Two-and three-dimensional HCN experiments for correlating base and sugar resonances in 15N, 13C-labeled RNA oligonucleotides

Summary New 2D and 3D ¹H-¹³C-¹⁵N triple resonance experiments are presented which allow unambiguous assignments of intranucleotide H1'-H8(H6) connectivities in ¹³C-and ¹⁵N-labeled RNA oligonucleotides. Two slightly different experiments employing double INEPT forward and back coherence transfers are optimized to obtain the H1'-C1'-N9/N1 and H8/H6-C8/C6-N9/N1 connectivities, respectively. The correlation of H1' protons to glycosidic nitrogens N9/N1 is obtained in a nonselective fashion. To correlate H8/H6 with their respective glycosidic nitrogens, selective ¹³C-refocusing and ¹⁵N-inversion pulses are applied to optimize the magnetization transfers along the desired pathway. The approach employs the heteronuclear one-bond spin-spin interactions and allows the 2D ¹H-¹⁵N and 3D¹H-¹³C-¹⁵N chemical shift correlation of nuclei along and adjacent to the glycosidic bond. Since the intranucleotide correlations obtained are based exclusively on through-bond scalar interactions, these experiments resolve the ambiguity of intra-and internucleotide H1'-H8(H6) assignments obtained from the 2D NOESY spectra. These experiments are applied to a 30-base RNA oligonucleotide which contains the binding site for Rev protein from HIV.     

Resolution of the 1H-1H NOE spectrum of RNA into three dimensions using 15N-1H two-bond couplings

The feasibility of using two-bond ¹⁵N-¹H couplings to resolve the ¹H-¹H nuclear Overhauser effect spectrum of RNA into a third dimension was investigated, using the 36-nucleotide gene 32 messenger RNA pseudoknot of bacteriophage T2 as an example. The two-bond ¹⁵N-¹H couplings present in adenosine and guanosine were found to be suitable for generating a three-dimensional ¹H-¹H-¹⁵N NOESY-HSQC spectrum with reasonably good sensitivity, as well as favorable chemical shift dispersion in the nitrogen dimension. The described NMR experiment provides a tool that can be used to complement other heteronuclear methods in the analysis of RNA structure.     

1H-15N Spin–spin couplings in alumichrome

We report the determination of two- and three-bond ¹H-¹⁵N spin–spin couplings in the nmr spectra of a polypeptide. The ¹H- and ¹⁵N-nmr spectra of 99.2% ¹⁵N-enriched alumichrome have been studied at 360 MHz and 10.1 MHz, repectively. While some ²J and ³J coupling are of the order of 5 Hz, most splitting resulting from the heteronuclear interaction are ?2 Hz, which introduces strigent requirements of spectral resolution. In the ¹H spectra these requirements were met by digital deconvolution with a sine bell routine combined with positive exponential filtering. Although the ¹⁵N spectra clearly exhibit features of fine structure, mainly because of the intrinsic higher nmir sensitivity of protons, observation of ¹H-¹⁵N spin–spin couplings was found to be more practical in the ¹H than in the ¹⁵N spectra. We find that the alumichrome data do not satisfy a simple cyclic relationship linking the heteronuclear couplings to the crystallographic ψ dihedral angles. It is suggested that a formal treatment of the ψ-related interresidue ¹H-¹⁵N coupling might have to take into account a more complex dependence of the intervening ³J on the overall local electronic structure, which is dependent on ?,ψ, and ω simultaneoulsy. In contrast, our analysis indicates that χ¹ can be readily determined from the measurement of the corresponding heteronuclear ³J coupling in the ¹H^β or in the amide ¹⁵N resonances. Karplus relationships are proposed that relate this heteronuclear ³J to the corresponding dihedral angle θ and which, on average, yield      

A simultaneous 15N,1H- and 13C,1H-HSQC with sensitivity enhancement and a heteronuclear gradient echo

