Proton-detected scalar coupling based assignment strategies in MAS solid-state NMR spectroscopy applied to perdeuterated proteins

Assignment of proteins in MAS (magic angle spinning) solid-state NMR relies so far on correlations among heteronuclei. This strategy is based on well dispersed resonances in the ¹⁵N dimension. In many complex cases like membrane proteins or amyloid fibrils, an additional frequency dimension is desirable in order to spread the amide resonances. We show here that proton detected HNCO, HNCA, and HNCACB type experiments can successfully be implemented in the solid-state. Coherences are sufficiently long lived to allow pulse schemes of a duration greater than 70 ms before incrementation of the first indirect dimension. The achieved resolution is comparable to the resolution obtained in solution-state NMR experiments. We demonstrate the experiments using a triply labeled sample of the SH3 domain of chicken α-spectrin, which was re-crystallized in H₂O/D₂O using a ratio of 1/9. We employ paramagnetic relaxation enhancement (PRE) using EDTA chelated Cu^II to enable rapid data acquisition.     

Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins

Previously, Ishii et al., could show that chelated paramagnetic ions can be employed to significantly decrease the recycle delay of a MAS solid-state NMR experiment [N.P. Wickramasinghe, M. Kotecha, A. Samoson, J. Past, Y. Ishii, Sensitivity enhancement in C-13 solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing H-1 T-1 relaxation, J. Magn. Reson. 184 (2007) 350-356]. Application of the method is limited to very robust samples, for which sample stability is not compromised by RF induced heating. In addition, probe integrity might be perturbed in standard MAS PRE experiments due to the use of very short duty cycles. We show that these deleterious effects can be avoided if perdeuterated proteins are employed that have been re-crystallized from D(2)O:H(2)O=9:1 containing buffer solutions. The experiments are demonstrated using the SH3 domain of chicken alpha-spectrin as a model system. The labeling scheme allows to record proton detected (1)H, (15)N correlation spectra with very high resolution in the absence of heteronuclear dipolar decoupling. Cu-edta as a doping reagent yields a reduction of the recycle delay by up to a factor of 15. In particular, we find that the (1)H T(1) for the bulk H(N) magnetization is reduced from 4.4s to 0.3s if the Cu-edta concentration is increased from 0mM to 250 mM. Possible perturbations like chemical shift changes or line broadening due to the paramagnetic chelate complex are minimal. No degradation of our samples was observed in the course of the experiments.     

Characteristics of zero-quantum correlation spectroscopy in MAS NMR experiments

Zero-quantum coherence generation and reconversion in magic-angle spinning solid-state NMR is analyzed. Two methods are discussed based on implementations using symmetry-based pulse sequences that utilize either isotropic J couplings or dipolar couplings. In either case, the decoupling of abundant proton spins plays a crucial role for the efficiency of the zero-quantum generation. We present optimized sequences for measuring zero-quantum single-quantum correlation spectra in solids, achieving an efficiency of 50% in ubiquitin. The advantages and disadvantages of zero-quantum single-quantum over single-quantum single-quantum correlation spectroscopy are explored, and similarities and differences with double-quantum single-quantum correlation spectroscopy are discussed. Finally, possible application of zero-quantum single-quantum experiments to polypeptides, where it can lead to better spectral resolution is investigated using ubiquitin, where we find high efficiency and high selectivity, but also increased line widths in the MQ dimension.     

Fast acquisition of multi-dimensional spectra in solid-state NMR enabled by ultra-fast MAS

The advantages offered by ultra-fast (>60 kHz) magic angle spinning (MAS) rotation for the study of biological samples, notably containing paramagnetic centers are explored.It is shown that optimal conditions for performing solid-state ¹³C NMR under 60 kHz MAS are obtained with low-power CW ¹H decoupling, as well as after a low-power ¹H,¹³C cross-polarization step at a double-quantum matching condition. Acquisition with low-power decoupling highlights the existence of rotational decoupling sidebands. The sideband intensities and the existence of first and second rotary conditions are explained in the framework of the Floquet–van Vleck theory.As a result, optimal ¹³C spectra of the oxidized, paramagnetic form of human copper zinc superoxide dismutase (SOD) can be obtained employing rf-fields which do not exceed 40 kHz during the whole experiment. This enables the removal of unwanted heating which can lead to deterioration of the sample. Furthermore, combined with the short ¹H T₁s, this allows the repetition rate of the experiments to be shortened from 3 s to 500 ms, thus compensating for the sensitivity loss due to the smaller sample volume in a 1.3 mm rotor. The result is that 2D ¹³C–¹³C correlation could be acquired in about 24 h on less than 1 mg of SOD sample.     

