Raman spectra and conformation of the glycerophosphorylcholine headgroup

The Raman and infrared spectra of L-α-glycerophosphylcholine (GPC) and its cadmium complex (GPC·CdCl₂) have been measured for both the anhydrous and hydrate crystals. The Raman spectra of GPC and GPC·CdCl₂ dissolved in water are also reported. All the spectra fall into three distinctive patterns, demonstrating the existence of at least three different kinds of rotational isomers; the first is stable in the anhydrous GPC crystal, the second is stable in the GPC hydrate and GPC·CdCl₂ hydrate crystals and the third is stable in the anhydrous GPC·CdCl₂ crystal and also in aqueous solutions of GPC·CdCl₂. The latter form is reported for the first time.Raman spectra of dimyristoyl-L-α-phosphatidylcholine (L-DMPC) and dipalmitoyl-(L and DL)-α-phosphatidylcholine (L and DL-DPPC) hydrates have been measured below, around and above the transition temperature T₁. The spectra below T₁ indicate that the conformation of the glycerophosphorylcholine (GPC) headgroup in the L-DMPC crystal is similar to that in the L-DPPC crystal. The headgroup conformation in the DL-DPPC crystal is quite different from that in the L-DPPC crystal, although they became the same aftera crystalline trasnformation of DL-DPPC. The changes of the Raman spectra through the T₁ transition for the above crystals show that the headgroup conformation remains unchanged at the beginning o the transition.     

NMR and modelling studies of disaccharide conformation

Long-range heteronuclear coupling constants were measured across the glycosidic linkages for a series of eight alpha- or beta-linked disaccharides in aqueous solution. Multiple 13C site-selective excitation experiments using 1H decoupling in conjunction with pulsed field gradient-enhanced spectroscopy were used to determine 3J(C,H) values. These were subsequently compared with the respective couplings calculated, using a Karplus relationship, from molecular dynamics simulations with the explicit inclusion of water.     

NMR structural analysis of a membrane protein: bacteriorhodopsin peptide backbone orientation and motion

In reconstituted vesicles above the lipid phase transition temperature, bacteriorhodopsin (BR) undergoes rotational diffusion about an axis perpendicular to the plane of the bilayer [Cherry, R. J., Muller, U., & Schneider, G. (1977) FEBS Lett. 80, 465]. This diffusion narrows the 13C NMR powder line shape of the BR peptide carbonyls. In contrast, BR in native purple membrane is relatively immobile and exhibits a rigid-lattice powder line shape. By use of the principal values of the rigid-lattice chemical shift tensor and the motionally narrowed line shape from the reconstituted system, the range of Euler angles of the leucine peptide groups relative to the diffusion axis has been calculated. The experimentally observed line shape is inconsistent with those expected for structures which consist entirely of either alpha helix or beta sheet perpendicular to the membrane or beta sheet tilted at angles up to about 60 degrees from the membrane normal. However, for two more complex structural models, the predicted line shapes agree well with the experimental one. These are, first, a structure consisting entirely of alpha1 helices tilted at 20 degrees from the membrane normal and, second, a combination of 60% alpha II helix perpendicular to the membrane plane and 40% antiparallel beta sheet tilted at 10-20 degrees from the membrane normal. The results also indicate that the peptide backbone of bacteriorhodopsin in native purple membrane is extremely rigid even at 40 degrees. The experiments presented here demonstrate a new approach, using solid-state nuclear magnetic resonance (NMR) methods, for structural studies of transmembrane proteins in fluid membrane environments, either natural or reconstituted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lamellar packing of a chiral N,N-dimethylphosphatidylethanolamine: electron diffraction. Evidence for a lecithin-type headgroup conformation

Lamellar electron diffraction intensity data from epitaxially crystallized 1,2-dipalmitoyl-sn-glycerophospho-N,N-dimethylethanolamine were used to determine the layer packing in order to compare the chiral structure to the crystal structure of a racemic homologue. After finding the chain orientation, the structure was determined by interpretation of the Patterson function, followed by independent crystallographic phase assignments with conventional direct methods (use of three phase structure invariants). The phase determination was verified by a translational search with a molecular model based on a similar lecithin structure. The final R-value is 0.29, and this is lowered to 0.18 after a correction is made for incoherent multiple electron scattering. The layer packing is found to be very much like that of a diacyl phosphatidylcholine with the N,N-dimethylethanolamine moiety parallel to the bilayer surface rather than the perpendicular arrangement of headgroups involved in an interdigitated layer, as seen for racemic homolog.     

