Biosupramolecular protein tectonics
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Taking a structural biology approach, this thesis describes in-depth investigations into protein bioconjugation (PEGylation), surface recognition (synthetic receptors), and ligand-mediated assembly (crystalline frameworks). NMR spectroscopy was used to investigate the effect of PEGylation on the structure, dynamics, and recognition properties of RSL, a model b-propeller lectin. RSL complexation with the globular glycopolymer Ficoll was characterised and developed as a model for protein binding. RSL-Ficoll binding was sterically impeded by PEGylation, as indicated by spectral broadening and supported by MD simulations. The binding of the synthetic receptor cucurbituril (Q6) to six model proteins was evaluated using NMR spectroscopy. Weak and strong Q6 N-terminal complexation was characterised in RSL and SAMP2, a ubiquitin-like protein. Installation of the high-affinity N- terminal SAMP2 Met-Lys motif into RSL resulted in similarly tight Q6 complexation. Co-crystallization of cationic proteins with sulfonato-calixarene (sclx8) has generated highly-porous frameworks amenable to engineering. Here, the reach of sclx8- mediated assembly is extended to the neutral proteins. RSL (pI 6.5) co-crystallized with sclx8 over a wide pH range. Porous RSL-sclx8 frameworks formed at low pH, apparently a consequence of protein cationisation. One framework formed spontaneously in batch mode without the need for precipitants. Cucurbituril (Q7)-directed sheet and cage assemblies of RSL were modulated via fusion of coiled coil and IDP tectons. N-terminal fusions functioned as ‘spacers’ within the sheet architecture, resulting in layers of varying rigidity. An IDP fusion was partially captured in the cage assembly via Q7 recognition of intrapeptide Phe side chains.