Sequence effects in structures of the dinucleotides d-pApT AND d-pTpA

The H1',H2' and H2″ regions of the 270-MHz PMR spectra of two deoxydinucleotides, d-pTpA and d-pApT, have been analyzed. The coupling constants in the sugar ring indicate that both A and T sugars have a tendency to acquire 2E conformations. There is also a marginal difference in the 2E populations of the T sugar in the two dinucleotides. The trends in the chemical shifts of base protons indicate different stacking of the bases in d-pApT and d-pTpA. The sequence effects on base stacking and pentose conformation are discussed.

A simple non-linear D/A converter

A simple generalized technique for realizing a non-linear digital to analogue converter (N-DAC), based on the principles of ' segment of equal digital interval ' is described. The simplicity of the proposed technique is demonstrated by realizing an N-DAC having a square law transfer function.

Intermolecular potentials in the dimer, the excimers, and the dimer ions of ethylene

Calculations are reported on the interaction energies in the dimer, the excimers, and the dimer ions of ethylene. The various a- and u-electron terms for different conformations of the dimeric species are determined by using the exchange perturbation method. The results predict that the singlet excimer and the dimer cation are stable primarily because of the large magnitude of the exciton-resonance and charge-resonance terms, respectively, while the neutral dimer, the triplet excimer, and the dimer anion are weakly stable. The variations of the various energy terms with conformations suggest that these dimeric species cannot have identifical structure.

Vibrational spectra of hydrazine carbothioamide and its 15N- and D-labelled species

Raman and infrared spectra of hydrazine carbothioamide (HCTA) and its three 15N-labelled molecules (H2N NH CS15NH2, H2 15N15NHCSNH2 and H2 15N15NHCS15NH2) and their deuterated compounds have been obtained. A complete normal coordinate analysis of HCTA has been made and revised assignments are presented. The factor group splittings of HCTA have been interpreted.

Dynamics of carbohydrateâlectin interaction The interaction p-nitrophenyl-β-D-galactose with a lectin from Ricinius communis

L&in-induced agglutination is a complex process determined by several factprs such as the nature of lectin (valency, binding constant) the properties of cell membrane (fluidity, distribution of lectin receptor sites) and the metabolic state of the cell (microvilli, microtubules, microfilament) [l-3].

D.c. and a.c. polarographic behaviour of vanadium-glycine complexes

A detailed investigation of the d.c. polarographic behaviour of vanadium(V),-(IV) and -(III) in glycine solutions has been made keeping the total glycine concentration at 0.1, 0.5 and 1.0 M and varying the pH of the solution. Experiments keeping the pH constant (using different ratios of glycine and glycine anion) and varying the glycine anion concentration, and also in predominantly anion solutions, have been made.

Conformation of polypeptide-chains containing both l-residues and D-residues .2. Double-helical structures of poly-ld-peptides

Polypeptides with alternating L- and D-amino acid residues can take up stereochemically satisfactory coaxial double-helical structures, both antiparallel and parallel, which are stabilized by systematic interchain NH O hydrogen bonds. Semiempirical energy calculations over allowed regions of conformational space have yielded the characteristics of these double-helices. There are four possible types of antiparallel double-helices - A3, A4, A5 and A6, with n, the number of LD peptide units per turn, around 2.8, 3.6, 4.5 and 5.5 respectively, while for the parallel double-helices there are two types, P3 and P4, having similar helical parameters as in A3 and A4. The hydrogen-bonding scheme restricts the pitch in all the models to the narrow range of 10.0 to 11.5 Å. All these helices have large central cores whose radii increase proportionately with n. In this respect, A3 and A4 are suitable models for the structure of gramicidin A. In terms of their relative energies, antiparallel double-helices are marginally more stable than those with parallel strands. Our results indicate that the energy differences amongst the members in the antiparallel family are not significant and thus provide an explanation for the polymorphism reported for poly(γ-benzyl-LD-glutamate).

Structure of a dimer of ageratochromene

A solid obtained in the dimerisation of ageratochromene (6,7-dimethoxy-2,2-dimethyl-1-benzopyran)(II) in the presence of acid has been shown to be 5,6,6a,6b,7,12b-hexahydro-1,2,10,11-tetramethoxy-5,5,7,7-tetramethylcyclopenta[1,2-c;5,4,3-de]bis[1]benzopyran (IV) by a study of its n.m.r. and mass spectra.

Reactivity of µ-Peroxo-Bridged Dimeric Vanadate in Bromoperoxidation

Diglycyl triperoxodivanadate [V2O2(O2)3(Gly H)2(H2O)2], a synthetic compound with μ-peroxo-bridge derived from H2O2and vanadate, oxidized bromide to a bromination-competent intermediate in phosphate buffer and physiological pH. This is in contrast to the requirement of acid medium with H2O2as the oxidant. Addition of its solid to bromide solution instantly produced a 262-nm-absorbing compound that converted phenol red (a trap) to its 592-nm-absorbing bromo-derivative. The high bromination activity was lost on dissolving this compound in water and the solution showed the presence of peroxovanadates (mono and di) and vanadates (V1and oligomeric V10) in51V-NMR spectrum. Of these, diperoxovanadate and vanadate together supported slow bromination activity by a second set of reactions including bromide-assisted reductive formation of vanadyl. Bromination activity dependent on vanadyl was sensitive to oxidation by excess H2O2and to complexation by EDTA, whereas that of triperoxodivanadate was relatively insensitive. Vanadyl and diperoxovanadate are capable of forming a μ-peroxo-bridged complex that is essentially similar to the synthetic vanadate dimer used in the present experiments. It appears that a μ-peroxo-intermediate is the proximal oxidant of bromide in vanadium-catalyzed bromoperoxidation.

