Measurement Of Dipolar Couplings Research Articles

Elucidation of the Mechanisms of Inter-domain Coupling in the Monomeric State of Enzyme I by High-pressure NMR

The catalytic cycle of Enzyme I (EI), a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate, is characterized by a series of local and global conformational rearrangements. This multistep process includes a monomer-to-dimer transition, followed by an open-to-closed rearrangement of the dimeric complex upon PEP binding. In the present study, we investigate the thermodynamics of EI dimerization using a range of high-pressure solution NMR techniques complemented by SAXS experiments. 1H-15N TROSY and 1H-13C methyl TROSY NMR spectra combined with 15N relaxation measurements revealed that a native-like engineered variant of full-length EI fully dissociates into stable monomeric state above 1.5 kbar. Conformational ensembles of EI monomeric state were generated via a recently developed protocol combining coarse-grained molecular simulations with experimental backbone residual dipolar coupling measurements. Analysis of the structural ensembles provided detailed insights into the molecular mechanisms driving formation of the catalytically competent dimeric state, and reveals that each step of EI catalytical cycle is associated with a significant reduction in either inter- or intra-domain conformational entropy. Altogether, this study completes a large body work conducted by our group on EI and establishes a comprehensive structural and dynamical description of the catalytic cycle of this prototypical multidomain, oligomeric enzyme.

Rheo-NMR at the Phase Transition of Liquid Crystalline Poly-γ-benzyl-l-glutamate: Phase Kinetics and a Valuable Tool for the Measurement of Residual Dipolar Couplings

Rheo-NMR at the Phase Transition of Liquid Crystalline Poly-γ-benzyl-<scp>l</scp>-glutamate: Phase Kinetics and a Valuable Tool for the Measurement of Residual Dipolar Couplings

Light-Controlled Lyotropic Liquid Crystallinity of Polyaspartates Exploited as Photo-Switchable Alignment Medium

Two polyaspartates bearing ortho-fluorinated azobenzenes (pFAB) as photo-responsive groups in the side chain were synthesized: PpFABLA (1) and co-polyaspartate PpFABLA-co-PBLA [11, 75%(n/n) PpFABLA content]. As a consequence of the E/Z-isomerization of the side chain, PpFABLA (1) undergoes a visible-light-induced reversible coil-helix transition in solution: Green light (525 nm) affords the coil, and violet light (400 nm) affords the helix. pFAB significantly increases the thermal stability of the Z-isomer at 20 °C (t1/2 = 66 d for the Z-isomer) and effectively counters the favored back formation of the helix. At 20%(w/w) polymer concentration, the helical polymer forms a lyotropic liquid crystal (LLC) that further orients unidirectionally inside a magnetic field, while the coil polymer results in an isotropic solution. The high viscosity of the polymer solution stabilizes the coexistence of liquid crystalline and isotropic domains, which were obtained with spatial control by partial light irradiation. When used as an alignment medium, PpFABLA (1) enables (i) the measurement of dipolar couplings without the need for a separate isotropic reference and (ii) the differentiation of enantiomers. PpFABLA-co-PBLA (11) preserves the helical structure, by intention, independently of the E/Z-isomerization of the side chain: Both photo-isomers of PpFABLA-co-PBLA (11) form a helix that─at a concentration of 16%(w/w)─form an LLC. Despite the absence of a change in the secondary structure, the E/Z-isomerization of the side chain changes the morphology of the liquid crystal and leads to different sets of dipolar coupling for the same probe molecule.

An Acrylonitrile-Based Copolymer Gel as an NMR Alignment Medium for Extraction of Residual Dipolar Couplings of Small Molecules in Aqueous Solution.

An NMR weakly-aligning polymer gel has been prepared by copolymerization of acrylonitrile and 2-acrylamide-2-methyl-1-propanesulfonic acid in the presence of 1,4-butanediol diacrylate as a cross-linker. The polymer readily swells in water in a large range of temperatures, although the swelling ratio is decreased in saline solutions. The swollen gel can be mechanically compressed, in a reversible way, generating anisotropy, as easily shown in 2 H NMR experiments, and allowing measurement of 1 DCH residual dipolar couplings (RDCs) through F1-coupled HSQC experiments. The performance of this gel as a NMR alignment medium was evaluated in several water-soluble organic molecules and, while it provided RDCs of proper size for sucrose and even such as small molecule as 5-norbornen-2-ol, in the case of azidothymidine and cefuroxime sodium salt the strong interaction of these molecules with the gel prevented successful extraction of the RDCs.

