HACACO revisited: residual dipolar coupling measurements and resonance assignments in proteins

The revisited version of the HACACO experiment here presented, is more robust and straightforward to implement and continues to be, to a greater extent, a convenient tool for protein backbone resonance assignment. Additionally, it turns out to be a sensitive and accurate method to measure C(alpha)-H(alpha) residual dipolar couplings (RDCs). The performance of our new pulse scheme for measurement of RDCs was tested on two proteins with different secondary structures: one characterized by a high beta-sheet content, the second dominated by the presence of alpha-helices. In both examples the new method provided significantly more accurate data, compared to all previously published 3D techniques.     

Determination of three-bond scalar coupling between 13C' and 1H(alpha) in proteins using an iHN(CA),CO(alpha/beta-J-COHA) experiment

Triple-resonance NMR experiments for measuring three-bond scalar coupling constant between 13C' (i-1) and 1H(alpha)(i) spins, defining the dihedral angle phi, are presented. The novel experiments enable the measurement of 3JC'H(alpha)) from simple two (or three)-dimensional 13C', (15N/13C(alpha)), 1H(N) correlation spectra with minimal resonance overlap, thanks to solely intraresidual coherence transfer pathway and spin-state-selection. The 3J(C'H(alpha)) values measured in human ubiquitin using the proposed intraresidual iHN(CA),CO(alpha/beta-J-COHA) TROSY method were compared with those determined previously utilizing the HCAN[C'] experiment.     

More line narrowing in TROSY by decoupling of long-range couplings: shift correlation and 1JNC' coupling constant measurements

Since the introduction of RDCs in high-resolution NMR studies of macromolecules, there is a growing interest in the development of accurate, and sensitive methods for determining coupling constants. Most methods for extracting these couplings are based on the measurement of the splitting between multiplet components in J-coupled spectra. However, these methods are often unreliable since undesired multiple-bond couplings can considerably broaden the multiplet components and consequently make accurate determination of their position difficult. To demonstrate one approach to this problem, G-BIRD((r)) decoupled TROSY sequences are proposed for the measurement of (1)J(NH) and (1)J(NC') coupling constants. Resolved or unresolved splittings due to remote protons are removed by a G-BIRD((r)) module employed during t(1) and as a result, spectra with narrow, well-resolved peaks are obtained from which heteronuclear one-bond couplings can be accurately measured. Moreover, introduction of a spin-state-selective alpha/beta-filter in the TROSY sequence allows the separation of the (1)J(NC') doublet components into two subspectra which contain the same number of peaks as the regular TROSY spectrum. The (1)J(NC') couplings are obtained from the displacement between the corresponding peaks in the subspectra.     

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.     

Assessment of protein alignment using 1H-1H residual dipolar coupling measurements

A quick and accurate method is described for assessing protein alignment from residual dipolar coupling (RDC) measurements. In contrast to observing D(2)O resonance splitting, which reflects the orientational order of the alignment medium, the degree of alignment of a protein of interest can be estimated directly from (1)H-(1)H RDCs. In this study, RDCs between aromatic protons in unlabeled Cp-rubredoxin were measured from proton homonuclear J-resolved experiments with high sensitivity, and the alignment was assessed without the need of extensive resonance assignment. Since labeled proteins are not needed, this method provides an efficient way for screening alignment media. In situations where the protein structure is known, as in the case of Cp-rubredoxin, a full set of order tensor parameters can be determined, allowing further studies, such as those of ligand alignment relative to a target protein.     

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.     

Measurement of one and two bond N–C couplings in large proteins by TROSY-based J-modulation experiments

Residual dipolar couplings (RDCs) between NC′ and NC^α atoms in polypeptide backbones of proteins contain information on the orientation of bond vectors that is complementary to that contained in NH RDCs. The ¹J_NC^α and ²J_NC^α scalar couplings between these atoms also display a Karplus relation with the backbone torsion angles and report on secondary structure. However, these N–C couplings tend to be small and they are frequently unresolvable in frequency domain spectra having the broad lines characteristic of large proteins. Here a TROSY-based J-modulated approach for the measurement of small ¹⁵N–¹³C couplings in large proteins is described. The cross-correlation interference effects inherent in TROSY methods improve resolution and signal to noise ratios for large proteins, and the use of J-modulation to encode couplings eliminates the need to remove frequency distortions from overlapping peaks during data analysis. The utility of the method is demonstrated by measurement of ¹J_NC′, ¹J_NC^α, and ²J_NC^α scalar couplings and ¹D_NC′ and ¹D_NC^α residual dipolar couplings for the myristoylated yeast ARF1·GTPγs protein bound to small lipid bicelles, a system with an effective molecule weight of 70 kDa.     

