Modeling 2hJiso(N, N) in nucleic acid base pairs: Ab initio characterization of the 2hJ(N, N) tensor in the methyleneimine dimer as a function of hydrogen bond geometry

The observation of ^2h J _iso(N, N) coupling has prompted considerable interest in this phenomenon from experimentalists and theoreticians due to the potential these couplings hold for the determination of secondary and tertiary structure in biologically important molecules. Here, we present an ab initio (MCSCF) study of the complete ^2h J(N, N) tensor for a model methyleneimine dimer system as a function of (i) the N-N separation, r _NN, and (ii) the hydrogen bond angle, . This simple system models the ^2h J(N, N) tensor of nucleic acid base pairs. Results indicate that although the Fermi-contact mechanism dominates ^2h J _iso(N, N), the coupling tensor is anisotropic due to contributions from the Fermi-contact spin-dipolar cross term. The variation in ^2h J _iso(N, N) as a function of r _NN is fit to an exponential decay. The influence of on the coupling constant is less pronounced but must be considered if experimental coupling constants are to be used for quantitative structure determination. Our results for this simple model system demonstrate that ^2h J _iso(N, N) is a valuable probe of hydrogen bonding in nucleic acid base pairs.     

1H-1H correlations across N–H•••N hydrogen bonds in nucleic acids

In 2HJ(NN)-COSY experiments, which correlate protons with donor/acceptor nitrogens across Nd...HNa bonds, the receptor nitrogen needs to be assigned in order to unambiguously identify the hydrogen bond. For many situations this is a non-trivial task which is further complicated by poor dispersion of (Na,Nd) resonances. To address these problems, we present pulse sequences to obtain direct, internucleotide correlations between protons in uniformly 13C/15N labeled nucleic acids containing Nd...HNa hydrogen bonds. Specifically, the pulse sequence H2(N1N3)H3 correlates H2(A,omega1):H3(U,omega2) protons across Watson-CrickA-U and mismatched G.A base pairs, the sequences H5(N3N1)H1/H6(N3N1)H1 correlate H5(C,omega1)/H6(C,omega1):H1(G,omega2) protons across Watson-Crick G-C base pairs, and the H2(N2N7)H8 sequence correlates NH2(G,A,C;omega1):H8(G,A;omega2) protons across G.G, A.A, sheared G.A and other mismatch pairs. These 1H-1H connectivities circumvent the need for independent assignment of the donor/acceptor nitrogen and related degeneracy issues associated with poorly dispersed nitrogen resonances. The methodology is demonstrated on uniformly 13C/15N labeled samples of (a) an RNA regulatory element involving the HIV-1 TAR RNA fragment, (b) a multi-stranded DNA architecture involving a G.(C-A) triad-containing G-quadruplex and (c) a peptide-RNA complex involving an evolved peptide bound to the HIV-1 Rev response element (RRE) RNA fragment.     

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.     

C, N CP MAS and high resolution multinuclear NMR study of methyl

Hydrogen bond networks stabilize RNA secondary and tertiary structure and are thus essentially important for protein recognition. During structure refinements using either NMR or X-ray techniques, hydrogen bonds were usually inferred indirectly from the proximity of donor and acceptor functional groups. Recently, quantitative heteronuclear J(N,N)-HNN COSY NMR experiments were introduced that allowed the direct identification of donor and acceptor nitrogen atoms involved in hydrogen bonds. However, protons involved in base pairing interactions in nucleic acids are often not observable due to exchange processes. The application of a modified quantitative J(N,N)-HNN COSY pulse scheme permits observation of ^2hJ(N,N) couplings via non-exchangeable protons. This approach allowed the unambiguous identification of the A27·U23 reverse Hoogsteen base pair involved in a U-A·U base triple in the HIV-2 transactivation response element–argininamide complex. Despite a wealth of NOE information, direct evidence for this interaction was lacking due to the rapid exchange of the U23 imino proton. The ability to directly observe hydrogen bonds, even in D₂O and in the presence of rapid exchange, should facilitate structural studies of RNA.     

