1H and 13C NMR investigation of the influence of nonligated residue contacts on the heme electronic structure in cyanometmyoglobin complexes reconstituted with centro- and pseudocentrosymmetric hemins.

The 1H and 13C chemical shifts for the heme methyls of low-spin, ferric sperm whale cyanometmyoglobin reconstituted with a variety of centrosymmetric and pseudocentrosymmetric hemins have been recorded and analyzed to shed light on the nature of heme-protein contacts, other than that of the axial His, that modulate the rhombic perturbation to the heme's in-plane electronic asymmetry. The very similar 1H dipolar shifts for heme pocket residues in all complexes yield essentially the same magnetic axes as in wild type, and the resultant dipolar shifts allow the direct determination of the heme methyl proton and 13C contact shifts in all complexes. It is demonstrated that, even when the magnetic axes and anisotropies are known, the intrinsic uncertainties in the orientational parameters lead to a sufficiently large uncertainty in dipolar shift that the methyl proton contact shifts are inherently significantly less reliable indicators of the unpaired electron spin distribution than the methyl 13C contact shifts. The pattern of the noninversion symmetry in 13C contact shifts in the centro- or pseudocentrosymmetric hemes is shown to correlate with the positions of aromatic rings of Phe43(CD1) and His97(FG3) parallel to, and in contact with, the heme. These results indicate that such pi-pi interactions significantly perturb the in-plane asymmetry of the heme pi spin distribution and cannot be ignored in a quantitative interpretation of the heme methyl 13C contact shifts in terms of the axial His orientation in b-type hemoproteins.

Dissociation and unfolding of cold-active alkaline phosphatase from atlantic cod in the presence of guanidinium chloride.

Cold-adaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a cold-active alkaline phosphatase (AP) from Atlantic cod, with the aim of understanding more clearly its structural stability in terms of subunit dissociation and unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size-exclusion chromatography and enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction could be made between dissociation of dimers into monomers and subsequent unfolding of monomers (fits a three-state model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a two-state model). The DeltaG for dimer dissociation of cod AP was 8.3 kcal.mol-1, and the monomer stabilization free energy was 2.2 kcal.mol-1, giving a total of 12.7 kcal.mol-1, whereas the total free energy of calf intestinal AP was 17.3 kcal.mol-1. Thus, dimer formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting dimer dissociation and stabilizing the monomers. Cod AP has reduced affinity for inorganic phosphate, the release of which is the rate-limiting step of the reaction mechanism. More flexible links at the interface between the dimer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.

Cryo-TEM and NMR studies of a micelle-forming phosphoglucolipid from membranes of Acholeplasma laidlawii A and B.

The chemical structure of a phosphoglucolipid from the membrane of the bacterium Acholeplasma laidlawii strain B-PG9 has been determined by high resolution NMR to be 1,2-diacyl-3-O-[glycerophosphoryl-6-O-(alpha-D-glucopyranosyl-(1 -->2)-O-alpha-D-glucopyranosyl)]-sn-glycerol (GPDGlcDAG). It was concluded that this lipid has exactly the same structure as one of the phosphoglucolipids from A. laidlawii strain A-EF22. By cryo transmission electron microscopy (cryo-TEM) and NMR diffusion techniques it was shown that, in highly diluted aqueous solutions, this membrane lipid forms long thread-like micelles in equilibrium with lipid vesicles. The cause of the occurrence of these different aggregates is discussed in terms of the varying molecular shapes of the lipid because of a heterogeneous composition of the acyl chains. A second membrane phosphoglucolipid from the bacterium, namely 1,2-diacyl-3-O-[glycerophosphoryl-6-O-(alpha-D- glucopyranosyl-(1 -->2)-monoacylglycerophosphoryl-6-O-alpha-D-glucopyranosyl)]-sn-gl ycerol (MABGPDGlcDAG), was found to form only a lamellar liquid crystalline phase coexisting with water.

Two-dimensional 1H-NMR of transmembrane peptides from Escherichia coli phosphatidylglycerophosphate synthase in micelles.