Magnetization Transfer via Residual Dipolar Couplings: Application to Proton–Proton Correlations in Partially Aligned Proteins

A novel three-dimensional NMR experiment is reported that allows the observation of correlations between amide and other protons via residual dipolar couplings in partially oriented proteins. The experiment is designed to permit quantitative measurement of the magnitude of proton–proton residual dipolar couplings in larger molecules and at higher degree of alignments. The observed couplings contain data valuable for protein resonance assignment, local protein structure refinement, and determination of low-resolution protein folds.     

Simultaneous extraction of multiple orientational constraints of membrane proteins by 13C-detected N-H dipolar couplings under magic angle spinning

A 13C-detected N-H dipolar coupling technique is introduced for uniaxially mobile membrane proteins for orientation determination using unoriented samples. For proteins undergoing rigid-body uniaxial rotation in the lipid bilayer, the intrinsic equality between the dipolar coupling constants measured in unoriented samples and the anisotropic coupling measured in static oriented samples has been shown recently. Here, we demonstrate that the orientation-sensitive backbone N-H dipolar couplings can be measured with 13C detection using 2D and 3D MAS correlation experiments, so that maximal site resolution can be achieved and multiple orientational constraints can be extracted from each experiment. We demonstrate this technique on the M2 transmembrane peptide of the influenza A virus, where the N-H dipolar couplings of various residues fit to a dipolar wave for a helical tilt angle of 37 degrees , in excellent agreement with data obtained from singly 15N-labeled samples.     

J-ONLY-TOCSY: efficient suppression of RDC-induced transfer in homonuclear TOCSY experiments using JESTER-1-derived multiple pulse sequences

The main purpose of homonuclear Hartmann-Hahn or TOCSY experiments is the assignment of spin systems based on efficient coherence transfer via scalar couplings. In partially aligned samples, however, magnetization is also transferred via residual dipolar couplings (RDCs) and therefore through space correlations can be observed in COSY and TOCSY experiments that make the unambiguous assignment of covalently bound spins impossible. In this article, we show that the JESTER-1 multiple pulse sequence, originally designed for broadband heteronuclear isotropic Hartmann-Hahn transfer, efficiently suppresses the homonuclear dipolar coupling Hamiltonian. This suppression can be enhanced even further by variation of the supercycling scheme. The application of the resulting element in homonuclear TOCSY periods results in coherence transfer via J-couplings only. As a consequence, the assignment of scalar coupled spin systems is also possible in partially aligned samples. The bandwidth of coherence transfer for the JESTER-1-derived sequences is comparable to existing TOCSY multiple pulse sequences. Results are demonstrated in theory and experiment.     

1D Determination of Long-Range Heteronuclear Coupling Constants by Gradient Enhanced SIMBA

Two¹H-detected one-dimensional gradient enhanced experiments and corresponding reference experiments are proposed for the measurement of heteronuclear long-rangeⁿJ_CHcoupling constants in organic molecules. These experiments use inverse detection and selective carbon excitation, provide excellent suppression of protons not coupled to¹³C, and are able to measure couplings from regions that are crowded in both the proton and carbon frequency ranges. The experiments are applied to determine the long-rangeⁿJ_CHcouplings in the sucrose molecule, including coupling across the glycosidic linkage. These pulse sequences are shown to provide accurate and efficient measurements of coupling constants that would be difficult or time-consuming to measure by other techniques.     

15N chemical shift anisotropy in protein structure refinement and comparison with NH residual dipolar couplings

Recent methods of aligning proteins which were developed in order to measure residual dipolar couplings (RDCs) in solution can also be used for additional applications such as measuring the 15N CSA in the form of chemical shift differences, Deltadelta. A new XPLOR-NIH module has been developed and implemented for NMR structure refinement using the 15N Deltadelta data as restraints. The results of this refinement are shown using the protein Bax. This method should be amenable to any protein which can be studied by NMR. An analysis comparing the structural information provided by NH RDCs and the 15N Deltadelta is included.     

基于纯化学位移的同核二维正交相敏J分解核磁共振波谱

二维J分解(2D J_RES)核磁共振(NMR)波谱是一种简单且用户友好的谱图表达形式,其将J偶合常数和化学位移信息分离到两个正交的频率维度上.自40年前首次被提出以来,2D J_RES技术在脉冲序列和方法的改进,以及实际应用的进展方面一直备受关注.本文回顾了新型2D J_RES脉冲序列,以及用于精确测量同核J偶合常数的2D J编辑谱的最近进展,特别是基于纯化学位移演化机制的正交相敏2D J_RES谱方法及其应用,并阐述其在克服强偶合效应和磁场不均匀性等方面的能力.     

