Utilizing Afterglow Magnetization from Cross-Polarization Magic-Angle-Spinning Solid-State NMR Spectroscopy to Obtain Simultaneous Heteronuclear Multidimensional Spectra

Abstract

The time required for data acquisition and subsequent spectral assignment are limiting factors for determining biomolecular structure and dynamics using solid state NMR spectroscopy. While strong magnetic dipolar couplings give rise to relatively broad spectra lines, the couplings also mediate the coherent magnetization transfer via the Hartmann Hahn cross polarization (HH-CP) experiment. This mechanism is used in nearly all backbone assignment experiments for carrying out polarization transfer between ¹H, ¹⁵N, and ¹³C. In this Article, we describe a general spectroscopic approach to use the residual or afterglow magnetization from the ¹⁵N to ¹³C selective HH-CP experiment to collect a second multidimensional heteronuclear dataset. This approach allowed for the collection of two multidimensional (2D NCA and NCO or 3D NCACX and NCOCX) datasets at the same time. These experiments were performed using instrumentation available on all standard solid state NMR spectrometers configured for magic angle spinning and were demonstrated on uniformly [¹³C,¹⁵N] and [1,3-¹³C] glycerol labeled ubiquitin. This method is compatible with several other sensitivity enhancement experiments and can be used as an isotopic filtering tool to reduce the spectral complexity and decrease the time needed for assigning spectra.

Introduction

Solid-state NMR magic-angle-spinning (SSNMR MAS) is a widely used method to probe structure and dynamics of hard and soft biological matter.^1-7 One of the main bottlenecks in this process is assigning the spectra, which can be especially challenging for membrane proteins and amyloid samples. To address this problem, a number of ways have been proposed to improve the spectral resolution and sensitivity of data acquisition. These can be broadly grouped into three categories: (1) spectroscopic based (e.g., pulse sequences, data acquisition), (2) sample preparation (e.g., isotopic labeling, paramagnetic labeling), and (3) advances in instrumentation (e.g., multiple receivers, cryogenic probes). Combined spectroscopic and instrumentation approaches such as simultaneous or sequential data acquisitions have been applied to the time-consuming process of spectral assignment through the improvement of these triple resonance techniques that shorten data acquisition times.^8-11

A novel solution NMR approach recently introduced by Kupce et al. utilizes the residual or “afterglow” ¹³C magnetization for the purpose of acquiring multiple heteronuclear spectra at the same time.⁹ In this pulse sequence, the ¹³C magnetization was directly detected to acquire a 2D (HA)CACO experiment nuclease A inhibitor in the standard way. The residual ¹³CO magnetization was then transferred to ¹⁵N and finally detected using the sensitive ¹H_N magnetization in a 3D (HA)CA(CO)NNH experiment. Since ¹³C and ¹H signals were detected, these experiments made use of parallel acquisition requiring two receivers. Unlike this solution NMR methodology that relied on J-couplings, the most important transfer mechanism in oriented and MAS SSNMR is the Hartmann Hahn cross-polarization (HH-CP).^12-15 This experiment utilizes matched spinlocks on two channels resulting in coherent polarization transfer from one nucleus to the other. Since the CP is the basic element for nearly all SSNMR applications, a tremendous amount of effort has been devoted to understanding and improving this experiment,^16-20 including the use of multiple contact pulses.^12,21 Transfer of magnetization among low frequency ¹⁵N and ¹³C most commonly utilizes the double CP^22-26 or other novel polarization transfer approaches.^27-32

A new application of the CP experiment was recently proposed and demonstrated using RNA in SSNMR spectroscopy. This triple resonance cross polarization method uses ¹H to simultaneously polarize ¹³C and ¹⁵N with subsequent parallel acquisition using two receivers.⁸ This scheme has been applied to obtain sequentially acquired datasets that give a ¹³C-¹³C correlation spectrum and a heteronuclear ¹³C-¹⁵N (NCA or NCO) correlation spectrum using a single receiver (DU-MAS).¹⁰ Careful optimization of the simultaneous cross-polarization will give only small losses on the transfer from ¹H to both ¹⁵N and ¹³C as compared to the double resonance CP experiment. These methods and others listed above rely on relatively long ¹⁵N and ¹³C T₁ and T_1ρ values in soft and hard matter, including membrane proteins in oriented lipid bilayers.^33,34

To improve the efficiency of data acquisition in SSNMR MAS, we describe an approach to detect residual or “afterglow” magnetization resulting from the double CP experiment involving selective transfer from ¹⁵N to ¹³CA. We show that this ¹⁵N magnetization is appreciable and can be used to obtain a second multidimensional heteronuclear correlation experiment with good sensitivity. In practice, the result is the detection of two 2D (NCA and NCO) or two 3D (NCACX and NCOCX) experiments at the same time (i.e., two for the price of one). These experiments do not require special instrumentation, and are therefore applicable to all spectroscopic approaches where residual coherence can be salvaged for increasing the speed of data acquisition.

