Experimental aspects of multidimensional solid-state NMR correlation spectroscopy.

The experimental parameters critical for the implementation of multidimensional solid-state NMR experiments that incorporate heteronuclear spin exchange at the magic angle are discussed. This family of experiments is exemplified by the three-dimensional experiment that correlates the (1)H chemical shift, (1)H-(15)N dipolar coupling, and (15)N chemical shift frequencies. The broadening effects of the homonuclear (1)H-(1)H dipolar couplings are suppressed using flip-flop (phase- and frequency-switched) Lee-Goldburg irradiations in both the (1)H chemical shift and the (1)H-(15)N dipolar coupling dimensions. The experiments are illustrated using the (1)H and (15)N chemical shift and dipolar couplings in a single crystal of (15)N-acetylleucine.     

Separating structure and dynamics in CSA/DD cross-correlated relaxation: a case study on trehalose and ubiquitin

We present two new sensitivity enhanced gradient NMR experiments for measuring interference effects between chemical shift anisotropy (CSA) and dipolar coupling interactions in a scalar coupled two-spin system in both the laboratory and rotating frames. We apply these methods for quantitative measurement of longitudinal and transverse cross-correlation rates involving interference of (13)C CSA and (13)C-(1)H dipolar coupling in a disaccharide, alpha,alpha-D-trehalose, at natural abundance of (13)C as well as interference of amide (15)N CSA and (15)N-(1)H dipolar coupling in uniformly (15)N-labeled ubiquitin. We demonstrate that the standard heteronuclear T(1), T(2), and steady-state NOE autocorrelation experiments augmented by cross-correlation measurements provide sufficient experimental data to quantitatively separate the structural and dynamic contributions to these relaxation rates when the simplifying assumptions of isotropic overall tumbling and an axially symmetric chemical shift tensor are valid.     

Characterization of 15N chemical shift tensors via 15N-13C REDOR and 1N-1H dipolar-shift CPMAS NMR spectroscopy

As part of our studies on the characterization of 15N chemical shift anisotropy (CSA) via magic angle spinning (MAS) NMR spectroscopy, we have investigated via numerical simulations the sensitivity of two different REDOR experimental protocols to the angles defining the orientation of the 15N-13C' bond vector in the principal axis system of the 15N CSA tensor of the amide nitrogen in a peptide bond. Additionally, employing polycrystalline samples of 15N and 13C', 15N-labeled acetanilide, we have obtained, in a first study of this type, the orientation of the 15N CSA tensor in the molecular frame by orienting the tensor with respect to the 15N-3C' and 15N-1H dipolar vectors via 15N-13C' REDOR and 15N-1H dipolar-shift MAS experiments, respectively.     

Separating Structure and Dynamics in CSA/DD Cross-Correlated Relaxation: A Case Study on Trehalose and Ubiquitin

We present two new sensitivity enhanced gradient NMR experiments for measuring interference effects between chemical shift anisotropy (CSA) and dipolar coupling interactions in a scalar coupled two-spin system in both the laboratory and rotating frames. We apply these methods for quantitative measurement of longitudinal and transverse cross-correlation rates involving interference of ¹³C CSA and ¹³C–¹H dipolar coupling in a disaccharide, α,α- -trehalose, at natural abundance of ¹³C as well as interference of amide ¹⁵N CSA and ¹⁵N–¹H dipolar coupling in uniformly ¹⁵N-labeled ubiquitin. We demonstrate that the standard heteronuclear T₁, T₂, and steady-state NOE autocorrelation experiments augmented by cross-correlation measurements provide sufficient experimental data to quantitatively separate the structural and dynamic contributions to these relaxation rates when the simplifying assumptions of isotropic overall tumbling and an axially symmetric chemical shift tensor are valid.     