Stabilization of small Aβ oligomers in SDS micelles.

Aβ fibrillation is a slow process without seeding or assisted nucleation. It was observed previously that SDS micelles promote Aβ40 fibrillation and stabilize Aβ42 small oligomers (in the dimer to tetramer range). Here we characterized small oligomers in SDS micelles by molecular dynamics simulations and solid-state NMR spectroscopy. Parallel β-sheets formed by the C-terminal region (Ala30 to the C-terminus, or C-strand) of Aβ40 and Aβ42 tetramers are unstable in SDS micelles, due to fraying starting at Gly37 and Gly38. In contrast, an antiparallel β-sheet formed by four Aβ42 C-strands is stable, with the tails of SDS molecules inserted next to the β-sheet in the voids above Gly33 of one strand and Gly37 and Gly38 one of the next strand. This deep insertion allows the SDS tails to form hydrophobic interactions with numerous nearby nonpolar sidechains. Measurements of intermolecular 13C-13C dipolar couplings show that the SDS-stabilized antiparallel tetramer is an on-pathway intermediate for a 150-kD oligomer formed on dialysis. These observations have implications for the stimulation of endogenous Aβ aggregation at cellular interfaces.

5-Formylcytosine weakens the G-C pair and imparts local conformational fluctuations to DNA duplexes.

DNA epigenetic modifications such as 5-methyl (5mC), 5-hydroxymethyl (5hmC), 5-formyl (5fC) and 5-carboxyl (5caC) cytosine have unique and specific biological roles. Crystallographic studies of 5mC containing duplexes were conducted in the A-, B- or the intermediate E-DNA polymorphic forms. 5fC-modified duplexes initially observed in the disputed F-DNA architecture were subsequently crystallized in the A-form, suggesting that epigenetic modifications enable DNA sequences to adopt diverse conformational states that plausibly contribute to their function. Solution-state studies of these modifications were found in the B-DNA form, with marked differences in the conformational flexibility of 5fC containing duplexes in comparison to C/5mC containing duplexes, compromising the DNA duplex's stability. Herein, we systematically evaluate sensitive and commonly inaccessible NMR parameters to map the subtle differences between C, 5mC, and their oxidized (5hmC/5fC) counterparts. We observe that 15N/1H chemical shifts effectively report on the weakening of 5fC-G Watson-Crick base-pair H-bonding, extending the instability beyond any achievable within the sequence-specific changes in DNA. Triple 5fC containing sequences propagate the destabilization farther from the site of modifications, explaining reduced duplex stability upon multiple modifications. Additionally, scalar and residual dipolar coupling measurements unravel local sugar pucker fluctuations. One-bond 13C-1H scalar coupling measurements point towards a significant deviation away from the anticipated C2'-endo pucker for the 5fC modified nucleotide. Structural models obtained employing 13C-1H residual dipolar couplings and inter-proton distances corroborate the sugar pucker's deviation for 5fC modified DNA duplexes. The changes in the sugar pucker equilibria remain local to the 5fC modified nucleotide sans additive/long-range effects arising from multiple contiguous modifications. These observations highlight the impact of a major groove modification that alters the physical properties of DNA duplex without disturbing the Watson-Crick face. The changes observed in our studies for the 5fC containing DNA contrast with the perturbations induced by damage/lesion highlight the varied conformational preferences that modified nucleobases impart to the DNA duplex. As sequence-specific DNA transactions are rooted in the base-pair stability and pucker deviations, the observed structural perturbations for 5fC-modified DNA potentially play critical functional roles, such as protein-DNA recognition and interactions.

Self-Assembled Oligopeptide (FK)4 as a Chiral Alignment Medium for the Anisotropic NMR Analysis of Organic Compounds.