Enhanced resolution in proton solid-state NMR with very-fast MAS experiments

We present a new smooth amplitude-modulated (SAM) method that allows to observe highly resolved ¹H spectra in solid-state NMR. The method, which works mainly at fast or ultra-fast MAS speed (ν_R > 25 kHz) is complementary to previous methods, such as DUMBO, FSLG/PMLG or symmetry-based sequences. The method is very robust and efficient and does not present line-shape distortions or fake peaks. The main limitation of the method is that it requires a modern console with fast electronics that must be able to define the cosine line-shape in a smooth way, without any transient. However, this limitation mainly occurs at ultra-fast MAS where the rotation period is very short.     

Through-bond heteronuclear single-quantum correlation spectroscopy in solid-state NMR, and comparison to other through-bond and through-space experiments

A new through-bond carbon-proton correlation technique, the MAS-J-HSQC experiment, is described for solid-state NMR. This new pulse scheme is compared experimentally with the previously proposed MAS-J-HMQC experiment in terms of proton resolution on a model sample of powdered L-alanine. We show that for natural abundance compounds, the MAS-J-HMQC and MAS-J-HSQC experiments give about the same proton resolution, whereas, for (13)C-labeled materials, narrower proton linewidths are obtained with the MAS-J-HSQC experiment. In addition we show that in scalar as well as in dipolar heteronuclear shift correlation experiments, when the proton chemical shift is encoded by the evolution of a single-quantum coherence, the proton resolution can be enhanced by simply adding a 180 degrees carbon pulse in the middle of the t(1) evolution time.     

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.     

Multiple-time correlation functions in spin-3/2 solid-state NMR spectroscopy

Stimulated echo spectroscopy of nonselectively excitable I = 3/2 nuclei offers new perspectives for the investigation of ultraslow motions predominantly in inorganic solids and solid-like materials. Conditions for the generation of pure, quadrupole modulated multipolar spin orders and for the detection of two- and four-time correlation functions are discussed. The case of spins I > 3/2 is also briefly considered. Copyright 2000 Academic Press.     

Low-power XiX decoupling in MAS NMR experiments

Low-power XiX proton decoupling under fast magic-angle spinning is introduced. The method is applicable if the MAS frequency exceeds the proton-proton interactions. For rigid organic solids this is the case for MAS frequencies above approximately 40 kHz. It is shown that the quality of the decoupling as well as the sensitivity to frequency offsets can be improved compared to low-power continuous-wave decoupling. The decoupling efficiency is somewhat reduced compared to optimized high-power decoupling: in a peptide sample investigated at an MAS frequency of 50 kHz a loss of about 10% in signal intensity for CH3 and CH groups, and of about 40% for CH2 groups was observed. Taking into consideration, that the rf amplitude in the low-power XiX was about 15 times lower than in high-power XiX decoupling, such a reduction in line intensity is sometimes tolerable.     

Proton-detected heteronuclear single quantum correlation NMR spectroscopy in rigid solids with ultra-fast MAS

In this article, we show the potential for utilizing proton-detected heteronuclear single quantum correlation (HSQC) NMR in rigid solids under ultra-fast magic angle spinning (MAS) conditions. The indirect detection of carbon-13 from coupled neighboring hydrogen nuclei provides a sensitivity enhancement of 3- to 4-fold in crystalline amino acids over direct-detected versions. Furthermore, the sensitivity enhancement is shown to be significantly larger for disordered solids that display inhomogeneously broadened carbon-13 spectra. Latrodectus hesperus (Black Widow) dragline silk is given as an example where the sample is mass-limited and the sensitivity enhancement for the proton-detected experiment is 8- to 13-fold. The ultra-fast MAS proton-detected HSQC solid-state NMR technique has the added advantage that no proton homonuclear decoupling is applied during the experiment. Further, well-resolved, indirectly observed carbon-13 spectra can be obtained in some cases without heteronuclear proton decoupling.     

