Synthesis and structural study of variously oxidized diastereomeric 5'-dimethoxytrityl-thymidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropyl]-phosphoramidite derivatives. Comparison of the effects of the P=O, P=S, and P=Se functions on the NMR spectral and chromatographic properties

R(P)- and S(P)-diastereomers of 5'-dimethoxytrityl-thymidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropyl]-phosphoramidite (T-CED) were separated by silica gel chromatography. Oxidation of both isomers with H(2)O(2), elemental sulfur and selenium, respectively, resulted in the corresponding oxidized analogues in nearly quantitative yields. All reactions were found to proceed with retention of P-configuration. This was confirmed by thorough NMR analysis which, in addition, aimed to study the spectral properties of the diastereomers with special respect to differences in the heteroatom effect of the O, S and Se atoms, double-bonded to the phosphorus, on the vicinal carbon-phosphorus couplings. It was found that the changes in the DeltaJ (=(3)J(P,C4') - (3)J(P,C2')) values were basically induced by the electronegativity of the heteroatoms, rather than differences in the rotational preferences about the C3'-O3' bond. The impact of the benzene solvent on the above couplings is also discussed. The effect of these heteroatoms on the chromatographic (normal and reverse phase HPLC) behavior of the compounds was also investigated and the reverse phase HPLC profiles showed an unambiguous correlation between the electronegativity of the heteroatoms and the chromatographic mobility of the analogues.     

Hydroxylamine and methoxyamine mutagenesis: displacement of the tautomeric equilibrium of the promutagen N6-methoxyadenosine by complementary base pairing

The imino-amino tautomeric equilibrium of the promutagenic adenosine analogue N6-methoxy-2',3',5'-tri-O-methyladenosine [OMe6A(Me)3], in solvents of various polarities, has been studied with the aid of 1H and 13C NMR spectroscopy. The high energy barrier (free enthalpy delta G = 80 +/- 5 kJ X mol-1) between the two tautomeric species renders possible direct observation of the independent sets of all 1H and 13C signals from each of them. The equilibrium ranges from 10% imino in CCl4 to 90% in aqueous medium. Thermodynamic parameters, including energy barriers and lifetimes, were calculated from the temperature dependence of the equilibrium. Essentially similar results prevail for the promutagenic N6-hydroxy analogue. The conformations of the sugar moieties, and of the base about the glycosidic bond, for both tautomers are similar to those for adenosine. The conformation of the exocyclic N6-OCH3 group, which determines the ability of each species to form planar associates (hydrogen-bonded base pairs), has also been evaluated. Formation of autoassociates of OMe6A(Me)3 and of heteroassociates with the potentially complementary 2',3',5'-tri-O-methyluridine and -cytidine, in chloroform solution, was also investigated. The amino form base pairs with uridine and the imino form with cytidine. Formation of a complementary base pair by a given tautomeric species was accompanied by an increase of up to 10% in the population of this species and a concomitant decrease in population of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbial synthesis of L-[15N]leucine L-[15N]isoleucine, and L-[3-13C]-and L-[3'-13C]isoleucines studied by nuclear magnetic resonance and gas chromatography-mass spectrometry

The preparation of leucine and isoleucine labeled with 15N and of site-specific 13C-labeled isoleucines is described. This method is based on the induction of the biosynthetic pathways specific for branched chain amino acids in glutamic acid producing bacteria, and controlled provision of stable isotope labeled precursors. Corynebacterium glutamicum (ATCC 13032), a glutamic acid overproducer, was incubated in leucine production medium which consisted of a basal medium supplemented with [15N]ammonium sulfate, glucose, and sodium alpha-ketoisocaproate. production of L-[15N]leucine reached 138 mumol/ml at an isotopic efficiency of 90%. It was purified and checked by proton NMR and GC-MS. The electron impact (EI) spectrum showed 95 atom% enrichment. The cultivation of C. glutamicum in a similar medium containing alpha-ketobutyrate yielded L-[15N]isoleucine at a concentration of 120 mumol/ml. The GC-MS EI and chemical ionization (CI) spectra confirmed enrichment of 96 atom% 15N as that of the labeled precursors. The biosynthesis of L-[13C]isoleucine was carried out by induced cells which were transferred to a similar medium in which [2-13C]- or [3-13C]pyruvic acid replaced glucose. 13C NMR of the product isoleucine revealed single-site enrichment at C-3 or at C-3' respective to the precursor [13C]pyruvate; i.e., C-3 was labeled from [2-13C]pyruvate and C-3' from [3-13C]pyruvate. Mass spectrometric analysis confirmed that all molecules were labeled only in one carbon. This site-specific incorporation of [13C]pyruvate is contrasted with the labeling pattern obtained when producing cells were supplied with [2-13C]acetate, instead of pyruvate, when most label was incorporated into carbons 3 and 3' of the same isoleucine molecule.     