Two 28-residue peptides, PTLLTLFRVILIPFFVLVFYKKKGKKKG [Pgs-(6-25)-peptidyl-KKKGKKKG; Pgs peptide A] and VEYAGIALFFVAAVLTLWSMLQYLSAAR [Pgs-(149-176)-peptide, Pgs peptide E], were synthesized and studied by CD and two-dimensional 1H-NMR spectroscopy. The first 20 amino acid residues of Pgs peptide A are identical to one predicted transmembrane segment (Pro6-Tyr25) of the integral membrane protein phosphatidylglycerophosphate synthase (Pgs) of Escherichia coli. Pgs peptide E is identical to another predicted transmembrane segment (Val149-Arg176), which is located in the C-terminal end of this lipid synthase. Pgs peptides A and E were dissolved in methanol or trifluoroethanol or were incorporated into solvent-free micelles of fully deuterated SDS. In all these systems, CD spectra of both peptides indicated an alpha-helical secondary structure. However, peptides that were solubilized in micelles exhibited the highest content of alpha-helix as judged from comparison of the CD spectra. Thermodynamically stable isotropic solutions at high peptide concentrations (1-3 mM) could only be obtained with the peptide incorporated in micelles; in organic solvents, significant peptide aggregation occurred. Relatively sharp peaks were obtained with 1H-NMR spectroscopy of the peptides in SDS micelles, which indicates rapid tumbling of the peptides in the micellar environment. Translational-diffusion coefficients of the micelles with and without peptide, determined by pulsed-field-gradient NMR, showed that the micellar size was unaffected by the solubilized peptide. The radius of the hydrated micelles was estimated to be about 2.7 nm (i.e. the mass of the aggregate is almost 30 kDa). Two-dimensional NMR spectroscopy of both peptides solubilized in the micelles indicated an alpha-helical conformation. This observation is strengthened by an investigation of the hydrogen exchange of the peptide amide protons, where significantly less exchange of the amide protons was observed in the middle of the peptides compared with the ends.

Structure of digalactosyldiacylglycerol from oats.

Digalactosyldiacylglycerol (DGalDAG) is one of the major lipids in the cells of higher plants. DGalDAG forms a lamellar liquid crystalline phase together with water. Lipid aggregates can thus be prepared which are of potential interest for use within the cosmetic and pharmaceutical industries. Oats may be an important source for preparation of DGalDAG, but the structure has not been determined for the DGalDAG lipid occurring in this plant. In the present study the structure of a high-purity DGalDAG preparation, isolated from commercial oat flakes, was determined by high resolution 1H- and 13C-NMR spectroscopy. The lipid was found to have the structure 1,2-diacyl-3-O-[alpha-galactopyranosyl-(1-->6)-O-beta-galactopyranosyl]- glycerol. By using 1H- and 13C-NMR spectroscopy techniques it is thus possible to determine, among other things, the identity of the glycosyl moieties in the polar head group, to establish the linkage positions between these moieties, and to establish the anomeric configurations of the moieties, without making any chemical modifications of the lipid.

Structures of glucolipids from the membrane of Acholeplasma laidlawii strain A-EF22. III. Monoglucosyldiacylglycerol, diglucosyldiacylglycerol, and monoacyldiglucosyldiacylglycerol.

The structures of three glucolipids from the membrane of Acholeplasma laidlawii, strain A-EF22, were determined by high resolution 1H-NMR and 13C-NMR spectroscopy. The two most abundant glucolipids in this organism were shown to be 1,2-diacyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerol (MGlcDAG) and 1,2-diacyl-3-O-[alpha-D-glucopyranosyl-(1 --> 2)-O-alpha-D- glucopyranosyl]-sn-glycerol (DGlcDAG). These structures agree with those determined previously by chemical analyses of the two most abundant glucolipids synthesized by the B strain of A. laidlawii. The structure of a newly discovered glucolipid in A. laidlawii strain A-EF22 was also determined. This lipid is an acylated derivative of DGlcDAG with the structure 1,2-diacyl-3-O-[alpha-D-glucopyranosyl-(1 --> 2)-O-(6-O-acyl-alpha-D- glucopyranosyl)]-sn-glycerol. The existence of this lipid was detected by 1H-NMR spectroscopy in preparations of MGlcDAG which had been judged by thin-layer chromatography to be pure. The biosynthesis of the glucolipids and their role in the metabolic lipid regulation are briefly discussed.

Structures of glucolipids from the membrane of Acholeplasma laidlawii strain A-EF22. II. Monoacylmonoglucosyldiacylglycerol.

The structure of one glucolipid from the membrane of Acholeplasma laidlawii, strain A-EF22, was determined. This glucolipid is synthesized only when a large fraction of saturated, straight-chain fatty acids are incorporated into the membrane lipids of strain A-EF22. The lipid was studied by 1H- and 13C-NMR spectroscopy. The structure of the lipid is 1,2-diacyl-3-O-[6-O-acyl-(alpha-D-glucopyranosyl)]-sn-glycerol. The result for this lipid shows that a previously published structure, based on incomplete chemical analyses, was incorrect. The phase equilibria for 1,2-diacyl-3-O-[6-O-acyl-(alpha-D-glucopyranosyl)]- sn-glycerol and the two dominating lipids in A. laidlawii, monoglucosyldiacylglycerol and diglucosyldiacylglycerol, are discussed and related to the chemical structure of the lipids.

