Protein-protein interactions in model systems : design, control of catalytic activity and biosensor applications

This thesis describes the design of polypeptides, unordered in the monomeric state but capable of folding into helix-loop-helix motifs and dimerise to form four-helix bundles. The goal of the design was to encode them with the capacity to form dimers highly selectively and the ability to carry out molecular functions in the folded state but not in the unordered state, and thus to establish a molecular link between recognition and function. The 42-residue sequences JR2E and JR2K were both shown by CD spectroscopy to adopt unordered conformations under single solute conditions at pH 7 but to form helical conformations in a 1:1 mixture. Analytical ultracentrifugation showed that JR2E and JR2K formed a clean heterodimer and the dissociation constant Kd, measured by CD spectroscopy, was found to be 5 ± 1 μM. Discrimination was enabled by the incorporation of charged residues at the dimer interface in the helical segments of the helix-loop-helix motif. Glutamic acids were incorporated in JR2E and lysines in JR2K, and charge repulsion prevented the monomeric subunits from forming homodimers. In mixtures, however, highly helical heterodimers were formed. The cooperative transition from unordered conformation to heterodimeric four-helix bundle was exploited in the design of a signal response system by incorporating a reactive site, capable of catalysing the hydrolysis of a m-nitrophenyl ester, into the negatively charged polypeptide. In the unfolded state the functionalised polypeptide was virtually inactive but in the folded state, induced by the interaction with JR2K, the substrate was hydrolysed approximately an order of magnitude more efficiently. Interactions between the designed polypeptides and a functionalised polythiophene polymer were studied and it was found that the conformation of the polymer was controlled by the polypeptides, largely by electrostatic interactions. The negatively charged JR2E forced the polymer to adopt a planar conformation while the positively charged JR2K induced a more twisted conformation of the polymer. The spectral changes coupled to the conformational transitions of the polymer were used to measure the binding of human Carbonic anhydrase II by JR2E functionalised with a benzenesulphonamide ligand, in demonstration of its use as a tool for high-throughput screening. JR2E immobilised on gold nanoparticles was shown to form homodimers reversibly under pH control, with affinities large enough to determine the state of aggregation of the gold nanoparticles