Glycine origin of the methyl substituent on C7'-N of octodiose for the biosynthesis of apramycin

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicating that glycine and/or serine might be involved in the biosynthesis of apramycin. The 13C-NMR analysis of [2-13C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH3 substituent of apramycin on the C7' of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes as previously reported for the case of rapamycin. The 15N NMR spectra of [2-13C,15N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the -NH2 substituents of apramycin.     

1H NMR conformational study of antiherpetic C5-substituted 2'-deoxyuridines: insight into the nature of structure-activity relationships

1H NMR study and conformational analysis of a broad series of biologically important C5-substituted 2'-deoxyuridines, including alkyl, halogen, vinyl, hydroxymethyl, and hydroxy derivatives as well as nitro, formyl, trifluoromethyl, and dimethylamino substituents, is presented. A thorough analysis of chemical shifts in correlation with C5-substituent electronegativity as well as calculations by SCF semi-empirical method of the formal charge localized on C6 carbon is discussed in terms of charge distribution for electron attracting and electron donating groups. Conformation of the sugar ring is determined from proton-proton coupling constants and described in terms of pseudorotation between two main puckering domains C2'endo (S) and C3'endo (N). Generally, electron donating groups destabilise the N conformation, simultaneously decreasing the mean pseudorotation amplitude. Absolute assignments of the H5' and H5' methylene protons in 1H NMR spectra permitted the unequivocal determination of molar fractions of the three classical exocyclic C4'-C5' rotamers gauche+, trans, and gauche-, and correlation of them with the sugar ring puckering domains. Conformation about the glycosidic bond is described in terms of equilibrium between two conformational regions, anti and syn. Finally, the role of the C5-substituent in the creation of cytotoxic activity is considered on the basis of a simplified model assuming that compound activity is a function of substituent polar surface, its molecular volume, and its molecule polarity defined at the relative partition of the polar atoms.     

Efficient methods for the synthesis of [2-15N]guanosine and 2'-deoxy[2-15N]guanosine derivatives

The nucleophilic addition-elimination reaction of 2',3',5'-tri-O-acetyl-2-fluoro-O6-[2-(4-nitrophenyl)ethyl]inosine (8) with [15N]benzylamine in the presence of triethylamine afforded the N2-benzyl[2-15N]guanosine derivative (13) in a high yield, which was further converted into the N2-benzoyl[2-15N] guanosine derivative by treatment with ruthenium trichloride and tetrabutylammonium periodate. A similar sequence of reactions of 2',3',5'-tri-O-acetyl-2-fluoro-06-[2-(methylthio)ethyl]inosine (9) and the 6-chloro-2-fluoro-9-(beta-D-ribofuranosyl)-9H-purine derivative (11), which were respectively prepared from guanosine, with potassium [15N]phthalimide afforded the N2-phthaloyl [2-15N]guanosine derivative (15; 62%) and 9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-6-chloro-2-[15N]phthalimido-9H-purine (17; 64%), respectively. Compounds 15 and 17 were then efficiently converted into 2',3',5'-tri-O-acetyl [2-15N]guanosine. The corresponding 2'-deoxy derivatives (16 and 18) were also synthesized through similar procedures.     