Response of the phosphatidylcholine headgroup to membrane surface charge in ternary mixtures of neutral, cationic, and anionic lipids: a deuterium NMR study.

Deuterium nuclear magnetic resonance (2H NMR) spectroscopy was used to investigate the response of the phosphatidylcholine headgroup of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) to changes in surface electrostatic charge in membranes consisting of ternary mixtures of lipids. DMPC was deuterated at the choline alpha- and beta-methylene segments. The membrane surface charge was manipulated by the simultaneous addition of cationic didodecyldimethylammonium bromide (DDAB) and anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) to neutral DMPC. Addition of increasing amounts of DDAB caused a progressive decrease (increase) in the 2H NMR quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Addition of increasing amounts of DMPG caused a progressive increase (decrease) in the quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Qualitatively, the 2H NMR quadrupole splitting charge response exhibited the same main features for ternary mixtures of DDAB/DMPG/DMPC and binary mixtures of DDAB/DMPC or DMPG/DMPC. Quantitatively, however, the 2H NMR quadrupole splittings obtained from ternary mixtures did not coincide with those obtained from binary mixtures of nominally identical surface charge densities. Hence, the quadrupole splitting did not respond directly to the net membrane surface charge. Instead, the quadrupole splitting measured for a given ternary lipid composition could be reproduced by summing the individual effects of the charged lipids in binary mixtures, weighted according to their appropriate mole fractions.     

Oligomeric structure, dynamics, and orientation of membrane proteins from solid-state NMR

Solid-state NMR is a versatile and powerful tool for determining the dynamic structure of membrane proteins at atomic resolution. I review the recent progress in determining the orientation, the internal and global protein dynamics, the oligomeric structure, and the ligand-bound structure of membrane proteins with both alpha-helical and beta sheet conformations. Examples are given that illustrate the insights into protein function that can be gained from the NMR structural information.     

Influence of lipid chemistry on membrane fluidity: tail and headgroup interactions

Membrane fluidity plays an important role in cell function and may, in many instances, be adjusted to facilitate specific cellular processes. To understand better the effect that lipid chemistry has on membrane fluidity the inclusion of three different lipids into egg phosphatidylcholine (eggPC) bilayers has been examined; the three lipids are egg phosphatidylethanolamine ((eggPE) made by transphosphatidylation of eggPC in the presence of ethanolamine), lyso-phosphatidylcholine (LPC), and lyso-phosphatidylethanolamine (LPE). The fluidity of the membranes was determined using fluorescence recovery after photobleaching and the intermolecular interactions were examined using attenuated total reflection Fourier transform infrared spectroscopy. It was observed that both headgroup and tail chemistry can significantly modulate lipid diffusion. Specifically, the inclusion of LPC and eggPE significantly altered the lipid diffusion, increased and decreased, respectively, whereas the inclusion of LPE had an intermediate effect, a slight decrease in diffusion. Strong evidence for the formation of hydrogen-bonds between the phosphate group and the amine group in eggPE and LPE was observed with infrared spectroscopy. The biological implications of these results are discussed.     

Solution conformation of a pectin fragment disaccharide using molecular modelling and nuclear magnetic resonance

In the present study, the conformational behaviour of methylated pectic disaccharide 4-O-alpha-D-galactopyranurosyl 1-O-methyl-alpha-D-galactopyranuronic 6,6'-dimethyl diester 1 has been completely characterized through combined n.m.r. and molecular modelling studies. The 1H-1H n.O.e. across the glycosidic bond was measured by both steady-state and transient 1D and 2D experiments. In parallel, the complete conformational analysis of the disaccharide has been achieved with the MM3 molecular mechanics method. The conformation of the pyranose ring is confirmed by the excellent agreement between the experimental and calculated intracyclic scalar coupling constants. The iso-energy contours displayed on the 'relaxed' map indicate an important flexibility about the glycosidic linkage. There is no significant influence of the methoxyl group on the conformational behaviour of the disaccharide. The theoretical n.m.r. data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for slow internal motions. 3JC-H coupling constants were calculated using an equation suitable for C-O-C-H segments. The agreement between experimental and theoretical data is excellent. Within the potential energy surface calculated for the disaccharide, several conformers can be identified. When these conformations are extrapolated to a regular polymer structure, they generate pectins with right- and left-handed chirality along with a two-fold helix. These different types of helical structure are the result of small changes in conformation, without any drastic variation of the fibre repeat.     

