Towards closing the gap: solid-state NMR and quantum mechanical calculations in microcrystalline proteins and organic solids
Date
2019
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Publisher
University of Delaware
Abstract
The overall theme of this thesis is the development and application of an integrated approach to accurately measure and compute anisotropic NMR parameters, chemical shift and dipolar tensors, in microcrystalline proteins and organic solids. Protein structures determined by solid state magic angle spinning (MAS) NMR use local dipolar correlations in conjunction with the qualitative torsion angle restraints derived from isotropic chemical shifts. Despite progress in the last two decades in the development of MAS NMR for protein structure determination, current methods are still time-consuming and error-prone. In large proteins and protein assemblies, multiple samples prepared with different isotopic labels and many multidimensional MAS NMR experiments on each of these samples are required for obtaining distance restraints. Even then, the distance restraints are local and do not exceed 5-7 Å. Existing protocols can potentially be improved, both in speed and accuracy, by adding information about chemical shift tensors and long-range distance restraints. ☐ NMR chemical shifts are sensitive probe of molecular structure and dynamics in molecules and supramolecular assemblies. While isotropic chemical shifts are easily measured with high accuracy and precision in conventional NMR experiments, they remain challenging to calculate quantum mechanically, particularly in inherently dynamic biological systems. Using a model benchmark protein, a 133-residue agglutinin from Oscillatoria agardhii (OAA), which has been extensively characterized by us previously, we have explored the integration of X-ray crystallography, solution NMR, MAS NMR, and quantum mechanics/molecular mechanics (QM/MM) calculations, for analysis of 13Cα and 15N isotropic chemical shifts. The influence of local interactions, crystal contacts, and dynamics, on the accuracy of calculated chemical shifts is analyzed. Our approach is broadly applicable and expected to be beneficial in chemical shift analysis and chemical-shift-based structure refinement for proteins and protein assemblies. ☐ Although the chemical shift is at the heart of NMR spectroscopy, the principal components of the shift tensor, which report on the asymmetry and anisotropy, contain a wealth of information. In this work, we report and compare accurately measured and calculated 15NH and 13Cα chemical shift tensors in proteins, using microcrystalline OAA as a model benchmark system. Experimental 13Cα and 15NH chemical tensors were obtained by solid-state NMR spectroscopy, employing RN-symmetry recoupling sequences, and for their QM/MM calculations different sets of functionals were evaluated. We show that 13Cα chemical shift tensors are primarily determined by backbone dihedral angles and dynamics, while 15NH tensors mainly depend on local electrostatic contributions from solvation and hydrogen bonding. In addition, the influence of including crystallographic waters, the molecular mechanics geometry optimization protocol, and the level of theory on the accuracy of the calculated chemical shift tensors is discussed. Specifically, the power of QM/MM calculations in accurately predicting the unusually upfield shifted 1HN G26 and G93 resonances is highlighted. Our integrated approach is expected to benefit structure refinement of proteins and protein assemblies. ☐ Long-range interatomic distance restraints are critical for the determination of molecular structures by NMR spectroscopy, both in solution and in the solid state. Fluorine is a powerful NMR probe in a wide variety of contexts, owing to its favorable magnetic properties, ease of incorporation into biological molecules, and ubiquitous use in synthetic organic molecules designed for diverse applications. Due to the large gyromagnetic ratio of the 100% naturally abundant 19F isotope, interfluorine distances as long as 20 Å are accessible in magic angle spinning (MAS) dipolar recoupling experiments. Herein, we present an approach for the determination of accurate interfluorine distances in multi-spin systems, using the finite pulse RFDR (fpRFDR) at high MAS frequencies of 40-60 kHz. We use a series of crystalline “molecular ruler” solids, difluorobenzoic acids and 7F-L-tryptophan, for which the intra- and intermolecular interfluorine distances are known. We describe the optimal experimental conditions for accurate distance determinations, including the choice of a phase cycle, the relative advantages of selective inversion 1D vs. 2D correlation experiments, and the appropriate numerical simulation protocols. A best strategy for the analysis of RFDR exchange curves in organic solids with extended spin interaction networks is presented, which, even in the absence of crystal structures, can be potentially incorporated into NMR structure determination.
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Keywords
Dipolar tensors, Microcrystalline proteins, Symmetry-based recoupling