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Title: Biophysical studies on gelsolin
Author: Isaacson, R. L.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2000
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Mutations D187N and D187Y in domain 2 (D2) of the actin-regulating protein gelsolin cause familial amyloidosis - Finnish type (FAF). Aberrant proteolysis in variant gelsolin D2 yields a 7.1 kDa amyloidogenic fragment. A recombinant version of gelsolin D2 which is sufficiently stable for kinetic and equilibrium measurements has been produced and crystallised. The wild-type (WT) domain and both amyloidogenic variants fold simple two-state kinetics without the accumulation of any intermediates. Unfolding kinetics exhibit significant curvature with increasing urea concentration indicating that the transition state for unfolding becomes more native-like under increasingly denaturing conditions in according with the Hammond postulate. A preliminary φ-value analysis of gelsolin D2 compares its transition state for folding with that of villin 14T, a domain with similar fold topology. Mutations D187D and D187Y destabilise gelsolin D2 by 1.22 and 2.16 kcal mol-1 respectively. The mutations do not prevent disulfide bond formation despite their direct contiguity with a cysteine residue involved in disulfide linkage. The destabilisation conferred on gelsolin domain 2 by the FAF mutations is sufficient to predict that an appreciable fraction is unfolded and, therefore, extremely susceptible to proteolysis, at body temperature. NMR experiments investigating structural and dynamic changes between WT and D187N gelsolin D2 show that upon mutation, no significant structural or dynamic changes occur at or near the FAF cleavage sites. Areas in conformational exchange are observed between β-strand 4 and α-helix 1 and within the loop region following β-strand 5. D187N mutation destabilises the C-terminal tail of D2 resulting in a more exposed cleavage site leading to the first proteolysis step in the formation of the amyloidogenic fragment.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available