A general strategy for the assignment of aliphatic side-chain resonances of uniformly 13C,15N-labeled large proteins

A general strategy is proposed to assign aliphatic side-chain resonances of large 13C,15N-labeled proteins without deuteration, using 4D 13C,15N-edited NOESY and MQ-(H)CCH-TOCSY experiments on the basis of prior assignments of backbone and 13Cbeta resonances. The strategy has been tested on a 214 residue protein (DdCAD-1) and applied to a chain-selectively 13C,15N-labeled hemoglobin (65 kDa). About 96 and 80% aliphatic side-chain spins in DdCAD-1 and hemoglobin have been assigned, respectively. The strategy proposed here will be very useful for the structure determination and dynamics characterization of large proteins by NMR.     

(13)C,(13)C]- and [(13)C,(1)H]-TROSY in a triple resonance experiment for ribose-base and intrabase correlations in nucleic acids.

A novel TROSY (transverse relaxation-optimized spectroscopy) element is introduced that exploits cross-correlation effects between (13)C-(13)C dipole-dipole (DD) coupling and (13)C chemical shift anisotropy (CSA) of aromatic ring carbons. Although these (13)C-(13)C effects are smaller than the previously described [(13)C,(1)H]-TROSY effects for aromatic (13)C-(1)H moieties, their constructive use resulted in further transverse relaxation-optimization by up to 15% for the resonances in a 17 kDa protein-DNA complex. As a practical application, two- and three-dimensional versions of the HCN triple resonance experiment for obtaining ribose-base and intrabase correlations in the nucleotides of DNA and RNA (Sklenar, V.; Peterson, R. D.; Rejante, M. R.; Feigon, J. J. Biomol. NMR 1993, 3, 721-727) have been implemented with [(13)C,(1)H]- and [(13)C,(13)C]-TROSY elements to reduce the rate of transverse relaxation during the polarization transfers between ribose (13)C1' and base (15)N1/9 spins, and between (13)C6/8 and N1/9 within the bases. The resulting TROSY-HCN experiment is user-friendly, with a straightforward, robust experimental setup. Compared to the best previous implementations of the HCN experiment, 2-fold and 5-fold sensitivity enhancements have been achieved for ribose-base and intrabase connectivities, respectively, for (13)C,(15)N-labeled nucleotides in structures with molecular weights of 10 and 17 kDa. TROSY-HCN experiments should be applicable also with significantly larger molecular weights. By using modified TROSY-HCN schemes, the origins of the sensitivity gains have been analyzed.     

Selective (15)N-labeling and analysis of (13)C-(15)N J couplings as an effective tool for studying the structure and azide-tetrazole equilibrium in a series of tetrazolo[1,5-b][1,2,4]triazines and tetrazolo[1,5-a]pyrimidines

Two general methods for the selective incorporation of an (15)N-label in the azole ring of tetrazolo[1,5-b][1,2,4]triazines and tetrazolo[1,5-a]pyrimidines were developed. The first approach included treatment of azinylhydrazides with (15)N-labeled nitrous acid, and the second approach was based on fusion of the azine ring to [2-(15)N]-5-aminotetrazole. The synthesized compounds were studied by (1)H, (13)C, and (15)N NMR spectroscopy in both DMSO and TFA solution, in which the azide-tetrazole equilibrium is shifted to tetrazole and azide forms, respectively. Incorporation of the (15)N-label led to the appearance of (13)C-(15)N J coupling constants (J(CN)), which can be measured easily using either 1D (13)C spectra with selective (15)N decoupling or with amplitude modulated 1D (13)C spin-echo experiments with selective inversion of the (15)N nuclei. The observed J(CN) patterns permit unambiguous determination of the type of fusion between the azole and azine rings in tetrazolo[1,5-b][1,2,4]triazine derivatives. Joint analysis of J(CN) patterns and (15)N chemical shifts was found to be the most efficient way to study the azido-tetrazole equilibrium.     

