One-bond and two-bond J couplings help annotate protein secondary-structure motifs: J-coupling indexing applied to human endoplasmic reticulum protein ERp18

NMR coupling constants, both direct one‐bond (¹J) and geminal two‐bond (²J), are employed to analyze the protein secondary structure of human oxidized ERp18. Coupling constants collected and evaluated for the 18 kDa protein comprise 1268 values of ¹J_CαHα, ¹J_CαCβ, ¹J_CαC′, ¹J_C′N′, ¹J_N′Cα, ¹J_N′HN, ²J_CαN′, ²J_HNCα, ²J_C′HN, and ²J_HαC′. Comparison with ¹J and ²J data from reference proteins and pattern analysis on a per‐residue basis permitted main‐chain φ,ψ torsion‐angle combinations of many of the 149 amino‐acid residues in ERp18 to be narrowed to particular secondary‐structure motifs. J‐coupling indexing is here being developed on statistical criteria and used to devise a ternary grid for interpreting patterns of relative values of J. To account for the influence of the varying substituent pattern in different amino‐acid sidechains, a table of residue‐type specific threshold values was compiled for discriminating small, medium, and large categories of J. For the 15‐residue insertion that distinguishes the ERp18 fold from that of thioredoxin, the J‐coupling data hint at a succession of five isolated Type‐I β turns at progressively shorter sequence intervals, in agreement with the crystal structure. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

The use of TROSY for detection and suppression of conformational exchange NMR line broadening in biological macromolecules

The interference between conformational exchange-induced time-dependent variations of chemical shifts in a pair of scalar coupled ¹H and ¹⁵N spins is used to construct novel TROSY-type NMR experiments to suppress NMR signal loss in [¹⁵N,¹H]-correlation spectra of a 14-mer DNA duplex free in solution and complexed with the Antp homeodomain. An analysis of double- and zero-quantum relaxation rates of base ¹H–¹⁵N moieties showed that for certain residues the contribution of conformational exchange-induced transverse relaxation might represent a dominant relaxation mechanism, which, in turn, can be effectively suppressed by TROSY. The use of the new TROSY method for exchange-induced transverse relaxation optimization is illustrated with two new experiments, 2D ^h1 J _HN,^h2 J _NN-quantitative [¹⁵N,¹H]-TROSY to measure ^h1 J _HN and ^h2 J _NN scalar coupling constants across hydrogen bonds in nucleic acids, and 2D (^h2 J _NN+^h1 J _NH)-correlation-[¹⁵N,¹H]-TROSY to correlate ¹H^N chemical shifts of bases with the chemical shifts of the tertiary ¹⁵N spins across hydrogen bonds using the sum of the trans-hydrogen bond coupling constants in nucleic acids.     

Conformational dependence of the vicinal proton coupling constant for the Cα?Cβ bond in peptides

We use the nmr data concerning the C^αH? C^βH fragment in eight peptides with rigid side chains to parametrize a Karplus correlation between the vicinal proton J_αβ coupling constant and the dihedral angle θ. When considering molecules containing the fragment C^αH^α? C^βH^βH^β′, the three-dimensional structure of the model peptides does not need to be known with accurate precision, since each set of J_αβ and J_αβ′ coupling constants is then related to the coefficients of the Karplus equation. A good correlation is observed with the Karplus equation, which is in substantial agreement with the J_αβ coupling constants reported for rigid as well as rotating C^α? C^β bonds in peptides.     

Quantitative f torsion angle analysis in Desulfovibrio vulgaris flavodoxin based on six f related 3 J couplings

