Measurement of small scalar and dipolar couplings in purine and pyrimidine bases.

A suite of spin-state-selective excitation (S3E) NMR experiments for the measurements of small one-bond (13C-13C, 15N-13C) and two-bond (1H-13C, 1H-15N) coupling constants in 13C,15N labeled purine and pyrimidine bases is presented. The incorporation of band-selective shaped pulses, elimination of the cross talk between alpha and beta sub-spectra, and accuracy and precision of the proposed approach are discussed. Merits of using S3E rather than alpha/beta-half-filter are demonstrated using results obtained on isotopically labeled DNA oligonucleotides.

NMR methodology for the study of nucleic acids.

During the past few years, NMR methodology for the study of nucleic acids has benefited from new developments that greatly improved state-of-the-art technology for the precise determination of three-dimensional structures. Substantial progress has been made in designing experimental protocols for the measurement of residual dipolar couplings, in sensitivity optimization of triple-resonance experiments and in detection of hydrogen bonds and in developing computational methods for structure refinement using NMR restraints.

Structural basis of pheromone binding to mouse major urinary protein (MUP-I).

The mouse major urinary proteins are pheromone-binding proteins that function as carriers of volatile effectors of mouse physiology and behavior. Crystal structures of recombinant mouse major urinary protein-I (MUP-I) complexed with the synthetic pheromones, 2-sec-butyl-4,5-dihydrothiazole and 6-hydroxy-6-methyl-3-heptanone, have been determined at high resolution. The purification of MUP-I from mouse liver and a high-resolution structure of the natural isolate are also reported. These results show the binding of 6-hydroxy-6-methyl-3-heptanone to MUP-I, unambiguously define ligand orientations for two pheromones within the MUP-I binding site, and suggest how different chemical classes of pheromones can be accommodated within the MUP-I beta-barrel.

Glycosylated major urinary protein of the house mouse: characterization of its N-linked oligosaccharides.

A minor component of the major urinary protein complex of the house mouse was chromatographically isolated and ascertained to be a previously suspected glycoprotein. Using highly sensitive mass-spectrometric techniques for sequencing and linkage analysis, the N-linked oligosaccharides of this glycoprotein were characterized. They were determined to be of the complex type with a wide heterogeneity. The heterogeneity was due to both the degree of sialylation and the presence of galactose residues in either beta(1-3) or beta(1-4) linkages. The biantennary structures were the most pronounced glycans, while tri- and tetraantennary entities were minor.

Positive identification of the puberty-accelerating pheromone of the house mouse: the volatile ligands associating with the major urinary protein.

Five structurally diverse small ligands, all binding to the major urinary protein (MUP) of the male house mouse, show individually puberty-accelerating pheromonal activity in the recipient females. A recombinant MUP (identical structurally to the natural protein) has shown no biological activity. While four of these ligands were previously implicated in oestrus synchronization (Whitten effect), the same chemosignals now appear responsible for both sexual maturation and cycling in adult females.

Increased protein backbone conformational entropy upon hydrophobic ligand binding.

For complexes between proteins and very small hydrophobic ligands, hydrophobic effects alone may be insufficient to outweigh the unfavorable entropic terms resulting from bimolecular association. NMR relaxation experiments indicate that the backbone flexibility of mouse major urinary protein increases upon binding the hydrophobic mouse pheromone 2-sec-butyl-4,5-dihydrothiazole. The associated increase in backbone conformational entropy of the protein appears to make a substantial contribution toward stabilization of the protein-pheromone complex. This term is likely comparable in magnitude to other important free energy contributions to binding and may represent a general mechanism to promote binding of very small ligands to macromolecules.

NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole.

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.

Reaction of N-acetylglycyllysine methyl ester with 2-alkenals: an alternative model for covalent modification of proteins.

Among the various reactions of lipid peroxidation products with proteins, 2-alkenals have been shown to react extensively with the epsilon-amino group of lysine residues [Zídek et al. (1997) Chem. Res. Toxicol. 10, 702-710]. To obtain additional information about the kinetic and mechanistic aspects of this modification, a model peptide (N-acetylglycyllysine O-methyl ester) was reacted with 2-hexenal. The reaction products were characterized through a combination of NMR and MS techniques. The structural elucidation efforts have shown the formation of pyridinium salts through the reaction of two or more alkenals with one amino group. Kinetic data were obtained using a continuous infusion of the reaction mixture into an electrospray ionization mass spectrometer. A mechanism is proposed that offers an alternative model for the formation of stable protein cross-links. The reaction progresses through a Schiff base intermediate to form a dihydropyridine species which can be alternatively reduced to form various 3,4- or 2,5-substituted pyridinium species or react with another Schiff base to form a trialkyl-substituted pyridinium structure. The stoichiometry of this structure (aldehyde/amine) is 3:2, in contrast to the widely accepted 1:2. Therefore, it represents another possible cross-linking mechanism for bifunctional products of lipid peroxidation.

Modification of horse heart cytochrome c with trans-2-hexenal.

Horse heart cytochrome c reacting with trans-2-hexenal was used as a simple model of the nonspecific interactions of proteins with 2-alkenals. The reaction mixtures containing relatively high concentrations of the protein and aldehyde were characterized using visible spectrophotometry, fluorescence, and circular dichroism measurement, capillary isoelectric focusing, size-exclusion chromatography, polyacrylamide gel electrophoresis, and mass-spectrometric techniques. The mass-spectrometric data indicate that cytochrome c becomes modified with one or two molecules of hexenal as the major reaction product. The modified species with a correspondingly lowered isoelectric point were detected through capillary isoelectric focusing. The results of proteolytic studies indicate nonspecific modifications. Significant quantities of the oligomeric forms of hexenal-modified protein were also observed electrophoretically.

Hydroperoxidic inhibitor of horse liver alcohol dehydrogenase activity, tightly bound to the enzyme-NAD+ complex, characteristically degrades the coenzyme.

The strong inhibition of horse liver alcohol dehydrogenase (HLAD) by p-methylbenzyl hydroperoxide (XyHP) is only transient, XyHP behaves also as a pseudo-substrate of the enzyme and in the presence of NAD+, is degraded by HLAD to (as yet unidentified) non-inhibiting products while the NAD+ is converted to a derivative similar to the "NADX", originally observed in an analogous reaction of HLAD with hydrogen peroxide. The apparent KM for XyHP is approximately 10(4) times smaller than that for H2O2. The catalytic constant kcat for HLAD degradation of XyHP is two orders of magnitude less than that for ethanol dehydrogenation. XyHP inhibits both directions of the alcohol-aldehyde interconversion with equal potency. The first step of the inhibition mechanism is a tight binding of XyHP to the binary HLAD-NAD+ complex.

Interactions of organic hydroperoxides with heme proteins.

The decomposition of p-methylbenzyl hydroperoxide by cytochrome c and other selected heme systems in the absence of reducing agents was investigated. p-Methylbenzaldehyde was identified as the major product. A mechanism for this reaction has been suggested. H2O2 and tertiary cumyl hydroperoxide do not react under these conditions. The ability of organic hydroperoxides to act as oxygen donors in the cytochrome-mediated 7-ethoxyresorufin-O-deethylation was studied. Cumyl and tert.butyl hydroperoxides are able to substitute oxygen in the absence of NADPH while p-methylbenzyl hydroperoxide is not.