G-matrix Fourier transform NOESY-based protocol for high-quality protein structure determination

A protocol for high-quality structure determination based on G-matrix Fourier transform (GFT) NMR is presented. Five through-bond chemical shift correlation experiments providing 4D and 5D spectral information at high digital resolution are performed for resonance assignment. These are combined with a newly implemented (4,3)D GFT NOESY experiment which encodes information of 4D 15N/15N-, 13C(alipahtic)/15N-, and 13C(aliphatic)/13C(aliphatic)-resolved [1H,1H]-NOESY in two subspectra, each containing one component of chemical shift doublets arising from 4D --> 3D projection at omega1:Omega(1H) +/- Omega(X) [X = 15N,13C(aliphatic)]. The peaks located at the centers of the doublets are obtained from simultaneous 3D 15N/13C(aliphatic)/13C(aromatic)-resolved [1H,1H]-NOESY, wherein NOEs detected on aromatic protons are also obtained. The protocol was applied for determining a high-quality structure of the 14 kDa Northeast Structural Genomics consortium target protein, YqfB (PDB ID ). Through-bond correlation and NOESY spectra were acquired, respectively, in 16.9 and 39 h (30 h for shift doublets, 9 h for central peaks) on a 600 MHz spectrometer equipped with a cryogenic probe. The rapidly collected highly resolved 4D NOESY information allows one to assign the majority of NOEs directly from chemical shifts, which yields accurate initial structures "within" approximately 2 angstroms of the final structure. Information theoretical "QUEEN" analysis of initial distance limit constraint networks revealed that, in contrast to structure-based protocols, such NOE assignment is not biased toward identifying additional constraints that tend to be redundant with respect to the available constraint network. The protocol enables rapid NMR data collection for robust high-quality structure determination of proteins up to approximately 20-25 kDa in high-throughput.     

15N NMR spectroscopy of labeled alkoxyamines. 15N-labeled model compounds for nitroxide-trapping studies in free-radical (Co)polymerization

Eight (15)N-labeled derivatives of 1-ethoxy-2,2,6,6-tetramethylpiperidine were synthesized in order to investigate the effects of their structural units on (15)N NMR spectra. A single peak is found for each alkoxyamine. The chemical shift depends extensively on the nature of the alpha carbon atom of the alkoxy group. The remote functional group attached to position 4 of the piperidine ring has a smaller but still significant effect. The results of the (15)N NMR measurements are supported by the detection of the N-H and N-C spin-spin coupling from the (1)H and (13)C NMR. The investigated alkoxyamines are model compounds for the radical-trapping products of styryl, methyl methacryloyl, alpha-methylstyryl, and methyl acryloyl radicals by (15)N-labeled nitroxides. The potential of (15)N NMR spectroscopy to analyze such products is discussed. In addition, it is shown that the (13)C chemical shifts of the alpha carbon atom of the alkoxy group fall in an empty part of the (13)C NMR spectrum, which allows the identification of trapped (macro)radicals via natural abundance (13)C NMR.     

Spectroscopic study of 3-methyl-2,4-diphenyl-3-azabicyclo[3.3.1]nonan-9-α(β)-ols

3-Methyl-2,4-diphenyl-3-azabicyclo[3.3.1]nonan-9-α(β)-ols have been synthesized and studied by ir, ¹H and ¹³C nmr spectroscopy. In deuteriochloroform and perdeuteriobenzene solutions, these compounds adopt a flattened chair-chair conformation in which the cyclohexane ring is more flattened. From the ¹H and ¹³C nmr data, several stereoelectronic effects have been deduced. The complete and unambiguous assignment of all protons of the 3-azabicyclo[3.3.1]nonane system, not described up to date, has been carried out.     

选择性远程DEPT技术用于吡咯里西啶类生物碱的结构测定和核磁共振研究

本文从峨眉千里光中分得二个吡咯里西啶类生物碱.经测定,一个为阔叶千里光碱,另一个为新阔叶千里光碱.采用选择性远程^13CDEPT技术对这二个化合物的结构及其^1H和^13CNMR谱峰归属作了研究.     

Spectroscopic characterization of the 1-substituted 3,3-diphenyl-4-(2'-hydroxyphenyl)azetidin-2-ones: application of (13)C NMR, (1)H-(13)C COSY NMR and mass spectroscopy

The article deals with spectroscopic characterization of azetidin-2-ones. The presence of substituents like hydroxyl, fluoro, methoxy and benzhydryl, etc., on the azetidin-2-one ring significantly affects the IR absorption and (13)C NMR frequencies of the carbonyl group present in these compounds. The presence of an ester carbonyl group or too many methine protons in the molecule has been observed to limit the scope of IR and (1)H NMR spectroscopy in unambiguous assignment of the structure. The application of (13)C NMR, 2D NMR ((1)H-(13)C COSY) and mass spectroscopy in characterization of complex azetidin-2-ones is discussed. An application of the latter two techniques is described in deciding unequivocally between an azetidin-2-one ring and chroman-2-one ring structure for the product obtained by treatment of the 1-substituted 3,3-diphenyl-4-[2'-(O-diphenylacyl)hydroxyphenyl]-2-azetidinones with ethanolic sodium hydroxide at room temperature.     