Quantitative φ-dihedral angle determinations of non-glycine and non-proline residues in Desulfovibrio vulgaris flavodoxin are carried out on the exclusive basis of ³ J coupling constants. In total 124 ³ JHNH _α , 123 ³ JHNC _′i , 118 ³ JHNC _β , 117 ³ JC′ _i–1Hα , 109 ³ JC′ _i–1C′i , and 103 ³ JC′ _i–1Hβ values form the experimental basis for translating J coupling data into geometry information using various combinations of Karplus parameters given in the literature. In addition, each backbone torsional angle φ is adjusted assuming different models of local geometry, either a rigid torsion, a Gaussian distribution centered at a distinct angle, or a two-site jump model. Numerical optimization is followed by a statistical significance evaluation to assess the results. It is found that experimental coupling constants of most of the residues involved in secondary structure elements agree best with those predicted from rigid local conformations. For dihedral angles in loop regions, mobility effects are not negligible, and a single torsion (Glu 42) is likely to adopt two distinct adjustments. However, α-helix conformations with –60° < φ < –45° give rise to an alternate solution with φ≈+170° with similar statistical significance when using the four traditionally determined proton-involved ³ J couplings. This ambiguity is efficiently avoided only when taking advantage of the complete data set comprising six available experimental ³ J coupling constants and of the degeneracy intrinsic to the Karplus relation. The optimized φ conformations are compared with reference values from the crystal structure of flavodoxin.     

Proton magnetic resonance studies of 9-(β-d-xylofuranosyl)adenine 3′,5′-cyclic monophosphate and 9-(β-d-arabinofuranosyl)adenine 2′,5′-cyclic monophosphate

A detailed ¹H 220-MHz n.m.r. study of 9-(β-d-xylofuranosyl)adenine 3′,5′-cyclic monophosphate (3′,5′-xylo-cAMP, 1) and 9-(?-d-arabinofuranosyl)adenine 2′,5′-cyclic monophosphate (2′,5′-ara-cAMP, 2) in D₂O solution is described. The sugar-ring conformations in 1 and 2 are shown to be ³E and ²E, respectively, and the phosphate rings are in a chair form. An unusual ⁴J_P,H coupling of 2.4 Hz is observed between H-4′ and phosphorus in 1 and a vicinal J_P,H of 30.8 Hz between H-5′ and phosphorus in 2. This latter coupling verifies a similar value found previously in the ara-cytidine analog of 2. A comparison of the conformational properties of cyclic nucleotides having fused phosphate and sugar rings has been made, together with an assessment of the use of the Karplus constants in such ring-systems.     

Nucleoside dimers analogues with a 1,2,3-triazole linkage: conjugation of floxuridine and thymidine provides novel tools for cancer treatment. Part II

Abstract

The fluorinated nucleoside dimers with a 1,2,3-triazole linkage are novel compounds within the field of bioorganic chemistry. We report on the synthesis and properties of two groups of nucleoside dimers analogs possessing a different arrangement of the 1,4-disubstituted 1,2,3-triazole linkage. Based on analysis of the ³J_HH, ³J_H1′C2, and ³J_H1′C6 we estimated conformational preferences of sugar part and orientation around glycosidic bond. These compounds show moderate anticancer activity, with cytostatic studies in three different cancer cell lines.     

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of ¹⁵N-¹⁵N and ¹H-¹⁵N trans-hydrogen bond scalar couplings, ^2h J _NN and ^1h J _HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.     

Conformational dependence of vicinal 13C′NCαH coupling constants in peptides: A dirac vector model investigation

Theoretical calculations of the heteronuclear vicinal coupling constant ³J(¹³C′NC^αH) in peptides have been carried out using the Dirac vector model. The results showed an angular dependence for this coupling constant, which can be expressed in the form ³J(¹³C′NC^αH) = A cos² θ + B cos θ + C, where A, B, and C are constants and θ is related to the torsional angle ? of the peptide backbone. The results of the present calculations are in very good agreement with those obtained using finite perturbation theory at the INDO level of approximation.     

1H- and 13C-nmr investigations on cis–trans isomerization of proline peptide bonds and conformation of aromatic side chains in H-Trp-(Pro)n-Tyr-OH peptides

