Measurement of multiple psi torsion angles in uniformly 13C,15N-labeled alpha-spectrin SH3 domain using 3D 15N-13C-13C-15N MAS dipolar-chemical shift correlation spectroscopy

We demonstrate the simultaneous measurement of several backbone torsion angles psi in the uniformly (13)C,(15)N-labeled alpha-Spectrin SH3 domain using two different 3D 15N-13C-13C-15N dipolar-chemical shift magic-angle spinning (MAS) NMR experiments. The first NCCN experiment utilizes double quantum (DQ) spectroscopy combined with the INADEQUATE type 13C-13C chemical shift correlation. The decay of the DQ coherences formed between 13C'(i) and 13C(alphai) spin pairs is determined by the "correlated" dipolar field due to 15N(i)-13C(alphai) and 13C'(i)-15N(i+1) dipolar couplings and is particularly sensitive to variations of the torsion angle in the regime |psi| > 140 degrees. However, the ability of this experiment to constrain multiple psi-torsion angles is limited by the resolution of the 13C(alpha)-(13)CO correlation spectrum. This problem is partially addressed in the second approach described here, which is an NCOCA NCCN experiment. In this case the resolution is enhanced by the superior spectral dispersion of the 15N resonances present in the 15N(i+1)-13C(alphai) part of the NCOCA chemical shift correlation spectrum. For the case of the 62-residue alpha-spectrin SH3 domain, we determined 13 psi angle constraints with the INADEQUATE NCCN experiment and 22 psi constraints were measured in the NCOCA NCCN experiment.     

Frequency selective heteronuclear dipolar recoupling in rotating solids: accurate (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled peptides

We describe a magic-angle spinning NMR experiment for selective (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled solids, where multiple (13)C-(15)N and (13)C-(13)C interactions complicate the accurate measurement of structurally interesting, weak (13)C-(15)N dipolar couplings. The new experiment, termed FSR (frequency selective REDOR), combines the REDOR pulse sequence with a frequency selective spin-echo to recouple a single (13)C-(15)N dipolar interaction in a multiple spin system. Concurrently the remaining (13)C-(15)N dipolar couplings and all (13)C-(13)C scalar couplings to the selected (13)C are suppressed. The (13)C-(15)N coupling of interest is extracted by a least-squares fit of the experimentally observed modulation of the (13)C spin-echo intensity to the analytical expression describing the dipolar dephasing in an isolated heteronuclear spin pair under conventional REDOR. The experiment is demonstrated in three uniformly (13)C,(15)N-labeled model systems: asparagine, N-acetyl-L-Val-L-Leu and N-formyl-L-Met-L-Leu-L-Phe; in N-formyl-[U-(13)C,(15)N]L-Met-L-Leu-L-Phe we have determined a total of 16 internuclear distances in the 2.5-6 A range.     

Determination of multiple torsion-angle constraints in U-(13)C,(15)N-labeled peptides: 3D (1)H-(15)N-(13)C-(1)H dipolar chemical shift NMR spectroscopy in rotating solids

