Improvement of resolution in solid state NMR spectra with J-decoupling: an analysis of lineshape contributions in uniformly 13C-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.     

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.     

<Superscript>33</Superscript>S MAS NMR of a disordered sulfur-doped silicate: Signal enhancement via RAPT,QCPMG and adiabatic pulses

Three different signal enhancement techniques have been applied to ³³S magic-angle spinning nuclear magnetic resonance (MAS NMR) of a disordered silicate containing 1.15 wt% ³³S. Partial saturation of the satellite transitions was achieved using a rotor-assisted population transfer (RAPT) pulse sequence, resulting in a signal enhancement of 1.63, albeit with a slight distortion of the line shape due to selective excitation. Adiabatic inversion of the satellite transitions by various amplitude-and frequency-modulated pulse shapes (such as hyperbolic secant and wideband uniform-rate smooth truncation) was also attempted, resulting in a signal enhancement of up to 1.85, with no apparent line shape distortion. Quadrupolar Carr-Purcell-Meiboom-Gill (QCPMG) and RAPT-QCPMG sequences were also used, both of which yielded spikelet spectra that accurately reflected the MAS line shape with a greatly improved signal-to-noise ratio. It is hoped that this study demonstrates that ³³S solid-state MAS NMR is now feasible even on disordered, low-sulfur-content systems.     

Double-quantum-filtered rotational-resonance MAS NMR in the presence of large chemical shielding anisotropies

Double-quantum filtration under rotational resonance MAS NMR conditions where the chemical shielding anisotropies involved exceed the differences in isotropic chemical shielding is considered by means of numerical simulations and (13)C MAS NMR experiments. The responses of two different pulse sequences, suitable for double-quantum filtration specifically under rotational resonance conditions, to large chemical shielding anisotropies are compared. In the presence of large chemical shielding anisotropies a very recently introduced pulse sequence (T. Karlsson, M. Edén, H. Luthman, and M. H. Levitt, J. Magn. Reson. 145, 95-107, 2000) suffers losses in double-quantum-filtration efficiencies. The double-quantum-filtration efficiency of another pulse sequence (N. C. Nielsen, F. Creuzet, R. G. Griffin, and M. H. Levitt, J. Chem. Phys. 96, 5668-5677, 1992) is less afflicted by the presence of large chemical shielding anisotropies. Both sequences deliver double-quantum-filtered lineshapes that sensitively reflect chemical shielding tensor orientations. It is further shown that double-quantum-filtered rotational-resonance lineshapes of spin systems composed of more than two spins offer a suitable experimental approach for determining chemical shielding tensor orientations for cases where conventional rotational-resonance experiments are not applicable due to the presence of additional background resonances.     

PFG-assisted selection and suppression of 1H NMR signals in the solid state under fast MAS

Under fast MAS conditions, techniques for 1H signal selection and suppression, which have originally been developed for solution-state NMR, become applicable to solids. In this work, we describe how WATERGATE and DANTE pulse sequences can be used under MAS to selectively excite or suppress peaks in 1H solid-state spectra. As known from the liquid-state analogues, signal selection and/or suppression is supported by pulsed-field gradients which selectively dephase and rephase transverse magnetisation. Under MAS, the required field gradients are provided by a simple pair of coils which have been built into a standard fast-MAS probe. PFG-assisted techniques enable efficient selection or suppression of 1H peaks in a single transient of the pulse sequence without the need for phase cycles. Therefore, these tools can readily be incorporated into solid-state MAS NMR experiments, which is demonstrated here for 1H-1H double-quantum NMR spectra of supramolecular systems. In the examples presented here, the 1H signals of interest are relatively weak and need to be observed despite the presence of the strong 1H signal of long alkyl sidechains. PFG-assisted suppression of this strong perturbing signal is shown to be particularly useful for obtaining unambiguous results.     

The 'DANTE' experiment

The selective excitation scheme known as ‘DANTE’ emerged from a confluence of several ideas for new NMR experiments, some more fanciful than others. DANTE offers a simple and effective way to restrict excitation to a very narrow frequency band, usually that of a single resonance line. Initially applied to the study of individual proton-coupled carbon-13 spin multiplets, the method has been extended to water presaturation, relaxation measurements, and chemical exchange studies. Through the imposition of a magnetic field gradient it offers a simple method to enhance resolution by restricting the effective volume of the sample. Multiple DANTE excitation (with Hadamard encoding) can speed up multidimensional spectroscopy by orders of magnitude. Applied to magnetic resonance imaging, the DANTE sequence has been used to superimpose a rectangular grid onto a cardiac image, permitting motional distortions to be monitored in real time.     