The roles of homonuclear line narrowing and the1H amide chemical shift tensor in structure determination of proteins by solid-state NMR spectroscopy

The seminal contributions of Ulrich Haeberlen to homonuclear line narrowing and the determination of¹H chemical shift tensors are crucial for protein structure determination by solid-state nuclear magnetic resonance spectroscopy. The¹H chemical shift is particularly important in spectra obtained on oriented samples of membrane proteins as a mechanism for providing dispersion among resonances that are not resolved with the¹H-¹⁵N dipolar coupling and¹⁵N chemical shift frequencies. This is demonstrated with three-dimensional experiments on uniformly¹⁵N-labeled samples of Magainin antibiotic peptide and the protein Vpu from HIV-1 in oriented lipid bilayers. These experiments enable resonances in two-dimensional¹H-¹⁵N dipolar coupling/¹⁵N chemical shift planes separated by¹H chemical shift frequencies to be resolved and analyzed. These three-dimensional spectra are compared to one-dimensional spectra of full-length Vpu, the cytoplasmic domain of Vpu, and Magainin, as well as to two-dimensional spectra of fd coat protein and Colicin El polypeptide. The¹H amide chemical shift tensor provides valuable structural information, and this is demonstrated with its contributions to orientational restrictions to one of the in-plane helical residues of Magainin.     

Protein Backbone N Relaxation Rates as a Tool for the Diagnosis of Structure Quality

In the work reported herein we define a structure validation factor that depends on protein backbone ¹⁵N relaxation rates. This is an alternative method to the previously defined quality factors derived from anisotropic chemical shifts or residual dipolar couplings. We have used the structure dependence of ¹⁵N relaxation rates of anisotropically tumbling proteins to calculate this structure diagnosis factor and have used it to demonstrate the improvement of protein structures refined with residual dipolar couplings.     

Obtaining molecular and structural information from 13C-14N systems with 13C FIREMAT experiments

The effect of dipolar coupling to 14N on 13C FIREMAT (five pi replicated magic angle turning) experiments is investigated. A method is developed for fitting the 13C FIREMAT FID employing the full theory to extract the 13C-14N dipolar and 13C chemical shift tensor information. The analysis requires prior knowledge of the electric field gradient (EFG) tensor at the 14N nucleus. In order to validate the method the analysis is done for the amino acids alpha-glycine, gamma-glycine, l-alanine, l-asparagine, and l-histidine on FIREMAT FIDs recorded at 13C frequencies of 50 and 100 MHz. The dipolar and chemical shift data obtained with this analysis are in very good agreement with the previous single-crystal 13C NMR results and neutron diffraction data on alpha-glycine, l-alanine, and l-asparagine. The values for gamma-glycine and l-histidine obtained with this new method are reported for the first time. The uncertainties in the EFG tensor on the resultant 13C chemical shift and dipolar tensor values are assessed.     

15N chemical shift tensor magnitude and orientation in the molecular frame of uracil determined via MAS NMR

The potential of heteronuclear MAS NMR spectroscopy for the characterization of (15)N chemical shift (CS) tensors in multiply labeled systems has been illustrated, in one of the first studies of this type, by a measurement of the chemical shift tensor magnitude and orientation in the molecular frame for the two (15)N sites of uracil. Employing polycrystalline samples of (15)N(2) and 2-(13)C, (15)N(2)-labeled uracil, we have measured, via (15)N-(13)C REDOR and (15)N-(1)H dipolar-shift experiments, the polar and azimuthal angles (θ, psi) of orientation of the (15)N-(13)C and (15)N-(1)H dipolar vectors in the (15)N CS tensor frame. The (θ(NC), psi(NC)) angles are determined to be (92 +/- 10 degrees, 100 +/- 5 degrees ) and (132 +/- 3 degrees, 88 +/- 10 degrees ) for the N1 and N3 sites, respectively. Similarly, (θ(NH), psi(NH)) are found to be (15 +/- 5 degrees, -80 +/- 10 degrees ) and (15 +/- 5 degrees, 90 +/- 10 degrees ) for the N1 and N3 sites, respectively. These results obtained based only on MAS NMR measurements have been compared with the data reported in the literature.     

An iterative fitting procedure for the determination of longitudinal NMR cross-correlation rates

We present a method to measure (15)N-(1)H dipolar/(15)N CSA longitudinal cross-correlation rates in protonated proteins. The method depends on the measurement of four observables: the cumulative proton-proton cross relaxation rates, the (15)N R(1) relaxation rate, the multiexponential decay of 2N(Z)H(N)(Z) spin-order, and multiexponential buildup of 2N(Z)H(N)(Z) spin-order. The (15)N-(1)H dipolar/(15)N CSA longitudinal cross-correlation rate is extracted from these measurements by an iterative fitting procedure to the solution of differential equations describing the coupled relaxation dynamics of the z-magnetization of the (15)N nucleus, the two-spin-order 2N(Z)H(N)(Z), and a two-spin-order term 2N(Z)H(Q)(Z) describing the interaction with remote protons. The method is applied to the microbial ribonuclease binase. The method can also extract longitudinal cross-correlation rates for those amide protons that are involved in rapid solvent exchange. The experiment that serves for extracting proton-proton cross-relaxation rates is a modification of 3D (15)N-resolved NOESY-HSQC. The experiment restores the solvent magnetization to its equilibrium state during data detection for all phase cycling steps and all values of NOE mixing times and is recommended for use in standard applications as well.     

