Levy, GR; (2014) Structural folding dynamics of an archetypal conformational disease using Nuclear Magnetic Resonance Spectroscopy. Doctoral thesis , UCL (University College London).

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Abstract

Members of the serpin (serine protease inhibitor) superfamily of proteins regulate key physiological processes through their ability to undergo major conformational transitions. In conformational diseases, native protein conformers convert to pathological species that polymerise. Structural characterization of these key transitions is challenging. Mechanistic intermediates are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. I use Nuclear Magnetic Resonance (NMR), and Circular Dichroism (CD) spectroscopy, to investigate the interrelated processes of serpin folding, misfolding and polymerisation in solution using the 45kDa prototypic serpin á1-antitrypsin, the recent assignment of the backbone resonances of á1-antitrypsin by our group, allows us to ask more sophisticated questions by a range of NMR techniques to study its structure and dynamics. In this study, I analysed early unfolding behaviour of á1-antitrypsin across a urea titration within what is apparently the largest two-states system yet characterised. In order to assess the dynamics of the native state, I have used hydrogen/deuterium exchange nuclear magnetic resonance spectroscopy (HDXNMR) to characterise motions on the slow (ms) timescale. I have conducted a detailed analysis of residue-specific changes in protection from exchange across a pH titration using SOFAST-HMQC. This is complemented by a detailed a preliminary analysis of fast motions (ps-ns) using NMR relaxation experiments. Moreover, a forme fruste deficiency variant of á1-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo was characterized in this study. Lys154Asn á1-antitrypsin populates an intermediate ensemble along the polymerisation pathway at physiological temperatures. Together, this study shows how the use of powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions, provides novel insights into pathological conformational behaviour.

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