Triple resonance solid state NMR experiments with reduced dimensionality evolution periods

Two solid state NMR triple resonance experiments which utilize the simultaneous incrementation of two chemical shift evolution periods to obtain a spectrum with reduced dimensionality are described. The CO N CA experiment establishes the correlation of (13)C(i-1) to (13)C alpha(i) and (15)N(i) by simultaneously encoding the (13)CO(i-1) and (15)N(i) chemical shifts. The CA N COCA experiment establishes the correlation (13)Ca(i) and (15)CO(i) to (13)C alpha(i-1) and (15)N(i-1) within a single experiment by simultaneous encoding of the (13)C alpha(i) and (15)N(i) chemical shifts. This experiment establishes sequential amino acid correlations in close analogy to the solution state HNCA experiment. Reduced dimensionality 2D experiments are a practical alternative to recording multiple 3D data sets for the purpose of obtaining sequence-specific resonance assignments of peptides and proteins in the solid state.     

The DQ-HN[CACB] and DQ-HN(CO)[CACB] sequences with evolution of double quantum Calpha-Cbeta coherences

The new variant of known HNCACB and HN(CO)CACB techniques is proposed that employs excitation and evolution of double quantum Calpha-Cbeta coherences. The most important features of the new method are: increased signal dispersion, lack of splittings due to 1J(Calpha-Cbeta) spin-spin couplings, and absence of accidental cancellations of positive and negative signals. The acquisition of both DQ-HN[CACB] and DQ-HN(CO)[CACB] techniques enables sequential assignment of protein backbone, using only Calpha-Cbeta DQ-frequencies. The determination of all Calpha and Cbeta chemical shifts requires, however, a comparison with HN(CO)CA or HNCA spectra. Examples of applications of the DQ-HN[CACB] and DQ-HN(CO)[CACB] experiments are presented, employing the 2D Reduced Dimensionality approach for 13C, 15N-labeled ubiquitin, and the 3D acquisition for 13C, 15N-double labeled Ca2+ -binding bovine S100A1 protein in the apo state (21 kDa) with overall correlation time of 8.1 ns.     

Sparsely sampled high-resolution 4-D experiments for efficient backbone resonance assignment of disordered proteins

Intrinsically disordered proteins (IDPs) play important roles in many critical cellular processes. Due to their limited chemical shift dispersion, IDPs often require four pairs of resonance connectivities (H(α), C(α), C(β) and CO) for establishing sequential backbone assignment. Because most conventional 4-D triple-resonance experiments share an overlapping C(α) evolution period, combining existing 4-D experiments does not offer an optimal solution for non-redundant collection of a complete set of backbone resonances. Using alternative chemical shift evolution schemes, we propose a new pair of 4-D triple-resonance experiments--HA(CA)CO(CA)NH/HA(CA)CONH--that complement the 4-D HNCACB/HN(CO)CACB experiments to provide complete backbone resonance information. Collection of high-resolution 4-D spectra with sparse sampling and FFT-CLEAN processing enables efficient acquisition and assignment of complete backbone resonances of IDPs. Importantly, because the CLEAN procedure iteratively identifies resonance signals and removes their associating aliasing artifacts, it greatly reduces the dependence of the reconstruction quality on sampling schemes and produces high-quality spectra even with less-than-optimal sampling schemes.     

Unambiguous Correlations of Backbone Amide and Aliphatic Gamma Resonances in Deuterated Proteins

Two 3D NMR pulse sequences that correlate aliphatic gamma carbon resonance frequencies to amide proton and nitrogen chemical shifts in perdeuterated proteins are presented. The HN(COCACB)CG provides only interresidue connectivities (NH_(i)and C_γ(i-1)) while the HN(CACB)CG detects both the inter- and intraresidue (NH_(i)and C_γ(i)or C_γ(i−1)) correlations. These two experiments are useful for sequential assignments and the identification of residue type from the C_γshifts. Spectra acquired on a perdeuterated 53-kDa protein illustrate the sensitivity and utility of these experiments.     

