Conformational Analysis of the Deoxyribofuranose Ring in DNA by means of Sums of Proton-proton Coupling Constants: A Graphical Method

Abstract

A graphical method is presented for the conformational analysis of the sugar ring in DNA fragments by means of proton-pro ton couplings. The coupling data required for this analysis consist of sums of couplings, which are referred to as Σ1′ (= J1′2′ + J1′2″), Σ2′ (= J1′2′ + J2′3′+J2′2″), Σ2″ (= J1′2″ + J 2″3′ + J2′2″) and Σ3′ (= J2′3′ + J2″3′ + J3′4′). These sums of couplings correspond to the distance between the outer peaks of the H1′, H2′, H2″ and H3′ {³¹P} resonances, respectively, (except for Σ2′ and Σ2″ in the case of a small chemical shift difference between the H2′ and H2″ resonances) and can often be obtained from ¹H-NMR spectra via first-order measurement, obviating the necessity of a computer-assisted simulation of the fine structure of these resonances. Two different types of graphs for the interpretation of the coupling data are discussed: the first type of graph serves to probe as to whether or not the sugar ring occurs as a single conformer, and if so to analyze the coupling data in terms of the geometry of this sugar ring. In cases where the sugar ring does not occur as a single conformer, but as a blend of N-and S-type sugar puckers, the second type of graph is used to analyze the coupling data in terms of the geometry and population of the most abundant form.

It is shown that the latter type of analysis can be carried out on the basis of experimental values for merely Σ1′, Σ2′ and Σ2″, without any assumptions or restrictions concerning a relation between the geometry of the N- and S-type conformer. In addition, the question is discussed as to how insight can be gained into the conformational purity of the sugar ring from the observed fine structure of the H1′ resonance. Finally, a comparison is made between experimental coupling data reported for single-stranded and duplex DNA fragments and covalent RNA-DNA hybrids on the one hand and the predicted couplings and sums of couplings presented in this paper on the other hand.     

Sex-specific differential survival of extra-pair and within-pair offspring in song sparrows, Melospiza melodia

It is widely hypothesized that the evolution of female extra-pair reproduction in socially monogamous species reflects indirect genetic benefits to females. However, a critical prediction of this hypothesis, that extra-pair young (EPY) are fitter than within-pair young (WPY), has rarely been rigorously tested. We used 18 years of data from free-living song sparrows, Melospiza melodia, to test whether survival through major life-history stages differed between EPY and WPY maternal half-siblings. On average, survival of hatched chicks to independence from parental care and recruitment, and their total lifespan, did not differ significantly between EPY and WPY. However, EPY consistently tended to be less likely to survive, and recruited EPY survived for significantly fewer years than recruited WPY. Furthermore, the survival difference between EPY and WPY was sex-specific; female EPY were less likely to survive to independence and recruitment and lived fewer years than female WPY, whereas male EPY were similarly or slightly more likely to survive and to live more years than male WPY. These data indicate that extra-pair paternity may impose an indirect cost on females via their female offspring and that sex-specific genetic, environmental or maternal effects may shape extra-pair reproduction.     

Electron tunneling in proteins: role of the intervening medium

Analysis of electron-transfer (ET) kinetics data obtained from experiments on Ru-modified proteins (azurin, cytochrome c, myoglobin) and the bacterial photosynthetic reaction center reveals that distant donor-acceptor electronic couplings depend upon the secondary structure of the intervening polypeptide matrix. The β-sheet azurin structure efficiently and isotropically mediates coupling with an exponential distance-decay constant of 1.1?Å^–1. The experimentally derived distance-decay constant of 1.4?Å^–1 for long-range ET in myoglobin and the reaction center suggests that hydrogen-bond couplings are weaker through α helices than across β sheets. The donor-acceptor interactions of systems with comparable tunneling energies fall into two coupling zones: the β zone (bounded by distance-decay constants of 0.9?and 1.15 Å^–1) includes all the β-sheet (azurin) couplings and all but one coupling in cytochrome c; the α zone (boundaries: 1.25 and 1.6?Å^–1) includes less strongly coupled donor-acceptor pairs in myoglobin and the reaction center as well as a relatively weakly coupled pair in cytochrome c.     