The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed, H134N and H147N SHMTs had K-m values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the k(cat) values were markedly decreased, H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme, Increasing concentrations of PLP (up to 500 mu M) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions, H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding, Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface, H150N SHMT was obtained in a tetrameric form with bound PLP, However, the mutant had very little enzyme activity (<2%). The k(cat)/K-m values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and SS-fold less compared with wild type enzyme, Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H-4-folate. However, it could form an external aldimine with serine and glycine, The wild type and the mutant enzyme had similar K-d values for serine and glycine, These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.

Synthesis of Specifically Deuteriated Derivatives of D-Galactose and D-Galactosamine

Simple and convenient methods for introducing deuterium label at C-3 and C-6 position of N-acetyl-D-galactosamine and D-galactose, respectively, are described. For the synthesis of 2-acetamido-2-deoxy-D-3-[2H] galactopyranose, benzyl 2-acetamido-2-deoxy-4,6-O-benzylidene-agr-D-galactopyranoside was oxidized with dimethyl sulfoxide- acetic anhydride and the product was reduced with sodium borodeuteride to introduce the deuterium at C-3. After benzylidene reduction, the mixture was subjected to hydrogenolysis and purified by column chromatography. 1,2:3,4-di-O-isopropylidene-agr-D-galactopyranoside was oxidized followed by reduction with sodium borodeuteride and deprotection to yield D-6-[2H] galactose.

Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group.

In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbance at 425 nm indicative of the presence of bound pyridoxal 5'-phosphate as an internal aldimine with a lysine residue. However, it had only 0.003% of the catalytic activity of the wild-type enzyme. It was also unable to perform reactions with glycine, beta-phenylserine or d-alanine, suggesting that the binding of these substrates to the mutant enzyme was affected. This was also evident from the interaction of amino-oxyacetic acid, which was very slow (8.4x10(-4) s-1 at 50 microM) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s-1 at 50 microM). In contrast, methoxyamine (which lacks the carboxy group) reacted with the mutant enzyme (1.72 s-1 at 250 microM) more rapidly than the wild-type enzyme (0.2 s-1 at 250 microM). Further, both wild-type and the mutant enzymes were capable of forming unique quinonoid intermediates absorbing at 440 and 464 nm on interaction with thiosemicarbazide, which also does not have a carboxy group. These results implicate Arg-401 in the binding of the substrate carboxy group. In addition, gel-filtration profiles of the apoenzyme and the reconstituted holoenzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wild-type enzyme underwent partial dissociation to a dimer, suggesting that the oligomeric structure was rendered more stable by the mutation of Arg-401. The increased stability of the mutant enzyme was also reflected in the higher apparent melting temperature (Tm) (61 degrees C) than that of the wild-type enzyme (56 degrees C). The addition of serine or serinamide did not change the apparent Tm of R401A mutant enzyme. These results suggest that the mutant enzyme might be in a permanently 'open' form and the increased apparent Tm could be due to enhanced subunit interactions.

NMR and X-ray crystallographic studies on cyclic tetrapeptide, cyclo (D-Phe-Pro-Sar-Gly)

The conformation of the synthetic cyclic tetrapeptide cyclo(D-Phe-Pro-Sar-Gly) has been determined in solution using the nuclear magnetic resonance technique and in the crystal state by X-ray crystallography. Results showed that the peptide exhibited two different conformations in solution, conformer 1 having cis-trans-cis-trans peptide bonds and conformer 2 having trans-cis-trans-cis peptide bonds. No intramolecular hydrogen bonds were observed in the structures. The X-ray diffraction studies showed the crystals to be orthorhombic with space group P2(1)2(1)2(1) with unit-cell dimensions, a = 5.790, b = 10.344, c = 31.446 A, Z = 4, R = 0.104 for 2301 observed reflections. The crystal structure showed only one type of conformer having cis-trans-cis-trans peptide bonds similar to the conformer 1 in solution.

Thermodynamics of Monosaccharide and Disaccharide Binding to Erythrina corallodendron Lectin

Isothermal titration calorimetry measurements of the binding of 2′-fucosyllactose, lactose, N-acetyllactosamine, galactopyranose, 2-acetamido-2-deoxygalactopyranoside, methyl α-N-dansylgalactosaminide (Me-α-DNS-GalN), methyl α-D-galactopyranoside, methyl β-D-galactopyranoside, and fucose to Erythrina corallodendron lectin (ECorL), a dimer with one binding site per subunit, were performed at 283-286 and 297-299 K. The site binding enthalpies, ΔHb, with the exception of Me-α-DNS-GalN, are the same at both temperatures and range from −47.1 ± 1.0 kJ mol−1 for N-acetyllactosamine to −4.4 ± 0.3 kJ mol−1 for fucose, and the site binding constants range from 3.82 ± 0.9 × 105 M−1 for Me-α-DNS-GalN at 283.2 K to 0.46 ± 0.05 × 103 M−1 for fucose at 297.2 K. The binding reactions are mainly enthalpically driven except for fucose and exhibit enthalpy-entropy compensation. The binding enthalpies of the disaccharides are about twice the binding enthalpies of the monosaccharides in contrast to concanavalin A where the binding enthalpies do not double for the disaccharides. Differential scanning calorimetry measurements show that denaturation of the ECorL dimer results in dissociation into its monomer subunits. The binding constants from the increase in denaturation temperature of ECorL in the presence of saccharides are in agreement with values from isothermal titration calorimetry results. The thermal denaturation of ECorL occurs around 333 K, well below the 344-360 K denaturation temperature of other legume lectins of similar size and tertiary structure, undoubtedly due to the difference in its quaternary structure relative to other legume lectins. This is also apparent from the independent unfolding of its two domains.