Improvement of resolution in solid state NMR spectra with J-decoupling: an analysis of lineshape contributions in uniformly C-enriched amino acids and proteins

In magic angle spinning (MAS) NMR spectra of highly and uniformly 13C,15N-enriched amino acids and proteins, homo-nuclear coupling interactions contribute significantly to the 13C linewidths, particularly for moderate applied magnetic field strengths and sample spinning frequencies. In this work, we attempted to dissect, analyze, and control the contributions of J-coupling and residual homo-nuclear dipolar coupling interactions to the linewidths of uniformly 13C,15N-enriched crystalline alanine; these studies were carried out at 9.4 T using a range of spinning frequencies from 5 to 15 kHz. The anisotropic second-order dipolar shifts and the J-splittings are comparable in their contribution to the linewidths, but behave very differently in terms of experimental protocols for line narrowing. In contrast to the J-coupling interactions, the second-order dipolar broadening cannot be refocused using selective pulses on the passively coupled spin. We carried out experiments to remove or refocus the 13C J-coupling interactions (omega1 J-decoupling) using a selective DANTE pulse in the center of the indirect evolution period. Inversion profiles and bandwidths of selective DANTE pulses acting on transverse magnetization, in the regime of moderate spinning frequencies, were characterized computationally and experimentally. A dramatic improvement in the resolution of the 2D spectrum was achieved when this decoupling protocol was employed.     

Homo-nuclear 13C J-decoupling in uniformly 13C-enriched solid proteins

Recently, we reported an analysis of carbon lineshapes in high resolution solid-state NMR spectra of uniformly 13C-enriched amino acids. Application of a 13C J-decoupling protocol during the carbon chemical shift evolution period allowed us to separate the contribution of the second-order dipolar shift from that of the 13C-13C J-coupling interactions to carbon linewidths. In this work, we have extended this approach to microcrystalline proteins. We describe the performance of the J-decoupling sequence applied to remove homo-nuclear 13C J-couplings in the 13C spectra of ubiquitin. Analysis of the J-decoupling efficiency for C(alpha) and carbonyl protein sites showed that a significant gain in resolution can be achieved.     

Two-dimensional chemical shift/heteronuclear dipolar coupling spectra obtained with polarization inversion spin exchange at the magic angle and magic-angle sample spinning (PISEMAMAS)

High-resolution two-dimensional ¹⁵N chemical shift/¹H-¹⁵N dipolar coupling polarization inversion spin exchange at the magic angle (PISEMA) spectra of a polycrystalline sample of ¹⁵N-acetylvaline were obtained with and without magic-angle sample spinning. These spectra demonstrate the advantages of the PISEMA experiment over conventional approaches to separated local-field spectroscopy, especially the high resolution in the dipolar dimension where the spinning sidebands have uniformly narrow linewidths.     

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.     

Applications of the CSA-amplified PASS experiment

The recently reported CSA-amplified PASS experiment correlates the spinning sidebands at the true spinning frequency omega(r) with the spinning sidebands that would be obtained at the effective spinning frequency omega(r)/N, where N is termed the scaling factor. The experiment is useful for the measurement of small chemical shift anisotropies, for which slow magic-angle spinning frequencies, required to measure several spinning sidebands, can be unstable. We have experimentally evaluated the reliability of this experiment for this application. In particular we have demonstrated that large scaling factors of the order of N=27 may be used, whilst still obtaining accurate chemical shift sideband intensities at the effective spinning frequency from the F(1) projection. Moreover, the sideband intensities are accurately obtained even in the presence of significant pulse imperfections. A second application of the CSA-amplified PASS experiment is the measurement of the chemical shift anisotropy of sites that experience homonuclear dipolar coupling, as may be found in uniformly labelled biological molecules, or for nuclei with a high natural abundance. The effects of homonuclear dipolar coupling on CSA-amplified PASS spectra has been investigated by numerical simulations and are demonstrated using uniformly (13)C enriched l-histidine monohydrochloride monohydrate.     

Homonuclear decoupled 13C chemical shift anisotropy in 13C doubly labeled peptides by selective-pulse solid-state NMR

We describe a new experiment for measuring homonuclear-decoupled anisotropic chemical shift patterns in doubly 13C-labeled compounds under magic-angle spinning. The experiment combines a pair of selective and non-selective 180 degrees pulses to suppress the 13C-13C scalar and dipolar interactions. This is combined with the recently developed SUPER technique to recouple the chemical shift anisotropy. Demonstrations on 13Calpha and 13CO-labeled amino acids and peptides show that accurate chemical shift powder patterns can be obtained. This permits the use of chemical shift anisotropy for conformational studies of suitably extensively 13C-labeled peptides and proteins.     