Structural dynamics of protein backbone φ angles: extended molecular dynamics simulations versus experimental 3 J scalar couplings

³ J scalar couplings report on the conformational averaging of backbone φ angles in peptides and proteins, and therefore represent a potentially powerful tool for studying the details of both structure and dynamics in solution. We have compared an extensive experimental dataset with J-couplings predicted from unrestrained molecular dynamics simulation using enhanced sampling available from accelerated molecular dynamics or using long timescale trajectories (200 ns). The dynamic fluctuations predicted to be present along the backbone, in agreement with residual dipolar coupling analysis, are compatible with the experimental ³ J scalar couplings providing a slightly better reproduction of these experimental parameters than a high-resolution static structure.     

HNCO-based measurement of one-bond amide <Superscript>15</Superscript>N-<Superscript>1</Superscript>H couplings with optimized precision

A pair of 3D HNCO-based experiments have been developed with the aim of optimizing the precision of measurement of ¹J_NH couplings. Both pulse sequences record ¹J_NH coupling evolution during the entire constant time interval that ¹⁵N magnetization is dephasing or rephasing with respect to the directly bonded ¹³C′ nucleus, with ¹⁵N¹³C′ multiple quantum coherence maintained during the ¹³C′ evolution period. The first experiment, designed for smaller proteins, produces an apparent doubling of the ¹J_NH coupling without any accompanying increases in line width. The second experiment is a J-scaled TROSY-HNCO experiment in which the ¹J_NH coupling is measured by frequency difference between resonances offset symmetrically about the position of the downfield component of the ¹⁵N doublet (i.e. the TROSY resonance). This experiment delivers significant gains in precision of ¹J_NH coupling measurement compared to existing J-scaled TROSY-HNCO experiments. With the proper choice of acquisition parameters and sufficient sensitivity to acquire a 3D TROSY-HNCO experiment, it is shown that ¹J_NH couplings can be measured with a precision which approaches or exceeds the precision of measurement with which the frequency of the TROSY resonance itself can be determined.     

Are extra-pair young better than within-pair young? A comparison of survival and dominance in alpine marmot

1. In socially monogamous species, females may seek extra-pair copulation to gain genetic benefits. In order to test this 'genetic quality' hypothesis, one must compare the performance of extra-pair young (EPY) and within-pair young (WPY). Such tests, however, are scarce and results published so far are inconclusive. 2. Here, we test the 'genetic quality' hypothesis using multistate capture-recapture models to compare age-specific survival and access to dominance between EPY and WPY in the alpine marmot Marmota marmota, a socially monogamous mammal showing extra-pair paternities. 3. When compared with WPY, survival of EPY was higher by 15%, 10% and 30%, for juveniles, yearlings and 2-year-old individuals, respectively. Survival at older ages did not differ. 4. Survival corresponded to true survival for yearlings and juveniles as dispersal does not occur before 2 years of age in marmots. For older individuals, survival estimates included a mixture of survival and dispersal. The 30% increase of the 2-year-old EPY survival might reflect delayed dispersal rather than high survival of EPY as compared with WPY. 5. WPY and EPY had the same probability (0.28) to access dominance at 2 years of age, but EPY were more successful at older ages than WPY (0.46 vs. 0.10). 6. Both survival and reproductive performance were higher in EPY than in WPY. The fitness advantages of adopting such a mixed mating tactic are thus likely to be high for marmot females. We suggest that obtaining genetic benefits is the main evolutionary force driving extra-pair paternity in alpine marmots.     

