1H,15N,13C-triple resonance NMR of very large systems at 900 MHz

We provide quantitative signal to noise data and feasibility study at 900 MHz for 1H-15N-13C triple resonance backbone assignment pulse sequences obtained from a medium sized 2H, 13C, 15N labeled protein slowed down in glycerol-water solution to mimic relaxation and spectroscopic properties of a much larger protein system with macromolecular tumbling correlation time of 52 and 80 ns, respectively, at 296 and 283 K (corresponding to molecular weights of 130 and 250 kDa). Comparisons of several different schemes for transferring magnetization from proton to nitrogen and back to proton confirms Yang and Kay's 1999 prediction that avoiding the unfavorable relaxation properties of 1H-15N multiple quantum coherence in the TROSY phase cycle of the final 15N-1H transfer before acquisition is crucial for maximal sensitivity from these very large molecular weight systems. We also show results which confirm some predictions regarding the superiority of TROSY at 900 MHz vs. 800 MHz especially as the molecular weights become very large.     

Evaluation of cross-correlation effects and measurement of one-bond couplings in proteins with short transverse relaxation times

Various strategies are described and compared for measurement of one-bond J(NH) and J(NC') splittings in larger proteins. In order to evaluate the inherent resolution obtainable in the various experiments, relaxation rates of (15)N-(1)H(N) coupled and heteronuclear decoupled resonances were measured at 600- and 800-MHz field strengths for both perdeuterated and protonated proteins. A comparison of decay rates for the two (15)N-?H(N)? doublet components shows average ratios of 4.8 and 3.5 at 800- and 600-MHz (1)H frequency, respectively, in the perdeuterated proteins. For the protonated proteins these ratios are 3.2 (800 MHz) and 2.4 (600 MHz). Relative to the regular HSQC experiment, the enhancement in TROSY (15)N resolution is 2.6 (perdeuterated; 800 MHz), 2.0 (perdeuterated; 600 MHz), 2.1 (protonated; 800 MHz), and 1.7 (protonated; 600 MHz). For the (1)H dimension, the upfield (1)H(N)-?(15)N? component on average relaxes slower than the downfield (1)H(N)-?(15)N? component by a factor of 1.8 (perdeuterated; 800 MHz) and 1.6 (perdeuterated; 600 MHz). The poor resolution for the upfield (15)N-?(1)H? doublet component in slowly tumbling proteins makes it advantageous to derive the J(NH) splitting from the difference in frequency between the narrow downfield (15)N doublet component and either the (1)H-decoupled (15)N resonance or the peak position in an experiment which J-scales the frequency of the upfield doublet component but maintains some of the advantages of the TROSY experiment.     

Suppression of diagonal peaks in TROSY-type 1H NMR NOESY spectra of 15N-labeled proteins.

A novel method for suppression of diagonal peaks in the amide region of NOESY NMR spectra of 15N-labeled proteins is presented. The method is particularly useful for larger proteins at high magnetic fields where interference between dipolar and chemical shift anisotropy relaxation mechanisms results in large TROSY effects, i.e. , large differences in 1HN linewidths depending on the spin state of attached 15N nuclei. In this limit the new TROSY NOESY method does not compromise sensitivity. It is demonstrated using a perdeuterated 15N-labeled protein sample, Neural Cell Adhesion Molecule 213-308 (NCAM) from rat, in H2O at 800 MHz.     

JE-TROSY: combined J- and TROSY-spectroscopy for the measurement of one-bond couplings in macromolecules

With the application of RDCs in high-resolution NMR studies of macromolecules, there has been an interest in the development of accurate, sensitive methods for measuring 15N-1H and 13C-1H one-bond coupling constants. Most methods for determining these couplings are based on the measurement of the displacement between cross-peak components in J-coupled spectra. However, for large macromolecules and macromolecular complexes, these methods are often unreliable since differential relaxation can significantly broaden one of the multiplet components (i.e., the anti-TROSY component) and thereby make accurate determination of its position difficult. To overcome this problem, a J-evolved transverse relaxation optimized (JE-TROSY) method is presented for the determination of one-bond couplings that involves J-evolution of the sharpest cross-peak multiplet component selected in a TROSY experiment. Couplings are measured from the displacement of the TROSY component in the additional J-evolution dimension relative to a zero frequency origin. The JE-TROSY method is demonstrated on uniformly labeled 15N, 13C-labeled RNA and peptide samples, as well as with an RNA-protein complex, in which the protein is uniformly 15N, 13C-labeled. In all cases, resolved, sensitive spectra are obtained from which heteronuclear one-bond J-couplings could be accurately and easily measured.     