Comparison of several hetero-nuclear dipolar recoupling NMR methods to be used in MAS HMQC/HSQC

We compare several hetero-nuclear dipolar recoupling sequences available for HMQC or HSQC experiments applied to spin-1/2 and quadrupolar nuclei. These sequences, which are applied to a single channel, are based either on the rotary resonance recoupling (R3) irradiation, or on two continuous rotor-synchronized modulations (SFAM1 and SFAM2), or on four symmetry-based sequences (R2(1)1,SR4(1)2,R12(3)5,R20(5)9), or on the REDOR scheme. We analyze systems exhibiting purely hetero-nuclear dipolar interactions as well as systems where homo-nuclear dipolar interactions need to be canceled. A special attention is given to the behavior of these sequences at very fast MAS. It is shown that R3 methods behave poorly due to the narrowness of their rf-matching curves, and that the best methods are SR4(1)2 and SFAM (SFAM1 or SFAM2 if homo-nuclear interactions are not negligible). REDOR can also recouple efficiently hetero-nuclear dipolar interactions, provided the sequence is sent on the non-observed channel and homo-nuclear dipolar interactions are negligible. We anticipate that at ultra-fast spinning speed, SFAM1 and SFAM2 will be the most efficient methods.     

Peptide torsion angle measurements: effects of nondilute spin pairs on carbon-observed, deuterium-dephased PM5-REDOR

Reintroducing dipolar coupling between spin-1/2 nuclei (e.g., (13)C, (15)N) and spin-1 (2)H, using phase-modulated deuterium dephasing pulses, provides a simple and efficient basis for obtaining peptide backbone torsion angles (phi, psi) in specific stable-isotope enriched samples. Multiple homonuclear spin-1/2 interactions due to isotopic enrichment can arise between neighboring molecules or within a multiply labeled protein after folding. The consequences of (13)C homonuclear interactions present during (13)C-observed, (2)H-dephased REDOR measurements are explored and the theoretical basis of the experimentally observed effects is investigated. Two tripeptides are taken to represent both the general case of (2)H(alpha)-alanine (in the tripeptide LAF) and the special case of (2)H(alpha)(2)-glycine (in the tripeptide LGF). The lyophilized tripeptides exhibit narrowed spectral linewidths over time due to reduced conformational dispersion. This is due to a hydration process whereby a small fraction of peptides is reorienting and the bulk peptide fraction undergoes a conformational change. The new molecular packing arrangement lacks homonuclear (13)C spin interactions, allowing determination of (phi, psi) backbone torsion angles.     

Determination of the backbone torsion psi angle by tensor correlation of chemical shift anisotropy and heteronuclear dipole-dipole interaction

We demonstrate that the backbone torsion psi angle of a uniformly labeled residue can be determined accurately by correlating the chemical shift anisotropy of the carbonyl carbon and the 13C-1H heteronuclear dipole-dipole interaction of the alpha carbon. To obtain the highest sensitivity for the psi angle determination, the following conditions are desired: (i) the recoupling pulse sequences for the CSA and the heteronuclear dipolar interactions are gamma encoded, in which the spatial parts of m=2 are selected; (ii) the homonuclear polarization transfer is based on the scalar spin-spin coupling. Experimental data were obtained for [U-13C, 15N]-alanine and N-acetyl-[U-13C, 15N]-d,l-valine under magic-angle spinning at 25kHz. Only three data points are required for the measurements and the dihedral angles determined are in excellent agreement with the diffraction data.     

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.     

微扰论与平均Hamiltonian理论Ⅰ.场相关化学位移

本文应用平均Hamiltonian理论讨论了异核偶极相互作用引起的场相关化学位移,对照二阶微拢论的结果,发现计入了"A"项的额外影响。说明在讨论非久期项效应时,一级平均Hamiltonian比只用二阶微扰论的结果更精确。文中,应用平均Hamiltonian理论还讨论了同核偶极相互作用产生的场相关化学位移。     

Homonuclear zero-quantum recoupling in fast magic-angle spinning nuclear magnetic resonance

Solid-state magic-angle-spinning NMR pulse sequences which implement zero-quantum homonuclear dipolar recoupling are designed with the assistance of symmetry theory. The pulse sequences are compensated on a short time scale by the use of composite pulses and on a longer time scale by the use of supercycles. (13)C dipolar recoupling is demonstrated in powdered organic solids at high spinning frequencies. The new sequences are compared to existing pulse sequences by means of numerical simulations. Experimental two-dimensional magnetization exchange spectra are shown for [U-(13)C]-L-tyrosine.     