Overall structure and sugar dynamics of a DNA dodecamer from homo- and heteronuclear dipolar couplings and 31P chemical shift anisotropy

The solution structure of d(CGCGAATTCGCG)₂ has been determined on the basis of an exceptionally large set of residual dipolar couplings. In addition to the heteronuclear ¹³C-¹H and ¹⁵N-¹H and qualitative homonuclear ¹H-¹H dipolar couplings, previously measured in bicelle medium, more than 300 quantitative ¹H-¹H and 22 ³¹P-¹H dipolar restraints were obtained in liquid crystalline Pf1 medium, and 22 ³¹P chemical shift anisotropy restraints. High quality DNA structures can be obtained solely on the basis of these new restraints, and these structures are in close agreement with those calculated previously on the basis of ¹³C-¹H and ¹⁵N-¹H dipolar couplings. In the newly calculated structures, ³¹P-¹H dipolar and ³Jsub H3 P sub couplings and ³¹P CSA data restrain the phosphodiester backbone torsion angles. The final structure represents a quite regular B-form helix with a modest bending of 10°, which is essentially independent of whether or not electrostatic terms are used in the calculation. Combined, the number of homo- and heteronuclear dipolar couplings significantly exceeds the number of degrees of freedom in the system. Results indicate that the dipolar coupling data cannot be fit by a single structure, but are compatible with the presence of rapid equilibria between C2-endo and C3-endo deoxyribose puckers (sugar switching). The C2-H2/H2 dipolar couplings in B-form DNA are particularly sensitive to sugar pucker and yield the largest discrepancies when fit to a single structure. To resolve these discrepancies, we suggest a simplified dipolar coupling analysis that yields N/S equilibria for the ribose sugar puckers, which are in good agreement with previous analyses of NMR J_HH couplings, with a population of the minor C3-endo form higher for pyrimidines than for purines.     

Assignment of cytosine N3 resonances in nucleic acids via intrabase three-bond coupling to amino protons

Coherences were observed between ¹⁵N3 of cytosine and its trans amino proton (H42) using a modified gradient-based heteronuclear single quantum coherence (HSQC) pulse sequence optimized for three-bond proton-nitrogen couplings. The method is demonstrated with a 22-nucleotide RNA fragment of the P5abc region of a group I intron uniformly labeled with ¹⁵N. Use of intraresidue ¹⁵ N3-amino proton couplings to assign cytosine ¹⁵ N3 signals complements the recently proposed J_NN HNN COSY [Dingley, A.J. and Grzesiek, S. (1998) J. Am. Chem. Soc., 120, 8293–8297] method of identifying hydrogen-bonded base pairs in RNA.     

Methyl β-lactoside: 600-MHz H- and 75-MHz C-n.m.r. studies of H- and C-enriched compounds

Using UDP-d-galactose : 2-acetamido-2-deoxy-d-glucose 4-β-d-galactosyltransferase (EC 2.4.1.22), several methyl β-lactosides have been prepared with ²H- and/or ¹³C-enrichment at specific sites to facilitate study by ¹³C (75 MHz) and ¹H (600 MHz) n.m.r. spectroscopy. ¹³C-Chemical shift assignments were verified and the ¹H-spectrum of β-lactoside was fully assigned. Sites of enrichment were selected to permit all of the potential three-bond C-C and C-H couplings through the glycosidic bond to be obtained. Replacement of H-3 of the d-glucose residue of methyl β-lactoside with ²H allowed resolution of C-1–H-4′ coupling in the 600-MHz ¹H-spectrum. Single or multiple ¹³C-enrichment at C-1, C-2, C-3, C-1′, C-3′, and/or C-4′ in the disaccharide allowed observation of intra- and inter-residue couplings. ¹³C-Spin-lattice relaxation-times (T₁) are interpreted in terms of molecular motion in solution. The data suggest that methyl β-lactoside has an extended conformation with little rotation about the glycosidic bond. Inter-residue couplings are best explained by tortion angles of φ ~ 40° and ψ ~ 15°, indicating that the conformations of β-lactoside in solution and in the crystal are similar.     

