High-resolution 1H NMR spectroscopy in the solid state: very fast sample rotation and multiple-quantum coherences.

A technique is presented to recouple homonuclear dipolar couplings between dilute spin pairs such as 13C–13C systems under very fast magic angle spinning (MAS) in solid-state nuclear magnetic resonance (NMR) spectroscopy. The presented technique, finite pulse rf driven recoupling (fpRFDR), restores homonuclear dipolar interactions based on constructive usage of finite pulse-width effects in a phase- and symmetry-cycled π-pulse train in which a rotor-synchronous π pulse is applied every rotation period. The restored effective dipolar interaction has the form of a zero-quantum dipolar Hamiltonian for static solids, whose symmetry in spin space is different from that obtained by conventional rf driven recoupling (RFDR) techniques. It is demonstrated that the efficiency of recoupling by fpRFDR is not strongly dependent on chemical shift differences or resonance offsets in contrast to previous recoupling methods under very fast MAS. To realize distance measurements without effects of spin relaxation, a constan...

13C-13C dipolar recoupling under very fast magic angle spinning in solid-state nuclear magnetic resonance: Applications to distance measurements, spectral assignments, and high-throughput secondary-structure determination

The changes in a solute's chemical shifts between an isotropic and a liquid crystalline phase provide information on the magnitude and orientation of the chemical shielding tensors relative to the molecule's alignment frame. Such chemical shift changes have been measured for the polypeptide backbone C‘, N, and HN resonances in the protein ubiquitin. Perdeuterated ubiquitin was dissolved in a medium containing a small volume fraction of phospholipid bicelles, which switches from an isotropic to a liquid crystalline phase at ca. 25 °C. The one-bond 1H−15N dipolar couplings provide a reference to determine the protein alignment tensor, using a vibrationally corrected 1H−15N bond length of 1.04 A, corresponding to rCN = 1.33 A. Assuming all atoms of a given type have the same chemical shielding anisotropy (CSA) tensor, the average C‘ tensor values are σ11 = −75 ppm, σ22 = −12 ppm, and σ33 = 87 ppm for 13C‘ with an angle between σ11 and the C‘−N bond of 38°, and σ33 orthogonal to the peptide plane. Similarly, ...

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Measurement of Proton, Nitrogen, and Carbonyl Chemical Shielding Anisotropies in a Protein Dissolved in a Dilute Liquid Crystalline Phase

Spin -spin coupling tensors as determined by experiment and computational chemistry

Amyloid aggregates of a C-truncated Y145Stop mutant of human prion protein, huPrP23-144, associated with a heritable amyloid angiopathy, have previously been shown to contain a compact, relatively rigid, and beta-sheet-rich approximately 30-residue amyloid core near the C-terminus under physiologically relevant conditions. In contrast, the remaining huPrP23-144 residues display considerable conformational dynamics, as evidenced by the absence of corresponding signals in cross-polarization (CP)-based solid-state NMR (SSNMR) spectra under ambient conditions and their emergence in analogous spectra recorded at low temperature on frozen fibril samples. Here, we present the direct observation of residues comprising the flexible N-terminal domain of huPrP23-144 amyloid by using 2D J-coupling-based magic-angle spinning (MAS) SSNMR techniques. Chemical shifts for these residues indicate that the N-terminal domain is effectively an ensemble of protein chains with random-coil-like conformations. Interestingly, a detailed analysis of signal intensities in CP-based 3D SSNMR spectra suggests that non-negligible molecular motions may also be occurring on the NMR time scale within the relatively rigid core of huPrP23-144 amyloid. To further investigate this hypothesis, quantitative measurements of backbone dipolar order parameters and transverse spin relaxation rates were performed for the core residues. The observed order parameters indicate that, on the submicrosecond time scale, these residues are effectively rigid and experience only highly restricted and relatively uniform motions similar to those characteristic for well-structured regions of microcrystalline proteins. On the other hand, significant variations in magnitude of transverse spin relaxation rates were noted for residues present at different locations within the core region and correlated with observed differences in spectral intensities. While interpreted only qualitatively at the present time, the extent of the observed variations in transverse relaxation rates is consistent with the presence of relatively slow, microsecond-millisecond time scale chemical exchange type phenomena within the huPrP23-144 amyloid core.

Conformational flexibility of Y145Stop human prion protein amyloid fibrils probed by solid-state nuclear magnetic resonance spectroscopy.

Vibrational effects on structural parameters obtained by solid-state NMR are studied by theoretical calculations and molecular-dynamics simulations. The structural parameters treated contain internuclear distances between directly bonded or remote nuclei including nonproton pairs in a molecule and bond and dihedral angles. In addition to the intramolecular normal mode vibrations, the libration of the whole molecule is considered in the theory. It is shown that the molecular libration as well as the intramolecular vibrations reduce dipolar interactions, and consequently lengthen the internuclear distances obtained from the dipolar interactions (RNMR). In contrast, the internuclear distances obtained by single crystal x-ray or neutron diffraction (Rcor) are proved to be shortened by the molecular libration. Molecular-dynamics simulations for glycine molecules in the crystal at room temperature reveal that RNMR are 1%–4% longer than Rcor, confirming the theoretical results. It is also demonstrated that the e...

Soichi Hayashi

Papers

Theory and simulation of vibrational effects on structural measurements by solid-state nuclear magnetic resonance