Selective saturation in strongly inhomogeneous magnetic fields

The possibility to produce selective saturation by nuclear magnetic resonance (NMR) sequences of low-power radio-frequency pulses in strongly inhomogeneous magnetic fields is explored. The saturation of parts of the sensitive volume is produced by a particular pulse sequence with reduced amplitude distribution and the spectrum of the recorded signal is compared with the simulated spectrum. The spectra of the recorded free induction decays and echo signals are in good agreement with the simulated spectra of the pulse sequence, which demonstrates the effect of the selective saturation. The results obtained are an important step towards the development of new mobile and lowpower NMR equipments operating with inhomogeneous magnetic fields.     

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.     

A method for detection of spectral spin diffusion from minor peaks, and its application to F MAS NMR of antimony(III)-doped fluorapatite

A new technique for detecting spectral spin diffusion in solids under MAS NMR conditions that is particularly well suited for accurately measuring cross-relaxation from minor spectral components is presented. The pulse sequence, SINK (Saturation Inter-Nuclear Kinetics), selectively saturates the magnetization of a minor spectral component with a series of rotor-synchronized DANTE pulse trains and monitors spin diffusion to other peaks with a non-selective 90° pulse. We have used SINK with ¹⁹F MAS NMR on samples of calcium fluorapatite doped with Sb³⁺ to measure spin diffusion between a weak peak at 68.6 ppm due to fluoride ions associated with Sb³⁺ and other peaks in the spectrum. The SINK experiment clearly demonstrates that spin diffusion from the former peak to the main resonance of fluorapatite at 64.0 ppm is faster than spin diffusion to a second antimony-related peak at 65.6 ppm. These results strengthen our previous conclusion that antimony(III) occupies a phosphate site in the apatite lattice, with an SbO₃³⁻ group replacing a PO₄³⁻ group. The SINK experiment also enables the detection of a “hidden” peak at approximately 62.9 ppm that is otherwise obscured by the intense main peak at 64.0 ppm.     

Amino-acid selective experiments on uniformly 13C and 15N labeled proteins by MAS NMR: Filtering of lysines and arginines

Amino-acid selective magic-angle spinning (MAS) NMR experiments can aid the assignment of ambiguous cross-peaks in crowded spectra of solid proteins. In particular for larger proteins, data analysis can be hindered by severe resonance overlap. In such cases, filtering techniques may provide a good alternative to site-specific spin-labeling to obtain unambiguous assignments that can serve as starting points in the assignment procedure. In this paper we present a simple pulse sequence that allows selective excitation of arginine and lysine residues. To achieve this, we make use of a combination of specific cross-polarization for selective excitation [M. Baldus, A.T. Petkova, J. Herzfeld, R.G. Griffin, Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems, Mol. Phys. 95 (1998) 1197-1207.] and spin diffusion for transfer along the amino-acid side-chain. The selectivity of the filter is demonstrated with the excitation of lysine and arginine side-chain resonances in a uniformly 13C and 15N labeled protein preparation of the alpha-spectrin SH3 domain. It is shown that the filter can be applied as a building block in a 13C-13C lysine-only correlation experiment.     

Slow-down of 13C spin diffusion in organic solids by fast MAS: a CODEX NMR Study.

One- and two-dimensional 13C exchange nuclear magnetic resonance experiments under magic-angle spinning (MAS) can provide detailed information on slow segmental reorientations and chemical exchange in organic solids, including polymers and proteins. However, observations of dynamics on the time scale of seconds or longer are hampered by the competing process of dipolar 13C spin exchange (spin diffusion). In this Communication, we show that fast MAS can significantly slow down the dipolar spin exchange effect for unprotonated carbon sites. The exchange is measured quantitatively using the centerband-only detection of exchange technique, which enables the detection of exchange at any spinning speed, even in the absence of changes of isotropic chemical shifts. For chemically equivalent unprotonated 13C sites, the dipolar spin exchange rate is found to decrease slightly less than proportionally with the sample-rotation frequency, between 8 and 28 kHz. In the same range, the dipolar spin exchange rate for a glassy polymer with an inhomogeneously broadened MAS line decreases by a factor of 10. For methylene groups, no or only a minor slow-down of the exchange rate is found.     