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.     

Determination of residual dipolar couplings in homonuclear MOCCA-SIAM experiments

In solutions with partial molecular alignment, anisotropic magnetic interactions such as the chemical shift anisotropy, the electric quadrupole interaction, and the magnetic dipole-dipole interaction are no longer averaged out to zero in contrast to isotropic solutions. The resulting residual anisotropic magnetic interactions are increasingly used in biological NMR studies for the determination of 3D structures of proteins and other biomolecules. In the present paper we propose a new approach allowing the measurement of residual H^N-H dipolar couplings of non-isotope enriched proteins based on the application of the MOCCA-SIAM experiment. This experiment allows the measurement of homonuclear coupling constants with an accuracy of ca. ±0.2 Hz and is therefore particularly well suited to determine residual dipolar couplings at relatively low degrees of molecular orientation. The agreement between experimentally determined residual H^N-H couplings and calculated values is demonstrated for BPTI.     

Facile measurement of <Superscript>1</Superscript>H–<Superscript>15</Superscript>N residual dipolar couplings in larger perdeuterated proteins

We present a simple method, ARTSY, for extracting ¹J_NH couplings and ¹H–¹⁵N RDCs from an interleaved set of two-dimensional ¹H–¹⁵N TROSY-HSQC spectra, based on the principle of quantitative J correlation. The primary advantage of the ARTSY method over other methods is the ability to measure couplings without scaling peak positions or altering the narrow line widths characteristic of TROSY spectra. Accuracy of the method is demonstrated for the model system GB3. Application to the catalytic core domain of HIV integrase, a 36 kDa homodimer with unfavorable spectral characteristics, demonstrates its practical utility. Precision of the RDC measurement is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC spectrum, and is approximately given by 30/(S/N) Hz.     

Determination of 3J(C,P) and 3J(H,P) coupling constants in nucleotide oligomers with FIDS-HSQC

Summary A method for measuring J(C,P) and J(H,P) coupling constants is presented, based on fitting a target multiplet containing the heteronuclear coupling to a reference multiplet that lacks the heteronuclear coupling. In DNA and RNA oligonucleotides, information on backbone torsion angles can be obtained from these couplings. Experimental multiplets are obtained from ³¹P-coupled and ³¹P-decoupled ¹H, ¹³C HSQC spectra of Rp-cyclic methylphosphonate. The accuracy to which the heteronuclear coupling constants can be determined depends on the signal-to-noise ratio of the experimental data and is analyzed in detail.Dedicated to Prof. R.R. Ernst on the occasion of his 60th birthday.     

Heteronuclear relayed E.COSY applied to the determination of accurate 3J(HN,C′) and 3J(Hβ,C′) coupling constants in Desulfovibrio vulgaris flavodoxin

Summary A simple constant-time 3D heteronuclear NMR pulse sequence has been developed to quantitatively determine the heteronuclear three-bond couplings ³J(H^N,C) and ³J(H,C) in uniformly ¹³C-enriched proteins. The protocols for measuring accurate coupling constants are based on ¹H,¹³C-heteronuclear relayed E.COSY [Schmidt, J.M., Ernst, R.R., Aimoto, S. and Kainosho, M. (1995) J. Biomol. NMR, 6, 95–105] in combination with numerical least-squares spectrum evaluation. Accurate coupling constants are extracted from 2D spectrum projections using 2D multiplet simulation. Confidence intervals for the obtained three-bond coupling constants are calculated from F-statistics. The three-bond couplings are relevant to the determination of and X ₁ dihedral-angle conformations in the amino acid backbone and side chain. The methods are demonstrated on the recombinant ¹³C, ¹⁵N-doubly enriched 147-amino acid protein Desulfovibrio vulgaris flavodoxin with bound flavin mononucleotide in its oxidized form. In total, 109 ³J(H^N,C) and 100 ³J(H,C) coupling constants are obtained from a single spectrum.Abbreviations ANOVA analysis of variances - COSY correlated spectroscopy - E.COSY exclusive correlation spectroscopy - FMN flavin mononucleotide - HMQC heteronuclear multiple-quantum coherence - HSQC heteronuclear single-quantum coherence     