Detection of N-H...N hydrogen bonding in RNA via scalar couplings in the absence of observable imino proton resonances

Hydrogen bond networks stabilize RNA secondary and tertiary structure and are thus essentially important for protein recognition. During structure refinements using either NMR or X-ray techniques, hydrogen bonds were usually inferred indirectly from the proximity of donor and acceptor functional groups. Recently, quantitative heteronuclear J(N,N)-HNN COSY NMR experiments were introduced that allowed the direct identification of donor and acceptor nitrogen atoms involved in hydrogen bonds. However, protons involved in base pairing interactions in nucleic acids are often not observable due to exchange processes. The application of a modified quantitative J(N,N)-HNN COSY pulse scheme permits observation of ^2hJ(N,N) couplings via non-exchangeable protons. This approach allowed the unambiguous identification of the A27·U23 reverse Hoogsteen base pair involved in a U-A·U base triple in the HIV-2 transactivation response element–argininamide complex. Despite a wealth of NOE information, direct evidence for this interaction was lacking due to the rapid exchange of the U23 imino proton. The ability to directly observe hydrogen bonds, even in D₂O and in the presence of rapid exchange, should facilitate structural studies of RNA.     

Identification of NH...N hydrogen bonds by magic angle spinning solid state NMR in a double-stranded RNA associated with myotonic dystrophy

RNA plays a central role in biological processes and exhibits a variety of secondary and tertiary structural features that are often stabilized via hydrogen bonds. The distance between the donor and acceptor nitrogen nuclei involved in NH…N hydrogen bonds in nucleic acid base pairs is typically in the range of 2.6–2.9 Å. Here, we show for the first time that such spatial proximity between ¹⁵N nitrogen nuclei can be conveniently monitored via magic angle spinning solid state NMR on a uniformly ¹⁵N-labelled RNA. The presence of NH…N hydrogen bonds is reflected as cross-peaks between the donor and acceptor nitrogen nuclei in 2D ¹⁵N dipolar chemical shift correlation spectra. The RNA selected for this experimental study was a CUG repeat expansion implicated in the neuromuscular disease myotonic dystrophy. The results presented provide direct evidence that the CUG repeat expansion adopts a double-stranded conformation.     

Characterization of the hydrogen bond network in guanosine quartets by internucleotide 3hJNC′ and 2hJNN scalar couplings

Scalar coupling correlations across hydrogen bonds with carbonyl groups as acceptors have been observed in a variety of proteins, but not in nucleic acids. Here we present a pulse scheme that allows such an observation and quantification of trans-hydrogen bond ^3hJ_NC correlations in nucleic acid base pairs, between the imino nitrogen ¹⁵N1 and the carbonyl ¹³C6 nuclei within the guanine quartets of the Oxy-1.5 DNA-quadruplex. Intra- and internucleotide N-H···O=C connectivities can be traced around each guanine quartet, allowing the hydrogen bonding partners to be unambiguously assigned. Absolute values of the ^3hJ_NC couplings are approximately 0.2 Hz as quantified by a selective long-range H(N)CO experiment and are thus on average smaller than the analogous ^3hJ_NC couplings observed in proteins. In addition, an improved version of the pseudo-heteronuclear H(N)N-COSY [Majumdar et al. (1999) J. Biomol. NMR, 14, 67–70] is presented which allows simultaneous detection of the ¹⁵N-donor and ¹⁵N-acceptor resonances connected by ^2hJ_NN couplings in hydrogen bonds involving amino groups. Using this experiment, values ranging between 6 and 8 Hz are determined for the ^2hJ_NN couplings between ¹⁵N2 and ¹⁵N7 nuclei in the guanine quartet. These values are not strongly influenced by the presence of a significant amount of chemical exchange broadening due to amino group rotations.     

Observation of internucleotide NH...N hydrogen bonds in the absence of directly detectable protons

Several structural motifs found in nucleic acids involve N-H...N hydrogen bonds in which the donor hydrogens are broadened to extinction due to chemical or conformational exchange. In such situations, it is impossible to use the well-established HNN-COSY or soft HNN-COSY experiments, which report the presence of the hydrogen bond directly on the donor proton(s). We present a pulse sequence, H(CN)N(H), for alleviating this problem in hydrogen bonds of the type NdH...Na-CH, in which the donor Nd nitrogen is correlated with the corresponding non-exchangeable C-H proton associated with the acceptor Na nitrogen. In this way, missing NdH...Na correlations in an HNN-COSY spectrum may be recovered from CH-Nd correlations in the H(CN)N(H) spectrum. By correlating a different set of nuclei relative to the HNN-COSY class of experiments, the H(CN)N(H) experiment also serves to remove ambiguities associated with degeneracies in HNN-COSY spectra. The technique is demonstrated on d(GGAGGAG)4,a quadruplex containing a novel A. (G.G.G.G). A hexad and on d(GGGCAGGT)4, containing a G.G.G.C tetrad, in which missing NH2...N7 correlations are retrieved via H8-(N2,N6) correlations in the H(CN)N(H) spectrum.     