Anisotropic NMR parameters have been proven to be powerful for the structural elucidation of organic molecules. Herein, we present an alignment medium based on the self-assembled (FK)4 oligopeptide, showing excellent properties in measurements of anisotropic NMR parameters in both D2O and CD3OD. The preparation of the (FK)4-based alignment medium is simple and rapid. The low viscosity of the anisotropic phase makes it easy to be transferred to the NMR tube. The alignment of the oligopeptide is fast, stable, and homogeneous, with weak background signals, permitting the acquirement of high-quality NMR spectra. The performance of this alignment medium in residual dipolar coupling measurements and diastereomer discriminations is demonstrated by analyzing several different analytes. The enantiodiscrimination property of the (FK)4 oligopeptide is revealed by the difference of residual chemical shift anisotropy of the two enantiomers in the 1D 13C spectrum, granting its potential use for the quantification and identification of enantiomers of small molecules.

Solution cis-Proline Conformation of IPCs Inhibitor Aureobasidin A Elucidated via NMR-Based Conformational Analysis.

Aureobasidin A (abA) is a natural depsipeptide that inhibits inositol phosphorylceramide (IPC) synthases with significant broad-spectrum antifungal activity. abA is known to have two distinct conformations in solution corresponding to trans- and cis-proline (Pro) amide bond rotamers. While the trans-Pro conformation has been studied extensively, cis-Pro conformers have remained elusive. Conformational properties of cyclic peptides are known to strongly affect both potency and cell permeability, making a comprehensive characterization of abA conformation highly desirable. Here, we report a high-resolution 3D structure of the cis-Pro conformer of aureobasidin A elucidated for the first time using a recently developed NMR-driven computational approach. This approach utilizes ForceGen's advanced conformational sampling of cyclic peptides augmented by sparse distance and torsion angle constraints derived from NMR data. The obtained 3D conformational structure of cis-Pro abA has been validated using anisotropic residual dipolar coupling measurements. Support for the biological relevance of both the cis-Pro and trans-Pro abA configurations was obtained through molecular similarity experiments, which showed a significant 3D similarity between NMR-restrained abA conformational ensembles and another IPC synthase inhibitor, pleofungin A. Such ligand-based comparisons can further our understanding of the important steric and electrostatic characteristics of abA and can be utilized in the design of future therapeutics.

Global Structure of the Intrinsically Disordered Protein Tau Emerges from Its Local Structure.

The paradigmatic disordered protein tau plays an important role in neuronal function and neurodegenerative diseases. To disentangle the factors controlling the balance between functional and disease-associated conformational states, we build a structural ensemble of the tau K18 fragment containing the four pseudorepeat domains involved in both microtubule binding and amyloid fibril formation. We assemble 129-residue-long tau K18 chains with atomic detail from an extensive fragment library constructed with molecular dynamics simulations. We introduce a reweighted hierarchical chain growth (RHCG) algorithm that integrates experimental data reporting on the local structure into the assembly process in a systematic manner. By combining Bayesian ensemble refinement with importance sampling, we obtain well-defined ensembles and overcome the problem of exponentially varying weights in the integrative modeling of long-chain polymeric molecules. The resulting tau K18 ensembles capture nuclear magnetic resonance (NMR) chemical shift and J-coupling measurements. Without further fitting, we achieve very good agreement with measurements of NMR residual dipolar couplings. The good agreement with experimental measures of global structure such as single-molecule Förster resonance energy transfer (FRET) efficiencies is improved further by ensemble refinement. By comparing wild-type and mutant ensembles, we show that pathogenic single-point P301L, P301S, and P301T mutations shift the population from the turn-like conformations of the functional microtubule-bound state to the extended conformations of disease-associated tau fibrils. RHCG thus provides us with an atomically detailed view of the population equilibrium between functional and aggregation-prone states of tau K18, and demonstrates that global structural characteristics of this intrinsically disordered protein emerge from its local structure.

Influence of Sulfur-Curing Conditions on the Dynamics and Crosslinking of Rubber Networks: A Time-Domain NMR Study.

