Analytic calculation of physiological acid-base parameters in plasma.

Analytic expressions for plasma total titratable base, base excess (DeltaCB), strong-ion difference, change in strong-ion difference (DeltaSID), change in Van Slyke standard bicarbonate (DeltaVSSB), anion gap, and change in anion gap are derived as a function of pH, total buffer ion concentration, and conditional molar equilibrium constants. The behavior of these various parameters under respiratory and metabolic acid-base disturbances for constant and variable buffer ion concentrations is considered. For constant noncarbonate buffer concentrations, DeltaSID = DeltaCB = DeltaVSSB, whereas these equalities no longer hold under changes in noncarbonate buffer concentration. The equivalence is restored if the reference state is changed to include the new buffer concentrations.

Occurrence, solution structure and stability of DNA hairpins stabilized by a GA/CG helix unit.

The occurrence and NMR solution structure of a class of biloop hairpins containing the sequence 5'-CGXYAG are presented. These hairpins, which are variations on a sequence found in the reverse transcript of the human T-cell leukemia virus 2 (HLV2), show elevated melting points and high chemical stability toward denaturation by urea. Hairpins with the 5'-CGXYAG configuration have melting points 18-20 degrees higher than hairpins with 5'-CAXYGG or 5'-GGXYAC configurations. The identities of the looping bases, X and Y above, play a negligible role in determining the stability of this DNA hairpin stability. This is very different from G-A based loops in RNA, where the third base must be a purine for high stability [the GNRA loops; V.P. Antao, S.Y. Lai and I. Tinoco, Jr (1991) Nucleic Acids Res., 19, 5901-5905]. We show that these properties are associated with a four base helix unit that contains both a sheared GA base pair and a Watson-Crick CG base pair upon which it is stacked. As an understanding of the significance of AG base pairs has become increasingly important in the structural biology of nucleic acids, we compute an 0.7-0.9 A precision ensemble of NMR solution structures using iterative relaxation matrix methods. Calculations performed on NMR-derived structures indicate that neither base-base electrostatic interactions, nor base-solvent dispersive interactions, are significant factors in determining the observed differences in hairpin stability. Thus the stability of the 5'-CGXYAG configuration would appear to derive from favorable base-base London/van der Waals interactions.

Overproduction and one-step purification of Escherichia coli 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase and oxygen transfer studies during catalysis using isotopic-shifted heteronuclear NMR.

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase catalyzes the condensation of D-arabinose 5-phosphate with phosphoenolpyruvate to give the unique 8-carbon acidic sugar 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) found only in Gram-negative bacteria and required for lipid A maturation and cellular growth. The Escherichia coli gene kdsA that encodes KDO 8-P synthase has been amplified by polymerase chain reaction methodologies and subcloned into the expression vector, pT7-7. A simple one-step purification yields 200 mg of homogeneous KDO 8-P synthase per liter of cell culture. [2-13C,18O]Phosphoenolpyruvate (PEP) was prepared by first, exchange of [2-13C]-3-bromopyruvate with 2H2 18O followed by reaction of the labeled bromopyruvate with trimethylphosphite. The fate of the enolic oxygen in this multilabeled PEP, during the course of the KDO 8-P synthase-catalyzed reaction with D-arabinose 5-phosphate, was monitored by 13C and 31P NMR spectroscopy. The inorganic phosphate formed during the reaction was further analyzed via mass spectral analysis of its trimethyl ester derivative. The 13C NMR spectrum of an incubation mixture of [2-13C]PEP and D-arabinose 5-phosphate in 2H2 18O in the presence of KDO 8-P synthase was also recorded. [2-13C]KDO 8-P was utilized to determine the extent of nonenzymatic incorporation of 18O into the C-2 position of KDO 8-P. The results indicate that the enolic oxygen of the PEP is recovered with the inorganic phosphate, and the C-2 oxygen of KDO 8-P originates from the solvent, H2O.

Structural characterization of the asparagine-linked oligosaccharides from Trypanosoma brucei type II and type III variant surface glycoproteins.

The complete primary structures of the major Asn-linked oligosaccharides from the type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the type III VSG, MITat 1.5, were determined using a combination of exo- and endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR spectroscopy. Each variant contained classical branched oligomannose-type and biantennary complex oligosaccharides, a proportion of the latter substituted with terminal alpha(1-3)-linked galactose residues, the first report of the presence of this epitope in Trypanosoma brucei. In addition both the type II variants contained relatively large amounts of the unusual small oligomannose-type oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to those from mammalian glycoproteins. These latter structures were also partially substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in the type II variants showed site specificity in that the poly-N-acetyllactosamine and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at Asn-glycosylation site 1 very close to the C terminus, whereas the Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively at site 2. This is the first report of the presence of poly-N-acetyllactosamine oligosaccharides in protozoa.

The conformational effects of N-glycosylation on the tailpiece from serum IgM.