Hydrogen bonding between cytosine and peptides of threonine or serine: is it relevant to the origin of the genetic code?

13C, 15N, and 1H nuclear magnetic resonance measurements indicate that chloroform-soluble threonine-containing tripeptide derivatives, such as t-Boc-Thr-Gly-Gly-OBz, form three strong hydrogen bonds to the cytosine moiety of 2',3'-O-isopropylidene-5'-O-t-butyldimethylsilylcytidine. The C = O and NH of the central peptide residue plus the OH of the threonine side chain appear to form bonds to the N(4')H2, N(3), and C(2) = O, respectively, of the pyrimidine. An association constant calculated from the cytidine 15N(4') nuclear magnetic resonance response to added peptide is four times larger than the corresponding cytosine-guanine constant. It is suggested that cytosine-peptide bonding was part of the primitive genetic coding mechanism early in evolution and accounts for the origin of the cytosine-centered codons for the hydroxy amino acids, serine and threonine, in the present code.     

Biological, biochemical and physicochemical evidence for the existence of the polyadenylate - polyuridylate - poly 2'-fluoro-2'-deoxyuridylate triple-stranded complex.

The interferon-inducing activity of the double-stranded complex poly(A) - poly(U) in primary rabbit kidney cell cultures is reduced when the cells are treated with poly(dUfl) either 1 h before, simultaneously with, or 1 h after the exposure to the double-stranded complex. It has been demonstrated in experiments involving sensitivity to hydrolysis by RNAase, UV absorbance-mixing curves, and UV absorbance-temperature profiles that this phenomenon is due to the formation of the triple-stranded complex poly(A) - poly(U) - poly(dUfl). The latter complex seems to be the principal product of interactions in the following systems: poly(A) - poly(U) + poly(dUfl); poly(A) - poly(dUfl) + poly(U); and poly(A) + poly(U) + poly (dUfl).     

New sequential-assignment routes of nucleic acid NMR signals using a [5'-(13)C]-labeled DNA dodecamer

NMR signal assignments for DNA oligomers have been performed by the well-established sequential assignment procedures based on NOESY and COSY. The H4'/H5'/H5' resonance region is congested and difficult to analyze without the use of isotope-labeled DNA oligomers. Here a DNA dodecamer constructed with 2'-deoxy[5'-(13)C]ribonucleotides, 5'-d(*C*G*C*G*A*A*T*T*C*G*CG)-3' (*N = [5'-(13)C]Nucleotide), was prepared in an effort to analyze the H4'/H5'/H5' resonance region by 2D 1H-13C HMQC-NOESY. In the C5' and H1' resonance region, weak and strong cross peaks for C5'(i)-H1'(i) and C5'(i)-H1'(i-1), respectively, were found, thus enabling the sequential assignment within this region. A similar sequential assignment route was found between C5' and H2'. Proton pair distances evaluated from the canonical B-DNA as well as A-DNA indicated that these sequential-assignment routes on a 2D 1H-13C HMQC-NOESY spectrum work for most nucleic acid stem regions.     

Probing the sequence effects on NarI-induced -2 frameshift mutagenesis by dynamic 19F NMR, UV, and CD spectroscopy

