Polar group interaction and molecular packing of membrane lipids: The crystal structure of lysophosphatidylethanolamine

The conformation and molecular packing of 3-palmitoyl-dl-glycerol-1-phosphoryl-ethanolamine has been determined by a single crystal analysis (R = 0.115); it crystallizes in the monoclinic space group $\text{P2}{}_1\text{a}$ with a unit cell of $\text{a = 7.66}\text{A}\text{?}\text{, b = 9.08}\text{A}\text{?}\text{, c = 37.08}\text{A}\text{?}\text{and}\text{β = 90.2 °}$, with four molecules per unit cell. The molecules exist as configurational and conformational enantiomers and pack in a bilayer arrangement. The phosphorylethanolamine groups have an orientation parallel to the layer surface. The hydrocarbon chains are arranged according to the T∥ chain packing mode and adopt an extreme tilt of 57.5 ° with respect to the layer normal. The free glycerol hydroxyl group forms an intramolecular hydrogen bond with, a phosphate oxygen and thus affects the conformation and orientation of the head group. The phosphorylethanolamine dipoles are oriented parallel to each other in double rows, while they are antiparallel and form a continuous network in dilauroylphosphatidylethanolamine (Elder et al., 1977). The area per molecule in 3-palmitoyl-dl-glycerol-1-phosphorylethanolamine (34.8 Ų) is less than in diacylphosphatidylethanolamine (38.6 Ų), indicating that in the latter the hydrocarbon chains determine the molecular cross-section. The significance of the interaction and space requirement of the phosphorylethanolamine group for the phase behaviour of phosphatidylethanolamine is discussed.     

Interactions and space requirement of the phosphate head group of membrane lipids: The single crystal structures of a triclinic and a monoclinic form of hexadecyl-2-deoxyglycerophosphoric acid monohydrate

The crystal structures of a triclinic form (HPA1) and a monoclinic form (HPA2) of hexadecyl-2-deoxyglycerophosphoric acid monohydrate were determined by single crystal analysis. The unit cell dimensions for HPA1 are $\text{a = 4.75, b = 5.72, c = 44.36}\text{A}\text{?}$ and α = 91.0, β = 101.5, γ = 100.5° (P$\text{1}$) and for HPA2, $\text{a = 4.75, b = 5.72, c = 88.72}\text{A}\text{?}$ and γ = 100.8° (P2₁). In both structures the molecules are fully extended and pack tail-to-tail in bilayers with tilting (47°) hydrocarbon chains. In HPA2, however, the chain tilt alternatingly changes direction in adjacent bilayers, giving rise to a doubled unit cell which spans two bilayers. The dihydrogen phosphate groups interact by hydrogen bonds and are arranged in rows. Laterally between these phosphate rows the water molecules are accommodated producing a compact two-dimensional network of hydrogen bonds. The packing cross-section in the layer plane of the dihydrogen phosphate monohydrate group is 26.7 Ų in both structures. The hydrocarbon chains pack according to the triclinic (T|) chain packing mode. In HPA2, however, the chain packing is somewhat less compact with accounts for a 2% increase in the molecular volume. In both structures the ether oxygen is accommodated into the hydrocarbon matrix without distortion of the chain packing.     

Conformation and packing properties of phosphatidic acid: The crystal structure of monosodium dimyristoylphosphatidate

The conformation and molecular packing of monosodium 1,2-dimyristoyl-sn-glycerophosphate (DMPA) has been determined by single crystal analysis (R = 0.107). The lipid crystallizes in the space group P2₁ with unit cell dimensions: $\text{a = 5.44, b = 7.95, c = 43.98}\text{A}\text{?}$ and β = 114.2°. The two molecules of the unit cell are related by a two-fold screw axis and pack tail-to-tail in a bilayer structure. The monosodium phosphate group packs with rather a small cross-section (24 Ų) relative to the two hydrocarbon chains. This unbalance in packing cross-section is overcome by an interdigitation of the phosphate head groups of adjacent bilayers and the formation of a single, common phosphate group layer at the bilayer interfaces. The phosphate groups are linked by hydrogen bonds to linear strands which laterally are separated by strands of sodium ions. The conformation of the molecules differs from that of other phospholipids. The glycerol chain is oriented parallel (instead of perpendicular) to the layer surface and the parallel stacking of the hydrocarbon chains is achieved by a bend of the γ-chain (instead of the β-chain). Otherwise the conformation of the glycerol dicarboxyl ester group displays the same preferred features as generally found in glycerophospholipids. The hydrocarbon chains pack according to the triclinic (T_∥) packing mode. The interaction and packing principles of the phosphate head group are discussed in relation to the structural behaviour of phosphatidic acid.     