1H-NMR spectroscopy has been used to study the conformation and dynamics of the isolated tailpiece from human serum immunoglobulin M, a 22-residue peptide containing a single asparagine glycosylation site. The peptide is isolated as a set of glycoforms, varying only in the sequence of the oligosaccharide attached at the glycosylation site. The oligosaccharides present have the general formula (Man)n(GlcNAc)2, with 45% having n = 6, 45% having n = 8 and 10% having n = 7 and/or 9. They have been identified and their NMR parameters compared to those found for the isolated oligosaccharides in free solution. The conformation and dynamics of the peptide component have also been studied, using NOE data and hydrogen-exchange experiments, and the results compared to those obtained from the aglycosyl peptide of the same sequence. The presence of the peptide is found to have no measurable effect on the conformation of the oligosaccharides. However, the presence of oligosaccharide causes a decrease in the conformational mobility of the backbone and sidechains of the peptide in the region of the glycosylation site. This is proposed to result from interactions between the oligosaccharide core and the amino acid side chains. Further, the conformation of the N-glycosidic linkage has been shown to be both rigid and planar. Thus, the conformational space available to an N-linked oligosaccharide in a glycoprotein relative to the protein may depend to a large extent upon the flexibility of the asparagine side chain. Various roles for the different glycoforms of the tail peptide are discussed.

Uncertainties in structural determinations of oligosaccharide conformation, using measurements of nuclear Overhauser effects.

A fundamental problem in the determination of molecular structure by n.m.r. spectroscopy is insufficient experimental constraints. This problem is particularly marked for oligosaccharides, where few constraints are available across glycosidic linkages. By calculating distances as a function of dihedral angle, it is shown that, in general, two n.O.e. constraints result in two possible conformations for each glycosidic linkage, one of which can usually be discarded on the basis of model building or energy calculations. Using these calculations, an estimate of the uncertainty in the structure can be obtained.

Characterisation of the asparagine-linked oligosaccharides from Trypanosoma brucei type-I variant surface glycoproteins.

The complete primary structures of the Asn-linked oligosaccharides from the conserved glycosylation site of the type-I variant surface glycoproteins of Trypanosoma brucei MITat 1.4 and MITat 1.6 were determined using a combination of exoglycosidase digestions, permethylation analysis, acetolysis and 1H NMR. Both variants contained almost exclusively oligomannose-type oligosaccharides, identical in structure to those of mammalian glycoproteins. The oligosaccharides ranged in size from (Man)9(GlcNAc)2 to (Man)5(GlcNAc)2. The relative abundance of each component was similar in both variants. The major components were (Man)8(GlcNAc)2 and (Man)7(GlcNAc)2 with slightly less (Man)9(GlcNAc)2 and (Man)6(GlcNAc)2 and much less (Man)5(GlcNAc)2. Both variants also contained the same structural isomers. The close similarity of the oligomannose series indicates identical processing at the conserved site in both variants.

Primary sequence dependence of conformation in oligomannose oligosaccharides.

The oligomannose series of oligosaccharides from bovine thyroglobulin (BTG) and the variant surface glycoprotein (VSG) of Trypanosoma brucei have been isolated and sequenced by 1H NMR. The structure of Man9GlcNAc2, the parent molecule of the series, is shown below. Structural isomerism occurs within this series through the removal of residues D1, D2, D3, and C. Using spin-spin coupling and chemical shift data the rotamer distributions about the dihedral angle omega for the Man alpha 1-6Man beta and Man alpha 1-6Man alpha linkages were determined for each member of the series. It is shown that the dihedral angle omega of the Man alpha 1-6Man beta linkage exhibits low flexibility with a preference for the omega = 180 degrees conformation when residue D2 is present and high flexibility when this residue is absent. Flexibility of omega for the Man alpha 1-6Man alpha is largely independent of primary sequence and is intermediate between the two Man alpha 1-6Man beta extremes, again with a preference for the omega = 180 degrees conformation. [see text] There are, however, data which indicate that removal of residue D3 may confer additional flexibility upon the dihedral angle omega of the Man alpha 1-6Man alpha linkage. Molecular graphics modelling, together with chemical and enzymatic modification studies, suggest that the origin of the observed primary sequence dependence of the Man alpha 1-6Man beta linkage arises from steric factors. On the basis of these observations taken together with previous work, it is postulated that recognition of individual oligomannose conformations may play a role in the control of N-linked oligosaccharide biosynthesis.

On the domain structure of antithrombin III. Tentative localization of the heparin binding region using 1H NMR spectroscopy.

The denaturation of human and bovine antithrombin III by guanidine hydrochloride has been followed by 1H NMR spectroscopy. The same unfolding transition seen previously from circular dichroism studies [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053] at low denaturant concentration was detected here by discontinuous changes in the chemical shifts of the C(2) protons of two of the five histidines in human antithrombin III and of three of the six histidines in bovine antithrombin III. These two histidines in human antithrombin III are assigned to residue 1 and, more tentatively, to residue 65. Two of the three histidines similarly affected in the bovine protein appear to be homologous to residues in the human protein. This supports the proposal of similar structures for the two proteins. In the presence of heparin, the discontinuous titration behavior of these histidine resonances is shifted to higher denaturant concentration, reflecting the stabilization of the easily unfolded first domain of the protein by bound heparin. From the tentative assignment of one of these resonances to histidine-1, it is proposed that the heparin binding site of antithrombin III is located in the N-terminal region and that this region forms a separate domain from the rest of the protein. The pattern of disulfide linkages is such that this domain may well extend from residue 1 to at least residue 128. Thermal denaturation also leads to major perturbation of these two histidine resonances in human antithrombin III, though stable intermediates in the unfolding were not detected.