选择性远程DEPT技术用于吡咯里西啶类生物碱的结构测定和核磁共振研究

本文从峨眉千里光(Sencic Fubcri Hensl)中分得二个吡咯里西啶类生物碱。经测定,一个为阔叶千里光碱(1),另一个为新阔叶千里光碱(2)。它们的~(1)H NMR研究尚未见报道。两者的~(13)C NMR研究已有报道,但1的许多~(13)C谱峰归属,不同的作者所得的结果不一致,而2的~(13)C谱峰归属与本文的二维NMR实验结果不一致。这二个化合物的NMR谱较为复杂,部分谱峰相互重选,其归属用一般方法不易确定。本文采用最近由我们提出的选择性远程~(13)CDEPT技术对这二个化合物的结构及其     

氧氟沙星的核磁共振波谱性质研究

结合1H, 13C NMR, DEPT, COSY, HSQC, HMBC谱和碳氟偶合裂分行为, 对酸性及碱性溶液中氧氟沙星(Ofloxacin, OFL)的1H和13C谱分别进行归属, 研究了哌嗪环亚甲基构成的AA'BB'复杂自旋体系中各H的化学位移. 发现噁嗪环上的甲基处于直立键; 5H在酸性溶液中化学位移移向低场, 这可能与形成C—H…O弱氢键有关; 在碱性溶液中, OFL的羧基变为羧酸根, 造成羧基和羰基周围碳原子上π电子重新分布, 导致相应C的化学位移和碳氟偶合常数发生明显变化.     

A study of the reaction mechanism of 3-nitro-4-R-furoxans formation by nitrosation of dipotassium salts of 1-hydroxyimino-2,2-dinitro-1-R-ethanes

The mechanism proposed earlier for explanation of the furoxan ring formation in the nitrosation of dipotassium salts of 1-hydroxyimino-2,2-dinitro-1-R-ethanes with NaNO₂/AcOH was confirmed experimentally by determining the ionization constants of the dinitromethyl and oxime fragments in the starting dipotassium salt and by examining ¹H, ¹³C, ¹⁴N, and ¹⁵N NMR and mass spectra of isomeric 3(4)-nitro-4(3)-R-furoxans with the ¹⁵N(5) and ¹⁵N(2) ring atoms, respectively, and 3,4-diarylfuroxan with both ¹⁵N-labeled ring atoms. A comparative analysis of the IR and Raman spectra of the ¹⁵N-labeled furoxan derivatives obtained and their unlabeled analogs allowed refinement of the literature data on interpretation of the vibrational spectra of furoxan derivatives.     

Differentiation between isomeric structures of benzazoles by 14N, 13C and 1H NMR

Nitrogen chemical shifts constitute an effective means of distinguishing between isomeric benzazole ring systems. There is a large difference in nitrogen shielding, usually more than 20 ppm, between isomeric benzenoid- and quinonoid-like structures. ¹³C and ¹H NMR is shown to provide unambiguous structure assignment only in cases of symmetric quinonoid ring systems of azoles.     

Solid-state NMR spectroscopy of human immunodeficiency virus fusion peptides associated with host-cell-like membranes: 2D correlation spectra and distance measurements support a fully extended conformation and models for specific antiparallel strand registries

The human immunodeficiency virus (HIV) is "enveloped" by a membrane, and infection of a host cell begins with fusion between viral and target cell membranes. Fusion is catalyzed by the HIV gp41 protein which contains a functionally critical approximately 20-residue apolar "fusion peptide" (HFP) that associates with target cell membranes. In this study, chemically synthesized HFPs were associated with host-cell-like membranes and had "scatter-uniform" labeling (SUL), that is, only one residue of each amino acid type was U-(13)C, (15)N labeled. For the first sixteen HFP residues, an unambiguous (13)C chemical shift assignment was derived from 2D (13)C/(13)C correlation spectra with short mixing times, and the shifts were consistent with continuous beta-strand conformation. (13)C-(13)C contacts between residues on adjacent strands were derived from correlation spectra with long mixing times and suggested close proximity of the following residues: Ala-6/Gly-10, Ala-6/Phe-11, and Ile-4/Gly-13. Specific antiparallel beta-strand registries were further tested using a set of HFPs that were (13)CO-labeled at Ala-14 and (15)N-labeled at either Val-2, Gly-3, Ile-4, or Gly-5. The solid-state NMR data were fit with 50-60% population of antiparallel HFP with either Ala-14/Gly-3 or Ala-14/Ile-4 registries and 40-50% population of structures not specified by the NMR experiments. The first two registries correlated with intermolecular hydrogen bonding of 15-16 apolar N-terminal residues and this hydrogen-bonding pattern would be consistent with a predominant location of these residues in the hydrophobic membrane interior. To our knowledge, these results provide the first residue-specific structural models for membrane-associated HFP in its beta-strand conformation.     

