From peptide oligomers to single-chain proteins

Abstract

If we truly understand the relationship between protein sequence and structure, we can solve the protein folding problem and, in the process, realise fully de novo proteins — that is, proteins that do not exist in nature. As fold determines function, de novo folds could yield novel functions, or simply offer the opportunity to probe known functions within new scaffolds.
The facile synthesis, hyper-thermostability, and tuneable pore size of type II α-helical coiled coils, also known as α-helical barrels (αHBs), predispose these quaternary structures to later-stage installation of function. However, the inherent symmetry of homomeric coiled coils may impact their utility. The Woolfson group have previously described the installation of rudimentary hydrolytic activity into a homomeric, seven-helix coiled coil barrel. Unfortunately, its efficiency pales in comparison to the natural hydrolase α-chymotrypsin. Computational transition state modelling suggests that the placement of the catalytic residues within the barrel core could be optimised by spreading them across adjacent helices. However, this cannot be achieved in the current homomeric αHB systems. As such, more sophisticated coiled coil scaffolds in which mutations can be made with pinpoint precision are sorely needed.
One possible route towards de-symmetrizing coiled coils is the construction of single-chain proteins with a coiled-coil core. We posit that controlled oligomerisation of polypeptide motifs with tertiary structure e.g. helix-loop helix motifs will allow access to such dark matter proteins. The monomeric PPα miniprotein comprises a three-heptad α helix buttressed by an antiparallel polyproline type-II (PPII) helix, and provides a plausible template for single-chain parallel αHBs.
This thesis describes the successful oligomerisation of the optimised PPα miniprotein, and the utility of these PPα-based oligomers as templates for single-chain α-helical bundles and barrels. These de novo scaffolds possess great potential for catalysis and other biotechnological applications.

Date of Award	27 Sept 2022
Original language	English
Awarding Institution	University of Bristol
Supervisor	Dek N Woolfson (Supervisor) & Matthew P Crump (Supervisor)