Molecular structure determination: solid-state NMR applications
Solid-state Nuclear Magnetic Resonance (ssNMR) has become one of the best techniques to determine molecular structure via inter/intra-molecular interactions. Unlike the well-known solution state NMR, solid-state NMR primarily elucidates the structural information by chemical shift anisotropy and dipolar coupling regardless of the solubility, crystallinity, and amorphousness of the target samples. This method has been applied in studying lyophilized samples, non-crystalline solids, insoluble aggregates, and biopolymer frozen solutions to determine their physical, chemical, and biological properties. This dissertation focuses on the applications of a variety of techniques of ssNMR spectroscopy in combination with other techniques to understand the molecular structures of substances of different kinds. The theoretical bases and the experimental introduction are reviewed first, and then experimental design and results are provided for the three projects separately: (1) The molecular structure of thin-film Boron carbide and the intermediate “X” in the PECVD fabrication process are elucidated mainly through ssNMR. Both13C and 11B direct excitation and cross polarization (CP) experiments, correlation experiments (13C-1H and 11B-1H), and 13C-11B distance measurements (REDOR) are applied to obtain connectivity information. FT-IR, elemental analysis, and ab initio calculations are used in addition to confirm the NMR results. The structure model of the intermediate “X” is proposed which is distinguished from the structure of the thin-film boron carbide, however, the structure model of the thin-film has not yet generated due to the weak signal from the natural abundance product. (2) The non-fibrillar and fibrillar C-terminal and K3 fragments of beta 2 microglobulin are evaluated. The homonuclear distances (CT-fpRFDR) measurements for the singly isotopically labeled protein samples are determined by comparing NMR results of the fibrillized fragments with the beta amyloid (G9V12) measurement standard. The fibril formation information is suggested by TEM, UV-Vis, and Fluorescence spectra. The CT and K3 fragments of beta 2 microglobulin are proved to form fibrils via parallel beta sheet structure. (3) The binding sites and/or the relative orientation of the dissociation drug of beta amyloid aggregates are investigated by ssNMR. Heteronuclear measurements (13C-2H REDOR) are mainly used to determine the distance between the deuterium label on the drug and the carbon label on Aβ1-42. The method upper limit is confirmed by lab made distance measurement standards,anthracene derivatives. However, the negative result is obtained from the triple labeled protein and singly labeled drug complex is not able to locate the drug-protein bound positions clearly. Therefore, different labeling schemes are also proposed for further confirm the assumption of binding sites.
Table of Contents
Introduction -- NMR basics -- Solid-state NMR experiments -- Structure determination of amorphous hydrogenated boron carbides -- Structure determination of beta-2 microglobulin protein fragments -- Structure determination of drug bound beta amyloid oligomer complex -- Appendix I. ab initio calculations and results -- Appendix II. REDOR measurement confirmation