Three-dimensional protein NMR TROSY-type (15)N-resolved (1)H(N)-(1)H(N) NOESY spectra with diagonal peak suppression

An earlier two-dimensional NOESY experiment with diagonal peak suppression in the (1)H(N)-(1)H(N) region is extended to three dimensions by including (15)N evolution while maintaining the TROSY approach throughout. The technique suppresses all anti-TROSY resonances by appropriate pulse sequence elements and for large molecules at high fields possible semi- and anti-TROSY artifacts are further suppressed by virtue of much shorter transverse relaxation times for these components. The new technique is demonstrated using an (15)N-labeled protein sample, RAP 17-97 (N-terminal domain of alpha2-macroglobulin Receptor Associated Protein), in H(2)O at 500 MHz.     

Determination of backbone angle psi in proteins using a TROSY-based alpha/beta-HN(CO)CA-J experiment

Transverse relaxation-optimized NMR experiment (TROSY) for the measurement of three-bond scalar coupling constant between (1)H(alpha)(i-1) and (15)N(i) defining the dihedral angle psi is described. The triple-spin-state-selective experiment allows measurement of (3)J(H(alpha)N) from (13)C(alpha), (15)N, and (1)H(N) correlation spectra H(2)O with minimum resonance overlap. Transverse relaxation of (13)C(alpha) spin is minimized by using spin-state-selective filtering and by acquiring a signal longer in (15)N-dimension in a manner of semi-constant-time TROSY evolution. The (3)J(H(alpha))(N) values obtained with the proposed alpha/beta-HN(CO)CA-J TROSY scheme are in good agreement with the values measured earlier from ubiquitin in D(2)O using the HCACO[N] experiment.     

Suppression of Diagonal Peaks in TROSY-Type H NMR NOESY Spectra of N-Labeled Proteins

A novel method for suppression of diagonal peaks in the amide region of NOESY NMR spectra of ¹⁵N-labeled proteins is presented. The method is particularly useful for larger proteins at high magnetic fields where interference between dipolar and chemical shift anisotropy relaxation mechanisms results in large TROSY effects, i.e., large differences in ¹H^N linewidths depending on the spin state of attached ¹⁵N nuclei. In this limit the new TROSY NOESY method does not compromise sensitivity. It is demonstrated using a perdeuterated ¹⁵N-labeled protein sample, Neural Cell Adhesion Molecule 213–308 (NCAM) from rat, in H₂O at 800 MHz.     

Suppression of diagonal peaks in three-dimensional protein NMR TROSY-type HCCH correlation experiments

A novel method for suppression of (13)C-(13)C diagonal peaks without sensitivity loss in three-dimensional HCCH TROSY-type NMR correlation experiments involving aromatic side chains in proteins (Pervushin et al., J. Am. Chem. Soc. 120, 6394-6400 (1998)) is presented. The key element is a spin-state-selective filter in the (13)C-(13)C mixing sequence with the dual effect of selecting the TROSY resonance in the preceding evolution period and interchanging TROSY and anti-TROSY resonances. The cross peaks are invariant to this filter but diagonal peak coherence gets concentrated on the anti-TROSY transition so that it can be eliminated by a (13)C --> (1)H TROSY transfer element. The new method is demonstrated using a (13)C,(15)N-labeled protein sample, RAP 18-112 (N-terminal domain of alpha(2)-macroglobulin receptor associated protein), at 750 MHz.     