On the stereochemistry of 2,3-dihydroxy fatty acids. The single crystal structure of potassium d-erythro-2,3-dihydroxyoctadecanoate

The erythro-2,3-dihydroxyoctadecanoic acid studied is a synthetic homologue of a natural occurring constituent of sphingolipids. The potassium salt of the acid crystallizes in the monoclinic space group P2, with the unit cell dimension: $\text{a = 5.39, b = 7.06, c = 26.26}\text{A}\text{?}\text{and}\text{β = 94.9°}$. The crystal structure was solved by direct methods and was refined to R = 0.062. The absolute configuration of the compound was determined by means of anomalous scattering effects, showing that the natural fatty acid has d-erythro configuration. The compound packs tail to tail in an unusual bilayer arrangement. The hydrocarbon chains have an extreme tilt of 60° and opposite inclination in the two halves of the bilayer. Laterally the hydrocarbon chains are arranged according to the monoclinic M∥ packing mode. The carbon chain makes a perpendicular bent at carbon atom 2. This places the 2-hydroxyl group in a preferred co-planar conformation towards the carboxylate group and at hydrogen bond distance to one of the carboxylate oxygens. The carboxylate group and the two hydroxyl groups are co-ordinated to K⁺ ions and together account for a large molecular cross-ection of 38 Ų. Monolayer studies show that the acid forms a phase with this spacious molecular area also in contact with water. On compression above 10 mN m^?1 transition to a more condensed state ($\text{S = 27}\text{A}\text{?}{}^2$) takes place accompanied by marked changes of the surface potential.     

X-ray diffraction analysis of dehydrated myelin

Improved X-ray diffraction data from dry nerve myelin are presented. In addition to the spacings of approx. 150 Å, 60 Å, 44 Å and 34.6 Å, which have been previously reported, we identify a 14 Å series. The data suggests that the hydrocarbon chains in the single bilayer ($\text{≈ 60}\text{A}\text{?}$) is ordered, whereas in the double bilayer ($\text{≈ 150}\text{A}\text{?}$) and in the fluid phase ($\text{≈ 44}\text{A}\text{?}$) it is disordered. It is shown that cholesterol ($\text{≈34.6}\text{A}\text{?}$) exists as a bilayer, and the 14 Å series is probably another cholesterol phase.     

Water structure in a protein crystal: rubredoxin at 1.2 A resolution

The model for rubredoxin based on X-ray diffraction data has been extensively refined with a 1.2 Å resolution data set. Water oxygen atoms were deleted from the model if B exceeded 50 Ų and occupancy was less than 0.3 eÅ^?3. The final water model consists of 127 sites with B values ranging from 15 to $\text{6}\text{?}$0 Ų and occupancies from unity down to 0.3, the most tightly bound water oxygen atoms being hydrogen bonded to two or more main-chain nitrogen or oxygen atoms. The water forms extensive hydrogen bond networks bridging the crevices on the molecular surfaces and between adjacent molecules. The minimum distances of the water sites from the protein surface are distributed about two distinct maxima, the major one at 2.5 to 3 Å and a minor one at 4 to 4.5 Å. Beyond $\text{5}\text{?}$ to 6 Å from the protein surface, the discrete water merges into the aqueous continuum.     

A preliminary crystallographic study of isocitrate dehydrogenase from Azotobacter vinelandii

Large single crystals of isocitrate dehydrogenase from Azotobacter vinelandii have been grown by vapor diffusion from ammonium sulfate and phosphate solutions. The crystals are tetragonal, space group P4₂2₁2 with cell dimensions $\text{a = 122.1}\text{A}\text{?}$, $\text{c = 163.9}\text{a}\text{?}$. There are two molecules of 80,000 molecular weight per asymmetric unit. Native data to 5.5 Å resolution have been collected on a diffractometer. A rotation function using data between 10 Å and 6 Å resolution indicates three possible orientations of the non-crystallographic 2-fold axis relating the two molecules.     