Sensitivity-enhanced static 15N NMR of solids by 1h indirect detection

A method for enhancing the sensitivity of 15N spectra of nonspinning solids through 1H indirect detection is introduced. By sampling the 1H signals in the windows of a pulsed spin-lock sequence, high-sensitivity 1H spectra can be obtained in two-dimensional (2D) spectra whose indirect dimension yields the 15N chemical shift pattern. By sacrificing the 1H chemical shift information, sensitivity gains of 1.8 to 2.5 for the 15N spectra were achieved experimentally. A similar sensitivity enhancement was also obtained for 2D (15)N-(1)H dipolar and 15N chemical shift correlation spectroscopy, by means of a 3D 1H/15N-1H/15N correlation experiment. We demonstrate this technique, termed PRINS for proton indirectly detected nitrogen static NMR, on a crystalline model compound with long 1H T(1rho) and on a 25-kDa protein with short 1H T(1rho). This 1H indirect detection approach should be useful for enhancing the sensitivity of 15N NMR of oriented membrane peptides. It can also be used to facilitate the empirical optimization of 15N-detected experiments where the inherent sensitivity of the sample is low.     

Three-dimensional solid-state NMR spectroscopy is essential for resolution of resonances from in-plane residues in uniformly (15)N-labeled helical membrane proteins in oriented lipid bilayers

Uniformly (15)N-labeled samples of membrane proteins with helices aligned parallel to the membrane surface give two-dimensional PISEMA spectra that are highly overlapped due to limited dispersions of (1)H-(15)N dipolar coupling and (15)N chemical shift frequencies. However, resolution is greatly improved in three-dimensional (1)H chemical shift/(1)H-(15)N dipolar coupling/(15)N chemical shift correlation spectra. The 23-residue antibiotic peptide magainin and a 54-residue polypeptide corresponding to the cytoplasmic domain of the HIV-1 accessory protein Vpu are used as examples. Both polypeptides consist almost entirely of alpha-helices, with their axes aligned parallel to the membrane surface. The measurement of three orientationally dependent frequencies for Val17 of magainin enabled the three-dimensional orientation of this helical peptide to be determined in the lipid bilayer.     

Structure determination in "shiftless" solid state NMR of oriented protein samples

An efficient formalism for calculating protein structures from oriented-sample NMR data in the torsion-angle space is presented. Angular anisotropies of the NMR observables are treated by utilizing an irreducible spherical basis of rotations. An intermediate rotational transformation is introduced that greatly speeds up structural fitting by rendering the dependence on the torsion angles Φ and Ψ in a purely diagonal form. Back-calculation of the simulated solid-state NMR spectra of protein G involving 15N chemical shift anisotropy (CSA), and 1H-15N and 1Hα-13Cα dipolar couplings was performed by taking into account non-planarity of the peptide linkages and experimental uncertainty. Even a relatively small (to within 1 ppm) random variation in the CSA values arising from uncertainties in the tensor parameters yields the RMSD's of the back-calculated structures of more than 10 ?. Therefore, the 15N CSA has been substituted with heteronuclear dipolar couplings which are derived from the highly conserved bond lengths and bond angles associated with the amino-acid covalent geometry. Using the additional 13Cα-15N and 13C'-15N dipolar couplings makes it possible to calculate protein structures entirely from "shiftless" solid-state NMR data. With the simulated "experimental" uncertainty of 15 Hz for protein G and 120 Hz for a helical hairpin derived from bacteriorhodopsin, back-calculation of the synthetic dipolar NMR spectra yielded a converged set of solutions. The use of distance restraints dramatically improves structural convergence even if larger experimental uncertainties are assumed.     

