Correction: Analysis of local molecular motions of aromatic sidechains in proteins by 2D and 3D fast MAS NMR spectroscopy and quantum mechanical calculations.

Correction for 'Analysis of local molecular motions of aromatic sidechains in proteins by 2D and 3D fast MAS NMR spectroscopy and quantum mechanical calculations' by Piotr Paluch et al., Phys. Chem. Chem. Phys., 2015, 17, 28789-28801.

Improving dipolar recoupling for site-specific structural and dynamics studies in biosolids NMR: windowed RN-symmetry sequences.

Experimental characterization of one-bond heteronuclear dipolar couplings is essential for structural and dynamics characterization of molecules by solid-state NMR. Accurate measurement of heteronuclear dipolar tensor parameters in magic-angle spinning NMR requires that the recoupling sequences efficiently reintroduce the desired heteronuclear dipolar coupling term, fully suppress other interactions (such as chemical shift anisotropy and homonuclear dipolar couplings), and be insensitive to experimental imperfections, such as radio frequency (rf) field mismatch. In this study, we demonstrate that the introduction of window delays into the basic elements of a phase-alternating R-symmetry (PARS) sequence results in a greatly improved protocol, termed windowed PARS (wPARS), which yields clean dipolar lineshapes that are unaffected by other spin interactions and are largely insensitive to experimental imperfections. Higher dipolar scaling factors can be attained in this technique with respect to PARS, which is particularly useful for the measurement of relatively small dipolar couplings. The advantages of wPARS are verified experimentally on model molecules N-acetyl-valine (NAV) and a tripeptide Met-Leu-Phe (MLF). The incorporation of wPARS into 3D heteronuclear or homonuclear correlation experiments permits accurate site-specific determination of dipolar tensors in proteins, as demonstrated on dynein light chain 8 (LC8). Through 3D wPARS recoupling based spectroscopy we have determined both backbone and side chain dipolar tensors in LC8 in a residue-resolved manner. We discuss these in the context of conformational dynamics of LC8. We have addressed the effect of paramagnetic relaxant Cu(ii)-EDTA doping on the dipolar coupling parameters in LC8 and observed no significant differences with respect to the neat sample permitting fast data collection. Our results indicate that wPARS is advantageous with respect to the windowless version of the sequence and is applicable to a broad range of systems including but not limited to biomolecules.

RF inhomogeneity and how it controls CPMAS.

In this report we discuss the effect of radiofrequency field (RF) inhomogeneity on cross-polarization (CP) under magic-angle spinning (MAS) by reviewing the dependence of the CP-detected signal intensity as a function of the position in the sample space. We introduce a power-function model to quantify the position-dependent RF-amplitude profile. The applicability of this model is experimentally verified by nutation spectra obtained by direct signal detection, as well as by CPMAS signal detection, in two commercial MAS probes with different degrees of RF inhomogeneity. A conclusion is that substantial sections of a totally filled rotor, even in a probe with rather good homogeneity, do not contribute at all to the detected spectra. The consequence is that in CPMAS-based recoupling experiments, such as the CP-with-variable-contact-time (CPVC), spatial selectivity of the Hartmann-Hahn matching condition overcomes complications that could be caused by RF inhomogeneity permitting determination of accurate spectral parameters even in cases with high inhomogeneity.

Analysis of local molecular motions of aromatic sidechains in proteins by 2D and 3D fast MAS NMR spectroscopy and quantum mechanical calculations.

We report a new multidimensional magic angle spinning NMR methodology, which provides an accurate and detailed probe of molecular motions occurring on timescales of nano- to microseconds, in sidechains of proteins. The approach is based on a 3D CPVC-RFDR correlation experiment recorded under fast MAS conditions (ν(R) = 62 kHz), where (13)C-(1)H CPVC dipolar lineshapes are recorded in a chemical shift resolved manner. The power of the technique is demonstrated in model tripeptide Tyr-(d)Ala-Phe and two nanocrystalline proteins, GB1 and LC8. We demonstrate that, through numerical simulations of dipolar lineshapes of aromatic sidechains, their detailed dynamic profile, i.e., the motional modes, is obtained. In GB1 and LC8 the results unequivocally indicate that a number of aromatic residues are dynamic, and using quantum mechanical calculations, we correlate the molecular motions of aromatic groups to their local environment in the crystal lattice. The approach presented here is general and can be readily extended to other biological systems.

Double and zero quantum filtered (2)H NMR analysis of D2O in intervertebral disc tissue.