Experimental Methods

Sample Preparation

Ubiquitin was expressed in BL21(DE3) E. coli bacteria in the presence of uniformly labeled ¹³C₆-glucose and ¹⁵N-ammonium chloride in minimal media (M9) and purified as previously described.³⁵ The glycerol labeling experiment was achieved by replacing glucose with [1,3-¹³C] glycerol (1 g/L) and natural abundance sodium carbonate (1 g/L).^36,37 For preparation of the solid-state NMR samples, 10 mg of ubiquitin was dissolved in 400 Fl 20 mM sodium citrate at pH 4.1. Crystallization was initiated by the dropwise addition of 2-methyl-2,4-pentanediol (MPD) to a final concentration of 60% and allowed to proceed overnight at 4 °C.³⁸ The samples were packed into 3.2 mm MAS rotors using samples spacers to prevent sample dehydration.

NMR Spectroscopy

All NMR experiments were carried out using a DDR2 Agilent NMR spectrometer operating at a ¹H frequency of 600 MHz. The temperature was set to 0 °C and the MAS rate was 12500 ± 5 Hz. The initial cross-polarization from ¹H to ¹⁵N utilized a contact time of 1 msec. The transfers between ¹⁵N to ¹³CA and ¹⁵N to ¹³CO utilized SPECIFIC-CP²³, a form of double CP,²⁵ where the ¹⁵N offset was set to 121 ppm, the ¹³CA offset to 57.5 ppm, and the ¹³CO offset to 175 ppm. The ¹⁵N to ¹³CA transfer used a tangent adiabatic ramp²² on the ¹⁵N channel, while the ¹³CO SPECIFIC-CP utilized the ramp on the carbon channel. The Δ/2π and β/2π parameters of the adiabatic cross-polarization were set to 1.2 kHz and 0.3 kHz for the ¹⁵N to ¹³CA transfer and 3.7 kHz and 0.9 kHz for the ¹⁵N to ¹³CO transfer (see Eqns. 1 and 2 of Franks et al.³⁹) with the total length of the cross-polarization time set to 4 msec. All parameters were optimized to obtain maximal signal intensity in the standard 1D NCO and NCA experiments. Both sequential data acquisition periods used a ¹³C spectral width of 100 kHz and an acquisition time of 20 msec. The indirect ¹⁵N dimension was acquired with a spectral width of 3125 Hz and 28 increments. A total of 64 and 128 scans were used for the [U-¹³C,¹⁵N] and [1,3-¹³C] glycerol labeled ubiquitin samples, respectively. For spin diffusion experiments between ¹³C spins, the DARR condition was set to the n=1 rotary resonance condition on ¹H (12.5 kHz).⁴⁰

Results

Sequential Acquisition of Residual ¹⁵N Magnetization

The standard experiments for obtaining resonance assignments and distance restraints in MAS utilize multiple transfers between ¹⁵N and ¹³CA or ¹⁵N and ¹³CO. The NCACX and NCOCX experiments are two of the most valuable that give correlations among the backbone nuclei. The former correlates intra-residue carbon atoms with the chemical shifts of ¹⁵N and ¹³CA. This involves a HH-CP element where ¹H polarization is transferred to ¹⁵N and followed by a ¹⁵N chemical shift evolution in t₁. Magnetization is then most commonly transferred to ¹³CA using a type of double cross polarization²⁵ called SPECIFIC CP.²³ After the transfer, ¹³CA chemical shifts are evolved in the t₂ time dimension and then allowed to undergo spin diffusion most commonly with a DARR mixing period.⁴⁰ Finally, the ¹³C magnetization is detected in the direct dimension. These are the standard experiments carried out and will be referred to as the STD-NCA or STD-NCACX (with DARR).