¹H and ¹³C high-resolution nmr spectra of cationic, zwitterionic, and anionic forms of the peptides: H-Trp-(Pro)_n-Tyr-OH, n = 0-5, and H-Trp-Pro-OCH₃ were obtained in D₂O solution. Analysis of H^α(Pro₁), H^α(Trp), C^γ(Pro), H^ε(Tyr), and H^δ(Trp) resonances provided evidence for the presence of two predominant backbone isomers: the all-trans one and another with the Trp-Pro peptide bond in cis conformation; the latter constituted about 0.8 molar fraction of the total peptide (n > 1) concentration. Relative content of these isomers varied in a characteristic way with the number of Pro residues and the ionization state of the peptides. The highest content of the cis (Trp-Pro) isomer, 0.74, was found in the anionic form of H-Trp-Pro-Tyr-OH; it decreased in the order of: anion ? zwitterion ≈ cation, and with the number of Pro residues to reach the value of 0.42 in the cationic form of H-Trp- (Pro)₅-Tyr-OH. Isomerization equilibria about Pro-Pro bond(s) were found to be shifted far (?0.9) in favor of the trans conformation. Interpretation of the measured vicinal coupling constants J_α?β′ and J_α?β″ for C^αH-C^βH₂ proton systems of Trp and Tyr side chains in terms of relative populations of g⁺, g^?, and t staggered rotamers around the χ₁ dihedral angle indicated that in all the peptides studied (a) rotation of Trp indole ring in cis (Trp-Pro) isomers is strongly restricted, and (b) rotation of Tyr phenol ring is relatively free. The most preferred χ₁ rotamer of Trp (0.8-0.9 molar fraction) was assigned as the t one on the basis of a large value of the vicinal coupling constant between the high-field H^β and carbonyl carbon atoms of Trp, estimated for the cis (Pro₁) form of H-Trp-Pro-Tyr-OH from a ¹H, ¹³C correlated spectroscopy ¹H detected multiple quantum experiment. This indicates that cis ? trans equilibrium in the Trp-Pro fragment is governed by nonbonding interactions between the pyrrolidine (Pro) and indole (Trp) rings. A molecular model of the terminal cis Trp-Pro dipeptide fragment is proposed, based on the presented nmr data and the results of our molecular mechanics modeling of low-energy conformers of the peptides, reported elsewhere. © 1993 John Wiley & Sons, Inc.     

Spin-state selection filters for the measurement of heteronuclear one-bond coupling constants

Novel α/β-half-filter elements are proposed for the separation of the high-field and low-field component of ¹ J _HC and ¹ J _HN splittings into different subspectra. The α/β-half-filter elements are of the same duration as the S³CT pulse sequence element and, like this, are less sensitive to cross talk between different subspectra than the original shorter α/β-half-filters. The filter elements are demonstrated with the measurement of ¹ J _HC coupling constants of C^αH groups in 2D and 3D experiments and the subspectral editing of the four different multiplet components observed in two-dimensional α/β-HSQC-α/β spectra recorded without heteronuclear decoupling in either dimension.     

Sesquiterpene Lactones from Centaurea zuccariniana and Their Antimicrobial Activity

Twenty‐nine compounds were isolated from the aerial parts of the Greek plant C. zuccariniana DC. The structures of the isolated compounds were established by means of NMR‐ (¹H,¹H‐COSY, ¹H,¹³C‐HSQC, HMBC, NOESY, and ROESY) and mass‐spectral analyses. These compounds comprise 13 sesquiterpene lactones, 14 flavonoids, two lignans, and one simple lactone. Among the isolated sesquiterpene lactones, three are new, namely one heliangolide, (1E,4Z)‐15‐hydroxy‐8α‐O‐(4′‐acetoxy‐3′‐hydroxy‐2′‐methylidenebutanoyl)‐6βH,7αH‐germacra‐1,4,11(13)‐trien‐6,12‐olide; and two eudesmanolides, 8α‐(4′,5′‐diacetoxyangeloyl)sonchucarpolide and one unusual eudesmanolide with an oxygenated bridge linking C(1) and C(4), named zuccarinin. The main sesquiterpene lactones were malacitenolide, cnicin, and 4′‐O‐acetylcnicin. These results are in agreement with those obtained from the previously studied Greek Centaurea sp. belonging to the section Acrolophus (Cass .) DC.; this finding could be of chemotaxonomic significance for the genus Centaurea. The in vitro antimicrobial activities of the isolated new sesquiterpene lactones were against eight bacteria and eight fungal species. A 96‐well microbioassay procedure for fast and easy evaluation of antibacterial and antifungal activities was applied to compare these compounds with commercial antibiotic and fungicide standards, and with previously isolated analogous sesquiterpene lactones tested by the same bioassay. All of the compounds tested showed moderate antibacterial, but significant antifungal activities; the present results corroborate with previous data, indicating that these types of compounds exhibit low or moderate antibacterial, but potent antifungal activities. The unusual eudesmanolide zuccarinin proved to be the most potent among the present tested sesquiterpene lactones, as well as among all previously tested eudesmanolides isolated from Greek Centaurea sp.     