We demonstrate constraint of peptide backbone and side-chain conformation with 3D (1)H-(15)N-(13)C-(1)H dipolar chemical shift, magic-angle spinning NMR experiments. In these experiments, polarization is transferred from (15)N[i] by ramped SPECIFIC cross polarization to the (13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1] resonances and evolves coherently under the correlated (1)H-(15)N and (1)H-(13)C dipolar couplings. The resulting set of frequency-labeled (15)N(1)H-(13)C(1)H dipolar spectra depend strongly upon the molecular torsion angles phi[i], chi1[i], and psi[i - 1]. To interpret the data with high precision, we considered the effects of weakly coupled protons and differential relaxation of proton coherences via an average Liouvillian theory formalism for multispin clusters and employed average Hamiltonian theory to describe the transfer of (15)N polarization to three coupled (13)C spins ((13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1]). Degeneracies in the conformational solution space were minimized by combining data from multiple (15)N(1)H-(13)C(1)H line shapes and analogous data from other 3D (1)H-(13)C(alpha)-(13)C(beta)-(1)H (chi1), (15)N-(13)C(alpha)-(13)C'-(15)N (psi), and (1)H-(15)N[i]-(15)N[i + 1]-(1)H (phi, psi) experiments. The method is demonstrated here with studies of the uniformly (13)C,(15)N-labeled solid tripeptide N-formyl-Met-Leu-Phe-OH, where the combined data constrains a total of eight torsion angles (three phi, three chi1, and two psi): phi(Met) = -146 degrees, psi(Met) = 159 degrees, chi1(Met) = -85 degrees, phi(Leu) = -90 degrees, psi(Leu) = -40 degrees, chi1(Leu) = -59 degrees, phi(Phe) = -166 degrees, and chi1(Phe) = 56 degrees. The high sensitivity and dynamic range of the 3D experiments and the data analysis methods provided here will permit immediate application to larger peptides and proteins when sufficient resolution is available in the (15)N-(13)C chemical shift correlation spectra.     

1H, 13C and 15N NMR spectra of ciprofloxacin

1H NMR assignment, including the values of delta(H) and J(H,H) for the cyclopropane moiety, and 13C NMR and 15N NMR spectral data for ciprofloxacin are presented.     

Use of 13C as an indirect tag in 15N specifically labeled nucleosides. Syntheses of [8-13C-1,7,NH2-15N3]adenosine, -guanosine, and their deoxy analogues

We have previously reported the use of a 13C tag at the C2 of 15N-multilabeled purine nucleosides to distinguish the adjacent-labeled 15N atoms from those in an untagged nucleoside. We now introduce the use of an indirect tag at the C8 of 15N7-labeled purine nucleosides. This tag allows unambiguous differentiation between a pair of 15N7-labeled purines in which only one is 13C8 labeled. Although the very small C8-N7 coupling (<1 Hz) precludes its direct detection in 1D 15N spectra, 2D 1H-15N NMR experiments display the large C8-H8 coupling (>200 Hz) because H8 is coupled to both N7 and C8. The 13C8 atom is introduced by means of a ring closure of the exocyclic amino groups of a pyrimidinone using [13C]sodium ethyl xanthate. Here, we present methods for the syntheses of [8-13C-1,7,NH2-15N3]adenosine, -guanosine, and their deoxy analogues.     

Complete 1H, 13C and 15N NMR assignment of tirapazamine and related 1,2,4-benzotriazine N-oxides

1H, 13C and 15N NMR measurements (1D and 2D including 1H--15N gs-HMBC) have been carried out on 3-amino-1, 2,4-benzotriazine and a series of N-oxides and complete assignments established. N-Oxidation at any position resulted in large upfield shifts of the corresponding N-1 and N-2 resonances and downfield shifts for N-4 with the exception of the 3-amino-1,2,4-benzotriazine 1-oxide in which a small upfield shift of N-4 was observed. Density functional GIAO calculations of the 15N and 13C chemical shifts [B3LYP/6-31G(d)//B3LYP/6-311+G(2d,p)] gave good agreement with experimental values confirming the assignments. The combination of 13C and 15N NMR provides an unambiguous method for assigning the 1H and 13C resonances of N-oxides of 1,2,4-benzotriazines.     

Selective chemical shift assignment of B800 and B850 bacteriochlorophylls in uniformly [13C,15N]-labeled light-harvesting complexes by solid-state NMR spectroscopy at ultra-high magnetic field