Summary New pulse sequences are introduced and discussed that allow for simultaneous acquisition of ¹⁵N,¹H-and ¹³C,¹H-HSQC correlations for fully ¹³C,¹⁵N-labeled biomacromolecules in combination with hetero-nuclear gradient echoes and sensitivity enhancement. The pulse sequence experimentally found to be optimal can be used as a building block, especially in time-consuming multidimensional NMR experiments. Due to the excellent solvent suppression obtained by employing heteronuclear gradient echoes, which allows detection of resonances under the water resonance, it would be possible to record two sensitivity-enhanced 4D experiments simultaneously on one sample dissolved in H₂O, e.g. a 4D ¹³C,¹H-HSQC-NOESY-¹⁵N, ¹H/¹³C,¹H-HSQC.     

Effective strategy to assign 1H-15N heteronuclear correlation NMR signals from lysine side-chain NH3 + groups of proteins at low temperature

Recent studies have shown that lysine side-chain NH₃ ⁺ groups are excellent probes for NMR investigations of dynamics involving hydrogen bonds and ion pairs relevant to protein function. However, due to rapid hydrogen exchange, observation of ¹H-¹⁵N NMR cross peaks from lysine NH₃ ⁺ groups often requires use of a relatively low temperature, which renders difficulty in resonance assignment. Here we present an effective strategy to assign ¹H and ¹⁵N resonances of NH₃ ⁺ groups at low temperatures. This strategy involves two new ¹H/¹³C/¹⁵N triple-resonance experiments for lysine side chains. Application to a protein-DNA complex is demonstrated.     

Gradient and sensitivity enhanced multiple-quantum coherence in heteronuclear multidimensional NMR experiments

Recent studies have indicated that the relaxation rate of the ¹H-¹³C multiple-quantum coherence is much slower than that of the ¹H-¹³C single-quantum coherence for non-aromatic methine sites in ¹³ C labeled proteins and in nucleic acids at the slow tumbling limit. Several heteronuclear experiments have been designed to use a multiple-quantum coherence transfer scheme instead of the single-quantum transfer method, thereby increasing the sensitivity and resolution of the spectra. Here, we report a constant time, gradient and sensitivity enhanced HMQC experiment (CT-g/s-HMQC) and demonstrate that it has a significant sensitivity enhancement over constant time HMQC and constant time gradient and sensitivity enhanced HSQC experiments (CT-g/s-HSQC) when applied to a ¹³C and ¹⁵ N labeled calmodulin sample in D₂O. We also apply this approach to 3D NOESY-HMQC and doubly sensitivity enhanced TOCSY-HMQC experiments, and demonstrate that they are more sensitive than their HSQC counterparts.     

Dipolar filtered 1H-13C heteronuclear correlation spectroscopy for resonance assignment of proteins

Resonance assignment is necessary for the comprehensive structure determination of insoluble proteins by solid-state NMR spectroscopy. While various 2D and 3D correlation techniques involving ¹³C and ¹⁵N spins have been developed for this purpose, ¹H chemical shift has not been exploited sufficiently. We demonstrate the combination of the regular ¹H-¹³C heteronuclear correlation (HETCOR) experiment and a dipolar filtered HETCOR technique to obtain better resolved ¹H chemical shift spectra. The dipolar filtered experiment, MELODI-HETCOR, simplifies the ¹H spectra by suppressing the directly bonded C-H correlation peaks and retaining only the medium- and long-range cross peaks. We apply this MELODI-HETCOR technique to several amino acids and proteins with various isotopic labeling patterns. The enhanced ¹H chemical shift resolution allows the assignment of overlapping H and H resonances in Ser, identifies the ¹H chemical shift differences between neutral and cationic imidazole rings of His, and permits the assignment of residues with side chain nitrogen atoms in ubiquitin. The potential utility of this dipolar filtered HETCOR technique to resonance assignment of extensively labeled proteins is discussed.     