Following the SPECIFIC-CP transfer to ¹³CA, there is residual ¹⁵N magnetization remaining that is typically discarded. Using [U-¹⁵N,¹³C] ubiquitin, we carried out the 1D NCA experiment detecting on ¹⁵N immediately following the SPECIFIC-CP transfer to ¹³CA. Relative to the ¹⁵N magnetization after CP from ¹H, ~40-45% of the ¹⁵N signal remained. Therefore, it is possible to detect this ¹⁵N polarization at the same time as the STD-NCA ¹³C detected experiment with a second receiver (parallel acquisition¹¹). However, 1D spectra do not provide the resolution required for resolving all the protein resonances. Instead, the pulse sequence in Figure 1 shows how afterglow (or residual) ¹⁵N signal can be salvaged to obtain a second 2D NCO or 3D NCOCX dataset. The first half of the pulse sequence in Figure 1 is identical to that of a standard NCA (or NCACX) experiment. To reuse the residual ¹⁵N magnetization, a 90° pulse is applied to ¹⁵N spins immediately following the SPECIFIC-CP transfer to ¹³CA. This stores the magnetization along the z-axis. Since the T₁ relaxation times for ¹⁵N in proteins are on the order of seconds, placing the spins along the z-axis results in only a small amount of magnetization lost due to longitudinal relaxation. Following the free-induction-decay acquisition for the 2D NCA (or 3D NCACX) experiment, a 90_x° pulse is applied to ¹⁵N placing it along the y-axis. Next, a second SPECIFIC-CP step is used to transfer magnetization to ¹³CO in the same way as a standard NCO experiment. The ¹³CO magnetization is then directly detected to obtain a 2D NCO or evolved in the indirect t₂’ dimension followed by a DARR mixing element to give a 3D NCOCX experiment. If evolved in the indirect dimension, t₂’ is arrayed concurrently with the ¹³CA t₂ evolution period for the NCACX experiment. Both simultaneously acquired datasets have an identical ¹⁵N chemical shift evolution period (t₁). For brevity, we will refer to the sequentially acquired datasets using the following nomenclature: SIM₁-NCA or SIM₁-NCACX (first dataset acquired) or SIM₂-NCO or SIM₂-NCOCX (second dataset acquired); the standard experiments will be called STD-NCA, STD-NCO, STD-NCACX, or STD-NCOCX.

Figure 1.

Pulse sequence with two acquisitions for simultaneous detection of 2D SIM₁-NCA and SIM₂-NCO or 3D SIM₁-NCACX and SIM₂-NCOCX spectra. The former are achieved by setting the DARR mixing time to zero. The DARR mixing can be replaced with a double quantum transfer for improved single bond transfers. The narrow rectangles correspond to 90° pulses. Phase are: ϕ₁=(x,−x), ϕ₂=(y), ϕ₃=(x,x,y,y), ϕ₄=(−y,−y,−x,−x), ϕ₅=(y,y,−x,−x), and ϕ_rec=(x,−x,−y,y). To obtain phase-sensitive data in t₁ and t₂, ϕ₂ and ϕ₃ were phase-shifted by 90°, respectively. After the first FID acquisition, a 5 msec time was allowed to dephase residual ¹³C magnetization; during this time a 90_x° pulse was applied ¹³C.

Application to Uniformly Labeled Ubiquitin

We carried out the pulse sequence in Figure 1 on selectively and uniformly labeled ubiquitin prepared in a microcrystalline state. [U-¹³C,¹⁵N] ubiquitin has 76 residues and therefore 76 ¹⁵N-¹³CA pairs. The 2D STD-NCA and SIM₁-NCA datasets on [U-¹³C,¹⁵N] ubiquitin are shown in Figure 2. These spectra give the same signal to noise, which is expected since the first half of the pulse sequence in Figure 1 is identical to the STD-NCA experiment. After the first transfer from ¹⁵N to ¹³CA, the amount of ¹⁵N magnetization available for the ¹⁵N to ¹³CO SPECIFIC-CP is reduced. This results in an overall signal intensity of the SIM₂-NCO 2D dataset of 32 ± 3% compared to that of the STD-NCO. However, the SIM₂-NCO gives ~50% the signal/noise as the SIM₁-NCA, which results from the slightly better efficiency of the ¹⁵N to ¹³CO vs. ¹⁵N to ¹³CA SPECIFIC-CP experiment.^39,41 Although the second dataset is lower in intensity (see 1D cross sections in Figure 3), the SIM₂-NCO is a free dataset since the preparation and subsequent detection of the afterglow ¹⁵N magnetization only adds ~25 msec to the overall pulse sequence. In practice, for a recycle delay of 2 sec and an acquisition period of 25 msec, the pulse sequence in Figure 1 only increases the experimental time by 1.2% relative to the STD-NCA. If a 3 sec recycle delay is used, this amounts to an increase of 0.8%.