The electronic ground states of the bacteriochlorophyll a type B800 and type B850 in the light-harvesting 2 complex of Rhodopseudomonas acidophila strain 10050 have been characterized by magic angle spinning (MAS) dipolar (13)C-(13)C correlation NMR spectroscopy. Uniformly [(13)C,(15)N] enriched light-harvesting 2 (LH2) complexes were prepared biosynthetically, while [(13)C,(15)N]-B800 LH2 complexes were obtained after reconstitution of apoprotein with uniformly [(13)C,(15)N]-enriched bacteriochlorophyll cofactors. Extensive sets of isotropic (13)C NMR chemical shifts were obtained for each bacteriochlorin ring species in the LH2 protein. (13)C isotropic shifts in the protein have been compared to the corresponding shifts of monomeric BChl a dissolved in acetone-d(6). Density functional theory calculations were performed to estimate ring current effects induced by adjacent cofactors. By correction for the ring current shifts, the (13)C shift effects due to the interactions with the protein matrix were resolved. The chemical shift changes provide a clear evidence for a global electronic effect on the B800 and B850 macrocycles, which is attributed to the dielectrics of the protein environment, in contrast with local effects due to interaction with specific amino acid residues. Considerable shifts of -6.2 < Deltasigma < +5.8 ppm are detected for (13)C nuclei in both the B800 and the B850 bacteriochlorin rings. Because the shift effects for the B800 and B850 are similar, the polarization of the electronic ground states induced by the protein environment is comparable for both cofactors and corresponds with a red shift of approximately 30 nm relative to the monomeric BChl dissolved in acetone-d(6). The electronic coupling between the B850 cofactors due to macrocycle overlap is the predominant mechanism behind the additional red shift in the B850.     

Study of stereospecificity of 1H, 13C, 15N and 77Se shielding constants in the configurational isomers of the selenophene-2-carbaldehyde azine by NMR spectroscopy and MP2-GIAO calculations

In the (1)H and (13)C NMR spectra of selenophene-2-carbaldehyde azine, the (1)H-5, (13)C-3 and (13)C-5 signals of the selenophene ring are shifted to higher frequencies, whereas those of the (1)H-1, (13)C-1, (13)C-2 and (13)C-4 are shifted to lower frequencies on going from the EE to ZZ isomer or from the E moiety to the Z moiety of EZ isomer. The (15)N chemical shift is significantly larger in the EE isomer relative to the ZZ isomer and in the E moiety relative to the Z moiety of EZ isomer. A very pronounced difference (60-65 mg/g) between the (77)Se resonance positions is revealed in the studied azine isomers, the (77)Se peak being shifted to higher frequencies in the ZZ isomer and in the Z moiety of EZ isomer. The trends in the changes of the measured chemical shifts are reasonably reproduced by the GIAO calculations at the MP2 level of the (1)H, (13)C, (15)N and (77)Se shielding constants in the energy-favorable conformation with the syn orientation of both selenophene rings relative to the C = N groups. The NBO analysis suggests that such an arrangement of the selenophene rings may take place because of a higher energy of some intramolecular interactions.     

hNCOcanH pulse sequence and a robust protocol for rapid and unambiguous assignment of backbone (1HN, 15N and 13C′) resonances in 15 N/13C‐labeled proteins

A three‐dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in ¹⁵N/¹³C‐labeled proteins. The experiment has been derived by a simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135–147]; t₂ evolution is used to frequency label ¹³C′ rather than ¹⁵N (similar trick has also been used in the design of hNCAnH pulse sequence from hNcaNH [Frueh et al., JACS, 131 (2009) 12880–12881]). The modification results in a spectrum equivalent to HNCO, but in addition to inter‐residue correlation peaks (i.e. H_i, C_i?1), the spectrum also contains additional intra‐residue correlation peaks (i.e. H_i?1, C_i?1) in the direct proton dimension which has maximum resolution. This is the main strength of the experiment and thus, even a small difference in amide ¹H chemical shifts (5–6 Hz) can be used for establishing a sequential connectivity. This experiment in combination with the HNN experiment described previously [Panchal et al., J. Biomol. NMR 20 (2001) 135–147] leads to a more robust assignment protocol for backbone resonances (¹H^N, ¹⁵N) than could be derived from the combination of HNN and HN(C)N experiments [Bhavesh et al., Biochemistry, 40 (2001) 14727–14735]. Further, this new protocol enables assignment of ¹³C′ resonances as well. We believe that the experiment and the protocol presented here will be of immense value for structural—and functional—proteomics research by NMR. Performance of this experiment has been demonstrated using ¹³C/¹⁵N labeled ubiquitin. Copyright © 2011 John Wiley & Sons, Ltd.     