RNA double-helical fragments at atomic resolution: II. The crystal structure of sodium guanylyl-3′,5′-cytidine nonahydrate

The crystal structure of sodium guanylyl-3′,5′-cytidine (GpC) nonahydrate has been determined by X-ray diffraction procedures and refined to an R value of 0.054. GpC crystallizes with four molecules per monoclinic unit cell, space group C2, with cell dimensions: $\text{a = 21.460, b = 16.297, c = 9.332}\text{A}\text{?}\text{and}\text{β = 90.54 °}$. Two molecules of GpC related by the 2-fold axis of the crystal form a small segment of right-handed, anti-parallel double-helical RNA in the crystal. Guanine is paired to cytosine through three hydrogen bonds of lengths 2.91, 2.95 and 2.86 Å. The bases along each strand are heavily stacked at a distance of about 3.4 Å. The fragments form skewed flattened rods within the lattice by the inter-molecular stacking of guanines with each other and the stacking of cytosine with the guanosine Ol′atom. The sodium cations are bound only to the ionized phosphate groups in this structure and exhibit face-sharing octahedral co-ordination. The sodium cations serve to bridge the rods of GpC fragments and organize them into sheets within the crystal. There are 18 water molecules per double-helical fragment which are all part of the first co-ordination shell of nitrogen, oxygen or sodium atoms.     

The crystal and molecular structure of cytosine-glycyl-glycine-copper(II) complex,a biologically important ternary coordination complex

The title compound was prepared and studied to gain some insight into the structural basis for the protein-nucleic acid-metal ion interaction. The crystal structure has been determined from three-dimensional diffractometer X-ray data using Cu Kα radiation. The crystals are monoclinic, space group $\text{P2}{}_1\text{c}$, with cell dimension; a=10.642(1)Å, b=8.081(1)Å, c=17.792(1)Å, β=124.29(1)^o, z=4. Amino and amide nitrogen, carboxyl O(8) of glycylglycine, N(3) of cytosine and O(2) of adjacent cytosine molecule coordinate to the central copper ion to form a square pyramid. An additional weak interaction in complex molecule between copper and O(2) of cytosine is also observed. The complex molecules are held together by hydrogen- and coordination-bonds in crystalline state.     

Quantitative analysis of the binding of melittin to planar lipid bilayers allowing for the discrete-charge effect

The interaction of melittin with lecithin bilayers was studied using the resulting surface potentials at the bilayer/water interfaces to monitor the association. Melittin added to the aqueous phase binds strongly to the interface but remains localized on that side of the bilayer to which it is added. The analysis of the binding curves reveals the inadequacy of the Gouy-Chapman theory for the fixed-charge surface potential in describing the electrostatic potential experienced by the adsorbed molecules. Calculations based on the Stern equation, modified for a discrete charge distribution, give a good fit to the experimental data. The thermodynamic analysis revealed different binding energies, Δ$\text{G}$°, at 10 and 100 mM ionic strength (?7.85 and ?8.26 kcal/mol, respectively). Binding saturates at an area of 650 Ų per melittin molecule. A change in the surface dipole potential corresponding to ?1.1 debye/?_a ($\text{?}{}_{\mathrm{a}}\text{= dielectric constant of the adsorption region}$) had to be postulated. The Debye-Hückel length for a charge bound to the membrane/solution interface was found to be about one-third smaller than in bulk solution.     

Deuterium magnetic resonance of selectively deuterated cholesteryl esters in dipalmitoyl phosphatidylcholine dispersions

Dispersions (50 wt% water) containing 95 mol% dipalmitoyl phosphatidylcholine/5 mol% deuterated cholesteryl palmitate (or stearate) were studied using ²H-NMR. Incorporation of ester into the phospholipid bilayer was found to be 0.5 mol% at 50°C. From the profile of ²H quadrupolar splitting vs. chain position, support for an average conformation resembling a ‘horseshoe’ within the bilayer is obtained. Quadrupolar relaxation times $\text{T}{}_{2e}$ of approx. 250 μs and approx. 850 μs are measured for cholesteryl palmitate-2,2-d₂ and cholesteryl palmitate-16,16,16-d₃, respectively, which are less than one-half those obtained for the corresponding positions in dipalmitoyl-d₆₂ phosphatidylcholine. This is ascribed to a slower rate of motion of the ester chain and/or an extra, slow motion of the molecule.     

A need for re-examination of the fibre diffraction data of A-DNA

A-DNA pattern, obtained using a flat plat camera, was indexed by Fuller $\text{et}\text{al}{}^6$ on the basis of a c-face centred monoclinic cell with a = 22.24 Å, b = 40.62 Å, c = 28.15 Å and β = 97.0°. A precession photograph of A-DNA which gives an undistorted picture of the lattice, showed that the unit cell parameters as given by Fuller $\text{et}\text{al}$ were not quite correct. The precession photograph showed a strong meridional reflection (R = 0.00 Å^?1) on the 11th layer line. But the occurrence of the meridional reflection on the 11th layer line could not be explained on the basis of the cell parameters given by Fuller $\text{et}\text{al}{}^6$; using those cell parameters the reflection which comes closest to the meridian on 11th layer line is at R = 0.025 Å^?1. However, a simple interchange of a and b values accounted for the meridional reflection on 11th layer line. The corrected cell parameter refined against 28 strong spots are a = 40.75 Å, b = 22.07 Å, c = 28.16 Å and β = 97.5°. In the new unit cell of A-DNA, the packing arrangement of the two molecules is different from that in the old one. Nonetheless, our earlier contention is again reaffirmed that both right and left-handed A-DNA are stereochemically allowed and consistent with the observed fibre pattern.     