The set of triple-resonance sequences with a multiple quantum coherence evolution period

The new pulse sequence building block that relies on evolution of heteronuclear multiple quantum coherences is proposed. The particular chemical shifts are obtained in multiple quadrature, using linear combinations of frequencies taken from spectra measured at different quantum levels. The pulse sequences designed in this way consist of small number of RF-pulses, are as short as possible, and could be applied for determination of coupling constants. The examples presented involve 2D correlations HNCO, HNCA, HN(CO)CA, and H(N)COCA via heteronuclear zero and double coherences, as well as 2D HNCOCA technique with simultaneous evolution of triple and three distinct single quantum coherences. Applications of the new sequences are presented for 13C,15N-labeled ubiquitin.     

Alanine check points in HNN and HN(C)N spectra

Rapid resonance assignment is a key requirement in structural genomics research by NMR. In this context we present here two new pulse sequences, namely, HNN-A and HN(C)N-A that have been developed by simple modification of the previously described pulse sequences, HNN and HN(C)N [S.C. Panchal, N.S. Bhavesh, R.V. Hosur, Improved 3D triple resonance experiments, HNN and HN(C)N, for H(N) and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins, J. Biomol. NMR, 20 (2001) 135-147]. These increase the number of start/check points in HNN and/or HN(C)N spectra and hence help in pacing up resonance assignment in proteins.     

Accelerated acquisition of high resolution triple-resonance spectra using non-uniform sampling and maximum entropy reconstruction

Non-uniform sampling is shown to provide significant time savings in the acquisition of a suite of three-dimensional NMR experiments utilized for obtaining backbone assignments of H, N, C', CA, and CB nuclei in proteins : HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB. Non-uniform sampling means that data were collected for only a subset of all incremented evolution periods, according to a user-specified sampling schedule. When the suite of six 3D experiments was acquired in a uniform fashion for an 11 kDa cytoplasmic domain of a membrane protein at 1.5 mM concentration, a total of 146 h was consumed. With non-uniform sampling, the same experiments were acquired in 32 h and, through subsequent maximum entropy reconstruction, yielded spectra of similar quality to those obtained by conventional Fourier transform of the uniformly acquired data. The experimental time saved with this methodology can significantly accelerate protein structure determination by NMR, particularly when combined with the use of automated assignment software, and enable the study of samples with poor stability at room temperature. Since it is also possible to use the time savings to acquire a greater numbers of scans to increase sensitivity while maintaining high resolution, this methodology will help extend the size limit of proteins accessible to NMR studies, and open the way to studies of samples that suffer from solubility problems.     

A solid-state NMR application of the anomeric effect in carbohydrates: galactosamine, glucosamine, and N-acetyl-glucosamine

Simple 2D 13C/15N heteronuclear correlation solid-state NMR spectroscopy was implemented to resolve the 15N resonances of the alpha and beta anomers of three amino monosaccharides: galactosamine (GalN), glucosamine hydrochloride (GlcN), and N-acetyl-glucosamine (GlcNAc) labeled specifically with 13C1/15N spin pairs. Although the 15N resonances could not be distinguished in normal 1D spectra, they were well resolved in 2D double CP/MAS correlation spectra by taking advantage of the 13C spectral resolution. The alpha and beta resonances shifted apart by 3-5 ppm in their 13C chemical shifts, and differed by 1-2 ppm in the extended 15N dimension. Aside from this, the detection of other 13C/15N correlations over short distances was also achieved arising from the C2, C3 and CO carbons present in natural abundance. 2D double CP/MAS chemical shift correlation NMR spectroscopy is a simple and powerful technique to characterize the anomeric effect of amino monosaccharides. Applications of the 2D method reveal well-resolved 15N and 13C chemical shifts might be useful for structural determination on carbohydrates of biological significance, such as glycopeptide or glycolipids.     

Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins. 1990

Four new and complementary three-dimensional triple-resonance experiments are described for obtaining complete backbone ¹H, ¹³C, and ¹⁵N resonance assignments of proteins uniformly enriched with ¹³C and ¹⁵N. The new methods all rely on ¹H detection and use multiple magnetization transfers through well-resolved one-bond J couplings. Therefore, the 3D experiments are sensitive and permit relatively rapid recording of 3D spectra (l–2 days) for protein concentrations on the order of 1 mM. One experiment (HNCO) correlates the amide ¹H and ¹⁵N shifts with the ¹³C shift of the carbonyl resonance of the preceding amino acid. A second experiment (HNCA) correlates the intraresidue amide ¹H and ¹⁵N shifts with the Cα chemical shift. This experiment often also provides a weak correlation between the amide NH and ¹⁵N resonances of one amino acid and the Ca resonance of the preceding amino acid. A third experiment (HCACO) correlates the Hα and Cα shifts with the intraresidue carbonyl shift. Finally, a 3D relay experiment, HCA(CO)N, correlates Ha and Cal resonances of one residue with the ¹⁵N frequency of the succeeding residue. The principles of these experiments are described in terms of the operator formalism. To optimize spectral resolution, special attention is paid to removal of undesired J splittings in the 3D spectra. Technical details regarding the implementation of these triple-resonance experiments on a commercial spectrometer are also provided. The experiments are demonstrated for the protein calmodulin (16.7 kDa).     

Sensitivity-enhanced IPAP experiments for measuring one-bond 13C'-13Calpha and 13Calpha-1Halpha residual dipolar couplings in proteins

Sensitivity-enhanced 2D IPAP experiments using the accordion principle for measuring one-bond 13C'-13Calpha and 1Halpha-13Calpha dipolar couplings in proteins are presented. The resolution of the resulting spectra is identical to that of the decoupled HSQC spectra and the sensitivity of the corresponding 1D acquisitions are only slightly lower than those obtained with 3D HNCO and 3D HN(COCA)HA pulse sequences due to an additional delay 2Delta. For cases of limited resolution in the 2D 15N-1HN HSQC spectrum the current pulse sequences can easily be modified into 3D versions by introducing a poorly digitized third dimension, if so desired. The experiments described here are a valuable addition to the suites available for determination of residual dipolar couplings in biological systems.     

Sensitivity-enhanced IPAP experiments for measuring one-bond C–C and C–H residual dipolar couplings in proteins

Sensitivity-enhanced 2D IPAP experiments using the accordion principle for measuring one-bond 13C'-13Calpha and 1Halpha-13Calpha dipolar couplings in proteins are presented. The resolution of the resulting spectra is identical to that of the decoupled HSQC spectra and the sensitivity of the corresponding 1D acquisitions are only slightly lower than those obtained with 3D HNCO and 3D HN(COCA)HA pulse sequences due to an additional delay 2Delta. For cases of limited resolution in the 2D 15N-1HN HSQC spectrum the current pulse sequences can easily be modified into 3D versions by introducing a poorly digitized third dimension, if so desired. The experiments described here are a valuable addition to the suites available for determination of residual dipolar couplings in biological systems.     

Correlation of backbone amide and side-chain (13)C resonances in perdeuterated proteins

Side-chain carbon resonance assignments are difficult to obtain for larger proteins. While standard methods require protons for excitation and detection of magnetization, their presence is often unacceptable and often leads to unacceptable relaxation losses at the directly bound carbon sites. In this paper, pulse sequences are presented which provide connectivities between aliphatic side-chain (13)C and amide (1)H and (15)N chemical shifts in fully deuterated, (13)C/(15)N-enriched proteins. Magnetization either starts off from carbons or from both nitrogens and protons and is passed along the side-chain via (13)C-(13)C isotropic mixing. Direct rather than (13)CO-relayed (15)N-->(13)C(alpha) or (13)C(alpha)-->(15)N transfer steps allow the detection of intraresidual as well as sequential correlations. To avoid ambiguities between these two types in the three-dimensional version of the experiments, a fourth dimension can be introduced to achieve their separation along a (13)C(alpha) frequency axis. The novel methods are demonstrated with the uniformly (2)H/(13)C/(15)N labeled 35-kDa protein diisopropylfluorophosphatase from Loligo vulgaris.     

