DISULPHIDE LOCKING: CONTRASTING EFFECTS ON DISPARATE PROTEINS
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This thesis details a series of structural and biophysical studies on two different proteins containing disulphide bonds: (1) soricidin and (2) engineered mutants of aciniform spider silk. All studies relied upon recombinant protein expression, purification, and refolding, with several biophysical and materials characterization techniques applied, including solution-state nuclear magnetic resonance (NMR) spectroscopy as the major tool. Soricidin is a venomous protein isolated from shrew saliva whose short derivatives without any disulphide linkages are reported as effective anticancer drugs. Here, I report drastic improvements in refolding to produce the paralytic conformer and present for the first time the atomic structure of the full-length bioactive protein. With one disulphide linking the N-terminal end of the protein to an a-helical segment in the otherwise disordered C-terminal tail to form an enclosed loop and two a-helices cross-linked by the other two disulphides, soricidin demonstrates a I–VI/II-IV/III–V disulphide connectivity. Although cysteine motifs and exposed Lys/Arg dyad are consistent with other venoms, soricidin adopts a rarely reported cysteine-stabilized helix-loop-helix fold. With the objective of identifying the trigger for fibrillogenesis in aciniform spider silk, conformation and dynamics were probed using engineered mutant forms of aciniform spider silk in the reduced (i.e., sulfhydryl-containing cysteine side chains) vs. disulphide-linked state. The reduced state of the aciniform silk mutant is capable of silk-like fibre formation and can be wet spun into silk-like fibres with improved mechanical properties relative to the wild-type protein. The disulphide-locked state, conversely, is unable to form silk-like fibres. These differences in functionality are correlated to solution-state conformation, dynamics and pre-fibre self-assembly behaviour. Specifically, the disulphide-locked aciniform silk protein loses some a-helical character around the disulphide but forms a more compact unit as a whole, correlating with more xxv heterogeneous and larger pre-fibre particle self-assembly. The reduced state, conversely, has unchanged structuring relative to the wildtype but exhibits slightly elevated ps-ns-timescale dynamics around the mutation site that correlates with pre-fibre self-assembly into smaller than the wildtype nanoparticles with similar homogeneity. These studies on two different classes of protein demonstrate that disulphide bridges have the potential to amend both native structure and dynamics with clear functional consequences.