1H NMR Assignments and Conformational Analysis of the Oligoribonucleotides CA,CAU, CAUG,ACAUG, and UCAUG: Observation of Pyrimidine H5-H1′ Long-Range Scalar Couplings

Abstract

Conformational analysis and ¹H NMR spectral assignments have been carried out using COSY and RELAY methods for a series of related oligoribonucleotides including two pen- tamers with 5′-dangling bases. Intraresidue long-range five bond scalar coupling was observed between pyrimidine H5 and H1′ protons in the COSY-45 spectra and this feature was useful for both assignment purposes and conformational analysis. The ribose ring conformations were predominantly C3′-endo with the C2′-endo population increasing at the 3′-terminus. The 5′-dangling bases were not stacked efficiently, exhibiting lower % C3′-endo values than their 3′-nearest neighbors. Backbone torsion angle populations, β′, γ⁺, ε′, were determined using ′H-′H, ′H-³¹P, and ¹³C-³¹P coupling constants. From β′ and γ⁺ populations the U3-G4 step in CAUG was found to be less efficiently stacked than the C1-A2 and A2-U3 steps. This observation in solution is consistent with the fiber diffraction A-RNA model (S. Arnott, D.W.L. Hukins, S.D. Dover, W. Fuller and A.R. Hodgson, J. Mol. Biol. 81, 107-122, 1973) which also predicts poor stacking in a U-G dinucleotide. The ε′ populations were >65% for all C3′- O3′ bonds and consistent with a right-handed A-RNA helix.     

Determination of h2JNN and h1JHN coupling constants across Watson–Crick base pairs in the Antennapedia homeodomain–DNA complex using TROSY

This paper describes NMR measurements of ¹⁵N–¹⁵N and ¹H–¹⁵N scalar couplings across hydrogen bonds in Watson–Crick base pairs, ^h2 J _NN and ^h1 J _HN, in a 17 kDa Antennapedia homeodomain–DNA complex. A new NMR experiment is introduced which relies on zero-quantum coherence-based transverse relaxation-optimized spectroscopy (ZQ-TROSY) and enables measurements of ^h1 J _HN couplings in larger molecules. The ^h2 J _NN and ^h1 J _HN couplings open a new avenue for comparative studies of DNA duplexes and other forms of nucleic acids free in solution and in complexes with proteins, drugs or possibly other classes of compounds.     

Evaluation of Biopolymer Conformations: Substituent Electronegativity in Nucleosides

Coupling constants J_ij for resonating vicinal protons i and j in NMR are studied theoretically and computationally. Electronegativity as a source of an additive correction for J_{1′, 2′} and J_{2′, 3′}, in two families of nucleosides is examined. Variations in J_{1′, 2′} + J_{3′, 4′} and J_{2′, 3′} are explained by conformational changes only. Variations in J_{1′, 2′} and J_{3′, 4′} are partially explained by a change of conformation, but mainly by variation in population caused by the 2′ substituents and by bee changes. Moreover, the changes of populations are quantifiable with respect to the substituent in 1′ or 2′. A computer program called SEARCH has been developed which optimizes the phase angle of pseudorotation P and the degree of pucker τ_m for a set of ring sugar coupling constants.     

Germacranolides from Viguiera microphylla

Three new germacranolides, including two heliangolides (niveusin C-2′,3′-epoxide and 1,2-dehydroniveusin C-2′,3′-epoxide) and a germacrolide (3β-hydroxy-8β-epoxyangeloyloxycostunolide-1β,10α-epoxide) were isolated from Viguiera microphylla. Their structures were established by spectroscopic analyses, including extensive ¹H NMR and ¹³C NMR decoupling experiments and chemical transformations. X-ray diffraction analysis confirmed the structure of niveusin C-2′,3′-epoxide.     