Measurement of one-bond 13Calpha-1Halpha residual dipolar coupling constants in proteins by selective manipulation of CalphaHalpha spins

We have developed new 2D and 3D experiments for the measurement of C(alpha)-H(alpha) residual dipolar coupling constants in (13)C and (15)N labelled proteins. Two experiments, 2D (HNCO)-(J-CA)NH and 3D (HN)CO-(J-CA)NH, sample the C(alpha)-H(alpha) splitting by means of C(alpha) magnetization, while 2D (J-HACACO)NH and 3D J-HA(CACO)NH use H(alpha) magnetization to achieve a similar result. In the 2D experiments the coupling evolution is superimposed on the evolution of the (15)N chemical shifts and the IPAP principle is used to obtain (1)H-(15)N HSQC-like spectra from which the splitting is determined. The use of a third dimension in 3D experiments reduces spectral overlap to the point where use of an IPAP scheme may not be necessary. The length of the sampling interval in the J-dimension of these experiments is dictated solely by the relaxation properties of C(alpha) or H(alpha) nuclei. This was made possible by the use of C(alpha) selective pulses in combination with either a DPFGSE or modified BIRD pulses. Inclusion of these pulse sequence elements in the J-evolution periods removes unwanted spin-spin interactions. This allows prolonged sampling periods ( approximately 25 ms) yielding higher precision C(alpha)-H(alpha) splitting determination than is achievable with existing frequency based methods.     

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.     

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.     

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.     

新药硫酸头孢匹罗的NMR研究

应用1D NMR、2D NMR （COSY、HMQC、HMBC、NOESY）及脉冲梯度场2D NMR技术[gHSQC（¹H-¹⁵N）、gHMBC（¹H-¹⁵N）]深入研究了第四代新型超广谱头孢菌素类抗生素硫酸头孢匹罗的结构,并首次对其¹H NMR、¹³C NMR谱和¹⁵N NMR谱的信号进行了全归属,通过NOESY实验对其立体结构提供有力依据.     

NMR assignment of protein side chains using residue-correlated labeling and NOE spectra

A new approach for the isotopic labeling of proteins is proposed that aims to facilitate side chain resonance assignments. Residue-correlated (RC) labeling is achieved by the expression of a protein on a medium containing a mixture of labeled, e.g., [U-13C,15N]amino acids, and NMR silent, [U-2H]amino acids. De novo synthesis of amino acids was suppressed by feedback inhibition by the amino acids in the growth medium and by the addition of beta-chloro-L-alanine, a transaminase inhibitor. Incorporation of these amino acids into synthesized proteins results in a relative diminution of inter-residue NOE interactions and a relative enhancement of intra-residue NOEs. Comparison of the resulting NOE spectra with those obtained from a uniformly labeled sample allows identification of intra-residue NOE peaks. Thus, this approach provides direct information for sidechain assignments in the NOE spectra, which are subsequently used for structural analysis. We have demonstrated the feasibility of this strategy for the 143 amino acid nuclease inhibitor NuiA, both at 35 degrees C, corresponding to a rotational correlation time of 9.5 ns, and at 5 degrees C, corresponding to a rotational correlation time of 22 ns.     

二蕊荷莲豆环肽B的NMR应用研究

植物环肽的¹H 和¹³C NMR图谱, 由于各种氨基酸自旋系统质子和碳的化学位移非常接近，谱峰高度重叠，结构解析比较困难. 文中以二蕊荷莲豆环肽B为例讨论了
现代2D NMR新技术，在植物环肽结构解析中的应用. HMQC-TOCSY图谱在氢谱方向和碳谱方向分别提供每一个氨基酸自旋系统内的氢和除季碳外碳的全相关信息，从而将每个氨基酸残基的NMR信号相互区分开来；结合¹H-¹H COSY 和 HMQC或HSQC图谱，就可以准确归属每个氨基酸的氢和碳的化学位移. 氨基酸残基之间的连接顺序可用HMBC、NOESY或ROESY图谱获得.