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.     

Improved narrowband dipolar recoupling for homonuclear distance measurements in rotating solids

Recovery of the magnetic dipolar interaction between nuclei bearing the same gyromagnetic ratio in rotating solids can be promoted by synchronous rf irradiation. Determination of the dipolar interaction strength can serve as a tool for structural elucidation in polycrystalline powders. Spinning frequency dependent narrow-band (nb) RFDR and SEDRA experiments are utilized as simple techniques for the determination of dipolar interactions between the nuclei in coupled homonuclear spin pairs. The magnetization exchange and coherence dephasing due to a fixed number of rotor-synchronously applied pi-pulses is monitored at spinning frequencies in the vicinity of the rotational resonance (R(2)) conditions. The powder nbRFDR and nbSEDRA decay curves of spin magnetizations and coherences, respectively, as a function of the spinning frequency can be measured and analyzed using simple rate equations providing a quantitative measure of the dipolar coupling. The effects of the phenomenological relaxation parameters in these rate equations are discussed and an improved methodology is suggested for analyzing nbRFDR data for small dipolar couplings. The distance between the labeled nuclei in the 1,3-(13)C(2)-hydroxybutyric acid molecule is rederived using existing nbRFDR results and the new simulation procedure. A nbSEDRA experiment has been performed successfully on a powder sample of singly labeled 1-(13)C-L-leucine measuring the dipolar interaction between the labeled carboxyl carbon and the natural abundant beta-carbon. Both narrowband techniques are employed for the determination of the nuclear distances between the side-chain carbons of leucine and its carbonyl carbon in a tripeptide Leu-Gly-Phe that is singly (13)C-labeled at the leucine carbonyl carbon position.     

Recoupling of heteronuclear dipolar interactions with rotational-echo double-resonance at high magic-angle spinning frequencies

Heteronuclear dipolar recoupling with rotational-echo double-resonance (REDOR) is investigated in the rapid magic-angle spinning regime, where radiofrequency irradiation occupies a significant fraction of the rotor period (10-60%). We demonstrate, in two model (13)C-(15)N spin systems, [1-(13)C, (15)N] and [2-(13)C, (15)N]glycine, that REDOR DeltaS/S(0) curves acquired at high MAS rates and relatively low recoupling fields are nearly identical to the DeltaS/S(0) curve expected for REDOR with ideal delta-function pulses. The only noticeable effect of the finite pi pulse length on the recoupling is a minor scaling of the dipolar oscillation frequency. Experimental results are explained using both numerical calculations and average Hamiltonian theory, which is used to derive analytical expressions for evolution under REDOR recoupling sequences with different pi pulse phasing schemes. For xy-4 and extensions thereof, finite pulses scale only the dipolar oscillation frequency by a well-defined factor. For other phasing schemes (e.g., xx-4 and xx-4) both the frequency and amplitude of the oscillation are expected to change.     

Band-selective recoupling of homonuclear double-quantum dipolar interaction with a generalized composite 0 degrees pulse: application to 13C aliphatic region-selective magnetization transfer in solids

Recoupling of homonuclear double quantum (DQ)-dipolar interactions is a useful technique for the structural analysis of molecules in solids. We have designed a series of elemental 0 degrees pulses for the recoupling sequences with the rf phase rotation about the z-axis, known as CN. The proposed 0 degrees pulses whose total flip angle >/=360 degrees provide spin rotation vectors in the xy-plane. Thus, the residual spin rotation can be canceled by rf phase rotation about the z-axis. An analysis by the coherent averaging theory showed that effective bandwidths of the recoupling sequences are limited not by the reduction in the dipolar scaling factor but by the increase in the residual spin rotation due to offset. A CN sequence with these elemental pulses provides an effective bandwidth of DQ-dipolar recoupling from ca. 0.5nu(R) to 4nu(R) for numerical simulations. Here, nu(R) is the sample spinning frequency. The 0 degrees pulses were applied to band-selective recoupling for the magnetization transfer in uniformly 13C-labeled molecules. Narrow-band recoupling enhances the magnetization transfer between spins within the effective range by decoupling the dipolar interactions between spins one of which is outside the range. The narrow band operation reduces rf field strength, which improves the CH decoupling. Increases in signal intensities by the use of the proposed 0 degrees pulses are experimentally shown for 13C-labeled amino acids.     

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.     