The characterization of the structural and dynamic properties of rubber networks is of fundamental importance in rubber science and technology to design materials with optimized mechanical properties. In this work, natural and isoprene rubber networks obtained by curing at three different temperatures (140, 150, and 170 °C) and three different sulfur contents (1, 2, and 3 phr) in the presence of a 3 phr accelerator were studied using a combination of low-field time-domain NMR (TD-NMR) techniques, including 1H multiple-quantum experiments for the measurement of residual dipolar couplings (Dres), the application of the Carr–Purcell–Meiboom–Gill pulse sequence for the measurement of the transverse magnetization decay and the extraction of 1H T2 relaxation times, and the use of field cycling NMR relaxometry for the determination of T1 relaxation times. The microscopic properties determined by TD-NMR experiments were discussed in comparison with the macroscopic properties obtained using equilibrium swelling, moving die rheometer, and calorimetric techniques. The obtained correlations between NMR observables, crosslink density values, maximum torque values, and glass transition temperatures provided insights into the effects of the vulcanization temperature and accelerator/sulfur ratio on the structure of the polymer networks, as well as on the effects of crosslinking on the segmental dynamics of elastomers. Dres and T2 were found to show linear correlations with the crosslink density determined by equilibrium swelling, while T1 depends on the local dynamics of polymer segments related to the glass transition, which is also affected by chemical modifications of the polymer chains occurring during vulcanization.

Saponins Form Nonionic Lipid Nanodiscs for Protein Structural Studies by Nuclear Magnetic Resonance Spectroscopy.

Structural studies of membrane proteins in native-like environments require the development of diverse membrane mimetics. Currently there is a need for nanodiscs formed with nonionic belt molecules to avoid nonphysiological electrostatic interactions between the membrane system and protein of interest. Here, we describe the formation of lipid nanodiscs from the phospholipid DMPC and a class of nonionic glycoside natural products called saponins. The morphology, surface characteristics, and magnetic alignment properties of the saponin nanodiscs were characterized by light scattering and solid-state NMR experiments. We determined that preparing nanodiscs with high saponin/lipid ratios reduced their size, diminished their ability to spontaneously align in a magnetic field, and favored insertion of individual saponin molecules in the lipid bilayer surface. Further, purification of saponin nanodiscs allowed flipping of the orientation of aligned nanodiscs by 90°. Finally, we found that aligned saponin nanodiscs provide a sufficient alignment medium to allow the measurement of residual dipolar couplings (RDCs) in aqueous cytochrome c.

Self‐Assembly of Helical Polymers and Oligomers to Create Liquid Crystalline Alignment for Anisotropic NMR Parameters

The measurement of anisotropic residual dipolar couplings (RDCs) parameters for the structure elucidation of organic molecules relies on suitable alignment media. Employment of self-assembled liquid crystalline systems to create anisotropic alignment can be an effective way to realize aligned samples and acquire RDCs. This Mini-review highlights the recent advances on amino acid-based helical polymers and supramolecular oligomers forming rigid, rod-like structures that aggregate into ordered liquid crystalline phases, including amino acid-based helical polyisocyanides, polyacetylenes, polypeptides, and oligopeptides assembled alignment media. The methodology for the determination of anisotropic liquid crystals is briefly discussed, and a summary of recent research progress in the enantiodifferentiation of helical polymers aligned media is followed. In addition, the self-assembled mechanism of oligopeptides and their RDCs structural analysis are also described.

Analysis of the orientation of cholesterol in high-density lipoprotein nanodiscs using solid-state NMR.

Cholesterol is an essential component of eukaryotic cellular membranes that regulates the order and phase behaviour of dynamic lipid bilayers. Although cholesterol performs many vital physiological roles, hypercholesterolaemia and the accumulation of cholesterol in atherosclerotic plaques can increase the risk of coronary heart disease morbidity. The risk is mitigated by the transportation of cholesterol from peripheral tissue to the liver by high-density lipoprotein (HDL), 6-20 nm-diameter particles of lipid bilayers constrained by an annular belt of the protein apolipoprotein A-I (apoA-I). Information on the dynamics and orientation of cholesterol in HDL is pertinent to the essential role of HDL in cholesterol cycling. This work investigates whether the molecular orientation of cholesterol in HDL differs from that in the unconstrained lipid bilayers of multilamellar vesicles (MLVs). Solid-state NMR (ssNMR) measurements of dynamically-averaged 13C-13C and 13C-1H dipolar couplings were used to determine the average orientation of triple 13C-labelled cholesterol in palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers in reconstituted HDL (rHDL) nanodiscs and in MLVs. Individual 13C-13C dipolar couplings were measured from [2,3,4-13C3]cholesterol in a one-dimensional NMR experiment, by using a novel application of a method to excite double quantum coherence at rotational resonance. The measured dipolar couplings were compared with average values calculated from orientational distributions of cholesterol generated using a Gaussian probability density function. The data were consistent with small differences in the average orientation of cholesterol in rHDL and MLVs, which may reflect the effects of the constrained and unconstrained lipid bilayers in the two environments. The calculated distributions of cholesterol in rHDL and MLVs that were consistent with the NMR data also agreed well with orientational distributions extracted from previous molecular dynamics simulations of HDL nanodiscs and planar POPC bilayers.