Structures of glucolipids from the membrane of Acholeplasma laidlawii strain A-EF22. I. Glycerophosphoryldiglucosyldiacylglycerol and monoacylbisglycerophosphoryldiglucosyldiacylglycerol.

The structures of two phosphoglucolipids from the membrane of Acholeplasma laidlawii, strain A-EF22 were determined by high resolution 13C-, 31P- and 1H-NMR. The lipids in question are 1,2-diacyl-3-O-[glycerophosphoryl-6-O-(alpha-D-glucopyranosyl- (1-->2)-O-alpha-D-glucopyranosyl)]-sn-glycerol (1) and 1,2-diacyl-3-O-[glycerophosphoryl-6-O-(alpha-D-glucopyranosyl-(1-- >2)- monoacyl-glycerophosphoryl-6-O-alpha-D-glucopyranosyl)]-sn-glycero l (2). Both lipids are thus derivatives of diglucosyldiacylglycerol. Previous reports on these lipids, based on insufficient chemical analyses, showed contradictory structures. A phosphoglycolipid having the structure of 2 has not been described previously.

Regulation and phase equilibria of membrane lipids from Bacillus megaterium and Acholeplasma laidlawii strain A containing methyl-branched acyl chains.

Phosphatidylethanolamine (PE) was isolated from Bacillus megaterium grown at 20 and 55 degrees C (PE-20 and PE-55). Iso and anteiso methyl-branched, saturated acyl chains are predominant in B. megaterium, and the value of the molar ratio of iso/anteiso acyl chains is more than 20-fold higher in PE-55 than in PE-20. Moreover, about 21 mol% of the acyl chains of PE-20 are monounsaturated. The phase equilibria differ between the two PE preparations: (1) PE-20 is more prone to form reversed nonlamellar phases than PE-55; (2) PE-20 forms both reversed cubic (I2) and reversed hexagonal (H(II)) phases while PE-55 forms only an HII phase; and (3) the lamellar liquid-crystalline (L alpha) phase of PE-20 takes up about 70% more water than the L alpha phase of PE-55. These differences can be explained by the differences in the acyl chain composition. When the growth temperature is raised, PE molecules with a reduced tendency to form nonlamellar phases are probably synthesized by B. megaterium in order to counteract the bilayer destabilizing effect of the temperature. The regulation of the acyl chain composition is not needed in order to regulate the temperature for the transition between gel/crystalline and L alpha phases of the membrane lipids. Acholeplasma laidlawii strain A-EF22 was grown at 37 degrees C on 15-(1,1,1(-2) H3)methylhexadecanoic acid, 14-(1,1,1(-2)H3)methylhexadecanoic acid or 13-(1,1,1(-2)H3)methylhexadecanoic acid, and these acids constituted 84-89 mol% of the acyl chains in the membrane lipids. The molar ratio between the two dominating lipids, monoglucosyldiacylglycerol (MGLcDAG) and diglucosyldiacylglycerol (DGlcDAG), decreased, and the molar fraction of the anionic lipids increased, when the methyl branch was moved from position 15 to position 13. Concomitantly, the order of the methyl branch increased in cells as well as in total lipid extracts. The phase equilibria of total lipid extracts (neutral lipids removed) were studied with 20 wt % of water, and HII and I2 phases were formed above 63-67 degrees C. These results indicate that the regulation of the polar head-group composition compensates for the difference in acyl chain packing introduced into the bilayer by the three branched-chain fatty acids. The regulation of the polar head-group composition of the A. laidlawii lipids cannot regulate the temperature for the transition between gel/crystalline and L alpha phases of the lipids, i.e. the transition to fluid acyl chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane lipid regulation in Acholeplasma laidlawii grown with saturated fatty acids. Biosynthesis of a triacylglucolipid forming reversed micelles.

The membrane lipid composition in several strains of Acholeplasma laidlawii is regulated upon a change in the growth conditions. Monoglucosyldiacylglycerol (MGlcDAG) and diglucosyldiacylglycerol (DGlcDAG) are the most abundant lipids in the A. laidlawii membrane. A third glucolipid, 3-O-acyl-monoglucosyldiacylglycerol (MAMGlcDAG) is synthesized by strain A-EF22 when the membrane lipids contain large amounts of saturated acyl chains. The lipid regulation can be understood from a simple theoretical model, in which the cells strive to maintain a balance between the lipids constituting a bilayer and those forming reversed non-lamellar liquid crystalline phases. Thus, the physical chemistry of membrane lipids, in particular their ability to form different aggregate structures, constitutes the basis for the lipid regulation, and therefore an understanding of the phase equilibria of membrane lipids is crucial. MGlcDAG and MAMGlcDAG isolated from A. laidlawii strain A-EF22 membranes were studied mainly by 2H NMR, 1H NMR, and 1H NMR diffusion measurements. MAMGlcDAG, containing 96 mol % saturated acyl chains formed a gel/crystalline phase up to about 80 degrees C, where a transition occurred to a reversed micellar (L2) phase. This is an unexpected finding for a membrane lipid. However, this lipid homogeneously mixes with the other membrane lipids at physiological temperatures. Previous and new data on MGlcDAG show that the lamellar phase is stabilized when the length and the degree of unsaturation of the acyl chains are decreased. The physicochemical properties of MAMGlcDAG and MGlcDAG were compared and found to be of great significance for the physiological regulation of the lipids in the membrane. MAMGlcDAG is synthesized under conditions when the phase equilibria of MGlcDAG are shifted from a non-lamellar toward a lamellar phase. Apart from MAMGlcDAG, MGlcDAG is the major lipid in A. laidlawii strain A-EF22 which is able to form reversed aggregate structures. MAMGlcDAG probably assists MGlcDAG in maintaining an optimal molecular packing, or negative curvature, of the lipids in the membrane.