Isotropic chemical shifts in magic-angle spinning NMR spectra of proteins

Here we examine the effect of magic-angle spinning (MAS) rate upon lineshape and observed peak position for backbone carbonyl (C') peaks in NMR spectra of uniformly-(13)C,15N-labeled (U-(13)C,15N) solid proteins. 2D N-C' spectra of U-(13)C,15N microcrystalline protein GB1 were acquired at six MAS rates, and the site-resolved C' lineshapes were analyzed by numerical simulations and comparison to spectra from a sparsely labeled sample (derived from 1,3-(13)C-glycerol). Spectra of the U-(13)C,15N sample demonstrate large variations in the signal-to-noise ratio and peak positions, which are absent in spectra of the sparsely labeled sample, in which most 13C' sites do not possess a directly bonded 13CA. These effects therefore are a consequence of rotational resonance, which is a well-known phenomenon. Yet the magnitude of this effect pertaining to chemical shift assignment has not previously been examined. To quantify these effects in high-resolution protein spectra, we performed exact numerical two- and four-spin simulations of the C' lineshapes, which reproduced the experimentally observed features. Observed peak positions differ from the isotropic shift by up to 1.0 ppm, even for MAS rates relatively far (a few ppm) from rotational resonance. Although under these circumstances the correct isotropic chemical shift values may be determined through simulation, systematic errors are minimized when the MAS rate is equivalent to approximately 85 ppm for 13C. This moderate MAS condition simplifies spectral assignment and enables data sets from different labeling patterns and spinning rates to be used most efficiently for structure determination.     

Proton-detected solid-state NMR spectroscopy of fully protonated proteins at 40 kHz magic-angle spinning

Remarkable progress in solid-state NMR has enabled complete structure determination of uniformly labeled proteins in the size range of 5-10 kDa. Expanding these applications to larger or mass-limited systems requires further improvements in spectral sensitivity, for which inverse detection of 13C and 15N signals with 1H is one promising approach. Proton detection has previously been demonstrated to offer sensitivity benefits in the limit of sparse protonation or with approximately 30 kHz magic-angle spinning (MAS). Here we focus on experimental schemes for proteins with approximately 100% protonation. Full protonation simplifies sample preparation and permits more complete chemical shift information to be obtained from a single sample. We demonstrate experimental schemes using the fully protonated, uniformly 13C,15N-labeled protein GB1 at 40 kHz MAS rate with 1.6-mm rotors. At 500 MHz proton frequency, 1-ppm proton line widths were observed (500 +/- 150 Hz), and the sensitivity was enhanced by 3 and 4 times, respectively, versus direct 13C and 15N detection. The enhanced sensitivity enabled a family of 3D experiments for spectral assignment to be performed in a time-efficient manner with less than a micromole of protein. CANH, CONH, and NCAH 3D spectra provided sufficient resolution and sensitivity to make full backbone and partial side-chain proton assignments. At 750 MHz proton frequency and 40 kHz MAS rate, proton line widths improve further in an absolute sense (360 +/- 115 Hz). Sensitivity and resolution increase in a better than linear manner with increasing magnetic field, resulting in 14 times greater sensitivity for 1H detection relative to that of 15N detection.     

13C-13C and (15)N-(13)C correlation spectroscopy of membrane-associated and uniformly labeled human immunodeficiency virus and influenza fusion peptides: amino acid-type assignments and evidence for multiple conformations