Magic Angle-Oriented Sample Spinning (MAOSS): A New Approach toward Biomembrane Studies

The application of magic angle sample spinning (MAS) NMR to uniformly aligned biomembrane samples is demonstrated as a new general approach toward structural studies of membrane proteins, peptides, and lipids. The spectral linewidth from a multilamellar lipid dispersion is dominated, in the case of protons, by the dipolar coupling. For low-γ or dilute spins, however, the chemical shift anisotropy dominates the spectral linewidth, which is reduced by the two-dimensional order in a uniformly aligned lipid membrane. The remaining line broadening, which is due to orientational defects (“mosaic spread”) can be easily removed at low spinning speeds. This orientational order in the sample also allows the anisotropic intermolecular motions of membrane components (such as rotational diffusion, τ_c= 10⁻¹⁰s) for averaging dipolar interactions to be utilized, e.g., by placing the membrane normal parallel to the rotor axis. The dramatic resolution improvement for protons which are achieved in a lipid sample at only 220 Hz spinning speed in a 9.4 T field is slightly better than any data published to date using ultra-high fields (up to 17.6 T) and high-speed spinning (14 kHz). Additionally, the analysis of spinning sidebands provides valuable orientational information. We present the first¹H,³¹P, and¹³C MAS spectra of uniformly aligned dimyristoylphosphatidylcholine (DMPC) bilayers. Also,¹H resolution enhancement for the aromatic region of the M13 coat protein reconstituted into DMPC bilayers is presented. This new method combines the high resolution usually achieved by MAS with the advantages of orientational constraints obtained by working with macroscopically oriented samples. We describe the general potential and possible perspectives of this technique.     

1H detected 1H,15N correlation spectroscopy in rotating solids

We describe new correlation experiments suitable for determining long-range 1H-1H distances in 2H,15N-labeled peptides and proteins. The approach uses perdeuteration together with back substitution of exchangeable protons during sample preparation as a means of attenuating the strong 1H-1H dipolar couplings that broaden 1H magic angle spinning (MAS) spectra of solids. In the approach described here, we retain 100% of the 1H sensitivity by labeling and detecting all exchangeable sites. This is in contrast to homonuclear multiple pulse decoupling sequences that are applied during detection and that compromise sensitivity because of the requirement of sampling between pulses. As a result 1H detection provides a gain in sensitivity of >5 compared to the 15N detected version of the experiment (at a MAS frequency of 13.5kHz). The pulse schemes make use of the favorable dispersion of the amide 15Ns resonances in the protein backbone. The experiments are demonstrated on a sample of the uniformly 2H,15N-labeled dipeptide N-Ac-Val-Leu-OH and are analogous to the solution-state suite of HSQC-NOESY experiments. In this compound the 1H amide linewidths at 750MHz vary from approximately 0.67 ppm at omega(r)/2pi approximately 5kHz to approximately 0.20 ppm at omega(r)/2pi approximately 30kHz, indicating that useful resolution is available in the 1H spectrum via this approach. Since the experiments circumvent the problem of dipolar truncation in the 1H-1H spin system, they should make it possible to measure long-range distances in a uniformly labeled environment. Thus, we expect the experiments to be useful in constraining the global fold of a protein.     

Recoupled polarization-transfer methods for solid-state (1)H--(13)C heteronuclear correlation in the limit of fast MAS

An in-depth account of the effects of homonuclear couplings and multiple heteronuclear couplings is given for a recently published technique for (1)H--(13)C dipolar correlation in solids under very fast MAS, where the heteronuclear dipolar coupling is recoupled by means of REDOR pi-pulse trains. The method bears similarities to well-known solution-state NMR techniques, which form the framework of a heteronuclear multiple-quantum experiment. The so-called recoupled polarization-transfer (REPT) technique is versatile in that rotor-synchronized (1)H--(13)C shift correlation spectra can be recorded. In addition, weak heteronuclear dipolar coupling constants can be extracted by means of spinning sideband analysis in the indirect dimension of the experiment. These sidebands are generated by rotor encoding of the reconversion Hamiltonian. We present generalized variants of the initially described heteronuclear multiple-quantum correlation (HMQC) experiment, which are better suited for certain applications. Using these techniques, measurements on model compounds with (13)C in natural abundance, as well as simulations, confirm the very weak effect of (1)H--(1)H homonuclear couplings on the spectra recorded with spinning frequencies of 25--30 kHz. The effect of remote heteronuclear couplings on the spinning-sideband patterns of CH(n) groups is discussed, and (13)C spectral editing of rigid organic solids is shown to be practicable with these techniques.