Effect of head-group structure and counterion condensation on phase equilibria in anionic phospholipid-water systems studied by 2H, 23Na, and 31P NMR and X-ray diffraction.

The phase equilibria, hydration, and sodium counterion association for the systems DOPA-2H2O, DOPS-2H2O, DOPG-2H2O, and DPG-2H2O were investigated with 2H, 23Na, and 31P NMR and X-ray diffraction. The following one-phase regions were found in the DOPA-water system: a reversed hexagonal liquid-crystalline (HII) phase up to about 35 wt % water and a lamellar liquid-crystalline (L alpha) phase between about 55 and 98 wt % water. The area per DOPA molecule was 36-65 A2 in the HII phase (10-40 wt % water) and 69 A2 in the L alpha phase (60 wt % water). DOPS and DOPG with 10-98 wt % water, and DPG with 20-95 wt % water formed an L alpha phase at temperatures between 25 and 55 degrees C. At temperatures above 55 degrees C, DPG with 20 and 30 wt % water formed a mixture of L alpha, HII, and cubic liquid-crystalline phases, the mole percent of lipid forming nonlamellar phases being smaller at 30 wt % water than at 20 wt % water. DPG with 10 wt % water probably formed a mixture of an L alpha phase and at least one nonlamellar liquid-crystalline phase at 25 and 35 degrees C, and a pure HII phase at 45 degrees C and higher temperatures. At water concentrations above about 50 wt % the 23Na quadrupole splitting was constant for all four lipid-water systems studied, implying that the counterion association to the charged lipid aggregates did not change upon dilution. These experimental observations can be described with an ion condensation model but not with a simple equilibrium model. The fraction of counterions located close to the lipid-water interface was calculated to be greater than 95%. The 2H and 23Na NMR quadrupole splittings of 2H2O and sodium counterions, respectively, indicate that the molecular order in the polar head-group region decreases for the L alpha phase in the order DOPA approximately DPG greater than DOPS greater than DOPG.

1H-NMR study of the mechanism of assembly and equilibrium heme orientation of sperm whale myoglobin reconstituted with protohemin type-isomers.

The products of the incorporation of various protohemin type-isomers into the heme pocket of sperm whale myoglobin were investigated by 1H-NMR in the met-cyano complexes, both immediately after reconstitution as well as at equilibrium. The type-isomers studied include those involving all possible interchanges of the two substituents on a given pyrrole. The protohemin-III and -XIII isomers, with true 2-fold symmetry, yielded only homogeneous products. Protohemins-XI, -XIV both exhibited two species after reconstitution, with one disappearing with time. Protohemin-I was the only asymmetric hemin that failed to exhibit two isomers initially. The orientation of the hemin within the pocket was established by nuclear Overhauser detected dipolar connectivities among heme substituents and between heme substituents and assigned heme pocket residues. At equilibrium, the heme orientations were dominated by the asymmetric propionate rather than vinyl dispositions on the hemin, with a clear preference for placing a propionate at the 8- vs. 5-methyl position of native myoglobin. For protohemin-XI, the propionates were found in the unexpected positions of the 7-propionate and 2-vinyl groups of native myoglobin, indicating that propionates can occupy positions well within the hydrophobic interior. The alternate heme orientation for the metastable intermediates detected for protohemin-XI and -XIV involved rotational isomerism about the alpha,gamma-meso axes bisecting the vinyl positions, but these two axes are at right angles to each other in the protein matrix. The fact that protohemin-XIV, but not protohemin-I, exhibits a reversed orientation as a reconstitution intermediate provides direct evidence that vinyl contacts, as well as propionate links, modulate the relative stabilities of the initial encounter complexes between hemin and apomyoglobin. The heme cavity molecular/electronic structure was found largely unperturbed for the complexes of the various protohemin type-isomers.