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Title: Insight into amyloidosis from structural studies of human lysozymes
Author: Johnson, R. J. K.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2006
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Reported here is the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5 Angstroms. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen, for residues 70-74, shows that the T70N substitution increased the flexibility of the peptide backbone around the site of mutation. In hydrogen/deuterium exchange experiments monitored by mass spectrometry, a transient but locally cooperative unfolding event was observed for the T70N variant (at 47°C) and the wild type protein (57°C), but at higher temperatures than for the amyloidogenic variants I56T and D67H (37°C). These findings reveal that such partial unfolding is an intrinsic property of the lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo. At physiological temperature, the protection factors for many of backbone amides in the β-domain of the T70N variant are decreased relative to those in the wild type protein, whereas those in the α-domain display wild type-like values. At the higher temperature a dramatic decrease in protection was observed for the β-domain and C-helix for the T70N variant. This clearly demonstrated that partial unfolding event affects the β-domain and C-helix is similar to that observed previously for the I56T and D67H variants.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available