Selective detection of single-enantiomer spectrum of chiral molecules aligned in the polypeptide liquid crystalline solvent: Transition selective one-dimensional 1H–1H COSY

One-dimensional (1D) proton NMR spectra of enantiomers are generally undecipherable in chiral orienting poly-γ-benzyl-l-glutamate (PBLG)/CDCl₃ solvent. This arises due to large number of couplings, in addition to superposition of spectra from both the enantiomers, severely hindering the ¹H detection. On the other hand in the present study the benefit is derived from the presence of several couplings among the entire network of interacting protons. Transition selective 1D ¹H–¹H correlation experiment (1D-COSY) which utilizes the coupling assisted transfer of magnetization not only for unraveling the overlap but also for the selective detection of enantiopure spectrum is reported. The experiment is simple, easy to implement and provides accurate eanantiomeric excess in addition to the determination of the proton–proton couplings of an enantiomer within a short experimental time (few minutes).     

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).     

Measurement ofJand Dipolar Couplings from Simplified Two-Dimensional NMR Spectra

Simple procedures are described for recording complementary in-phase and antiphaseJ-coupled NMR spectra. The sum and difference of these spectra contain only the upfield and the downfield components of a doublet, making it possible to measure theJsplitting directly from these combinations without an increase in resonance overlap relative to the decoupled spectrum. The approach is demonstrated for measurement of¹J_NHsplittings and²J_HNC′splittings in oriented and isotropic ubiquitin. Dipolar couplings obtained from differences in the splittings measured in the oriented and isotropic phases are in excellent agreement with dipolar couplings obtained from direct measurement of the splitting or from a conventional E.COSY-type measurement.     

Measurement of homonuclear (2)J-couplings from spin-state selective double-/zero-quantum two-dimensional NMR spectra.

(1)H-detected two-dimensional double-/zero-quantum experiments are described for measurement of homonuclear (2)J(HH)-couplings of NH(2) or CH(2) groups in proteins. These experiments utilize multiple-quantum coherence for determination of the size and the absolute sign of the geminal scalar and dipolar couplings in the presence of broad lines. Spectra are simplified by gradient selection and spin-state selective filters.     

A Consistent Grid Coupling Method for Lattice-Boltzmann Schemes

A method of coupling grids of different mesh size is developed for classical Lattice-Boltzmann (LB) algorithms on uniform grids. The approach is based on an asymptotic analysis revealing suitable quantities equalized along the grid interfaces for exchanging information between the subgrids. In contrast to other couplings the method works without overlap zones. Moreover the grid velocity (Mach number) is not kept constant, as the time step depends not linearly but quadratically on the grid spacing. To illustrate the basic idea we use a simple LB algorithm solving the advection-diffusion equation. The proposed grid coupling is validated by numerical convergence studies indicating, that the coupling does not affect the second-order convergence behavior of the LB algorithm which is observed on uniform grids. In order to demonstrate its principal applicability to other LB models, the coupling is generalized to the standard D2P9 model for (Navier-)Stokes flow and tested numerically. As we use analytic tools different from the Chapman-Enskog expansion, the theoretical background material is given in two appendices. In particular, the results of numerical experiments are confirmed with a consistency analysis.     

3D NMR spectroscopy for resonance assignment and structure elucidation of proteins under MAS: novel pulse schemes and sensitivity considerations

Two types of 3D MAS NMR experiments are introduced, which combine standard (NC,CC) transfer schemes with (1H,1H) mixing to simultaneously detect connectivities and structural constraints of uniformly 15N,13C-labeled proteins with high spectral resolution. The homonuclear CCHHC and CCC experiments are recorded with one double-quantum evolution dimension in order to avoid a cubic diagonal in the spectrum. Depending on the second transfer step, spin systems or proton-proton contacts can be determined with reduced spectral overlap. The heteronuclear NHHCC experiment encodes NH-HC proton-proton interactions, which are indicative for the backbone conformation of the protein. The third dimension facilitates the identification of the amino acid spin system. Experimental results on U-[15N,13C]valine and U-[15N,13C]ubiquitin demonstrate their usefulness for resonance assignments and for the determination of structural constraints. Furthermore, we give a detailed analysis of alternative multidimensional sampling schemes and their effect on sensitivity and resolution.     

液体核磁共振中由分子间偶极耦合和分子内标量耦合共同作用后的谱图裂分模式

在高极化多自旋液体样品中,同时存在着分子间偶极（D）耦合和分子内标量（J）耦合,它们的共同作用产生了一些原来观测不到的分子间多量子相干信号。而且,信号的裂分模式与只存在J耦合的多自旋体系中观测到的多量子相干信号的裂分模式不同。本文从理论和实验上研究了这些禁阻的共振峰及其独特的裂分模式。为了比较验证,我们以I2S3+X自旋体系为例,结合使用选择和非选择性的射频脉冲序列来获得分子间双量子相干信号的五种裂分模式。进而归纳出对IpSq+Xk (p, q, k = 1, 2, 3,…)自旋体系普适的裂分模式规则。并指出,它们中如(1:0:-1)的裂分模式会放大J耦合裂分,使得J耦合常数的测量更精确,特别在J耦合常数很小或不均匀场中的J耦合常数的测量中具有诱人的应用前景。结果表明理论预测,计算机模拟和实验观测结果三者吻合的很好。