The NarI recognition sequence (5'-G1G2CG3CN-3') is the most vulnerable hot spot for frameshift mutagenesis induced by the carcinogen 2-aminofluorene and its analogues in Escherichia coli. Lesioning of the guanine in the G3 position induces an especially high frequency of -2 deletion mutations; vulnerability to these mutations is modulated by the nature of the nucleotide in the N position (C approximately A > G > T). The objective of the present study was to probe the structural basis of this N-mediated influence on the propensity of the G3 lesion to form a slipped mutagenic intermediate (SMI) during translesion synthesis. We studied NarI-based fully paired [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNCGGCCGAG-3'), N = dC or dT] and -2 deletion [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNGCCGAG-3'), N = dC or dT] duplexes, in which G* was either AF [N-(2'-deoxyguanosin-8-yl)-2-aminofluorene] or the 19F probe FAF [N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene]. The latter sequences mimic the bulged SMI for -2 deletion mutations. Dynamic 19F NMR, circular dichroism, and UV melting results indicated that the NarI-dC/-2 deletion duplex adopts exclusively an intercalated conformer, whereas the NarI-dT/-2 deletion duplex exists as multiple conformers. The data support the presence of a putative equilibrium between a carcinogen-intercalated and a carcinogen-exposed SMI for the dT/-2 duplex. A similar dT-induced conformational heterogeneity was observed for the fully paired duplexes in which all three guanines were individually modified by AF or FAF. The frequency of the carcinogen stacked S-conformation was found to be highest (69-75%) at the G3 hot spot in NarI-dC duplexes. Taken together, our results support the hypothesis that the conformational stability of the SMI is a critical determinant for the efficacy of -2 frameshift mutagenesis in the NarI sequence. We also provide evidence for AF/FAF conformational compatibility in the NarI sequences.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed 13C NMR

13C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane alphaII-helices. Surprisingly, the 13C NMR spectra of [3-(13)C]Ala-D85N turned out to be very similar to those of [3-(13)C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting 13C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane alphaII-helices of the M-like state are suppressed already by fluctuation motions in the order of 10(4)-10(5) Hz interfered with frequencies of magic angle spinning or proton decoupling. However, 13C NMR signal from the cytoplasmic alpha-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl 13C NMR signals of the loop and transmembrane alpha-helices followed by Pro residues in [1-(13)C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, 13C NMR spectral feature of reconstituted [1-(13)C]Val and [3-(13)C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.     

Conformation of the common purine (beta) ribosides in solution: further evidence for a correlation between N-S state of the ribose moiety and syn-anti equilibrium.

The solution conformations of adenosine, guanosine and inosine in liquid ND3 have been determined by NMR. Comparison of the Karplus analysis of the proton HR spectra of the ribose moiety obtained in this solvent with the data from aqueous solutions of A and I proves that the conformations of the nucleosides are very similar in both liquids. From the analysis of the vicinal coupling constants of the ring protons it has been deduced that the S state C(2')-endo is slightly preferred. The mole fraction in S approximates 0.6 for all three nucleosides. C-13 relaxation measurements have been applied in the determination of the correlation times for rotational diffusion. Only at temperatures below - 40 degrees C is the pseudo-rotation of the furanoside ring slowed down sufficiently for it not to contribute to the measured relaxation rates. From NOE studies and T1 measurements on the individual protons it is derived that the N, C(3')-endo, form of the ribose is correlated with an anti conformation of the base (Y approximately 210 degrees to 220 degrees) and the S, C(2')-endo, form of the ribose with a syn conformation of the base (Y approximately 30 degrees to 50 degrees). The glycosyl torsion angles derived for the two conformations of A, G, and I are equal within the limits of accuracy.     

Conformationally restricted 2-substituted wyosine derivatives. 1H, 13C, and 15N NMR study

It was found by 1H, 13C and 15N NMR study that substitution of 4,9-dihydro-4,6-dimethyl-9-oxo-3-(2',3',5'-tri-O-acetyl-beta-D-ribofuranosyl) imidazo [1,2-a]purine (wyosine triacetate, 1) at C2 position with electronegative groups CH30 and C6H5CH2O results in a noticeable electron distribution disturbance in the "left-hand" imidazole ring and a significant increase in the North conformer population of the sugar moiety.     