1H, 13C and 15N NMR assignments of phenazopyridine derivatives

Phenazopyridine hydrochloride (1), a drug in clinical use for many decades, and some derivatives were studied by one- and two-dimensional (1)H, (13)C and (15)N NMR methodology. The assignments, combined with DFT calculations, reveal that the preferred protonation site of the drug is the pyridine ring nitrogen atom. The chemoselective acetylation of phenazopyridine (2) and its influence on the polarization of the azo nitrogen atoms were evidenced by the (15)N NMR spectra. Molecular calculations of the phenazopyridines 2-4 show that the pyridine and phenyl groups are oriented in an antiperiplanar conformation with intramolecular hydrogen bonding between the N-b atom and the C-2 amino group preserving the E-azo stereochemistry.     

1H-13C/1H-15N heteronuclear dipolar recoupling by R-symmetry sequences under fast magic angle spinning for dynamics analysis of biological and organic solids

Fast magic angle spinning (MAS) NMR spectroscopy is becoming increasingly important in structural and dynamics studies of biological systems and inorganic materials. Superior spectral resolution due to the efficient averaging of the dipolar couplings can be attained at MAS frequencies of 40 kHz and higher with appropriate decoupling techniques, while proton detection gives rise to significant sensitivity gains, therefore making fast MAS conditions advantageous across the board compared with the conventional slow- and moderate-MAS approaches. At the same time, many of the dipolar recoupling approaches that currently constitute the basis for structural and dynamics studies of solid materials and that are designed for MAS frequencies of 20 kHz and below, fail above 30 kHz. In this report, we present an approach for (1)H-(13)C/(1)H-(15)N heteronuclear dipolar recoupling under fast MAS conditions using R-type symmetry sequences, which is suitable even for fully protonated systems. A series of rotor-synchronized R-type symmetry pulse schemes are explored for the determination of structure and dynamics in biological and organic systems. The investigations of the performance of the various RN(n)(v)-symmetry sequences at the MAS frequency of 40 kHz experimentally and by numerical simulations on [U-(13)C,(15)N]-alanine and [U-(13)C,(15)N]-N-acetyl-valine, revealed excellent performance for sequences with high symmetry number ratio (N/2n > 2.5). Further applications of this approach are presented for two proteins, sparsely (13)C/uniformly (15)N-enriched CAP-Gly domain of dynactin and U-(13)C,(15)N-Tyr enriched C-terminal domain of HIV-1 CA protein. Two-dimensional (2D) and 3D R16(3)(2)-based DIPSHIFT experiments carried out at the MAS frequency of 40 kHz, yielded site-specific (1)H-(13)C/(1)H-(15)N heteronuclear dipolar coupling constants for CAP-Gly and CTD CA, reporting on the dynamic behavior of these proteins on time scales of nano- to microseconds. The R-symmetry-based dipolar recoupling under fast MAS is expected to find numerous applications in studies of protein assemblies and organic solids by MAS NMR spectroscopy.     

Determination of pKa values using 15N and 13C nuclear magnetic resonance spectroscopy. The case of apramycin

By comparison of pK^a values derived from ¹⁵N and ¹³C nuclear magnetic resonance (NMR) spectroscopies, the assignment of the ¹⁵N resonances of apramycin is completed. ¹³C NMR spectra appear to provide accurate pK_a determinations with this aminoglycoside. Hydroxylation adjacent to one of the basic nitrogens of apramycin appears to change the pK_a values of all five amines of the molecule.     

1H, 13C and 15N NMR spectral assignments of adenosine derivatives with different amino substituents at C6-position

The complete (1)H, (13)C and (15)N NMR signals assignment of adenosine derivatives differently substituted at C(6)-position was achieved using one- and two-dimensional experiments (gs-COSY, gs-NOESY, gs-HSQC and gs-HMBC).     

