NMR methods for characterising protein structure in solution and solid state
Creators: name: Orton, Henry
Publisher: name: The Australian National University
Year: 2020

description: Unpaired electrons give rise to spatially dependent paramagnetic phenomena observable by NMR spectroscopy which can be utilised for molecular structure determination. Paramagnetic lanthanide protein-complexes display distance-dependent relaxation enhancements of nuclear spins which can be used to refine molecular structure, an area of great interest to structural biology and drug discovery. Such relaxation enhancements are usually measured using spin labels with slowly relaxing unpaired electrons. The relaxation enhancement driven by paramagnetic lanthanide ions with fast relaxing electrons is theoretically well described by a magnetic susceptibility tensor, however, poor agreement with experiment has been observed previously. Chapter 1 of this thesis reports the development of a unified theoretical framework for calculation of paramagnetic NMR phenomena and describes its implementation as a software tool for fitting magnetic susceptibility tensors. A coordinate-free matrix representation was derived for calculation of all paramagnetic NMR observables, including pseudocontact shifts, residual dipolar couplings, paramagnetic relaxation enhancements and cross-correlation effects between Curie-spin relaxation, chemical shift anisotropy and dipole-dipole relaxation. The software was then used to expose the shortcomings of established methods for measurement of paramagnetic relaxation enhancements with an experimental investigation of the model protein calbindin D9k. It was found that residual dipolar couplings and intermolecular relaxation enhancements contaminate the experimental measurement of relaxation, leading to artificially short distances as derived from theory. Novel NMR experiments were established to eliminate these artefacts, leading to reliable measurements and drastically improved distance predictions in proteins up to 25 Angstrom from the paramagnetic centre. To study higher molecular weight biological systems including amyloid fibrils, viral assemblies and membrane proteins in liposomes, solid-state NMR achieves distance measurements by methods of dipolar recoupling. Recent developments in ultra-fast magic angle spinning technology have allowed for proton-detected higher-dimensional NMR spectroscopy, and with it, long-range inter-nuclear distance measurements for structure determination. An essential step towards structure analysis requires assignment of NMR chemical shifts to atoms of the molecule. Chapter 2 of this thesis pushes the limits of spin coherence transfer in NMR with the design and demonstration of a five-dimensional NMR experiment in the solid state for complete protein backbone assignment. Automated projection spectroscopy was used as a method for sparse sampling of the 5D experiment which was acquired for micro-cyrstalline GB1 protein, micro-crystalline superoxide dismutase and fibrillar B2 microglobulin. Fully automated assignment of chemical shifts was then demonstrated, showing that the new 5D experiment performs robust measurements that require very little human interpretation to complete an otherwise labour-intensive process. Chapter 3 of this thesis describes efforts in establishing equipment for ultra-fast magic angle spinning at the Australian National University. The chapter includes protocols for sample packing and handling of 1.2 mm and 0.5 mm rotors as well as protocols for magic angle spinning at 60 kHz and 150 kHz respectively.