Figure 2.

Comparison of the standard vs. sequentially acquired datasets on [U-¹³C,¹⁵N] ubiquitin. The STD-NCA and STD-NCO datasets (A and C) used identical experimental parameters as the sequentially acquired ones (B and D). The spectra in panels A and B are each plotted starting at a contour level of 18.75. This is a factor of 1.5 higher than the SIM₂-NCO dataset (panel D; first contour: 12.5). The SIM₂-NCO spectrum (panel C) is plotted at 3 times the signal level compared to the STD-NCO (first contour: 37.5), which accounts for the signal loss in the SIM₂-NCO from the SPECIFIC-CP transfer to ¹³CA. For the listed contour levels, the standard deviation of the noise is 1.0. The peaks in the dotted rectangles indicate side chain residues that have 75% intensity retention. The 2D datasets for the STD-NCA and STD-NCO together required 2 times longer acquisition than that for the SIM₁-NCA and SIM₂-NCO.

Figure 3.

1D cross-sections of the 2D spectra shown in Figure 2 for [U-¹³C,¹⁵N] ubiquitin. The ¹⁵N frequencies are given in each panel. The STD-NCA 1D cross sections (A, C) can be directly compared with those of the SIM1-NCA dataset (B, D). As expected these peak intensities are identical. The STD-NCO cross sections (E, G) are directly compared to those of the SIM2-NCO (F, H). The noise level is the same in all 1D spectra. The spectra in panels E and F correspond to side chain ¹⁵NH₂ resonances.

There are ~45-50 resonances within the NCA spectra shown in Figure 2 out of the 76 residues in ubiquitin. It has been reported that nine peaks do not appear as a result of residual motion in the loop encompassing residues 8-10 and the C-terminus (residues 71-76) at 0 °C.³⁸ In addition, some of the peaks have reduced signal intensity based on crystallization with MPD or polyethylene glycol (PEG).⁴² For example, in the 2D N-CA spectrum reported by Seidel et al. crystallized from PEG, there are four clearly resolved Gly peaks (G10, G35, G47, and G53), however, in the ubiquitin N-CA spectrum reported by Schubert et al. prepared by crystallization using MPD (same as the ubiquitin preparations used in this study), only G35 and G47 were observed.⁴³ The lack of the Gly10 peak is consistent with the cross-polarization based results from Igumenova et al.³⁸ These observations from the literature as well as our detection of an additional ~3-5 peaks at a reduced contour level from the data in Figure 2 account for the large majority of the expected resonances in ubiquitin. To identify the remaining spin systems, experiments at a higher magnetic field and/or 3D spectroscopy can be used.⁴⁴

The 3D version of the SIM₂-NCOCX and SIM₁-NCACX datasets evolve the ¹³CO and ¹³CA dimensions together in t₂’ and t₂, respectively. It is important to realize that the dwell times for the ¹³CO and ¹³CA dimensions can be different, which is beneficial since the ¹³CA chemical shift range is ~30 ppm, while the ¹³CO region is ~10 ppm (a factor of three). Therefore, it is possible to reduce the number of increments in the indirect ¹³CO dimension by a factor of three and increase the number of scans by the same factor in order to obtain the same maximal indirect acquisition time in t₂ and t₂’. Alternatively, one may acquire additional indirect points in t₂ or t₂’.¹⁰ For example, ¹³CO nuclei typically have longer T₂ relaxation time in proteins relative to ¹³CA, allowing for increased sampling of the maximum t₂’ in the SIM₂-NCOCX experiment, which would increase the resolution. These linear correction factors have previously been used in the DU-MAS technique.¹⁰

It should also be stated that while the first DARR mixing time is intended for only the ¹³C spins, the ¹⁵N nuclei that have been stored along the z-axis may also undergo spin diffusion. However, this will be relatively minor (< 5%) for typical mixing times of ~200 msec.⁴² It is also noted that the order of the sequentially acquired experiments can be inverted to detect the NCO first (i.e., SIM₁-NCO), followed by the SIM₂-NCA experiment. In this case, the SIM₁-NCO will give the same signal/noise as the STD-NCO, while the SIM₂-NCA spectrum will have ~33% the signal/noise as the STD-NCA experiment.