13C/15N distance determination by CPMAS NMR in uniformly 13C labeled molecules

The REDOR and CPMAS techniques are applied for measuring 13C-15N dipolar coupling constants in glycine. It is shown that the selective CP or SPECIFIC CP technique removes the coherent evolution of the spin system under homonuclear 13C-13C J couplings. While the large coupling constant (approximately 900 Hz) is readily determined because of the presence of large oscillations in the CPMAS dynamics, their absence precludes the measurement of the small coupling constant (approximately 200 Hz). The experimental results and numerical simulations demonstrate that the determination of 13C-15N coupling constants of medium size (<1 kHz) by the CPMAS technique is mainly limited by the strength of the 1H decoupling field and the size of the 13C and 15N chemical shift anisotropies.     

Complete 1H and 13C NMR spectral assignment of symmetric and asymmetric bis-spiropyran derivatives

(1)H and (13)C NMR spectra of symmetric and asymmetric bis-spiropyrans, Series 1-3, were completely assigned. Especially, the (1)H assignment of asymmetric spiropyrans was achieved by utilizing (1)H-(1)H COSY and nOe experiments. All of the carbons in the dye molecules were investigated through a combination of heteronuclear 2D-shift correlation spectroscopy (HETCOR), together with an attached proton test (APT).     

Ultrahigh field MAS NMR dipolar correlation spectroscopy of the histidine residues in light-harvesting complex II from photosynthetic bacteria reveals partial internal charge transfer in the B850/His complex.

Low-temperature 15N and 13C CP/MAS (cross-polarization/magic angle spinning) NMR has been used to analyze BChl-histidine interactions and the electronic structure of histidine residues in the light-harvesting complex II (LH2) of Rhodopseudomonas acidophila. The histidines were selectively labeled at both or one of the two nitrogen sites of the imidazole ring. The resonances of histidine nitrogens that are interacting with B850 BChl a have been assigned. Specific 15N labeling confirmed that it is the tau-nitrogen of histidines which is ligated to Mg2+ of B850 BChl molecules (beta-His30, alpha-His31). The pi-nitrogens of these Mg2+-bound histidines were found to be protonated and may be involved in hydrogen bond interactions. Comparison of the 2-D MAS NMR homonuclear (13C-13C) dipolar correlation spectrum of [13C6,15N3]-histidines in the LH2 complex with model systems in the solid state reveals two different classes of electronic structures from the histidines in the LH2. In terms of the 13C isotropic shifts, one corresponds to the neutral form of histidine and the other resembles a positively charged histidine species. 15N-13C double-CP/MAS NMR data provide evidence that the electronic structure of the histidines in the neutral BChl a/His complexes resembles the positive charge character form. While the Mg...15N isotropic shift confirms a partial positive charge transfer, its anisotropy is essentially of the lone pair type. This provides evidence that the hybridization structure corresponding to the neutral form of the imidazole is capable of "buffering" a significant amount of positive charge.     

Application of unsymmetrical indirect covariance NMR methods to the computation of the (13)C <--> (15)N HSQC-IMPEACH and (13)C <--> (15)N HMBC-IMPEACH correlation spectra

Utilization of long-range (1)H--(15)N heteronuclear chemical shift correlation has continually grown in importance since the first applications were reported in 1995. More recently, indirect covariance NMR methods have been introduced followed by the development of unsymmetrical indirect covariance processing methods. The latter technique has been shown to allow the calculation of hyphenated 2D NMR data matrices from more readily acquired nonhyphenated 2D NMR spectra. We recently reported the use of unsymmetrical indirect covariance processing to combine (1)H--(13)C GHSQC and (1)H--(15)N GHMBC long-range spectra to yield a (13)C--(15)N HSQC-HMBC chemical shift correlation spectrum that could not be acquired in a reasonable period of time without resorting to (15)N-labeled molecules. We now report the unsymmetrical indirect covariance processing of (1)H--(13)C GHMBC and (1)H--(15)N IMPEACH spectra to afford a (13)C--(15)N HMBC-IMPEACH spectrum that has the potential to span as many as six to eight bonds. Correlations for carbon resonances long-range coupled to a protonated carbon in the (1)H--(13)C HMBC spectrum are transferred via the long-range (1)H--(15)N coupling pathway in the (1)H--(15)N IMPEACH spectrum to afford a much broader range of correlation possibilities in the (13)C--(15)N HMBC-IMPEACH correlation spectrum. The indole alkaloid vincamine is used as a model compound to illustrate the application of the method.     