Selective excitation in Fourier transform nuclear magnetic resonance. 1978

The applications of frequency-selective excitation methods in Fourier transform NMR are discussed, and a simple technique is described for selective excitation of a narrow frequency region of a high-resolution NMR spectrum in a Fourier transform spectrometer. A regular sequence of identical radiofrequency pulses of small flip angle exerts a strong cumulative effect on magnetizations close to resonance with the transmitter frequency or one of a set of equally spaced sidebands separated by the pulse repetition rate. All other magnetizations precess through an incomplete number of full rotations between pulses, and are caught by successive pulses at an ever changing phase of their precession, which destroys the cumulative effect. The motion of the various nuclear magnetization vectors may be described pictorially according to the Bloch equations, neglecting relaxation during the pulse sequence. A general theory is presented for selective or “tailored” excitation by an arbitrary modulation of the radiofrequency transmitter signal. It confirms earlier conclusions that the frequency-domain excitation spectrum corresponds to the Fourier transform of the transmitter modulation pattern, provided that the NMR response remains linear. The excitation spectra calculated for the selective pulse sequence by these two alternative approaches show good agreement within their respective limitations. A number of practical applications of selective excitation are explored, including solvent peak suppression, the detection of partial spectra from individual chemical sites, selective studies of relaxation and slow chemical exchange, and holeburning or localized saturation.     

13C PGSE NMR experiment with heteronuclear dipolar decoupling to measure diffusion in liquid crystals and solids

A new PGSE NMR experiment, designed to measure molecular diffusion coefficients in systems with nonvanishing static dipolar coupling, is described. The fast static dipolar dephasing of the single-quantum (13)C coherences is removed by multiple-pulse heteronuclear decoupling. The resulting slow dephasing of the (13)C coherences allows for inserting appropriate gradient pulses into the pulse sequence. The presence of the large magnetic field gradient reduces the efficiency of the decoupling sequences which is compensated for by introducing a scheme of sequential slice selection across the sample. The method is demonstrated by (19)F-decoupled (13)C PGSE NMR experiments in a lyotropic nematic and lamellar liquid crystal.     

Shaped pulses for selective inversion of magnetization in solids spinning at the magic angle

In order to achieve selective inversion of a chosen family of sidebands in NMR spectra of solids rotating at the magic angle (MAS spectra), a sequence of so-called DANTE pulses can be applied in synchronization with the sample rotation period. It is shown by simulation and experiment that the profiles of the offset dependence can be greatly improved by varying the amplitudes of the DANTE pulses, so that the envelope corresponds to that of a G³ Gaussian cascade. Alternatively, the DANTE pulses may be replaced by sandwiches composed of four hard pulses, adjusted so that the time-dependence of the nutation angles matches the envelope of a G³ Gaussian cascade. It is shown by simulations that in solids the profiles are affected by homogeneous decay of magnetization, in analogy to the influence of transverse relaxation in liquids. Applications to the intramolecular rearrangement (tautomerism) of polycrystalline tropolone illustrate that dynamic processes in the solid state can be monitored with great accuracy.     

Heteronuclear dipolar recoupling in liquid crystals and solids by PISEMA-type pulse sequences

A pulse sequence is described for the recoupling of heteronuclear dipolar interactions under MAS. The method is similar to the PISEMA experiment, but employs a well-defined amplitude modulation of one of the two radio-frequency fields. The technique is used for measurements of 1H-13C dipolar couplings in unoriented solid and liquid-crystalline samples.     

