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Title: Furthering structural insight into complement active fragments of DAF using NMR spectroscopy
Author: Brook, Eve
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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This thesis describes the reassignment of original two- and three-dimensional NMR data, acquisition of additional data sets and subsequent recalculation of DAF23 solution structure. Additional data included acquisition of three new sets of residual dipolar couplings (RDCs); these provide additional restraints for structure calculation. New resonance assignment highlighted both wrong and missing assignments in the original work. New structures of two conformers are presented. Their intermodular orientations are not congruent with original solution structures, but with the crystal structures; this observation is analysed and discussed. Dynamics of DAF23 are described using re-acquired 15N relaxation data and the model-free method of analysis. DAF23 shows very limited intermodular movement, with consistent orientation. Key interface residues to not appear to be moving on timescales different to the rest of the modules. Conflicting interpretation of mutagenesis data is now resolved; key residues at the 2/3 interface are important in stabilising structure, not solvent exposed and directly involved in binding as previously thought. This thesis also describes the purification of DAF fragment DAF12 and DAF234, to a suitable quality for study by NMR spectrometry. Use of a strong cation exchange column and a gradient elution provided the level of purification required.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available