Conformation of 4-thio-l-lyxono-1,4-lactone in solution and in the crystalline state

The conformation in ²H₂O of 4-thio-l-lyxono-1,4-lactone (1) was studied by nuclear magnetic resonance spectroscopy, by means of homonuclear (J_1H,1H) and heteronuclear (J_1H,13C) coupling constants. The couplings were directly measured by a two-dimensional heteronucleus-coupled ω₁ hetero-half-filtered proton-proton correlation (HETLOC) experiment, which does not require ¹³C isotopic enrichment. In solution, the thiolactone ring of 1 adopts preferentially the E₃ conformation, and its hydroxymethyl group populates mainly the gt rotamer. The X-ray diffraction data of a single crystal of 1 indicates that also in the solid state the thiolactone ring adopts an E₃ conformation, with a puckering somewhat larger than that observed for aldono-1,4-lactones and furanose rings. The molecules are linked by hydrogen bonds, which form chains. Particularly, O-5 is fully engaged as donor and acceptor in hydrogen bonding and the rotameric conformation of the hydroxymethyl group of 1 is fixed in the tg form.     

Conformation of 4-thio- -lyxono-1,4-lactone in solution and in the crystalline state

The conformation in ²H₂O of 4-thio- -lyxono-1,4-lactone (1) was studied by nuclear magnetic resonance spectroscopy, by means of homonuclear (J_1H,1H) and heteronuclear (J_1H,13C) coupling constants. The couplings were directly measured by a two-dimensional heteronucleus-coupled ω₁ hetero-half-filtered proton-proton correlation (HETLOC) experiment, which does not require ¹³C isotopic enrichment. In solution, the thiolactone ring of 1 adopts preferentially the E₃ conformation, and its hydroxymethyl group populates mainly the gt rotamer. The X-ray diffraction data of a single crystal of 1 indicates that also in the solid state the thiolactone ring adopts an E₃ conformation, with a puckering somewhat larger than that observed for aldono-1,4-lactones and furanose rings. The molecules are linked by hydrogen bonds, which form chains. Particularly, O-5 is fully engaged as donor and acceptor in hydrogen bonding and the rotameric conformation of the hydroxymethyl group of 1 is fixed in the tg form.     

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.     

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.     

Sensitivity enhanced NMR spectroscopy by quenching scalar coupling mediated relaxation: Application to the direct observation of hydrogen bonds in 13C/15N-labeled proteins

In this paper, we demonstrate that the sensitivity of triple-resonance NMR experiments can be enhanced significantly through quenching scalar coupling mediated relaxation by using composite-pulse decoupling (CPD) or an adiabatic decoupling sequence on aliphatic, in particular alpha-carbons in ¹³C/¹⁵N-labeled proteins. The CPD-HNCO experiment renders 50% sensitivity enhancement over the conventional CT-HNCO experiment performed on a 12 kDa FK506 binding protein, when a total of 266 ms of amide nitrogen–carbonyl carbon defocusing and refocusing periods is employed. This is a typical time period for the direct detection of hydrogen bonds in proteins via trans-hydrogen bond ^3h J _NC couplings. The experimental data fit theoretical analysis well. The significant enhancement in sensitivity makes the experiment more applicable to larger-sized proteins without resorting to perdeuteration.     

Simultaneous CT-13C and VT-15N chemical shift labelling: Application to 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH

Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) ¹³C and variable time (VT) ¹⁵N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/J_CC=28.6 ms). The magnetisation originating from NH protons is initially stored in the 2H_zN_z state, then prior to the VT chemical shift labelling period is converted into 2H_zN_y coherence. The VT -¹⁵N mode eliminates the effect of ¹ J _N,CO and ^1,2 J _N,CA coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH₂ groups, thus removing any potential overlap with the CH₃ signals. The CT-¹³CH₃,VT-¹⁵N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH₃NH and 3D ¹³C,¹⁵N HSQC-NOESY-CH₃NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH₃ and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH₃ and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin.     

