Mackenzie, Harold Wallace; (2021) NMR Methods to Characterise Arginine Side-Chains in Proteins using 13C-Detection. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

An understanding of the behaviour of biomolecules in their native physiological environment is the cornerstone of drug discovery and is crucial in the fight to address the unmet medical needs of a growing population. Whilst the flexible and dynamic nature of proteins has long been known to play an important role in human biology, these molecular motions often significantly hamper efforts to study the most interesting systems. The research presented in this thesis discusses the development of several new pulse sequences and aims to demonstrate the utility of NMR spectroscopy to the study of dynamic systems, with a particular focus on 13C-detected techniques. Historically eschewed in favour of the more sensitive nuclei, recent hardware developments have enabled the exploitation of the favourable relaxation properties of 13C to design novel pulse sequences that overcome some of the inherent limitations of 1H-detection. A significant section of this research is devoted to the arginine side-chain, an amino acid that is routinely implicated in a wide range of intramolecular interactions within proteins. Firstly, a pair of pulse sequences is presented, in which 15Nη double-quantum magnetisation is generated and evolved to provide high-resolution correlation spectra of the functional guanidinium group, the part of the amino acid that is most often obscured by motions within the side-chain. Secondly, the development of the ASHDEX experiment – an arginine-specific method to monitor hydrogen exchange ¬– is discussed. Data obtained using the novel experiment shows that the rate with which hydrogen nuclei undergo chemical exchange with neighbouring solvent molecules correlates with the presence of intramolecular interactions observed in the crystal structure of T4 lysozyme. A final application to arginine interrogates the atom-specific deuterium isotope shifts and illustrates that this easily measured parameter is a powerful indicator of intramolecular salt-bridge formation. The utility of the method is exemplified by application to T4 lysozyme and bacterial ribonuclease. The final part of this thesis concerns the study of hydrogen exchange in intrinsically disordered proteins. IDPs have become the focus of many research groups owing to their implication in numerous disease models but the lack of a stable three-dimensional structure challenges the long-held view of a structure-activity relationship. Whilst the absence of tertiary structure hampers traditional 1H NMR observation, the switch to 13C-detection provides increased signal dispersion and enables the collection of high-resolution NMR spectra. The development of the CARBEX pulse sequence is presented and successfully demonstrated on human αSynuclein, the molecule implicated in Parkinson’s Disease.

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