31P NMR Studies on the Putative Chemoselective Activation of Nucleoside-3′-thiophosphate-O-esters

Abstract

P-diastereomerically pure O-esters of N^Bz-5′-DMT-dA-3′-monothiophosphate, having charged S?P?O^? moiety, have been synthesized. Chemoselectivity of their activations by formation of different mixed anhydrides, followed by couplings with N^Bz-3′-levulinyl-dA, were studied by ³¹P NMR spectroscopy.     

Interference between Cross-correlated Relaxation and the Measurement of Scalar and Dipolar Couplings by Quantitative J

The effects of cross-correlated relaxation in Quantitative J methods are analyzed. One-bond ¹H–¹³C scalar and dipolar couplings of protein methine and methylene sites are obtained by monitoring proton and carbon magnetization in Quantitative J experiments. We find that scalar and dipolar couplings of the same pair of nuclei vary depending on the type of magnetization involved. These discrepancies can be as large as several Hz for methylene moieties. The contribution of dynamic frequency shifts, which are known to affect J couplings, is too small to explain the observed differences. We show that processes of magnetization transfer originated by cross-correlated relaxation are largely responsible for these discrepancies. We estimate the error transferred to methylene J values by cross-correlation interference, and show that is close to the experimentally observed one. Furthermore, this analysis indicates that cross-correlated relaxation effects under isotropic and anisotropic media differ, indicating that errors are not cancelled in residual dipolar coupling measurements.     

HIFI-C: a robust and fast method for determining NMR couplings from adaptive 3D to 2D projections

We describe a novel method for the robust, rapid, and reliable determination of J couplings in multi-dimensional NMR coupling data, including small couplings from larger proteins. The method, “High-resolution Iterative Frequency Identification of Couplings” (HIFI-C) is an extension of the adaptive and intelligent data collection approach introduced earlier in HIFI-NMR. HIFI-C collects one or more optimally tilted two-dimensional (2D) planes of a 3D experiment, identifies peaks, and determines couplings with high resolution and precision. The HIFI-C approach, demonstrated here for the 3D quantitative J method, offers vital features that advance the goal of rapid and robust collection of NMR coupling data. (1) Tilted plane residual dipolar couplings (RDC) data are collected adaptively in order to offer an intelligent trade off between data collection time and accuracy. (2) Data from independent planes can provide a statistical measure of reliability for each measured coupling. (3) Fast data collection enables measurements in cases where sample stability is a limiting factor (for example in the presence of an orienting medium required for residual dipolar coupling measurements). (4) For samples that are stable, or in experiments involving relatively stronger couplings, robust data collection enables more reliable determinations of couplings in shorter time, particularly for larger biomolecules. As a proof of principle, we have applied the HIFI-C approach to the 3D quantitative J experiment to determine N-C′ RDC values for three proteins ranging from 56 to 159 residues (including a homodimer with 111 residues in each subunit). A number of factors influence the robustness and speed of data collection. These factors include the size of the protein, the experimental set up, and the coupling being measured, among others. To exhibit a lower bound on robustness and the potential for time saving, the measurement of dipolar couplings for the N-C′ vector represents a realistic “worst case analysis”. These couplings are among the smallest currently measured, and their determination in both isotropic and anisotropic media demands the highest measurement precision. The new approach yielded excellent quantitative agreement with values determined independently by the conventional 3D quantitative J NMR method (in cases where sample stability in oriented media permitted these measurements) but with a factor of 2–5 in time savings. The statistical measure of reliability, measuring the quality of each RDC value, offers valuable adjunct information even in cases where modest time savings may be realized. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pressure alters electronic orbital overlap in hydrogen bonds