Geometric characteristics of hydrogen bonds involving sulfur atoms in proteins

Sulfur atoms have been known to participate in hydrogen bonds (H‐bonds) and these sulfur‐containing H‐bonds (SCHBs) are suggested to play important roles in certain biological processes. This study aims to comprehensively characterize all the SCHBs in 500 high‐resolution protein structures (≤1.8 Å). We categorized SCHBs into six types according to donor/acceptor behaviors and used explicit hydrogen approach to distinguish SCHBs from those of nonhydrogen bonding interactions. It is revealed that sulfur atom is a very poor H‐bond acceptor, but a moderately good H‐bond donor. In α‐helix, considerable SCHBs were found between the sulphydryl group of cysteine residue i and the carbonyl oxygen of residue i‐4, and these SCHBs exert effects in stabilizing helices. Although for other SCHBs, they possess no specific secondary structural preference, their geometric characteristics in proteins and in free small compounds are significantly distinct, indicating the protein SCHBs are geometrically distorted. Interestingly, sulfur atom in the disulfide bond tends to form bifurcated H‐bond whereas in cysteine‐cysteine pairs prefer to form dual H‐bond. These special H‐bonds remarkably boost the interaction between H‐bond donor and acceptor. By oxidation/reduction manner, the mutual transformation between the dual H‐bonds and disulfide bonds for cysteine‐cysteine pairs can accurately adjust the structural stability and biological function of proteins in different environments. Furthermore, few loose H‐bonds were observed to form between the sulphydryl groups and aromatic rings, and in these cases the donor H is almost over against the rim rather than the center of the aromatic ring. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

A spin-state-selective experiment for measuring heteronuclear one-bond and homonuclear two-bond couplings from an HSQC-type spectrum

Recently, a set of selective 1D experiments with spin-state-selective excitation for CH spin systems was introduced by Parella and Belloc (J. Magn. Reson., 148, 78–87 (2001)). We have expanded and generalized this concept further, and demonstrated that a very simple experiment utilizing spin-state-selective filtering can be used for simultaneous measurement of heteronuclear ¹ J _NH (or ¹ J _CH) and geminal ² J _HH couplings from two-dimensional ¹⁵N-¹H (or ¹³C-¹H) correlation spectrum. The experiment has very high sensitivity owing to the preservation of equivalent coherence transfer pathways analogous to the sensitivity and gradient enhanced HSQC experiment. However, overall length of the pulse sequence is 1/(2J) shorter than the gradient selected SE-HSQC experiment. Furthermore, the spin-state-selection can be utilized between NH and NH₂ (or CH and CH₂) moieties by changing the phase of only one pulse. The pulse scheme will be useful for the measurement of scalar and residual dipolar couplings in wide variety of samples, due to its high sensitivity and artifact suppression efficiency. The method is tested on NH₂ and CH₂ moieties in ¹⁵N- and ¹⁵N/¹³C–labeled ubiquitin samples.     

HNCAN pulse sequences for sequential backbone resonance assignment across proline residues in perdeuterated proteins

A TROSY-based triple-resonance pulse scheme is described which correlates backbone ¹H and ¹⁵N chemical shifts of an amino acid residue with the ¹⁵N chemical shifts of both the sequentially preceding and following residues. The sequence employs ¹ J _NC and ² J _NC couplings in two sequential magnetization transfer steps in an `out-and-back' manner. As a result, N,N connectivities are obtained irrespective of whether the neighbouring amide nitrogens are protonated or not, which makes the experiment suitable for the assignment of proline resonances. Two different three-dimensional variants of the pulse sequence are presented which differ in sensitivity and resolution to be achieved in one of the nitrogen dimensions. The new method is demonstrated with two uniformly ²H/¹³C/¹⁵N-labelled proteins in the 30-kDa range.     

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.     

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.     

Magnetic field induced residual dipolar couplings of imino groups in nucleic acids from measurements at a single magnetic field

For base-paired nucleic acids, variations in ¹ J _NH and the imino ¹H chemical shift are both dominated by hydrogen bond length. In the absence of molecular alignment, the ¹ J _NH coupling for the imino proton then can be approximated by ¹ J _NH = (1.21Hz/ppm)δ_H − 103.5 ± 0.6 Hz, where δ_H represents the chemical shift of the imino proton in ppm. This relation permits imino residual dipolar couplings (RDCs) resulting from magnetic susceptibility anisotropy (MSA) to be extracted from measurement of (¹ J _NH + RDC) splittings at a single magnetic field strength. Magnetic field-induced RDCs were measured for tRNA^Val and the alignment tensor determined from magnetic-field alignment of tRNA^Val agrees well with the tensor calculated by summation of the MSA tensors of the individual nucleobases. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jinfa Ying, Alexander Grishaev and Michael P. Latham contributed equally to this work.