The analysis of double and zero quantum filtered (2)H NMR spectra obtained from D2O perfused in the nucleus pulposus of human intervertebral disc tissue samples is reported. Fitting the spectra with a three-site model allows for residual quadrupolar couplings and T2 relaxation times to be measured. The analysis reveals changes in both the couplings and relaxation times as the tissue begins to show signs of degradation. The full analysis demonstrates that information about tissue hydration, water collagen interactions, and sample heterogeneity can be obtained and used to better understand the biochemical differences between healthy and degraded tissue.

A Magic-Angle Spinning NMR Method for the Site-Specific Measurement of Proton Chemical-Shift Anisotropy in Biological and Organic Solids.

Proton chemical shifts are a rich probe of structure and hydrogen bonding environments in organic and biological molecules. Until recently, measurements of 1H chemical shift tensors have been restricted to either solid systems with sparse proton sites or were based on the indirect determination of anisotropic tensor components from cross-relaxation and liquid-crystal experiments. We have introduced an MAS approach that permits site-resolved determination of CSA tensors of protons forming chemical bonds with labeled spin-1/2 nuclei in fully protonated solids with multiple sites, including organic molecules and proteins. This approach, originally introduced for the measurements of chemical shift tensors of amide protons, is based on three RN-symmetry based experiments, from which the principal components of the 1H CS tensor can be reliably extracted by simultaneous triple fit of the data. In this article, we expand our approach to a much more challenging system involving aliphatic and aromatic protons. We start with a review of the prior work on experimental-NMR and computational-quantum-chemical approaches for the measurements of 1H chemical shift tensors and for relating these to the electronic structures. We then present our experimental results on U-13C,15N-labeled histdine demonstrating that 1H chemical shift tensors can be reliably determined for the 1H15N and 1H13C spin pairs in cationic and neutral forms of histidine. Finally, we demonstrate that the experimental 1H(C) and 1H(N) chemical shift tensors are in agreement with Density Functional Theory calculations, therefore establishing the usefulness of our method for characterization of structure and hydrogen bonding environment in organic and biological solids.

Accurate measurement of heteronuclear dipolar couplings by phase-alternating R-symmetry (PARS) sequences in magic angle spinning NMR spectroscopy.

We report a Phase-Alternating R-Symmetry (PARS) dipolar recoupling scheme for accurate measurement of heteronuclear (1)H-X (X = (13)C, (15)N, (31)P, etc.) dipolar couplings in MAS NMR experiments. It is an improvement of conventional C- and R-symmetry type DIPSHIFT experiments where, in addition to the dipolar interaction, the (1)H CSA interaction persists and thereby introduces considerable errors in the dipolar measurements. In PARS, phase-shifted RN symmetry pulse blocks applied on the (1)H spins combined with π pulses applied on the X spins at the end of each RN block efficiently suppress the effect from (1)H chemical shift anisotropy, while keeping the (1)H-X dipolar couplings intact. Another advantage over conventional DIPSHIFT experiments, which require the signal to be detected in the form of a reduced-intensity Hahn echo, is that the series of π pulses refocuses the X chemical shift and avoids the necessity of echo formation. PARS permits determination of accurate dipolar couplings in a single experiment; it is suitable for a wide range of MAS conditions including both slow and fast MAS frequencies; and it assures dipolar truncation from the remote protons. The performance of PARS is tested on two model systems, [(15)N]-N-acetyl-valine and [U-(13)C,(15)N]-N-formyl-Met-Leu-Phe tripeptide. The application of PARS for site-resolved measurement of accurate (1)H-(15)N dipolar couplings in the context of 3D experiments is presented on U-(13)C,(15)N-enriched dynein light chain protein LC8.

Multidimensional magic angle spinning NMR spectroscopy for site-resolved measurement of proton chemical shift anisotropy in biological solids.

The proton chemical shift (CS) tensor is a sensitive probe of structure and hydrogen bonding. Highly accurate quantum-chemical protocols exist for computation of (1)H magnetic shieldings in the various contexts, making proton chemical shifts potentially a powerful predictor of structural and electronic properties. However, (1)H CS tensors are not yet widely used in protein structure calculation due to scarcity of experimental data. While isotropic proton shifts can be readily measured in proteins even in the solid state, determination of the (1)H chemical shift anisotropy (CSA) tensors remains challenging, particularly in molecules containing multiple proton sites. We present a method for site-resolved measurement of amide proton CSAs in fully protonated solids under magic angle spinning. The approach consists of three concomitant 3D experiments yielding spectra determined by either mainly (1)H CSA, mainly (1)H­(15)N dipolar, or combined (1)H CSA and (1)H­(15)N dipolar interactions. The anisotropic interactions are recoupled using RN-sequences of appropriate symmetry, such as R12(1)(4), and (15)N/(13)C isotropic CS dimensions are introduced via a short selective (1)H­(15)N cross-polarization step. Accurate (1)H chemical shift tensor parameters are extracted by simultaneous fit of the lineshapes recorded in the three spectra. An application of this method is presented for an 89-residue protein, U-(13)C,(15)N-CAP-Gly domain of dynactin. The CSA parameters determined from the triple fits correlate with the hydrogen-bonding distances, and the trends are in excellent agreement with the prior solution NMR results. This approach is generally suited for recording proton CSA parameters in various biological and organic systems, including protein assemblies and nucleic acids.