Secondary structure and orientation of the pore-forming toxin lysenin in a sphingomyelin-containing membrane

Lysenin is a sphingomyelin-recognizing toxin which forms stable oligomers upon membrane binding and causes cell lysis. To get insight into the mechanism of the transition of lysenin from a soluble to a membrane-bound form, surface activity of the protein and its binding to lipid membranes were studied using tensiometric measurements, Fourier-transform infrared spectroscopy (FTIR) and FTIR-linear dichroism. The results showed cooperative adsorption of recombinant lysenin-His at the argon-water interface from the water subphase which suggested self-association of lysenin-His in solution. An assembly of premature oligomers by lysenin-His in solution was confirmed by blue native gel electrophoresis. When a monolayer composed of sphingomyelin and cholesterol was present at the interface, the rate of insertion of lysenin-His into the monolayer was considerably enhanced. Analysis of FTIR spectra of soluble lysenin-His demonstrated that the protein contained 27% β-sheet, 28% aggregated β-strands, 10% α-helix, 23% turns and loops and 12% different kinds of aggregated forms. In membrane-bound lysenin-His the total content of α-helices, turns and loops, and β-structures did not change, however, the 1636cm⁻¹ β-sheet band increased from 18% to 31% at the expense of the 1680cm⁻¹ β-sheet structure. Spectral analysis of the amide I band showed that the α-helical component was oriented with at 41° to the normal to the membrane, indicating that this protein segment could be anchored in the hydrophobic core of the membrane.     

Secondary structure and orientation of the pore-forming toxin lysenin in a sphingomyelin-containing membrane

Lysenin is a sphingomyelin-recognizing toxin which forms stable oligomers upon membrane binding and causes cell lysis. To get insight into the mechanism of the transition of lysenin from a soluble to a membrane-bound form, surface activity of the protein and its binding to lipid membranes were studied using tensiometric measurements, Fourier-transform infrared spectroscopy (FTIR) and FTIR-linear dichroism. The results showed cooperative adsorption of recombinant lysenin-His at the argon-water interface from the water subphase which suggested self-association of lysenin-His in solution. An assembly of premature oligomers by lysenin-His in solution was confirmed by blue native gel electrophoresis. When a monolayer composed of sphingomyelin and cholesterol was present at the interface, the rate of insertion of lysenin-His into the monolayer was considerably enhanced. Analysis of FTIR spectra of soluble lysenin-His demonstrated that the protein contained 27% beta-sheet, 28% aggregated beta-strands, 10% alpha-helix, 23% turns and loops and 12% different kinds of aggregated forms. In membrane-bound lysenin-His the total content of alpha-helices, turns and loops, and beta-structures did not change, however, the 1636cm(-1) beta-sheet band increased from 18% to 31% at the expense of the 1680cm(-1) beta-sheet structure. Spectral analysis of the amide I band showed that the alpha-helical component was oriented with at 41 degrees to the normal to the membrane, indicating that this protein segment could be anchored in the hydrophobic core of the membrane.     

The structure and orientation of class-A amphipathic peptides on a phospholipid bilayer surface

The amphipathic α-helix is a recognised structural motif that is shared by membrane-associating proteins and peptides of diverse function. The aim of this paper is to determine the orientation of an α-helical amphipathic peptide on the bilayer surface. We use five amphipathic 18-residue peptide analogues of a class A amphipathic peptide that is known to associate with a bilayer surface. Tyrosine and tryptophan are used as spectroscopic probes to sense local environments in the peptide in solution and when bound to the surface of unilamellar phosphatidylcholine vesicles. In a series of peptides, tryptophan is moved progressively along the sequence from the nonpolar face (positions 3, 7, 4) to the polar face of the peptide (positions 2, 12). The local environment of the tryptophan residue at each position is determined using fluorescence spectroscopy employing quantum yield, and the wavelength of the emission maximum as indicators of micropolarity. The exposure of the tryptophan residues at each site is assessed by acrylamide quenching. On association with vesicles, the tryptophan residues at positions 3, 7 and 14 are in nonpolar water-shielded environments, and the tryptophan at position 12 is in an exposed polar environment. The tryptophan at position 2, which is located near the bilayer-water interface, exhibits intermediate behaviour. Analysis of the second-derivative absorption spectrum confirmed that the tyrosine residue at position 7 is in a nonpolar water-shielded environment in the peptide-lipid complex. We conclude that these class A amphipathic peptides lie parallel to the lipid surface and penetrate no deeper than the ester linkages of the phospholipids. Received: 8 April 1998 / Revised version: 6 July 1998 / Accepted: 7 August 1998     