Poly(arylisocyanides) as Versatile, Enantiodiscriminating Alignment Media for Small Molecules.

Lyotropic liquid crystalline (LLC) phases of amino acid derived polyarylisocyanides were employed as chiral alignment media for the measurement of residual dipolar couplings (RDCs) of small chiral organic molecules. Anisotropic samples in CDCl3 displayed quadrupolar splittings of the deuterium signal in the range of several hundreds of Hertz. The LLC phases showed excellent orienting properties for a broad range of analytes bearing various functional groups. The precise extraction of RDCs in the range of up to ±40 Hertz from F2-coupled HSQC spectra was possible. Additionally, the chiral environment offers the opportunity for diastereomorphous interactions with the enantiomers of chiral analytes leading to two different sets of RDCs. This differential order effect was particularly pronounced with ketones and alcohols.

Spatial arrangement of dynamic surface species from solid-state NMR and machine learning-accelerated MD simulations.

The surface arrangement of motional organic functionalities is explored by experimental dipolar coupling measurements and the prediction of motionally-averaged coupling constant from molecular dynamics simulations. The use of machine learning potentials was key to reaching the timescale required. The distance between dynamic surface species are important in cooperative heterogeneous catalysis.

Measurement of Residual Dipolar Couplings Using Magnetically Aligned and Flipped Nanodiscs.

Recent developments in lipid nanodisc technology have successfully overcome the major challenges in the structural and functional studies of membrane proteins and drug delivery. Among the different types of nanodiscs, the use of synthetic amphiphilic polymers created new directions including the applications of solution and solid-state NMR spectroscopy. The ability to magnetically align large-size (>20 nm diameter) polymer nanodiscs and flip them using paramagnetic lanthanide ions has enabled high-resolution studies on membrane proteins using solid-state NMR techniques. The use of polymer-based macro-nanodiscs (>20 nm diameter) as an alignment medium to measure residual dipolar couplings (RDCs) and residual quadrupole couplings by NMR experiments has also been demonstrated. In this study, we demonstrate the use of magnetically aligned and 90°-flipped polymer nanodiscs as alignment media for structural studies on proteins by solution NMR spectroscopy. These macro-nanodiscs, composed of negatively charged SMA-EA polymers and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids, were used to measure residual 1H-15N dipolar couplings (RDCs) from the water-soluble ∼21 kDa uniformly 15N-labeled flavin mononucleotide binding domain (FBD) of cytochrome-P450 reductase. The experimentally measured 1H-15N RDC values are compared with the values calculated from the crystal structures of cytochrome-P450 reductase that lacks the transmembrane domain. The N-H RDCs measured using aligned and 90°-flipped nanodiscs show a modulation by the function (3 cos2 θ - 1), where θ is the angle between the N-H bond vector and the applied magnetic field direction. This successful demonstration of the use of two orthogonally oriented alignment media should enable structural studies on a variety of systems including small molecules, DNA, and RNA.

Advances in NMR Spectroscopy of Weakly Aligned Biomolecular Systems.

The measurement and application of residual dipolar couplings (RDCs) in solution NMR studies of biological macromolecules has become well established over the past quarter of a century. Numerous methods for generating the requisite anisotropic orientational molecular distribution have been demonstrated, each with its specific strengths and weaknesses. In parallel, an enormous number of pulse schemes have been introduced to measure the many different types of RDCs, ranging from the most widely measured backbone amide 15N-1H RDCs, to 1H-1H RDCs and couplings between low-γ nuclei. Applications of RDCs range from structure validation and refinement to the determination of relative domain orientations, the measurement of backbone and domain motions, and de novo structure determination. Nevertheless, it appears that the power of the RDC methodology remains underutilized. This review aims to highlight the practical aspects of sample preparation and RDC measurement while describing some of the most straightforward applications that take advantage of the exceptionally precise information contained in such data. Some emphasis will be placed on more recent developments that enable the accurate measurement of RDCs in larger systems, which is key to the ongoing shift in focus of biological NMR spectroscopy from structure determination toward gaining improved understanding of how molecular flexibility drives protein function.