Application of Correlated Residual Dipolar Couplings to the Determination of the Molecular Alignment Tensor Magnitude of Oriented Proteins and Nucleic Acids

Residual dipolar couplings (RDC) between nuclear spins in partially aligned samples offer unique insights into biomacromolecular structure and dynamics. To fully benefit from the RDC data, accurate knowledge of the magnitude ( D (a)) and rhombicity ( R ) of the molecular alignment tensor, A, is important. An extended histogram method (EHM) is presented which extracts these parameters more effectively from dipolar coupling data. The method exploits the correlated nature of RDCs for structural elements of planar geometry, such as the one-bond (13)C'(i)-(13)C(i)(alpha), (13)C'(i)-(15)N(i+1), and (15)N(i+1)-(1)H(N)(i+1) couplings in peptide bonds of proteins, or suitably chosen combinations of (1) D (C1'H1'), (1) D (C2'H2'), (1) D (C1'C2'), (2) D (C2'H1'), (2) D (C1'H2'), and (3) D (H1'H2') couplings in nucleic acids, to generate an arbitrarily large number of synthetic RDCs. These synthetic couplings result in substantially improved histograms and resulting values of D (a) and R, compared with histograms generated solely from the original sets of correlated RDCs, particularly when the number of planar fragments for which couplings are available is small. An alternative method, complementary to the EHM, is also described, which uses a systematic grid search procedure, based on least-squares fitting of sets of correlated RDCs to structural elements of known geometry, and provides an unambiguous lower limit for the degree of molecular alignment.     

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.     

On the structures of flavoprotein D-amino acid oxidase purple intermediates. A resonance Raman study

Resonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of D-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3,3,3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C = N stretching mode of an imino acid derived from D-alanine, i.e., alpha-iminopropionate. The 1658 cm-1 band with D-proline was also assigned to the C = N stretching mode of an imino acid derived from D-proline, i.e., delta 1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C = N+1H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3 symmetric deformation modes in alpha-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359 cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C = N+1-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton(s) exchangeable by deuterium(s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4,10a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida have been reconstituted with 13C- and 15N-enriched FAD. The protein preparations were studied by 13C-NMR, 15N-NMR and 31P-NMR techniques in the oxidized and in the two-electron-reduced states. The chemical shift values are compared with those of free flavin in water or chloroform. It is shown that the pi electron distribution in oxidized free p-hydroxybenzoate hydroxylase is comparable to free flavin in water, and it is therefore suggested that the flavin ring is solvent accessible. Addition of substrate has a strong effect on several resonances, e.g. C2 and N5, which indicates that the flavin ring becomes shielded from solvent and also that a conformational change occurs involving the positive pole of an alpha-helix microdipole. In the reduced state, the flavin in p-hydroxybenzoate hydroxylase is bound in the anionic form, i.e. carrying a negative charge at N1. The flavin is bound in a more planar configuration than when free in solution. Upon binding of substrate the resonances of N1, C10a and N10 shift upfield. It is suggested that these upfield shifts are the result of a conformational change similar, but not identical, to the one observed in the oxidized state. The 13C chemical shifts of FAD bound to apo(salicylate hydroxylase) indicate that in the oxidized state the flavin ring is also fairly solvent accessible in the free enzyme. Addition of substrate has a strong effect on the hydrogen bond formed with O4 alpha. It is suggested that this is due to the exclusion of water from the active site by the binding of substrate. In the reduced state, the flavin is anionic. Addition of substrate forces the flavin ring to adopt a more planar configuration, i.e. a sp2-hybridized N5 atom and a slightly sp3-hybridized N10 atom. The NMR results are discussed in relation to the reaction catalyzed by the enzymes.     

Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides

An effective in vitro enzymatic synthesis is described for the production of nucleoside triphosphates (NTPs) which are stereo-specifically deuterated on the H5" position with high selectivity (>98%), and which can have a variety of different labels (13C, 15N, 2H) in other positions. The NTPs can subsequently be employed in the enzymatic synthesis of RNAs using T7 polymerase from a DNA template. The stereo-specific deuteration of the H5" immediately provides the stereo-specific assignment of H5' resonances in NMR spectra, giving access to important structural parameters. Stereo-chemical H-exchange was used to convert commercially available 1,2,3,4,5,6,6-2H-1,2,3,4,5,6-13C-D-glucose (d7-13C6-D-glucose) into [1,2,3,4,5,6(R)-2H-1,2,3,4,5,6-13C]-D-glucose (d6-13C6-D-glucose). [1',3',4',5"-2H-1',2',3',4',5'-13C]GTP (d4-13C5-GTP) was then produced from d6-13C6-D-glucose and guanine base via in vitro enzymatic synthesis employing enzymes from the pentose-phosphate, nucleotide biosynthesis and salvage pathways. The overall yield was approximately 60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d4-13C5-GTP, together with in vitro synthesised d5-UTP, d5-CTP and non-labelled ATP, were used in the synthesis of a 31 nt RNA derived from the primer binding site of hepatitis B virus genomic RNA. (13C,1H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly 13C/15N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with 13C-labelled non-deuterated RNA.     