Lipid-protein surface films generated from membrane vesicles: selfassembly,composition, and film structure

Lipid-protein films at the air-water interface were generated from a variety of native vesicles and from vesicles derived from lipid extracts. A technique is described which is particularly suitable for the generation of films from small amounts of material at high yield and velocity. In all instances, 10 l vesicle suspensions containing 25 g protein yield at least 50 cm² film area at a constant surface pressure of 12 mN/m within minutes. Upon formation, surface films are separated from vesicles by use of shear forces. Complete separation is demonstrated by electron microscopy and surface pressure-area diagrams. The latter confirms previous conclusions that surface films generated from lipid vesicles are organized as a monolayer. Analysis of lipid-protein surface layers reveals that their lipid to protein ratios match those of the vesicles used, within a factor of two, irrespective of whether films are generated at high or low surface pressure. Surface denaturation of membrane proteins is shown to be effectively prevented when the film is generated and held at high surface pressure ( 15 mN/m). Upon surface pressure jumps from high to low values, denaturation kinetics revealed activation areas of 1.5 (±0.2) nm². Offprint requests to: H. Schindler     

Glycerol conformation and molecular packing of membrane lipids: The crystal structure of 2,3-dilauroyl-d-glycerol

The single-crystal structure of 2,3-dilauroyl-d-glycerol has been determined by Patterson rotation and translation methods and refined to R = 0.069. 2,3-dilauroyl-d-glycerol crystallizes in the monoclinic space group P2₁, with unit cell dimensions: $\text{a = 5.46}\text{A}\text{?}\text{, b = 7.59}\text{A}\text{?}\text{, c = 34.2}\text{A}\text{?}\text{and}\text{β = 93.1 °}$, and with two molecules per unit cell. The molecules have their hydrocarbon chains aligned parallel, and are arranged in a bilayer structure. The chain stacking is achieved by a bend in the fatty acid. The hydrocarbon chains pack according to the orthorhombic perpendicular chain packing mode, and are tilted 26.5 ° from the layer normal.The structural features of 2,3-dilauroyl-d-glycerol have been analysed with reference to the corresponding hydrophobic moieties in the crystal structures of different membrane lipids. The glycerol group in 2,3-dilauroyl-d-glycerol is oriented parallel to the layer plane, but changes to an approximately layer-perpendicular orientation when a polar group is attached. The molecular conformation of the glycerol-dicarboxylic ester group, however, is identical in both the absence and presence of a head group, indicating extensive conformational restrictions for this group due to both intrinsic properties and chain stacking. The gathered data provide detailed information on the structural properties of the hydrophobic moiety of membrane lipids.     

The effect of headgroup class on the conformation of membrane lipids in Acholeplasma Laidlawii: A 2H-NMR study

[2-²H₂]Oleic, [2-²H₂]palmitic, [2-²H₂]dihydrosterculic and [3-²H₂]oleic acids were biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B. ²H-NMR spectroscopy and spectral ‘de-Parking” (M. Bloom, J.H. Davis and M.I. Valic, Can. J. Phys., 58 (1980) 1510) were used to study the effect of lipid headgroup class on the conformational order in the vicinity of the C-2 position of the acyl chains of lipids in the liquid crystalline phase. The results indicate that although the orientation and conformations of the membrane lipids in the region of the C-2 position of the chains are qualitatively very similar among the various lipid classes, quantitatively there are some differences, particularly between the glycolipids and the phospholipids. These differences do not exted to the C-3 position. Unlike the headgroup class, the membrane proteins appear to have little if any effect on the molecular ordering of the lipids.