Correlation of nucleotide base and sugar protons in a 15N-labeled HIV-1 RNA oligonucleotide by 1H-15N HSQC experiments

Summary The advent of methods for preparing ¹⁵N- and ¹³C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studies by NMR spectroscopy. A practical limitation is the expense of the ¹³C label. It may therefore sometimes be desirable to prepare a relatively inexpensive ¹⁵N-labeled sample only. Here we show that the two-bond ¹H-¹⁵N HSQC experiment can be used on ¹⁵N-labeled RNA to correlate the intranucleotide H1 and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected ¹H-¹⁵N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RNA oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1 and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.     

Amino acid type-selective backbone 1H-15N-correlations for Arg and Lys

Four novel amino acid type-selective triple resonance experiments to identify the backbone amino proton and nitrogen resonances of Arg and Lys and of their sequential neighbors in (¹³C,¹⁵N)-labeled proteins are presented: the R(i+1)-HSQC and R(i,i+1)-HSQC select signals originating from Arg side chains, the K(i+1)-HSQC and K(i,i+1)-HSQC select signals originating from Lys side chains. The selection is based on exploiting the characteristic chemical shifts of a pair of carbon atoms in Arg and Lys side chains using selective 90° pulses. The new experiments are recorded as two-dimensional ¹H-¹⁵N-correlations and their performance is demonstrated with the application to a protein domain of 83 amino acids.     

Identification of metabolites from 2D 1H-13C HSQC NMR using peak correlation plots

Background

Identification of individual components in complex mixtures is an important and sometimes daunting task in several research areas like metabolomics and natural product studies. NMR spectroscopy is an excellent technique for analysis of mixtures of organic compounds and gives a detailed chemical fingerprint of most individual components above the detection limit. For the identification of individual metabolites in metabolomics, correlation or covariance between peaks in ¹H NMR spectra has previously been successfully employed. Similar correlation of 2D ¹H-¹³C Heteronuclear Single Quantum Correlation spectra was recently applied to investigate the structure of heparine. In this paper, we demonstrate how a similar approach can be used to identify metabolites in human biofluids (post-prostatic palpation urine).

Results

From 50 ¹H-¹³C Heteronuclear Single Quantum Correlation spectra, 23 correlation plots resembling pure metabolites were constructed. The identities of these metabolites were confirmed by comparing the correlation plots with reported NMR data, mostly from the Human Metabolome Database.

Conclusions

Correlation plots prepared by statistically correlating ¹H-¹³C Heteronuclear Single Quantum Correlation spectra from human biofluids provide unambiguous identification of metabolites. The correlation plots highlight cross-peaks belonging to each individual compound, not limited by long-range magnetization transfer as conventional NMR experiments.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0413-z) contains supplementary material, which is available to authorized users.     

Long-Range 1H-15N Heteronuclear Shift Correlation at Natural Abundance: a Tool To Study Benzothiazole Biodegradation by Two Rhodococcus Strains

The biodegradation of benzothiazole and 2-hydroxybenzothiazole by two strains of Rhodococcus was monitored by reversed phase high-pressure liquid chromatography and by ¹H nuclear magnetic resonance (NMR). Both xenobiotics were biotransformed into a hydroxylated derivative of 2-hydroxybenzothiazole by these two strains. The chemical structure of this metabolite was determined by a new NMR methodology: long-range ¹H-¹⁵N heteronuclear shift correlation without any previous ¹⁵N enrichment of the compound. This powerful NMR tool allowed us to assign the metabolite structure to 2,6-dihydroxybenzothiazole.     

Three-dimensional 15N-1H-1H and 15N-13C-1H nuclear-magnetic resonance studies of HPr a central component of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Assignment of backbone resonances.