Resonance Raman spectra of carbon-13- and nitrogen-15-labeled riboflavin bound to egg-white flavoprotein.

The resonance Raman spectra of [2-13C]-, [4a-13C]-, [4-13C]-8 [10a-13C]-, [2,4,4a, 10a-13C]-, [5-15N]-, [1,3-15N]-, and [1,3,5-15N]riboflavin bound to egg-white proteins were observed for N(3)-H and N(3)-D forms with spontaneous Raman technique by using the 488.0-nm excitation line of an argon ion laser. The fluorescence of riboflavin was quenched by forming a complex with egg-white riboflavin binding protein. The in-plane displacements of the C(2), C(4a), N(1), N(3), and N(5) atoms during each Raman active vibration were calculated from the observed isotopic frequency shifts. The 1252-cm-1 mode of the N(3)-H form was found to involve large vibrational displacements of the C(2) and N(3) atoms and to be strongly coupled with the N(3)-H bending mode. This line can be used as an indicator for state of N(3)-H...protein interaction. The 1584-cm-1 mode, which is known to be resonance-enhanced upon excitation near the 370-nm absorption band, was accompanied by the displacement of the N(5) atom in particular. The 1355-cm-1 mode was most strongly resonance-enhanced by the 450-nm absorption band and involved the displacements of all carbon atoms of ring III. Both lines can be used as structure probes for elucidating the structure of electronically excited states of isoalloxazine.     

DNA adduct bypass polymerization by Sulfolobus solfataricus DNA polymerase Dpo4: analysis and crystal structures of multiple base pair substitution and frameshift products with the adduct 1,N2-ethenoguanine

1,N(2)-Etheno(epsilon)guanine is a mutagenic DNA lesion derived from lipid oxidation products and also from some chemical carcinogens. Gel electrophoretic analysis of the products of primer extension by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) indicated preferential incorporation of A opposite 3'-(1,N(2)-epsilon-G)TACT-5', among the four dNTPs tested individually. With the template 3'-(1,N(2)-epsilon-G)CACT-5', both G and A were incorporated. When primer extension was done in the presence of a mixture of all four dNTPs, high pressure liquid chromatography-mass spectrometry analysis of the products indicated that (opposite 3'-(1,N(2)-epsilon-G)CACT-5') the major product was 5'-GTGA-3' and the minor product was 5'-AGTGA-3'. With the template 3'-(1,N(2)-epsilon-G)TACT-5', the following four products were identified by high pressure liquid chromatography-mass spectrometry: 5'-AATGA-3', 5'-ATTGA-3', 5'-ATGA-3', and 5'-TGA-3'. An x-ray crystal structure of Dpo4 was solved (2.1 A) with a primer-template and A placed in the primer to be opposite the 1,N(2)-epsilon-G in the template 3'-(1,N(2)-epsilon-G)TACT 5'. The added A in the primer was paired across the template T with classic Watson-Crick geometry. Similar structures were observed in a ternary Dpo4-DNA-dATP complex and a ternary Dpo4-DNA-ddATP complex, with d(d)ATP opposite the template T. A similar structure was observed with a ddGTP adjacent to the primer and opposite the C next to 1,N(2)-epsilon-G in 3'-(1,N(2)-epsilon-G)CACT-5'. We concluded that Dpo4 uses several mechanisms, including A incorporation opposite 1,N(2)-epsilon-G and also a variation of dNTP-stabilized misalignment, to generate both base pair and frameshift mutations.