Sign determination of dipolar couplings in liquid crystals by off-magic-angle sample spinning

Dipolar NMR spectroscopy is a powerful analytical tool to study structural and dynamic properties of rigid and soft solids at the molecular level. While magnitudes of dipolar couplings can be determined from spectral splittings, signs of the constants are unavailable. A method to determine the signs of the dipolar couplings in liquid crystals is presented. Sign information is directly obtained from one-dimensional NMR spectra in samples spinning at an angle slightly misset from the magic angle. The approach is used to develop an experimental strategy for sign-sensitive measurements of heteronuclear dipolar couplings in anisotropic soft materials.

The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation.

We report data on the structural dynamics of the neuropeptide Y (NPY) G-protein-coupled receptor (GPCR) type 1 (Y1R), a typical representative of class A peptide ligand GPCRs, using a combination of solid-state NMR and molecular dynamics (MD) simulation. First, the equilibrium dynamics of Y1R were studied using 15N-NMR and quantitative determination of 1H-13C order parameters through the measurement of dipolar couplings in separated-local-field NMR experiments. Order parameters reporting the amplitudes of the molecular motions of the C-H bond vectors of Y1R in DMPC membranes are 0.57 for the Cα sites and lower in the side chains (0.37 for the CH2 and 0.18 for the CH3 groups). Different NMR excitation schemes identify relatively rigid and also dynamic segments of the molecule. In monounsaturated membranes composed of longer lipid chains, Y1R is more rigid, attributed to a higher hydrophobic thickness of the lipid membrane. The presence of an antagonist or NPY has little influence on the amplitude of motions, whereas the addition of agonist and arrestin led to a pronounced rigidization. To investigate Y1R dynamics with site resolution, we conducted extensive all-atom MD simulations of the apo and antagonist-bound state. In each state, three replicas with a length of 20 μs (with one exception, where the trajectory length was 10 μs) were conducted. In these simulations, order parameters of each residue were determined and showed high values in the transmembrane helices, whereas the loops and termini exhibit much lower order. The extracellular helix segments undergo larger amplitude motions than their intracellular counterparts, whereas the opposite is observed for the loops, Helix 8, and termini. Only minor differences in order were observed between the apo and antagonist-bound state, whereas the time scale of the motions is shorter for the apo state. Although these relatively fast motions occurring with correlation times of ns up to a few µs have no direct relevance for receptor activation, it is believed that they represent the prerequisite for larger conformational transitions in proteins.

A modification of γ-encoded RN symmetry pulses for increasing the scaling factor and more accurate measurements of the strong heteronuclear dipolar couplings

Symmetry based γ-encoded RNnν elements are broadly used in magic-angle spinning solid-state NMR experiments to achieve selective recoupling of the heteronuclear dipolar interactions. The recoupled dipolar couplings in such experiments are scaled by a factor, Ksc, which theoretically depends on the chosen symmetry numbers N, n, and ν. However, the maximum theoretical value of Ksc for γ-encoded RNnν pulses is limited to ~0.25, resulting in long RNnν experiment times. Also, the dependence of Ksc on the experimental parameters can result in systematic errors in the experimental determination of the dipolar couplings, especially at low and moderate MAS rates. In this manuscript, we investigate the use of MODifiEd RNnν symmetry (MODERNnν(ϕM)) pulses that increase the dipolar scaling factor by at least 1.45 fold compared to γ-encoded RNnν. The second advantage of MODERNnν(ϕM) pulses with respect to traditional RNnν pulses is the reduced influence of experimental parameters on Ksc, which allows for more accurate measurement of short-range distances. The robustness of MODERNnν(ϕM) is compared with γ-encoded R1423 symmetry pulses. The enhanced performance is demonstrated on two uniformly-13C-enriched samples, N-acetyl valine and the microcrystalline protein GB1, at a 31.111 kHz MAS rate.