Time resolved spectroscopy of the tryptophyl fluorescence of the E. coli LAC repressor.

A new crystal form of a mitogenic lectin from pea seeds ($\text{Pisum sativum}$) has been obtained which is suitable for high resolution structural work. The crystals are orthorhombic, space group P2₁2₁2₁, with unit cell dimensions: $\text{a}$ = 64.2Å, $\text{b}$ = 72. 7Å, $\text{c}$ = 108. 3Å. The asymmetric unit contains one protein molecule.     

ESR studies on the orientation of cholesteryl ester in phosphatidylcholine multilayers

The alignment of cholesteryl esters in multilayer phosphatidylcholine membranes was investigated using two spin-labelled cholesteryl esters: 10 : 3 ester ($\text{I}$) and 1 : 14 ester ($\text{II}$). The nitroxide label of $\text{I}$ is aligned in the membrane with a very large angle of tilt (47° ± 1.5°) with respect to the normal to the membrane surface; $\text{II}$ does not show such a tilt. $\text{I}$ gives spectra corresponding to immobilized label while $\text{II}$ gives nearly isotropic spectra. Ascorbate treatment of the multilayers shows that the labels in $\text{I}$ and $\text{II}$ are not present at the phosphatidylcholine-water interphase.The data supports a ‘horseshoe’ configuration for the cholesteryl ester in the bilayer, with both the fatty acid chain and the cholesteryl moiety extending deep into the hydrophobic region of the membrane and with the ester linkage near the surface.     

Small-angle x-ray scattering study of lysine transfer RNA ligase from yeast

The scattered X-ray intensities from dilute solutions of lysine transfer RNA ligase, in 0.1 m-phosphate buffer at pH 7.0, have been measured at 21 °. The radius of gyration R (37.5 Å), the molecular weight M (114,000), and the volume V (295,000 ų) were determined.A comparison between the scattering curves obtained from the enzyme and the theoretical scattering curves of different triaxial bodies shows that the shape of the molecule can be represented by an oblate ellipsoid with the semiaxes A = 62.7, B = 50.1 and $\text{C = 23.5}\text{A}\text{?}$.     

Crystallographic study of plastocyanins

Crystals of plastocyanins from pea and corn leaves have been obtained. Both are suitable for X-ray structure analysis with a resolution up to 1.8 Å. The crystal form of plastocyanin from pea leaves belongs to the space group P2₁2₁2₁ with unit cell dimensions: $\text{a = 49.0}\text{A}\text{?}\text{, b = 53.3}\text{A}\text{?}\text{, c = 82.6}\text{A}\text{?}$. The assumed number of protein molecules per asymmetric unit of the unit cell is two. Crystals of the oxidized (Cu²⁺) and reduced (Cu⁺) forms are isomorphic. No essential differences in spot intensities for the main zone with a resolution of 3 Å were revealed. The crystal form of plastocyanin from corn leaves belongs to the space group P1 with unit cell parameters: $\text{a = 24.8}\text{A}\text{?}\text{, b = 30.0}\text{A}\text{?}\text{, c = 58.5}\text{A}\text{?}$ and α = 96° 10′, β = 87°08′, γ = 78°40′. The assumed number of protein molecules per asymmetric unit is two.     

Molecular arrangements in sphingolipids. The crystal structure of cerebroside

The single crystal analysis of a complex, membrane glycolipid is described. Cerebroside (β-D-galactosyl-N-(2-D-hydroxyoctadecanoyl)-D-dihydrosphingosine, $\text{C}{}_{42}\text{H}{}_{32}\text{O}{}_9\text{N}\text{·}\text{1}\text{2}\text{C}{}_2\text{H}{}_5\text{OH}$) — an important constituent of plasma membranes — crystallizes in the monoclinic spacegroup P2, with a = 11.202, b = 9.262, c = 46.46 A and β = 99.00^°. There are two independent molecules in the asymmetric unit partly related by a non-crystallographic symmetry. The structure was determined by direct methods and refined to R = 0.116.The molecules pack in a typical bilayer arrangement with adjacent double layers separated by ethanol molecules of crystallization. The planes of the sugar rings are turned almost parallel to the layer interface which gives the molecules the shape of a shovel. Together with the polar ceramide part, the galactose head groups form an extensive lateral network of hydrogen bonds within the polar region of each layer. The chains tilt by an angle of 49^° towards this polar boundary. A parallel stacking of the chains is achieved by a bend of the sphingosine chain as far up as carbon atom 5 and 6 in the two independent molecules. The lateral hydrocarbon chain packing is of an earlier unknown hybrid type (HS2). Chains with parallel zigzag planes are arranged in pleated shoets. These sheets contain alternatively fatty acid and sphingosine chains which have a mutually perpendicular chain plane orientation.     