Distinguishing Side Chain NH₂ Peaks in the Spectrum

In the SIM₂-NCO spectrum, it was also possible to easily distinguish the side chain NH₂ peaks from those of backbone amides in [U-¹³C,¹⁵N] samples. For the side chain Asn or Gln residues, the ¹⁵N to ¹³CA SPECIFIC-CP does not transfer amine ¹⁵N magnetization to ¹³C spins, since the covalent C_γ (Asn) or C_δ (Gln) nuclei have chemical shifts of ~175 ppm (¹³CA offset for the experiment is ~57.5 ppm). Therefore, a relatively small loss in side chain signal (~25%) was observed in the SIM₂-NCO spectrum relative to that of the STD-NCO. The side chain amine peaks are highlighted in the SIM₂-NCO experiment in Figure 2 and displayed as 1D cross-sections in Figure 3E and F. For [U-¹³C,¹⁵N] samples, this means these peaks are the most intense in the spectrum and are easily distinguishable from those of the backbone NCO cross peaks. Since the ¹⁵N amine resonances can overlap with those from the amide ¹⁵N of Ser, Gly, and Thr, the intensity of the peaks can be used to easily distinguish these residue types for the purpose of spectral assignment.

Application to Selective Glycerol Labeling in Ubiquitin

A common way to improve resolution and reduce spectral congestion is the use of selective labeling schemes.^45,46 One of the preferred methods is glycerol labeling ([1,3-¹³C] or [2-¹³C]) that significantly reduces pairwise ¹³C labels (i.e., few ¹³C-¹³C covalent bonds). This decreases resonance linewidths by removing one-bond carbon-carbon J-couplings and eliminates many of the resonances in a given spectrum.^36,37 It is also common to use glycerol or reverse labeling⁴⁷ in conjunction with double CP^48,49 or REDOR based dephasing^50-53 to assist in the assignment process.^48,49 We applied our sequential acquisition pulse sequence to ubiquitin labeled with [1,3-¹³C] glycerol and detected SIM₁-NCA and SIM₂-NCO 2D spectra at the same time (Figure 4). Selected 1D cross sections are shown in Figure 5 for a simpler comparison. In general, we found that several of the peaks in the NCA and NCO based spectra were missing as expected from the isotopic labeling pattern (e.g., Gly resonances at 45 ppm in the SIM₁-NCA).^36,37 In fact, on average we observed that the SIM₂-NCO gave 63 ± 9% of the sensitivity compared to the STD-NCO spectrum (Figure 6). The primary advantage of our approach for partially labeled samples is to maintain no loss in sensitivity for the SIM₁-NCA dataset, while also obtaining a complementary dataset compared to that for [U-¹³C,¹⁵N] samples. In other words, the ¹⁵N to ¹³CA SPECIFIC-CP element acts as an isotope filter that can be used to assign peaks.⁴⁸ For uniformly labeled samples, the spectral information for backbone amide peaks between STD-NCO and SIM₂-NCO contains identical information, since all sites have ¹³C labeling. However, for the [1,3-¹³C] glycerol sample, the incorporation at the ¹³CA depends on the residue type.^36,37 As a way to illustrate this, we plot the intensity retention as a histogram in Figure 6 comparing the uniform vs. glycerol labeling. The retention was calculated as the SIM₂-NCO peak intensity divided by the same resonance in the STD-NCO. Only resolved peaks were used in this calculation and all resonances analyzed are the same for the glycerol and uniform labeling. The wide distribution of intensity retentions compared to that obtained for the [U-¹³C,¹⁵N] ubiquitin sample shows that the sequential acquisition approach can be used to assign resonances in a similar way as done previously.^48,54

Figure 4.

