluid, than in controls. A cross-over point for the perturbation occurred at C8-9, which is about 12-14 A into the membrane. This is the approximate depth of the hydrophobic pocket shown in pancreatic phospholipase A2 [Drenth et al. (1976) Nature (Lond.) 264, 373-377], a protein likely to be homologous to the basic subunit of the toxin. At all temperatures, rotational lipid motion was inhibited by the toxin as indicated by the steroid probe. The electron spin-resonance spin-label results are interpreted in terms of the partial penetration of the basic subunit of the intact toxin into the membrane, disordering the ordered chains at low temperature and ordering the disordered chains at physiological temperatures. The purified individual toxin subunits did not perturb the membrane lipids at physiological temperatures implying that both subunits must be associated for activity of the toxin which is confirmed by toxicity studies.     

Membrane-bound conformation and topology of the antimicrobial peptide tachyplesin I by solid-state NMR

The conformation and membrane topology of the disulfide-stabilized antimicrobial peptide tachyplesin I (TP) in lipid bilayers are determined by solid-state NMR spectroscopy. The backbone (phi and psi) torsion angles of Val(6) are found to be -133 degrees and 142 degrees , respectively, and the Val(6) CO-Phe(8) H(N) distance is 4.6 A. These constrain the middle of the N-terminal strand to a relatively ideal antiparallel beta-sheet conformation. In contrast, the phi angle of Gly(10) is +/-85 degrees , consistent with a beta-turn conformation. Thus, TP adopts a beta-hairpin conformation with straight strands, similar to its structure in aqueous solution but different from a recently reported structure in DPC micelles where bending of the two beta-strands was observed. The Val(6) and Gly(10) CO groups are both 6.8 A from the lipid (31)P, while the Val(6) side chain is in (1)H spin diffusion contact with the lipid acyl chains. These results suggest that TP is immersed in the glycerol backbone region of the membrane and is oriented roughly parallel to the plane of the membrane. This depth of insertion and orientation differs from those of the analogous beta-sheet antimicrobial peptide protegrin-1 and suggest the importance of structural amphiphilicity in determining the location and orientation of membrane peptides in lipid bilayers.     

Membrane hydration and structure on a subnanometer scale as seen by high resolution solid state nuclear magnetic resonance: POPC and POPC/C12EO4 model membranes.

The position on a subnanometer scale and the dynamics of structurally important water in model membranes was determined using a combination of proton magic-angle spinning NMR (MAS) with two-dimensional NOESY NMR techniques. Here, we report studies on phosphocholine lipid bilayers that were then modified by the addition of a nonionic surfactant that is shown to dehydrate the lipid. These studies are supplemented by 13C magic-angle spinning NMR investigations to get information on the dynamics of segmental motions of the membrane molecules. It can be shown that the hydrophilic chain of the surfactant is positioned at least partially within the hydrophobic core of the lipid bilayer. With the above NMR approach, we are able to establish molecular contacts between water and the lipid headgroup as well as with certain groups of the hydrocarbon chains and the glycerol backbone. This is possible because high resolution proton and 13C-NMR spectra of multilamellar bilayer membranes are obtained using MAS. A phase-sensitive NOESY must also be applied to distinguish positive and negative cross-peaks in the two-dimensional plot. These studies have high potential to investigate membrane proteins hydration and structural organization in a natural lipid bilayer surrounding.     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0-70 degrees C. In the low-temperature (LC) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (LC) phase to the intermediate-temperature (L beta) phase at 25 degrees C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L beta) to the high-temperature (L alpha) phase at 37 degrees C is a gel-to-liquid-crystalline phase transition analogous to the 41.5 degrees C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the LC phase. In the L beta and L alpha phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C = O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A2 on the basis of a preferred head group conformation.