We have performed three-dimensional NMR studies on a central component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli, denoted as HPr. The protein was uniformly enriched with 15N and 13C to overcome spectral overlap. Complete assignments were obtained for the backbone 1H, 15N and 13C resonances, using three-dimensional heteronuclear 1H NOE 1H-15N multiple-quantum coherence spectroscopy (3D-NOESY-HMQC) and three-dimensional heteronuclear total correlation 1H-15N multiple-quantum coherence spectroscopy (3D-TOCSY-HMQC) experiments on 15N-enriched HPr and an additional three-dimensional triple-resonance 1HN-15N-13C alpha correlation spectroscopy (HNCA) experiment on 13C, 15N-enriched HPr. Many of the sequential backbone 1H assignments, as derived from two-dimensional NMR studies [Klevit, R.E., Drobny, G.P. & Waygood, E.B. (1986) Biochemistry 25, 7760-7769], were corrected. Almost all discrepancies are in the helical regions, leaving the published antiparallel beta-sheet topology almost completely intact.     

1H, 13C and 15N backbone and side-chain chemical shift assignments for oxidized and reduced desulfothioredoxin

Based on sequence homology, desulfothioredoxin (DTrx) from Desulfovibrio vulgaris Hildenborough has been identified as a new member of the thioredoxin superfamily. Desulfothioredoxin (104 amino acids) contains a particular active site consensus sequence, CPHC probably correlated to the anaerobic metabolism of these bacteria. We report the full ¹H, ¹³C and ¹⁵N resonance assignments of the reduced and the oxidized form of desulfothioredoxin (DTrx). 2D and 3D heteronuclear NMR experiments were performed using uniformly ¹⁵N-, ¹³C-labelled DTrx. More than 98% backbone and 96% side-chain ¹H, ¹³C and ¹⁵N resonance assignments were obtained. (BMRB deposits with accession number 16712 and 16713).     

Assignment of amide proton signals by combined evaluation of HN,NN and HNCA MAS-NMR correlation spectra

In this paper, we present a strategy for the ¹H^N resonance assignment in solid-state magic-angle spinning (MAS) NMR, using the -spectrin SH3 domain as an example. A novel 3D triple resonance experiment is presented that yields intraresidue H^N-N-C correlations, which was essential for the proton assignment. For the observable residues, 52 out of the 54 amide proton resonances were assigned from 2D (¹H-¹⁵N) and 3D (¹H-¹⁵N-¹³C) heteronuclear correlation spectra. It is demonstrated that proton-driven spin diffusion (PDSD) experiments recorded with long mixing times (4 s) are helpful for confirming the assignment of the protein backbone ¹⁵N resonances and as an aid in the amide proton assignment.     

NMR experiments for the sign determination of homonuclear scalar and residual dipolar couplings

A modified version of the JHH-TOCSY experiment, `signed COSY', is presented that allows the determination of the sign of residual dipolar ¹H-¹H coupling constants with respect to the sign of one-bond ¹H-X coupling constants in linear three-spin systems X-¹H-¹H, where X = ¹³C or ¹⁵N. In contrast to the original JHH-TOCSY experiments, the signs of J _HH couplings may be determined for CH₂-CH₂ moieties and for uniformly ¹³C/¹⁵N-labelled samples. In addition, sensitivity is enhanced, diagonal peaks are suppressed and cross peaks are observed only between directly coupled protons, as in a COSY spectrum.     

Synthesis of Biotin-Containing Phosphoramidite Linker with Polyether Spacer Arm

A phosphoramidite linker unit, based on glycerol backbone and containing a biotin residue attached through a tetraethylene glycol spacer arm, was synthesized. DMTr-Glycidol and tetraethylene glycol were used as starting materials. After conversion of one of hydroxy groups in tetraethylene glycol into an amino group, the epoxy cycle in DMTr-glycidol was opened by this amino alcohol, resulting in the corresponding ether and some quantity of secondary amine. After attaching of biotin residue to the ether followed by phosphitylation, the desirable linker was obtained. The structure of the linker was confirmed by ¹H-¹H COSY, ¹H-¹³C HSQC, ¹H-¹³C HMBC, ¹H-¹⁵N HSQC, and ¹H-¹⁵N HMBC spectra. The resulted phosphoramidite linker unit is suitable for use in common DNA synthesizers. This approach can be used for preparation of various modifiers containing reporter groups attached to the primary amino function using conventional procedures.     