3hJ coupling between C(alpha) and H(N) across hydrogen bonds in proteins

J couplings between (13)C(alpha) and (1)H(N) across hydrogen bonds in proteins are reported for the first time, and a two- or three-dimensional NMR technique for their measurement is presented. The technique exploits the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms, for sensitivity enhancement. The 2D or 3D spectra exhibit E.COSY patterns where the splittings in the (13)CO and (1)H(N) dimensions are (1)J((13)C(alpha), (13)CO) and the desired (3h)J((13)C(alpha), (1)H(N)), respectively. A demonstration of the new method is shown for the (15)N,(13)C-labeled protein chymotrypsin inhibitor 2 where 17 (3h)J((13)C(alpha), (1)H(N)) coupling constants ranging from 0 to 1.4 Hz where identified and all of positive sign.     

Clean TROSY: compensation for relaxation-induced artifacts

TROSY pulse sequences for recording, e.g., (1)H-(15)N chemical shift correlation spectra of proteins are designed to select only one of four two-dimensional multiplet components. However, all of the variants published so far are prone to relaxation-induced artifacts at the positions of two of the other multiplet components. This article introduces modifications to the two spin-state-selective coherence transfer building blocks of the TROSY mixing sequence resulting in a clean TROSY spectrum with the artifacts largely suppressed. It works by having the new mixing sequence generate peaks of opposite phase at the positions of the relaxation artifacts. The clean TROSY pulse sequence is marginally shorter than the original one and contains the same pulses. Experimental demonstration is presented for the (15)N-labeled proteins RAP 17-97 (N-terminal domain of alpha(2)-macroglobulin receptor associated protein) and EQT, equinatoxin II, from the Mediterranean anemone Actinia equina.     

J-modulated TROSY experiment extends the limits of homonuclear coupling measurements for larger proteins.

This paper describes the use of a TROSY experimental scheme and its variant extended with a scaled J-modulation spin-echo sequence for accurate and sensitive measurement of homonuclear 3J(H(N)H(alpha)) coupling constants in larger proteins with uniform 15N labeling. Exclusive selection of the most slowly relaxing component of a 15N-1H multiplet by the TROSY approach leads to substantial improvement in resolution; this is a prerequisite for accurate measurement of couplings from the 1H multiplets directly along the 1H frequency dimension or from the J-scaled doublets along the 15N frequency dimension.     

Improved measurement of (15)N-[(1)H] NOEs in the presence of H(N)-water proton chemical exchange.

A simple method is presented to accurately determine (15)N-[(1)H] NOEs in biomolecules in the presence of H(N)-water proton chemical exchange. Three measurements are required: one with nonselective proton saturation and two with different water saturation conditions to determine the equilibrium value of the (15)N signal. This approach is exemplified with data on two peptides, one helix-forming 17-mer and one compactly folded 56-mer. Results indicate that (15)N-[(1)H] NOEs determined using the standard approach with short recycle times (3 to 4 s) can be significantly in error when H(N)-water proton chemical exchange is relatively rapid, water proton relaxation is relatively slow, and (15)N-[(1)H] NOEs are away from the value of -1. This new method avoids such inaccuracies resulting from the use of short recycle times.     

On the accurate measurement of amide one-bond 15N-1H couplings in proteins: effects of cross-correlated relaxation, selective pulses and dynamic frequency shifts

Amide one-bond 15N-1H scalar couplings of 15N- and [15N,2H]-isotopically enriched ubiquitin have been measured with the Quantitative J approach by monitoring NMR signal intensity modulation. Scalar couplings of the non-deuterated protein are in average approximately 0.6 Hz larger than values of deuterated ubiquitin. This deviation is 30 times the error derived from experiment reproducibility. Refocusing dipole/dipole cross-correlated relaxation decreases the discrepancy to approximately 0.1 Hz, suggesting that it likely originates from relaxation interference. Alternatively, the subtraction of J values obtained at different magnetic fields largely reduces the relaxation effects. In contrast, the dynamic frequency shift whose main contribution to 1J(15N-1H) arises from 15N chemical shielding anisotropy/NH dipole cross-correlation, is not eliminated by refocusing spin evolution under this interaction. Furthermore, the average difference of 1J(15N-1H) values at two magnetic fields closely agrees with the theoretical expected difference in the dynamic frequency shift.     