Many viruses which cause disease including human immunodeficiency virus (HIV) and influenza are "enveloped" by a membrane and infection of a host cell begins with joining or "fusion" of the viral and target cell membranes. Fusion is catalyzed by viral proteins in the viral membrane. For HIV and for the influenza virus, these fusion proteins contain an approximately 20-residue apolar "fusion peptide" that binds to target cell membranes and plays a critical role in fusion. For this study, the HIV fusion peptide (HFP) and influenza virus fusion peptide (IFP) were chemically synthesized with uniform (13)C, (15)N labeling over large contiguous regions of amino acids. Two-dimensional (13)C-(13)C and (15)N-(13)C spectra were obtained for the membrane-bound fusion peptides and an amino acid-type (13)C assignment was obtained for the labeled residues in HFP and IFP. The membrane used for the HFP sample had a lipid headgroup and cholesterol composition comparable to that of host cells of the virus, and the (13)C chemical shifts were more consistent with beta strand conformation than with helical conformation. The membrane used for the IFP sample did not contain cholesterol, and the chemical shifts of the dominant peaks were more consistent with helical conformation than with beta strand conformation. There were additional peaks in the IFP spectrum whose shifts were not consistent with helical conformation. An unambiguous (13)C and (15)N assignment was obtained in an HFP sample with more selective labeling, and two shifts were identified for the Leu-9 CO, Gly-10 N, and Gly-10 Calpha nuclei. These sets of two shifts may indicate two beta strand registries such as parallel and antiparallel. Although most spectra were obtained on a 9.4 T instrument, one (13)C-(13)C correlation spectrum was obtained on a 16.4 T instrument and was better resolved than the comparable 9.4 T spectrum. More selective labeling and higher field may, therefore, be approaches to obtaining unambiguous assignments for membrane-associated fusion peptides.     

Laccase-catalyzed phenol oxidation. Rapid assignment of ring-proton deficient polycyclic benzofuran regioisomers by experimental 1H-13C long-range coupling constants and DFT-predicted product formation

Laccase-catalyzed oxidation of substituted catechols followed by reaction with 4-hydroxy-pyrone/-benzopyrone afforded substituted benzofuran regioisomers whose structures with only two aromatic protons in total prevent a rapid structural assignment. Based on the evaluation of (1)H-(13)C long-range coupling constants a rule of thumb could be deduced for an easy and unambiguous differentiation between the possible regioisomers formed. DFT frontier orbital calculations of the reactants offer an interesting tool to explain the regioselectivity of the key reaction.     

A (13)C INADEQUATE and HF-GIAO study of C(60)H(2) and C(60)H(6) identification of ring currents in a 1,2-dihydrofullerene

The hydrofullerenes C(60)H(2) (1) and C(60)H(6) (2) have been prepared in (13)C-enriched form and 2D INADEQUATE NMR spectra were measured. These spectra have provided unambiguous (13)C assignments for 2, and nearly unambiguous assignments for 1. In both cases, the most downfield resonances are immediately adjacent to the sp(3) carbons, despite the fact that these carbons are the least pyramidalized carbons in the molecule. Typically, (13)C chemical shifts move downfield with increasing pyramidalization (THETA(p)), but in these systems there is no strong correlation between THETA(p) and delta. HF-GIAO calculations are able to predict the chemical shifts, but provide little chemical insight into the origin of these chemical shifts. London theory reveals a significant paramagnetic ring current in 1, a feature that helps explain the (1)H shifts in these compounds and may contribute to the (13)C chemical shifts as well.     

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.     

Toward direct determination of conformations of protein building units from multidimensional NMR experiments VI: chemical shift analysis of his to gain 3D structure and protonation state information

NMR--chemical shift structure correlations were investigated by using GIAO-RB3LYP/6-311++G(2d,2p) formalism. Geometries and chemical shifts (CSI values) of 103 different conformers of N'-formyl-L-histidinamide were determined including both neutral and charged protonation forms. Correlations between amino acid torsional angle values and chemical shifts were investigated for the first time for an aromatic and polar amino acid residue whose side chain may carry different charges. Linear correlation coefficients of a significant level were determined between chemical shifts and dihedral angles for CSI[(1)H(alpha)]/phi, CSI[(13)C(alpha)]/phi, and CSI[(13)C(alpha)]/psi. Protonation of the imidazole ring induces the upfield shift of CSI[(13)C(alpha)] for positively charged histidines and an opposite effect for the negative residue. We investigated the correspondence of theoretical and experimental (13)C(alpha), (13)C(beta), and (1)H(alpha) chemical shifts and the nine basic conformational building units characteristic for proteins. These three chemical shift values allow the identification of conformational building units at 80% accuracy. These results enable the prediction of additional regular secondary structural elements (e.g., polyProlineII, inverse gamma-turns) and loops beyond the assignment of chemical shifts to alpha-helices and beta-pleated sheets. Moreover, the location of the His residue can be further specified in a beta-sheet. It is possible to determine whether the appropriate residue is located at the middle or in a first/last beta-strand within a beta-sheet based on calculated CSI values. Thus, the attractive idea of establishing local residue specific backbone folding parameters in peptides and proteins by employing chemical shift information (e.g., (1)H(alpha) and (13)C(alpha)) obtained from selected heteronuclear correlation NMR experiments (e.g., 2D-HSQC) is reinforced.