Spatial configuration of single-stranded polynucleotides. Calculations of average dimensions and Nmr coupling constants

The probability distributions of the torsional angles (Φ′, ω′, ω, Φ, and ψ), which fix the structure of nucleotide backbone, have been calculated using the results of energy calculations based on extended Huckel theory (EHT), complete neglect of differential overlap (CNDO), perturbative configuration interaction using localized orbitals (PCILO), and classical potential functions (CPF) methods. Statistical average values of the vicinal ¹H? ¹H, ¹H? ³¹P, and ¹³C? ³¹P nmr coupling constants 〈J〉 have been calculated from the generalized Karplus relations using the probability distribution in the Φ′, Φ, and ψ space. Experimental 〈J〉 values for polyribouridylic acid (polyU) support the theoretical predictions for these torsional angles. Using Monte Carlo technique, random coils of single-stranded polynucleotides have been simulated and the mean-square end-to-end distance 〈r²〉 has been calculated. Molecular orbital methods (EHT, CNDO, and PCILO) suggest considerable flexibility around O? P bonds, leading to fairly small values for the characteristic ratio (C_∞ ～ 4). Observed values of the unperturbed characteristic ratio for polynucleotides are quite large (C_∞ ～ 18) suggesting a relatively rigid nucleotide backbone. The results based on molecular orbital calculations can be reconciled with the experimental values by introducing an additional stabilization of ～2 kcal mol^?1 for the predicted minimum energy ragion (Φ′ ～ 240°, ω′ ～ 290°, ω 290°, Φ 180°, and ψ 60°). Such a stabilization may arise from the association of water molecules and metal ions with the phosphate group and (or) Coulomb interaction between neighboring phosphate groups. The calculations provide a semiquantitative estimate of torsional rigidity in the nucleotide backbone.     

Conformational study of ribonucleotides, 2′-deoxyribonucleotides,and arabinonucleotides by carbon-13 nuclear magnetic resonance

The geminal and vicinal ¹³C-³¹P coupling constants have been monitored, as a function of pH, for a series of uracil and cytosine 3′- and 5′-nucleotides with a ribose, arabinose, or 2′-deoxyribose sugar. Data were also obtained for two 3′,5′-diphosphates in the ribose and arabinose series. The geminal J(C5′-P5′) and J(C3′-P3′) couplings show only a small dependence on the ionization state of the phosphate, decreasing by < 0.5 Hz in the pH 5–7 range. For the ribose and arabinose 3′-nucleotides, the vicinal J(C4′-P3′) increase (up to 1.5 Hz) on secondary phosphate ionization in the pH 5–7 range, whereas their J(C2′-P3′) couplings decrease (up to 1.5 Hz) over the same pH range. In contrast for the 2′-deoxyribose molecules, both couplings decrease (～0.5 Hz) on phosphate ionization. The titration curves provide information about the influence of the sugar on the conformation about the C3′? O3′ bond. Some conformational trends could be rationalized by consideration of the sugar-puckerdependent contact interactions between the 3′-phosphate and the substituents on the furanose ring.     

Conformationally Restricted 2-Substituted Wyosine Derivatives. 1H, 13C,and 15N NMR Study

Abstract

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-β-D-ribofuranosyl) imidazo [1,2-a]purine (wyosine triacetate, 1) at C2 position with electronegative groups CH₃O 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.     

Proton magnetic resonance study of nucleosides,nucleotides, and dideoxynucleoside monophosphates containing a syn pyrimidine base

Proton magnetic resonance data have been obtained for 6-methyl-2′-deoxyuridine (dT*), its 3′- and 5′-monophosphates, and its 3′,5′-diphosphate, as well as for the corresponding thymine derivatives. The synthesis of the dideoxynucleoside monophosphates—d(TpT), d(T*pT), d(TpT*), and d(T*pT*)—was accomplished, and spectral data were obtained for these four dimers. The data show that the 6-methyluracil base prefers the syn conformation about the N-glycosyl bond at the monomer and dimer levels. The presence of the syn base leads to increases in the cis couplings of the sugar ring, J_1′2″ and J_2′3′, which indicate a trend towards eclipsing of the substituents on the C1′-C2′ and C2′-C3′ fragments. This trend is discussed in terms of changes in the pseudorotational parameters which describe the pucker of the ring. The syn base destabilizes the g⁺ conformer about the C4′-C5′ bond, leading to a preference for the t conformer in all dT* residues at the monomer and dimer levels. Preliminary work on the formation of cyclobutane-type photodimers in d(T*pT) and d(T*pT*) is discussed and presented as evidence for the capability of the syn 6-methyluracil base to form base-stacked complexes.