Evaluation and optimization of coherence transfer in high molecular weight systems

For very large proteins in the highest magnetic fields, the large chemical shift anisotropy (CSA) of carbonyl carbon deteriorates coherence transfer efficiency in experiments designed for unambiguous sequential backbone assignment. In this communication, coherence throughput of several TROSY experiments is evaluated. Two new experiments, MP-HNCA and HN(CO)CANH, are also introduced as attractive alternatives for sequential assignment purposes of large proteins with correlation time over 50 ns. Their theoretical coherence transfer efficiencies for the interresidual (13)C(alpha) correlations are significantly better than in recently introduced MP-CT-HNCA and sequential HNCA experiments. The improvement with the new experiments is observed already on 60.8 kDa homodimer of protein Cel6A at 800 (1)H MHz.     

A compact high-speed mechanical sample shuttle for field-dependent high-resolution solution NMR

Analysis of NMR relaxation data has provided significant insight on molecular dynamic, leading to a more comprehensive understanding of macromolecular functions. However, traditional methodology allows relaxation measurements performed only at a few fixed high fields, thus severely restricting their potential for extracting more complete dynamic information. Here we report the design and performance of a compact high-speed servo-mechanical shuttle assembly adapted to a commercial 600 MHz high-field superconducting magnet. The assembly is capable of shuttling the sample in a regular NMR tube from the center of the magnet to the top (fringe field ～0.01 T) in 100 ms with no loss of sensitivity other than that due to intrinsic relaxation. The shuttle device can be installed by a single experienced user in 30 min. Excellent 2D-(15)N-HSQC spectra of (u-(13)C, (15)N)-ubiquitin with relaxation at low fields (3.77 T) and detection at 14.1T were obtained to illustrate its utility in R(1) measurements of macromolecules at low fields. Field-dependent (13)C-R(1) data of (3,3,3-d)-alanine at various field strengths were determined and analyzed to assess CSA and (1)H-(13)C dipolar contributions to the carboxyl (13)C-R(1).     

柚皮素7-O-葡萄糖苷非对映异构体的NMR波谱分析

二氢黄酮糖苷化后产生的R与S构型非对映异构体在¹H NMR谱上会呈现一些差别,但文献对其差别描述非常有限.为便于利用¹H NMR谱图判断二氢黄酮糖苷的R与S构型非对映异构体,本文首先在植物药皂荚提取物中分离得到一种二氢黄酮苷-柚皮素7-O-葡萄糖苷R与S构型混合物,分析其氘代二甲亚砜（DMSO-d₆）溶液的¹H NMR、¹³C NMR、¹H-¹H COSY、¹H-¹³C HSQC和¹H-¹³C HMBC谱,对其¹H和¹³C NMR谱峰进行了归属;然后,采用手性色谱柱对该混合物进行分离,结合圆二色光谱（CD）技术确定构型;最后,为鉴别R与S构型柚皮素7-O-葡萄糖苷在¹H NMR谱中特征差别谱峰,避免葡萄糖残基质子对二氢黄酮苷元质子化学位移的影响,采集了R与S构型柚皮素7-O-葡萄糖苷及其混合物氘代乙腈（CD₃CN）溶液的NMR谱,结果显示葡萄糖残基端基质子H-1″化学位移差别最为明显,为9.4 Hz;5-位酚羟基质子化学位移差别为5.8 Hz,C环上3个质子化学位移差也较明显．     

Efficient measurement of (3)J(N,Cgamma) and (3)J(C',Cgamma) coupling constants of aromatic residues in (13)C, (15)N-labeled proteins

An NMR pulse sequence is proposed for the simultaneous determination of side chain chi1 torsion-angle related (3)J(N,Cgamma) and (3)J(C', Cgamma) couplings in aromatic amino acid spin systems. The method is of the quantitative J correlation type and takes advantage of attenuated (15)N and (1)H transverse relaxation by means of the TROSY principle. Unlike previously developed schemes for the measurement of either of the two coupling types, spectra contain internal reference peaks that are usually recorded in separate experiments. Therefore, the desired information is extracted from a single rather than four data sets. The new method is demonstrated with uniformly (13)C/(15)N labeled Desulfovibrio vulgaris flavodoxin, which contains 14 aromatic out of 147 total amino acid residues.     