23Na TQF NMR imaging for the study of spinal disc tissue.

A method for acquiring triple quantum filtered (TQF) (23)Na NMR images is proposed that takes advantage of the differences in transverse relaxation rates of sodium to achieve positive intensity, PI, NMR signal. This PITQF imaging sequence has been used to obtain spatially resolved one-dimensional images as a function of the TQF creation time, tau, for two human spinal disc samples. From the images the different parts of the tissue, nucleus pulposus and annulus fibrosus, can be clearly distinguished based on their signal intensity and creation time profiles. These results establish the feasibility of (23)Na TQF imaging and demonstrate that this method should be applicable for studying human disc tissues as well as spinal disc degeneration.

The application of 23Na double-quantum-filter (DQF) NMR spectroscopy for the study of spinal disc degeneration.

Degenerative disc disease is an irreversible process that leads to a loss of mechanical integrity and back pain in millions of people. In this report, (23)Na double-quantum-filtered (DQF) NMR spectroscopy is used to study disc tissues in two stages of degeneration. Initial results indicate that the (23)Na DQF signal may be useful for determining the degree of degeneration. The spectral analysis reveals the presence of sodium environments with different residual quadrupolar couplings and T(2) relaxation times that we attribute to different regions, or compartments, corresponding to different biochemical regions in the tissue. In general it is found that there are compartments with no residual quadrupolar couplings, compartments with moderate couplings (200 to 1000 Hz), and compartments with couplings ranging from 1500 to 3000 Hz. The results indicate that (23)Na DQF NMR spectroscopy provides a probe of the degenerative state of the intervertebral disc tissues, and might hold potential as a novel diagnostic method for detection of disc degeneration.

The relationship between 207Pb NMR chemical shift and solid-state structure in Pb(II) compounds.

The analysis of heavy-metal solids with NMR spectroscopy provides a means of investigating the electronic environment through the dependence of the chemical shift on structure. We have investigated the relation of the 207Pb NMR isotropic chemical shift, span, and skew of a series of solid Pb(II) compounds to lattice parameters. Complementary relativistic spin-orbit density functional calculations on clusters such as PbI64- that model the local environment in the dihalides show a dependence of NMR properties on the local structure in good agreement with experimental results.

Internuclear (31)P-(51)V distance measurements in polyoxoanionic solids using rotational echo adiabatic passage double resonance NMR spectroscopy.

We report the first results establishing rotational echo adiabatic passage double resonance (REAPDOR) experiments for distance measurements between a spin-1/2 (31P) and spin-7/2 (51V) pair in a series of vanadium-substituted polyoxoanionic solids from the Keggin and Wells-Dawson families. We have quantitatively measured 31P-51V distances in monovanadium substituted K4PVW11O40, 1-K7P2VW17O62, and 4-K7P2VW17O62. Numerical simulations of the experimental data yield very good agreement with the averaged P-W/P-V distances determined from the X-ray diffraction measurements in the same or related compounds. REAPDOR is therefore a very sensitive P-V distance probe anticipated to be especially useful in the absence of long-range order. Our results suggest that REAPDOR spectroscopy could be broadly applicable for interatomic distance measurements in other spin-7/2-spin-1/2 nuclear pairs.

2H double quantum filtered (DQF) NMR spectroscopy of the nucleus pulposus tissues of the intervertebral disc.

Deuterium (2H) double-quantum filtered (DQF) NMR spectroscopy of nucleus pulposus (NP) tissues from human intervertebral discs is reported. The DQF spectral intensities, DQ build-up rates, and DQF-detected rotating-frame spin-lattice relaxation times are sensitive to the degree of hydration of the NP tissue, and display a monotonous correlation with age between 15 and 80 years. The implications of this work are that the changes in water dynamics as detected via DQF NMR spectroscopy may be used as a probe of tissue degeneration in NP, particularly in the early stages of degeneration to which most standard NMR methods are not sensitive.

51V solid-state magic angle spinning NMR spectroscopy of vanadium chloroperoxidase.