Efficient resonance assignment of proteins in MAS NMR by simultaneous intra- and inter-residue 3D correlation spectroscopy

Resonance assignment is the first step in NMR structure determination. For magic angle spinning NMR, this is typically achieved with a set of heteronuclear correlation experiments (NCaCX, NCOCX, CONCa) that utilize SPECIFIC-CP ¹⁵N–¹³C transfers. However, the SPECIFIC-CP transfer efficiency is often compromised by molecular dynamics and probe performance. Here we show that one-bond ZF-TEDOR ¹⁵N–¹³C transfers provide simultaneous NCO and NCa correlations with at least as much sensitivity as SPECIFIC-CP for some non-crystalline samples. Furthermore, a 3D ZF-TEDOR-CC experiment provides heteronuclear sidechain correlations and robustness with respect to proton decoupling and radiofrequency power instabilities. We demonstrate transfer efficiencies and connectivities by application of 3D ZF-TEDOR-DARR to a model microcrystalline protein, GB1, and a less ideal system, GvpA in intact gas vesicles.     

Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of ¹H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond ¹H–¹⁵N or ¹H–¹³C couplings. However, by filtering against protons directly bonded to ¹³C or ¹⁵N nuclei, signals from slowly-exchanging hydroxyls can be observed in the ¹H-NMR spectrum of a uniformly ¹³C/¹⁵N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with ¹³C/¹⁵N-serine or ¹³C/¹⁵N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via ³J_C–OH couplings in long-range ¹³C-HSQC spectra. These scalar couplings (~5–7 Hz) were measured in a sample of uniformly ¹³C/¹⁵N-labeled BcX using a quantitative ¹³C/¹⁵N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a ¹³C/¹⁵N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.     

Synthesis and Structure Assignment of 1-(2-Acetoxyethoxy)methyl Derivatives of 5-Chloro-6-azauracil and 5-Bromo-6-azaisocytosine

Abstract

1-[(2-Acetoxyethoxy)methyl]-5-chloro-6-azauracil has been prepared and its unambiguous assignment of ¹H and ¹³C peaks through the ¹H-¹³C heteronuclear correlation (HETCOR) NMR experiments is described. The isosteric 1-[(2-acetoxyethoxy)methyl]-5-bromo-6-azaisocytosine has also been synthesized. The X-Ray crystallographic analysis reveals unambiguously the site of glycosylation at N¹. Deacetylation of both acyclonucleosides provided 5-chloro-1-[(2-hydroxyethoxy)methyl]-6-azauracil and 5-bromo-1-[(2-hydroxyethoxy)methyl]-6-azaisocytosine respectively. Their structures have been well established by the NMR spectra and the elemental analyses.     

Line narrowing in spectra of proteins dissolved in a dilute liquid crystalline phase by band-selective adiabatic decoupling: Application to 1HN-15N residual dipolar coupling measurements

Residual heteronuclear dipolar couplings obtained from partially oriented protein samples can provide unique NMR constraints for protein structure determination. However, partial orientation of protein samples also causes severe ¹ H line broadening resulting from residual ¹ H-¹H dipolar couplings. In this communication we show that band-selective ¹H homonuclear decoupling during data acquisition is an efficient way to suppress residual ¹H-¹H dipolar couplings, resulting in spectra that are still amenable to solution NMR analysis, even with high degrees of alignment. As an example, we present a novel experiment with improved sensitivity for the measurement of one-bond ¹ H^N-¹⁵N residual dipolar couplings in a protein sample dissolved in magnetically aligned liquid crystalline bicelles.