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Title: Studies of the binding interactions of human lysozyme with camelid antibody fragments
Author: Chan, P.-H.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2006
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We report the studies of three new camelid antibody fragments denoted cAb-HuL3, cAb-HuL5, and cAb-HuL22 raised against wild-type human lysozyme. These antibody fragments are made of a single polypeptide chain that constitutes a single antigen-binding domain. Of these antibody fragments, the cAb-HuL5 fragment has the highest conformational stability against urea, acid, alkaline, and heat-induced denaturation whereas the cAb-HuL3 fragment has the lowest. The effects of binding of the cAb-HuL22 fragment on the structural cooperativity of the I56T and D67H variants were studied. Nuclear magnetic resonance studies reveal that the cAb-HuL22 fragment interacts with the region corresponding to the B- and D- helices within the α-domain, the 310 helix adjacent to the C-helix as well as the β-strands and the long loop within the β-domain of human lysozyme, which encompasses the catalytic pocket. Surface plasmon resonance studies show that this antibody fragment competes effectively with the inhibitor, N, N’, N”-triaceylchitotriose, for the catalytic pocket of wild-type human lysozyme. Pulse-labelling hydrogen/deuterium exchange experiments monitored by mass spectrometry reveal that the cAb-HuL22 fragment suppresses strongly the locally cooperative unfolding event of the β-domain and the adjacent C-helix of both the I56T and D67H variants. These observations, therefore, highlight the fact that the structural cooperativity of the lysozyme variants can be restored to that characteristic of the wild-type protein through the occupation of the catalytic pocket. The effects of binding of the cAb-HuL5 fragment on the structural cooperativity of the lysozyme variants and on their propensities to form fibrils were studied. The results indicate that antibody binding domains can protect against the formation of pathogenic aggregates by several different mechanisms, reinforcing their value as potential therapies against amyloid diseases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available