先进固体NMR技术研究高分子结构与动力学

随着固体NMR理论和谱仪硬件技术的不断发展,近年来固体NMR技术在高分子多尺度结构与动力学研究领域中正发挥着越来越重要的作用. 多脉冲及高速魔角旋转（MAS）等质子高分辨技术的发展使得高灵敏度的¹H谱可有效地用于高分子化学结构与链间相互作用的检测;基于化学键（J-耦合）相关和通过空间（偶极耦合）相互作用的各种二维异核相关谱NMR新技术,使得复杂高分子的链结构得以严格解析. 基于MAS下同核和异核偶极-偶极相互作用、化学位移各向异性等各向异性相互作用重聚的系列新技术,使得研究者可在采用高分辨¹H或¹³C 检测信号的同时检测准静态下的各向异性相互作用,进而获得与之密切相关的结构和动力学信息. 通过质子偶极滤波技术可有效检测多相聚合物中的界面相与相区尺寸、高分子共混物中的相容性等问题. 在动力学的研究中,通过质子间自旋扩散的有效压制技术和化学位移各向异性的重聚,目前已经可以有效地获取链段上单个化学键的快速局域运动以及链段的超慢分子运动. 上述丰富的多尺度NMR技术可以使研究者在不同空间和时间尺度上对高分子聚合物的微观结构、相分离和动力学行为等进行详细的研究,进而阐明高分子微观结构与宏观性能的关联. 该文以固体NMR中最主要的2类核（¹H和¹³C）的检测技术为主线,简单介绍近年来固体NMR领域的一些最新研究进展及其在高分子结构和动力学研究中的应用.     

Asymmetric MRI magnet design using a hybrid numerical method

Two-dimensional 1H/13C polarization inversion spin exchange at the magic angle experiments were applied to single crystal samples of amino acids to demonstrate their potential utility on oriented samples of peptides and proteins. High resolution is achieved and structural information obtained on backbone and side chain sites from these spectra. A triple-resonance experiment that correlates the 1H-13Calpha dipolar coupling frequency with the chemical shift frequencies of the alpha-carbon, as well as the directly bonded amide 15N site, is also demonstrated. In this experiment the large 1H-13Calpha heteronuclear dipolar interaction provides an independent frequency dimension that significantly improves the resolution among overlapping 13C resonances of oriented polypeptides, while simultaneously providing measurements of the 13Calpha chemical shift, 1H-13C dipolar coupling, and 15N chemical shift frequencies and angular restraints for backbone structure determination.     

Triple resonance experiments for aligned sample solid-state NMR of (13)C and (15)N labeled proteins

Initial steps in the development of a suite of triple-resonance (1)H/(13)C/(15)N solid-state NMR experiments applicable to aligned samples of (13)C and (15)N labeled proteins are described. The experiments take advantage of the opportunities for (13)C detection without the need for homonuclear (13)C/(13)C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are approximately 20% randomly labeled with (13)C in all backbone and side chain carbon sites and approximately 100% uniformly (15)N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are (13)C labeled at only the alpha-carbon and (15)N labeled at the amide nitrogen of a few residues. The requirement for homonuclear (13)C/(13)C decoupling while detecting (13)C signals is avoided in the first case because of the low probability of any two (13)C nuclei being bonded to each other; in the second case, the labeled (13)C(alpha) sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the (13)C chemical shift and (1)H-(13)C and (15)N-(13)C heteronuclear dipolar coupling frequencies associated with the (13)C(alpha) and (13)C' backbone sites, which provide orientation constraints complementary to those derived from the (15)N labeled amide backbone sites. (13)C/(13)C spin-exchange experiments identify proximate carbon sites. The ability to measure (13)C-(15)N dipolar coupling frequencies and correlate (13)C and (15)N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the (13)C chemical shift and (1)H-(13)C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach.     

A 15N NMR Study of Some Imidazolidine-2,4-Dichalcogen Derivatives

The ¹⁵N NMR chemical shifts and ¹(¹⁵N-¹H) coupling constants of a series of imidazolidine-2,4-dichalcogen (O, S) derivatives are reported.The ¹⁵N NMR chemical shifts show a linear correlatlon wlth the vNH stretchlng vlbratlons. The influence of the substitution of the oxygen at C₂ and/or C₄ with the sulphur, and of the hydrogen at C₅ wlth the methyls and phenyls has been considered. The ¹J(¹⁵N-¹H)'s found In thls serles of molecules agrees well with the expected values.     

