Labelling proteins for paramagnetic NMR Spectroscopy and EPR Spectroscopy.
Creators: name: Mahawaththage Don, Mithun
Publisher: name: The Australian National University
Year: 2019

description: Paramagnetic nuclear paramagnetic resonance (NMR) spectroscopy and Double Electron-Electron Resonance (DEER) experiments provide powerful tools to study the structures of biological macromolecules, especially proteins. This thesis focuses on two major objectives. (1) It demonstrates the application of these techniques to answer questions in structure biology. (2) A second focus is on the development of new spectroscopic labels and labelling strategies to improve the sensitivity, accuracy and scope of NMR and EPR spectroscopy experiments of proteins. Chapter 2 describes an investigation of the solution conformation of a Zika virus NS2B-NS3 protease construct, where NS2B and NS3 are covalently linked by a flexible linker peptide, in the presence and absence of inhibitor. The study relied to a major extent on pseudocontact shifts (PCS) generated by attaching lanthanide ions to the protein. It showed that the protease exists in a closed conformation in the presence of inhibitor as observed in crystal structures, whereas it is in an open conformation in the absence of inhibitor. Chapters 3 and 4 focus on new labelling strategies for paramagnetic NMR spectroscopy. Chapter 3 demonstrates a pnictogen-mediated self-assembly strategy to attach a lanthanide ion to the target protein via a double-cysteine motif. This approach proved selective as the assembly was shown not to form with single solvent-exposed cysteine residues. It appeared, however, that the approach cannot be generalized, as it depends on additional amino acid side chains to immobilize the lanthanide ion with respect to the protein backbone. Chapter 4 demonstrates a novel approach of introducing a lanthanide ion via a genetically incorporated unnatural amino acid, phosphoserine. Two phosphoserine residues in close proximity provide a sufficient number of negative charges to attract lanthanide ions. Remarkably, the scheme is capable of tightly immobilizing the lanthanide ion near the protein backbone, resulting in fairly large magnetic susceptibility anisotropy tensors with record-low quality factors. Unfortunately, the scheme was successful for only two protein mutants, whereas many other attempts with different sites and proteins failed, often due to lack of protein expression, which may be explained by premature protein degradation as too many negative charges in close proximity may prevent the protein from folding. Further experiments will need to be performed to generalize this approach. Chapter 5 discusses the use of two Gd3+ tags, propargyl-DO3A and C11, which are small, neutral and hydrophilic, for distance measurements in proteins by double electron-electron resonance (DEER) experiments in a high-field (W-band) electron paramagnetic resonance (EPR) spectrometer. The accuracy of the distances measured is compared with that of a previously published tag. The propargyl-DO3A tag is particularly attractive, as it can be attached to a genetically encoded unnatural amino acid, 4-azidophenylalanine, in a Cu+-catalyzed click reaction. The perfomance of the C11 and propargyl-DO3A tags is demonstrated with two model proteins. Chapter 6 illustrates the ability of the propargyl-DO3A-Gd tag to detect multiple protein conformations. The study comprised five different related proteins, three of which were created by ancestral reconstruction. The proteins span the range from the lysine/arginine/ornithine binding protein (LAOBP) to the structurally related enzyme cyclohexadienyl dehydratase (CDT). LAOBP and some of the ancestral proteins showed clear evidence of two different conformational species, open and closed, which had previously been documented by X-ray crystallography. DEER experiments confirmed the presence of these structural conformers in solution, but also showed that open and closed conformers can co-exist in solution even in the absence of ligand molecules.