A 13C solid-state NMR investigation of the alkynyl carbon chemical shift tensors for 2-butyne-1,4-diol

The alkynyl carbon chemical shift (CS) tensors for 2-butyne-1,4-diol are reported, based on analyses of the carbon-13 NMR spectra of stationary-powder and slow magic-angle spinning (MAS) samples for which the alkynyl carbon nuclei are enriched in 13C. NMR spectra of slow MAS samples exhibit spinning-frequency-dependent fine structure typical of crystallographically equivalent but magnetically distinct nuclei. Simulated spectra of slow MAS samples of this two-spin system are particularly sensitive to the relative orientations of the CS tensors. In addition, the value of 1J(13C, 13C), +175 +/- 10 Hz, is determined by examination of the total NMR lineshape of slow MAS samples. The CS tensors are almost axially symmetric, delta11 = 158.9 +/- 1.0 ppm and delta22 = 155.7 +/- 1.0 ppm; the direction of greatest shielding is approximately along the alkynyl C-C bond, delta33 = -57.8 +/- 2.0 ppm. Both the magnitudes of the principal components of the CS tensors and their orientations are in agreement with those predicted from first-principles calculations at the HF and MP2 levels of theory. This study demonstrates the importance of examining the NMR spectra of homonuclear two-spin systems with and without MAS under a variety of conditions (e.g., two or more applied magnetic fields and slow MAS).     

Methylene spectral editing in solid-state 13C NMR by three-spin coherence selection

A robust new solid-state nuclear magnetic resonance (NMR) method for selecting CH2 signals in magic-angle spinning (MAS) 13C NMR spectra is presented. Heteronuclear dipolar evolution for a duration of 0.043 ms, under MREV-8 homonuclear proton decoupling, converts 13C magnetization of CH2 groups into two- and three-spin coherences. The CH2 selection in the SIJ (C H H) spin system is based on the three-spin coherence S(x)I(z)J(z), which is distinguished from 13C magnetization (S(x)) by a 1H 0 degrees/90 degrees pulse consisting of two 45 degrees pulses. The two-spin coherences of the type S(y)I(z) are removed by a 13C 90 degrees x-pulse. The three-spin coherence is reconverted into magnetization during the remainder of the rotation period, still under MREV-8 decoupling. The required elimination of 13C chemical-shift precession is achieved by a prefocusing 180 degrees pulse bracketed by two rotation periods. The selection of the desired three-spin coherence has an efficiency of 13% theoretically and of 8% experimentally relative to the standard CP/MAS spectrum. However, long-range couplings also produce some three-spin coherences of methine (CH) carbons. Therefore, the length of the 13C pulse flipping the two-spin coherences is increased by 12% to slightly invert the CH signals arising from two-spin coherences and thus cancel the signal from long-range three-spin coherences. The signal intensity in this cleaner spectrum is 6% relative to the regular CP/TOSS spectrum. The only residual signal is from methyl groups, which are suppressed at least sixfold relative to the CH2 peaks. The experiment is demonstrated on cholesteryl acetate and applied to two humic acids.     

13C/14N heteronuclear multiple-quantum correlation with rotary resonance and REDOR dipolar recoupling

A two-dimensional (13)C/(14)N heteronuclear multiple quantum correlation (HMQC) experiment using dipolar recoupling under magic-angle spinning (MAS) is described. The experiment is an extension of the recent indirect (13)C detection scheme for measuring (14)N quadrupolar coupling under MAS. The recoupling allows the direct use of the much larger dipolar interaction instead of the small J and residual dipolar couplings for establishing (13)C/(14)N correlations. Two recoupling methods are incorporated into the HMQC sequence, both applying rf only to the observed (13)C spin. The first one uses the REDOR sequence with two pi-pulses per rotor cycle. The second one uses a cw rf field matching the spinning frequency, known as rotary resonance. The effects of CSA, T(2)(') signal loss, MAS frequency and stability and t(1)-noise are compared and discussed.     

Photo-CIDNP MAS NMR beyond the T1 limit by fast cycles of polarization extinction and polarization generation

In nanosecond-laser flash photo-CIDNP MAS NMR, polarization generation (PG) proceeds much faster than longitudinal spin relaxation. With a nanosecond-laser setup linked to the NMR console the repetition time of the experiment is then limited by the minimum recycle delay of the NMR spectrometer and the maximum repetition rate of laser flashes. These limits can only be reached if polarization left after the NMR experiment is completely canceled before the next laser flash. We introduce a presaturation pulse sequence, based on three (pi/2) (13)C pulses and optimized timing and phase cycling that allows for such efficient polarization extinction (PE). The technique is demonstrated on selectively isotope labeled bacterial reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides wildtype (WT). High-quality (13)C photo-CIDNP MAS NMR spectra are obtained using cycle rates up to 4 Hz. The PE-PG strategy proposed here provides a general experimental scheme for reduction of measurement time in magnetic resonance experiments based on fast PG.