Pressure-induced changes in ^3h J _NC scalar couplings through hydrogen bonds were investigated in the immunoglobulin binding domain of streptococcal protein G. ¹H, ¹⁵N and ¹³C triple-resonance NMR spectroscopy coupled with the on-line high pressure cell technique was used to monitor ^3h J _NC scalar couplings at 30 and 2000 bar in uniformly labeled ¹⁵N and ¹³C protein isotopes. Both increased and decreased ^3h J _NC scalar couplings were observed at high pressure. No correlation with secondary structure was apparent. The difference in coupling constants as well as pressure-induced chemical shift data suggests a compaction of the helix ends and an increase of the helix pitch at its center in response to pressure. Our data provides the first direct evidence that the electronic orbital overlap in protein backbone hydrogen bonds is altered by pressure.     

Solid-State NMR Evidence for β-Hairpin Structure within MAX8 Designer Peptide Nanofibers

MAX8, a designer peptide known to undergo self-assembly following changes in temperature, pH, and ionic strength, has demonstrated usefulness for tissue engineering and drug delivery. It is hypothesized that the self-assembled MAX8 nanofiber structure consists of closed β-hairpins aligned into antiparallel β-sheets. Here, we report evidence from solid-state NMR spectroscopy that supports the presence of the hypothesized β-hairpin conformation within the nanofiber structure. Specifically, our ¹³C-¹³C two-dimensional exchange data indicate spatial proximity between V3 and K17, and ¹³C-¹³C dipolar coupling measurements reveal proximity between the V3 and V18 backbone carbonyls. Moreover, isotopic dilution of labeled MAX8 nanofibers did not result in a loss of the ¹³C-¹³C dipolar couplings, showing that these couplings are primarily intramolecular. NMR spectra also indicate the existence of a minor conformation, which is discussed in terms of previously hypothesized nanofiber physical cross-linking and possible nanofiber polymorphism.     

Observation of H-bond mediated 3hJH2H3 coupling constants across Watson-Crick AU base pairs in RNA

^3hJ_H2H3trans-hydrogen bond scalar coupling constants have been observed for the first time in Watson-Crick AU base pairs in uniformly ¹⁵N-labeled RNA oligonucleotides using a new ^2hJ_NN-HNN-E. COSY experiment. The experiment utilizes adenosine H2 (AH2) for original polarization and detection, while employing ^2hJ_NNcouplings for coherence transfer across the hydrogen bonds (H-bonds). The H3 protons of uracil bases are unperturbed throughout the experiment so that these protons appear as passive spins in E. COSY patterns. ^3hJ_H2H3coupling constants can therefore be accurately measured in the acquisition dimension from the displacement of the E. COSY multiplet components, which are separated by the relatively large ¹J_H3N3coupling constants in the indirect dimension of the two-dimensional experiment. The ^3hJ_H2H3scalar coupling constants determined for AU base pairs in the two RNA hairpins examined here have been found to be positive and range in magnitude up to 1.8 Hz. Using a molecular fragment representation of an AU base pair, density functional theory/finite field perturbation theory (DFT/FPT) methods have been applied to attempt to predict the relative contributions of H-bond length and angular geometry to the magnitude of ^3hJ_H2H3coupling constants. Although the DFT/FPT calculations did not reproduce the full range of magnitude observed experimentally for the ^3hJ_H2H3coupling constants, the calculations do predict the correct sign and general trends in variation in size of these coupling constants. The calculations suggest that the magnitude of the coupling constants depends largely on H-bond length, but can also vary with differences in base pair geometry. The dependency of the ^3hJ_H2H3coupling constant on H-bond strength and geometry makes it a new probe for defining base pairs in NMR studies of nucleic acids.