2,2'-二(对胺苯甲酯)-1,1'联萘的核磁共振谱解析

利用~1HNMR,~(13)CNMR谱研究了2,2’-二(对胺苯甲酯)-1,1’联萘的结构,并通过~1H-~1HCOSY,~(13)C-~1H异核相关及~(13)C-~1H异核远程相关谱进一步地确定了~1H谱和~(13)C谱中各谱峰的归属,为同类化合物的表征提供了一个依据.     

13C,15N]2-Acetamido-2-deoxy-D-aldohexoses and their methyl glycosides: synthesis and NMR investigations of J-couplings involving 1H, 13C, and 15N

[reaction: see text] A series of 2-amino-2-deoxy-D-[1-13C]aldohexoses and their methyl glycosides was prepared with use of a simplified cyanohydrin reduction route. Four d-aldopentosylamines (arabino, lyxo, ribo, xylo) were prepared from the corresponding D-aldopentoses by reaction with NH3(g) in MeOH solvent, isolated in solid form, and characterized by 13C and 1H NMR. Hydrolysis of beta-D-xylopyranosylamine was studied using 13C-labeled substrates to establish optimal solution conditions for cyanohydrin formation. Major hydrolytic intermediates were observed and identified by time-lapse 1D and 2D NMR analyses of reaction mixtures. The aldopentosylamines were subsequently employed in cyanohydrin reduction reactions with K13CN to yield C2-epimeric [1-13C]2-aminosugars, which were separated by chromatography on ion-exchange columns. N-Acetylation and methyl glycosidation followed by chromatography gave pure 2-acetamido-2-deoxy-D-[1-13C]aldohexopyranosides. J(CH) and J(CC) spin-spin coupling constants involving the labeled anomeric carbon were measured and compared to those observed previously in methyl D-[1-13C]aldohexopyranosides. In parallel studies, theoretical J-couplings were calculated in model N-acetylated aldopyranosides using density functional theory (DFT) to predict the effect of OH vs NHCOCH(3) substitution at C2 on J(CH) and J(CC) values in aldopyranosyl rings. The synthetic method was also modified to accommodate (15)N- and (13)C-labeling within the N-acetyl side-chain, and some J-couplings involving 1H, 13C, and 15N atoms in 2-[1,2-13C2;15N]acetamido-2-deoxy-D-[1-13C]glucose were measured and interpreted.     

13C/15N-19F intermolecular REDOR NMR study of the interaction of TAR RNA with Tat peptides

The complex of the HIV TAR RNA with the viral regulatory protein Tat is of considerable interest, but the plasticity of this interaction has made it impossible so far to establish the structure of that complex. In order to explore a new approach to obtain structural information on protein-RNA complexes, we performed (13)C/(15)N-(19)F REDOR NMR experiments in the solid state on TAR bound to a peptide comprising the RNA-binding section of Tat. A critical arginine in the peptide was uniformly (13)C and (15)N labeled, and 5-fluorouridine was incorporated at the U23 position of TAR. REDOR irradiation resulted in dephasing of the (13)C and (15)N resonances, indicating the proximity of the U23(5F)-C and U23(5F)-N spin pairs. Best fits to the REDOR data show the U23(5F)-C distances and the U23(5F)-N distances are in good agreement with the distances obtained from solution NMR structures of partial complexes of Tat with TAR. These results demonstrate that it is possible to study protein-RNA complexes using solid-state REDOR NMR measurements, adding to a growing list of solid state techniques for studying protein-nucleic acid complexes.