Crystallisation of tRNALeuCUG from Escherichia coli after purification with hydroxyapatite.

tRNA_CUG^Leu from E. Coli was purified by column chromatography on benzoylated DEAE-cellulose, followed by hydroxyapatite prepared by an improved method. Crystals obtained by vapour diffusion gave X-ray diffraction out to 7 Å in the hk0 projection and 10 Å in h0?. The space group was P4₂2₁2 with $\text{a = b = 133}\text{A}\text{?}$, $\text{c = 66}\text{A}\text{?}$ and 8 molecules in the unit cell. Birefringence showed preferred orientation of RNA helical regions in the ab plane.     

Identification of a structural hairpin in the filamentous chimeric phage M13Gori1

Crystals of alkaline phosphatase (EC 3.1.3.1; M_r 94,000) grown at pH 9.5 from 2.25 m-(NH₄)₂SO₄ with 5 × 10^?5 m-Zn and 10^?2 m-Mg present were analyzed by X-ray diffraction at pH 7.5 in 2.66 m-(NH₄)₂SO₄ with 10^?2 m-Zn and 10^?2 m-Mg present. The crystals are orthorhombic with $\text{a = 195.5}\text{A}\text{?}\text{, b = 168.3}\text{A}\text{?}\text{and}\text{c = 76.33}\text{A}\text{?}$, and the space group is I222. X-ray phases were determined by the multiple isomorphous replacement and anomalous dispersion method using K₂PtCl₄, KAu(CN)₂ and K₂OsO₄ derivatives. The electron density maps and analysis of metal binding sites reveal one molecule per asymmetric unit with an internal, non-crystallographic, 2-fold rotation axis relating the subunits. Each subunit contains a major $\text{α}\text{β}$ domain with a seven-stranded β-sheet flanked by helices. The sheets are roughly coplanar but the general direction of the strands in each is at 20 ° to the rotation axis and thus 40 ° from each other. The helical content of the $\text{α}\text{β}$ domain is approximately 27% of the 459 residues in the monomer and the β content is approximately 7%. The chains in a smaller domain are more convoluted and less easily characterized than in the $\text{α}\text{β}$ domain. In both there is extensive monomer-monomer contact.Removal of the zinc and magnesium from the parent crystal produces a stable apoenzyme crystal and addition of cobalt at 10^?2 m or cadmium at 10^?2 or 5 × 10^?2 m reveals seven metal binding sites per dimer. The active centers are 32 Å apart and each is shown by anomalous dispersion data to contain two metal binding sites, A and B. The cadmium derivative refinement determined the A-B separation to be 4.9 Å. Comparison of the parent and apo structures by means of difference maps reveals the double metal site with Zn at A and probably Mg at B. A prominent, partially resolved peak centered 7 Å away is interpreted as a stabilization of the backbone in this position by the metal ion co-ordination to a side-chain. Several negative peaks within 10 Å of the metals indicate local differences between apo and native structures but no significant differences are seen in the other parts of the molecule. At 5 × 10^?2 m-Cd two metal sites (D and D′) are found 25.5 Å from the active center, on the surface of the minor domain. They are related to each other by the molecular 2-fold axis with a D-D′ distance of 25 Å. The seventh Cd site, E, is 20 Å from the active center, on the major domain, near a crystalline contact region, and devoid of any molecular symmetry mate.The apparent dissociation constants for cadmium at the A, B and D sites (and A′, B′, D′) are 3 × 10^?3 m, 1.5 × 10^?1 m and 1.3 × 10^?2 m, respectively. Thus in these conditions cadmium is seen to distribute between A and B sites when the combined stoichiometry is two metal ions per dimer.     

Crystallographic studies on concanavalin B

Concanavalin B has been grown as hexagonal needles and analyzed by x-ray diffraction and electron microscopy. The crystals are of space group P6₁ with $\text{a}$ = 81Å and $\text{c}$ = 101Å. There is one molecule of 30, 000 molecular weight as the asymmetric unit of the crystal. Electron micrographs demonstrate that the crystals maintain considerable order after dehydration and exhibit large intersticial solvent regions.