We report 51V solid-state NMR spectroscopy of the 67.5-kDa vanadium chloroperoxidase, at 14.1 T. We demonstrate that, despite the low concentration of vanadium sites in the protein (one per molecule, 1 mumol of vanadium spins in the entire sample), the spinning sideband manifold spanning the central and the satellite transitions is readily detectable. The quadrupolar and chemical shift anisotropy tensors have been determined by numerical simulations of the spinning sideband envelopes and the line shapes of the individual spinning sidebands corresponding to the central transition. The observed quadrupolar coupling constant C(Q) of 10.5 +/- 1.5 MHz and chemical shift anisotropy delta(sigma) of -520 +/- 13 ppm are sensitive reporters of the geometric and electronic structure of the vanadium center. Density functional theory calculations of the NMR spectroscopic observables for an extensive series of active site models indicate that the vanadate cofactor is most likely anionic with one axial hydroxo- group and an equatorial plane consisting of one hydroxo- and two oxo- groups. The work reported in this manuscript is the first example of 51V solid-state NMR spectroscopy applied to probe the vanadium center in a protein directly. This approach yields the detailed coordination environment of the metal unavailable from other experimental measurements and is expected to be generally applicable for studies of diamagnetic vanadium sites in metalloproteins.

Controlling the effects of pulse transients and RF inhomogeneity in phase-modulated multiple-pulse sequences for homonuclear decoupling in solid-state proton NMR.

The effects of pulse imperfections and RF inhomogeneity on NMR spectra obtained with phase-modulated multiple-pulse NMR sequences are analyzed. The emphasis is on the combined effects of frequency offset, RF inhomogeneity, and pulse phase transients. To enable a theoretical description of the transients associated with phase changes under continuous RF irradiation, the nature of the transients is investigated in depth. As monitored in our 300 MHz spectrometer, they are found to be caused by linear elements of the RF circuitry. The validity of their representation as delta-function pulses and the significance of their decomposition into antisymmetric and symmetric components are discussed. A practical method for quantitative control of the antisymmetric phase transients is proposed. The linearity property allows the development of a theoretical description of the spin dynamics caused by the transients. This leads to a vector-Hamiltonian model for phase-modulated Lee-Goldburg experiments. It quantitatively predicts both the frequency shift and the line broadening caused by antisymmetric phase transients and their coupling with RF inhomogeneity. The model is shown to be equally applicable to frequency-switched Lee-Goldburg experiments. A noteworthy discovery is that for a given magnitude of the antisymmetric phase transients a frequency offset exists at which the inhomogeneity broadening is essentially canceled. This explains the common observation that for best resolution one side of resonance is preferred over the other. It also suggests a strategy for enhancing resolution without having to resort to severe sample volume restriction. Numerical calculations verified the theoretical predictions and allowed extension of the model to BLEW-12 and DUMBO-1. Experimental verification is presented. The deviations from theoretical predictions are discussed.

Interatomic distance measurement in solid-state NMR between a spin- 1/2 and a spin- 5/2 using a universal REAPDOR curve.

A universal curve for the solid-state NMR REAPDOR experiment on an isolated spin-1/2-spin-5/2 pair is proposed that provides a simple means to measure their interatomic distance. REAPDOR data were obtained at three separate REAPDOR experiments using different values of the rotor spinning frequency. All points were fitted simultaneously to the universal formula without a need for full density matrix calculations. The 13C-17O distance of 2.45 A was measured between the C6 carbon and the 17O label in a tyrosine sample. The error of 8% in the dipolar coupling (Dfit = 278 Hz) is well within the 15% theoretical tolerance of this curve.

Internuclear distance determination of S = 1, I = 1/2 spin pairs using REAPDOR NMR.

A universal function is proposed to describe REAPDOR dephasing curves of an observed spin-1/2 nucleus dipole-recoupled to a spin-1 quadrupolar nucleus ((2)H or (14)N). Previous work had shown that, in contrast to REDOR, the shape of the dephasing curve depends on a large number of parameters including the quadrupolar coupling constant and asymmetry parameter, the sample rotation speed, the RF amplitude, and the relative orientations of the quadrupole tensor and the internuclear vector. Here we demonstrate by numerical simulations that the actual dispersion of REAPDOR dephasing curves is quite small, provided the rotation speed and the RF amplitude applied to the quadrupolar nucleus satisfy an adiabaticity condition. The condition is easily met for (2)H and is also practically achievable for virtually any (14)N-containing compound. This allows the REAPDOR curves to be approximated by a simple universal gaussian-type function, comparison of which with experimental data yields internuclear distances with less than 4% error. The spin dynamics of the recoupling mechanism is discussed. The critical importance of a stable spinning speed for optimizing the signal-to-noise ratio of the (13)C echoes is demonstrated and practical suggestions for achieving high stability are presented. Examples of applications of the universal curve are given for (2)H/(13)C and (14)N/(13)C REAPDOR in alanine.