Optimal control based NCO and NCA experiments for spectral assignment in biological solid-state NMR spectroscopy

We present novel pulse sequences for magic-angle-spinning solid-state NMR structural studies of (13)C,(15)N-isotope labeled proteins. The pulse sequences have been designed numerically using optimal control procedures and demonstrate superior performance relative to previous methods with respect to sensitivity, robustness to instrumental errors, and band-selective excitation profiles for typical biological solid-state NMR applications. Our study addresses specifically (15)N to (13)C coherence transfers being important elements in spectral assignment protocols for solid-state NMR structural characterization of uniformly (13)C,(15)N-labeled proteins. The pulse sequences are analyzed in detail and their robustness towards spin system and external experimental parameters are illustrated numerically for typical (15)N-(13)C spin systems under high-field solid-state NMR conditions. Experimentally the methods are demonstrated by 1D (15)N-->(13)C coherence transfer experiments, as well as 2D and 3D (15)N,(13)C and (15)N,(13)C,(13)C chemical shift correlation experiments on uniformly (13)C,(15)N-labeled ubiquitin.     

Rotor-synchronized dipolar-filter sequence at fast MAS in solid-state NMR

Dipolar filters are of considerable importance for eliminating the ¹H NMR signal of the rigid components of heterogeneous compounds while selecting the signal of their mobile parts. On the basis of such filters, structural and dynamical information of these compounds can often be acquired through further manipulations (e.g. spin diffusion) on the spin systems. To overcome the destructive interferences between the magic angle spinning (MAS) speed and the cycle-time of the widely-used Rotor-Asynchronized Dipolar Filter (RADF) sequence, we introduce a new method called Rotor-Synchronized Dipolar Filter (RSDF). This communication shows that this sequence does not present any interference with the spinning speed and is more compatible than RADF with high MAS frequencies (ν_R > 12 kHz). This new pulse sequence will potentially contribute to future researches on heterogeneous materials, such as multiphase polymer and membrane systems.     

Quantitative characterization of coal structure by high-resolution CP/MAS 13C solid-state NMR spectroscopy

A method for quantitatively characterizing the carbon skeletal structure of coal by variable contact time experiment using high-resolution CP/MAS ¹³C solid-state NMR spectroscopy is proposed in this paper. The initial polarization transfer intensity from protons directly bonded with carbons, instead of dipolar-dephasing techniques which had to run on a lower frequency NMR spectrometer (100.02 MHz for proton), was used to divide the bridgehead and protonated aromatic carbons, making all the NMR data in this paper obtained on a high frequency NMR spectrometer (500.12 MHz for proton). On this basis, the fractions of different carbons in coal were further divided by the initial polarization transfer intensity from spin diffusion of protons unbonded with carbons. The structure of Naomaohu coal, a subbituminous coal from China, was measured. The change of polarization transfer with contact time was analyzed quantitatively. The fractions of aromatic, aliphatic, carboxyl and carbonyl carbons, and corrective aromaticity are 0.61, 0.39, 0.1 and 0.51, respectively. The fractions of protonated and bridgehead aromatic carbons are 0.22 and 0.09, respectively. These results agreed with literatures, and bond concentration calculated by the carbon skeletal structure distribution of coal was reasonable.     

A thermometer for nonspinning solid-state NMR spectroscopy

We discuss a method to determine temperature in a static NMR experiment from the temperature variation of the lead nitrate peak shift.     

Aromatic spectral editing techniques for magic-angle-spinning solid-state NMR spectroscopy of uniformly 13C-labeled proteins

The four aromatic amino acids in proteins, namely histidine, phenylalanine, tyrosine, and tryptophan, have strongly overlapping ¹³C chemical shift ranges between 100 and 160 ppm, and have so far been largely neglected in solid-state NMR determination of protein structures. Yet aromatic residues play important roles in biology through π–π and cation–π interactions. To better resolve and assign aromatic residues' ¹³C signals in magic-angle-spinning (MAS) solid-state NMR spectra, we introduce two spectral editing techniques. The first method uses gated ¹H decoupling in a proton-driven spin-diffusion (PDSD) experiment to remove all protonated ¹³C signals and retain only non-protonated carbon signals in the aromatic region of the ¹³C spectra. The second technique uses chemical shift filters and ¹H–¹³C dipolar dephasing to selectively detect the Cα, Cβ and CO cross peaks of aromatic residues while suppressing the signals of all aliphatic residues. We demonstrate these two techniques on amino acids, a model peptide, and the microcrystalline protein GB1, and show that they significantly simplify the 2D NMR spectra and both reveal and permit the ready assignment of the aromatic residues' signals.     