Comparison of the standard vs. sequentially acquired datasets on [1,3-¹³C] glycerol labeled ubiquitin. The STD-NCA (not shown) is identical to that of the SIM₁-NCA (panel A). All comparable experimental parameters were the same between the STD-NCO (panel C) and the SIM₂-NCO (panel B). The sequentially acquired datasets in panels A and B are plotted at the same noise level (first contour: 11.0). The SIM₂-NCO spectrum (panel B) is plotted at 1.5 times the signal level compared to the STD-NCO (panel C; first contour: 16.5); this accounts for the average signal loss in the SIM₂-NCO due to the SPECIFIC-CP transfer to ¹³CA. For the listed contour levels, the standard deviation of the noise is 1.0.

Figure 5.

1D cross sections of the 2D spectra shown in Figure 4 for the [1,3-¹³C] glycerol labeled ubiquitin sample. The ¹⁵N frequencies indicated in each panel are directly comparable for the STD-NCO (A, C) and SIM₂-NCO spectra (B, D). The noise level is the same for the four 1D cross-sections.

Figure 6.

Histogram of intensity retention for the [1,3-¹³C] glycerol vs. [U-¹³C,¹⁵N] labeling ubiquitin samples. The intensity retention is calculated by dividing the intensity of the resolved peak in the 2D SIM₂-NCO spectrum by the corresponding resonance in the 2D STD-NCO. In total 24 resolved peaks from the 2D spectra from Figures 2 and 4 were used for this analysis. The average ± standard deviation for the two samples are: [U-¹³C,¹⁵N] = 32 ± 3%, [1,3-¹³C] glycerol = 63 ± 9%.

Discussion

Developments in magnetic resonance spectroscopy aim to improve the rate of characterizing biomolecular structure, dynamics, and imaging. We introduced a simple approach to utilizing residual or afterglow magnetization from the ¹⁵N double CP experiment to obtain a second multidimensional dataset that can be used to assign biomolecules by SSNMR. In current practice this residual magnetization is neither refocused nor directly detected. Since one of the most important factors determining experimental length is the recycle time (time needed to obtain equilibrium), methods such as RELOAD⁵⁵ and use of paramagnetic agents^56-58 strive to reduce or eliminate the need for the long recycle delays. Our method detects two heteronuclear correlation spectra (NCA and NCO) back to back with no recycle delay between experiments. This means two datasets can be acquired for the same amount of time as the standard acquisition that gives only a single dataset. For [U-¹³C,¹⁵N] ubiquitin, we have shown that the SIM₂-NCO gives 33% of the sensitivity as the STD-NCO dataset and 50% the signal/noise as the SIM₁-NCA. This means that an additional dataset with excellent signal/noise can be obtained with no loss in sensitivity to the first dataset when compared to the standard method. Related methods have utilized multiple cross-polarization elements to improve sensitivity⁵⁹ or cross depolarization filtering techniques.⁶⁰ Note that it is possible to carry novel non-selective ¹⁵N to ¹³C double CP transfer,²⁶ but this compromises the sensitivity of the NCA region by ~30%.

Our sequential acquisition method is also amenable to selective glycerol or reverse labeling approaches. Since these samples have dilute ¹³C spins, it is possible to obtain two datasets with nearly the same signal/noise without compromising the sensitivity of the spectrum acquired first. An application of our approach is the use of selectively labeled protein samples to aide in the assignment of overlapped spectra. This is important for poorly dispersed ¹⁵N signals such as membrane proteins that often do not allow for robust assignments from triple resonance experiments.^52,61 While some well-ordered membrane proteins give excellent spectra,^62-64 several other membrane proteins have biologically relevant conformational disorder^65-69 that gives rise to broader spectral lines. Future developments such as TROSY based methods may aide in reducing ¹⁵N line-widths that are broadened from N-H motion on the nsec to μsec timescale.⁷⁰

In summary, the presented method uses “afterglow” ¹⁵N magnetization remaining from the initial double CP step to obtain a second high quality dataset. The acquisition of these 2D or 3D datasets relies on long T_1ρ and T₁ relaxation times that exist for ¹⁵N in SSNMR.⁷¹ Our approach is also compatible with several existing techniques, including the DU-MAS method,^10,72 paramagnetic-based sensitivity enhancement methods,^56-58 dynamic nuclear polarization experiments,⁷³ applications utilizing time-resolved MAS techniques,^74-76 and oriented SSNMR. This method requires no additional SSNMR hardware and will result in no loss in sensitivity for the first dataset. The second dataset is free and can be detected with good sensitivity for biomolecular assignments in MAS SSNMR spectroscopy.