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

Abstract

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.

Expanding the horizons for structural analysis of fully protonated protein assemblies by NMR spectroscopy at MAS frequencies above 100 kHz

“…RN-symmetry based experiments and their improved versions developed by our group for accurate measurements of heteronuclear dipolar and 1 H CSA tensors at MAS frequencies of 40 kHz and below have been applied to organic and biological systems, including proteins and protein assemblies [27; 28; 29; 34; 42; 48]. In these reports, we have presented in-depth analysis of the performance of the RN-symmetry based experiments and validated their applications in protein assemblies, including the HIV-1 CA assemblies, using various means, such as empirical correlations between 1 H CSA and hydrogen bond length using electrostatic models [29], quantum chemical (DFT) calculations of CSA tensors [49], as well as a combined MD/DFT approach [44]. Others have demonstrated that 1 H CSA tensors can be recorded in small molecules at the MAS frequency of 65 kHz [50], albeit no validation was reported on the measured CSAs from quantum chemical calculations.…”

“…Others have demonstrated that 1 H CSA tensors can be recorded in small molecules at the MAS frequency of 65 kHz [50], albeit no validation was reported on the measured CSAs from quantum chemical calculations. Indeed, 1 H CSA tensors, are another sensitive probe of dynamics, which also report on hydrogen bonding interactions [29; 49]. To extract dynamics information from CSA tensors, quantum mechanical Density Functional Theory (DFT) calculations as well as combined MD/DFT calculations can be employed, as we have demonstrated recently [44].…”

Fast magic angle spinning NMR with heteronucleus detection for resonance assignments and structural characterization of fully protonated proteins

“…We have recently developed an experimental approach for deriving 1 H CSA tensors in proteins and other biological molecules, in a residue-specific way (Hou et al 2014; Hou et al 2013), and corroborated that the principal components are strongly correlated with hydrogen bonding environments, providing very useful structural probe. At the same time, we could not record 1 H isotropic chemical shifts under moderate MAS conditions and had to rely on solution values, an approach, which is likely to be error-prone due to the different environments of the solid-like and solution states.…”

NMR of Macromolecular Assemblies and Machines at 1 GHz and Beyond: New Transformative Opportunities for Molecular Structural Biology

“…With the use of an appropriate R-symmetry element one can select for the 1 H CSA and 1 H-X heteronuclear dipole while eliminating contributions from 1 H- 1 H homonuclear couplings for measurements in fully protonated proteins. Site-specific CSAs can be determined by incorporating the R-symmetry recoupling as one dimension in a 3D experiment with heteronuclear detection (120, 121). Two methods for the measurement of 1 H CSAs in solution are CSA-dipolar cross-correlated relaxation, and residual CSA (RCSA) measurements in aligned media (122–124).…”