A large volume flat coil probe for oriented membrane proteins

15N detection of mechanically aligned membrane proteins benefits from large sample volumes that compensate for the low sensitivity of the observe nuclei, dilute sample preparation, and for the poor filling factor arising from the presence of alignment plates. Use of larger multi-tuned solenoids, however, is limited by wavelength effects that lead to inhomogeneous RF fields across the sample, complicating cross-polarization experiments. We describe a 600 MHz 15N-1H solid-state NMR probe with large (580 mm3) RF solenoid for high-power, multi-pulse sequence experiments, such as polarization inversion spin exchange at the magic angle (PISEMA). In order to provide efficient detection for 15N, a 4-turn solenoidal sample coil is used that exceeds 0.27 lambda at the 600 MHz 1H resonance. A balanced tuning-matching circuit is employed to preserve RF homogeneity across the sample for adequate magnetization transfer from 1H to 15N. We describe a procedure for optimization of the shorted 1/4 lambda coaxial trap that allows for the sufficiently strong RF fields in both 1H and 15N channels to be achieved within the power limits of 300 W 1H and 1 kW 15N amplifiers. The 8 x 6 x 12 mm solenoid sustains simultaneous B1 irradiation of 100 kHz at 1H frequency and 51 kHz at 15N frequency for at least 5 ms with 265 and 700 W of input power in the respective channels. The probe functionality is demonstrated by 2D 15N-1H PISEMA spectroscopy for two applications at 600 MHz.     

Proton-detected separated local field spectroscopy

PISEMO, a separated local field experiment that can be performed with either direct (15)N (or (13)C) detection or indirect (1)H detection, is demonstrated on a single crystal of a model peptide. The (1)H signals modulated by (1)H-(15)N heteronuclear dipole-dipole couplings are observed stroboscopically in the windows of the multiple-pulse sequence used to attenuate (1)H-(1)H homonuclear dipole-dipole couplings. (1)H-detection yields spectra with about 2.5 times the signal to noise ratio observed with (15)N-detection under equivalent conditions. Resolution in both the (15)N chemical shift and (1)H-(15)N heteronuclear dipole-dipole coupling dimensions is similar to that observed with PISEMA, however, since only on-resonance pulses are utilized, the bandwidth is better.     

Amide proton relaxation measurements employing a highly deuterated protein

Proton NMR longitudinal and transverse relaxation rates of unlabelled proteins are generally dominated by the many 1H-1H dipolar interactions so that spin diffusion, rather than molecular or internal motions, governs longitudinal relaxation. Here, relaxation measurements of backbone amide proton (1H(N)) magnetisations have been carried out employing the 99% 2H, 98% 15N labelled, small 2F2 protein domain in 10%/90% H(2)O/D(2)O solution. Under these conditions, the longitudinal relaxation rates exhibit time constants, T(1)*=1/R(1)* if described by a mono-exponential, within the range of 3.0 to 18.7s-a wide range which indicates that the phenomenon of spin diffusion has been greatly reduced. The majority of 1H(N) nuclei in this sample (pH 4.0 and 5 degrees C) exhibit chemical exchange with solvent that couples their longitudinal relaxation to that of the solvent. For the subset of 1H(N) nuclei not undergoing detectable solvent chemical exchange, the R(1)* rates correlate well with their individual 1H(N,O)/2H(N,O) structural environments. The correlation for corresponding transverse relaxation rates, R(2)* was found to be less good. Longitudinal relaxation measurements in 1%/99% H(2)O/D(2)O solution identify a further subset of 1H(N) nuclei which exhibit essentially indistinguishable R(1)* rates in both 1% and 10% H(2)O, implying that averaging of rates from spin diffusion processes and different 2F2 isotopomer populations are negligible for these 1H(N) sites. In addition to a high sensitivity to structural parameters, model calculations predict 1H(N) relaxation rates to exhibit pronounced sensitivity to internal dynamics.