Rotational diffusion of membrane proteins in aligned phospholipid bilayers by solid-state NMR spectroscopy

Solid-state NMR experiments on mechanically aligned bilayer and magnetically aligned bicelle samples demonstrate that membrane proteins undergo rapid rotational diffusion about the normal in phospholipid bilayers. Narrow single-line resonances are observed from 15N labeled sites in the trans-membrane helix of the channel-forming domain of the protein Vpu from HIV-1 in phospholipid bilayers with their normals at angles of 0 degrees, 20 degrees, 40 degrees, and 90 degrees, and bicelles with their normals at angles of 0 degrees and 90 degrees with respect to the direction of the applied magnetic field. This could only occur if the entire polypeptide undergoes rotational diffusion about the bilayer normal. Comparisons between experimental and simulated spectra are consistent with a rotational diffusion coefficient (DR) of approximately 10(5)s-1.     

Coherence selection in double CP MAS NMR spectroscopy

Applications of double cross-polarization (CP) magic-angle spinning (MAS) NMR spectroscopy, via (1)H/(15)N and then (15)N/(13)C coherence transfers, for (13)C coherence selection are demonstrated on a (15)N/(13)C-labeled N-acetyl-glucosamine (GlcNAc) compound. The (15)N/(13)C coherence transfer is very sensitive to the settings of the experimental parameters. To resolve explicitly these parameter dependences, we have systematically monitored the (13)C{(15)N/(1)H} signal as a function of the rf field strength and the MAS frequency. The data reveal that the zero-quantum coherence transfer, with which the (13)C effective rf field is larger than that of the (15)N by the spinning frequency, would give better signal sensitivity. We demonstrate in one- and two-dimensional double CP experiments that spectral editing can be achieved by tailoring the experimental parameters, such as the rf field strengths and/or the MAS frequency.     

Indirectly detected heteronuclear correlation solid-state NMR spectroscopy of naturally abundant 15N nuclei

Two-dimensional indirectly detected through-space and through-bond ¹H{¹⁵N} solid-state NMR experiments utilizing fast magic angle spinning (MAS) and homonuclear multipulse ¹H decoupling are evaluated. Remarkable efficiency of polarization transfer can be achieved at a MAS rate of 40 kHz by both cross-polarization and INEPT, which makes these methods applicable for routine characterizations of natural abundance solids. The first measurement of 2D ¹H{¹⁵N} HETCOR spectrum of natural abundance surface species is also reported.     

Sensitivity enhancement in natural-abundance solid-state 33S MAS NMR spectroscopy employing adiabatic inversion pulses to the satellite transitions

The WURST (wideband uniform rate smooth truncation) and hyperbolic secant (HS) pulse elements have each been employed as pairs of inversion pulses to induce population transfer (PT) between the four energy levels in natural abundance solid-state (33)S (spin I=3/2) MAS NMR, thereby leading to a significant gain in intensity for the central transition (CT). The pair of inversion pulses are applied to the satellite transitions for a series of inorganic sulfates, the sulfate ions in the two cementitious materials ettringite and thaumasite, and the two tetrathiometallates (NH(4))(2)WS(4) and (NH(4))(2)MoS(4). These materials all exhibit (33)S quadrupole coupling constants (C(Q)) in the range 0.1-1.0 MHz, with precise C(Q) values being determined from analysis of the PT enhanced (33)S MAS NMR spectra. The enhancement factors for the WURST and HS elements are quite similar and are all in the range 1.74-2.25 for the studied samples, in excellent agreement with earlier reports on HS enhancement factors (1.6-2.4) observed for other spin I=3/2 nuclei with similar C(Q) values (0.3-1.2 MHz). Thus, a time saving in instrument time by a factor up to five has been achieved in natural abundance (33)S MAS NMR, a time saving which is extremely welcome for this important low-gamma nucleus.