Magnitudes and Orientations of the N Chemical Shift Tensor of [1-N]-2′-Deoxyguanosine Determined on a Polycrystalline Sample by Two-Dimensional Solid-State NMR Spectroscopy

The magnitudes and orientations of the ¹⁵N chemical shift tensor of [1-¹⁵N]-2′-deoxyguanosine were determined from a polycrystalline sample using the two-dimensional PISEMA experiment. The magnitudes of the principal values of the ¹⁵N chemical shift tensor of the N1 nitrogen of [1-¹⁵N]-2′-deoxyguanosine were found to be ς₁₁ = 54 ppm, ς₂₂ = 148 ppm, and ς₃₃ = 201 ppm with respect to (¹⁵NH₄)₂SO₄ in aqueous solution. Comparisons of experimental and simulated two-dimensional powder pattern spectra show that ς_33N is approximately collinear with the N–H bond. The tensor orientation of ς_33N for N1 of [1-¹⁵N]-2′-deoxyguanosine is similar to the values obtained for the side chain residues of ¹⁵N_ε1-tryptophan and ¹⁵N_π-histidine even though the magnitudes differ significantly.     

An NMR study of the origin of dioxygen-induced spin-lattice relaxation enhancement and chemical shift perturbation

Due to its depth-dependent solubility, oxygen exerts paramagnetic effects which become progressively greater toward the hydrophobic interior of micelles, and lipid bilayer membranes. This paramagnetic gradient, which is manifested as contact shift perturbations (19F and 13C NMR) and spin-lattice relaxation enhancement (19F and 1H NMR), has been shown to be useful for precisely determining immersion depth, membrane protein secondary structure, and overall topology of membrane proteins. We have investigated the influence of oxygen on 19F and 13C NMR spectra and spin-lattice relaxation rates of a semiperfluorinated detergent, (8,8,8)-trifluoro (3,3,4,4,5,5,6,6,7,7)-difluoro octylmaltoside (TFOM) in a model membrane system, to determine the dominant paramagnetic spin-lattice relaxation and shift-perturbation mechanism. Based on the ratio of paramagnetic spin-lattice relaxation rates of 19F and directly bonded 13C nuclei, we conclude that the dominant relaxation mechanism must be dipolar. Furthermore, the temperature dependence of oxygen-induced chemical shift perturbations in 9F NMR spectra suggests a contact interaction is the dominant shift mechanism. The respective hyperfine coupling constants for 19F and 13C nuclei can then be estimated from the contact shifts <(deltav/v0)19F> and <(deltav/v0)13C>, allowing us to estimate the relative contribution of scalar and dipolar relaxation to 19F and 13C nuclei. We conclude that the contribution to spin-lattice relaxation from the oxygen induced paramagnetic scalar mechanism is negligible.     

Two Simple NMR Experiments for Measuring Dipolar Couplings in Asparagine and Glutamine Side Chains

Residual dipolar couplings are now widely used for structure determination of biological macromolecules. Until recently, the main focus has been on measurement of dipolar couplings in the protein main chain. However, with the aim of more complete protein structure, it is also essential to have information on the orientation of protein side chains. In addition, residual dipolar couplings can potentially be employed to study molecular dynamics. In this Communication, two simple NH(2) and spin-state edited experiments are presented for rapid and convenient determination of five residual dipolar couplings from (15)N, (1)H correlation spectrum in asparagine and glutamine side chains. The pulse sequences are demonstrated on two proteins, 30.4-kDa Cel6A in diluted liquid crystal phase and 18-kDa human cardiac troponin C in water.     

A 2D MAS solid-state NMR method to recover the amplified heteronuclear dipolar and chemical shift anisotropic interactions

A two-dimensional solid-state NMR method for the measurement of chemical shift anisotropy tensors of X nuclei (15N or 13C) from multiple sites of a polypeptide powder sample is presented. This method employs rotor-synchronized pi pulses to amplify the magnitude of the inhomogeneous X-CSA and 1H-X dipolar coupling interactions. A combination of on-resonance and magic angle rf irradiation of protons is used to vary the ratio of the magnitudes of the 1H-X dipolar and X-CSA interactions which are recovered under MAS, in addition to suppressing the 1H-1H dipolar interactions. The increased number of spinning sidebands in the recovered anisotropic interactions is useful to determine the CSA tensors accurately. The performance of this method is examined for powder samples of N-acetyl-(15)N-L-valine (NAV), N-acetyl-15N-L-valyl-15N-L-leucine (NAVL), and alpha-13C-L-leucine. The sources of experimental errors in the measurement of CSA tensors and the application of the pulse sequences under high-field fast MAS operations are discussed.