Abstract
A principle aim of synthetic biology is the design of new biological parts that can be applied to replace or augment natural components in biological systems to produce entirely original functions. A major challenge in this field is the design of ‘switchable’ protein-protein interaction (PPI) domains that could be used to bring together other proteins in a controllable fashion. One approach is the design of components from first principles, or de novo. Coiled coils (CCs) are a protein structural motif that are well adapted to de novo design approaches given the available and well understood sequence-to-structure relationships. Taking inspiration from natural systems, our approach to produce novel switchable PPI domains is through the post-translational modification of CC components. To this end, here we incorporate phosphorylation motifs into de novo designed heterotetrameric CC assemblies and demonstrate their phospho-switching behaviours in vitro. This system elicits rapid and reversible switching between tetramer and monomer state in response to enzyme-catalysed phosphorylation and dephosphorylation.Next, we describe the application of this system in vivo, with the aim of producing a switchable PPI domain that can function orthogonally to host cell signalling processes. The designed switchable CCs function in E. coli cells. However, toxicity caused by the expression of the kinase ‘switch’ prevents the system functioning as anticipated in vivo. To overcome this, we reversed the switching mechanism by directly incorporating phosphoserine during protein expression in vivo, and expressing a phosphatase to trigger the switch between states. Promising preliminary results have been obtained. Such minimal switches have great potential to deliver dynamic functions to designed proteins, including the control of synthetic signalling networks, amongst other important synthetic biology applications.
This project has also delivered an entirely new CC design for an antiparallel heterotetramer, apCC-Tet-A2B2; and a split-fluorescent-protein assay that is able to distinguish the orientation of CCs modules directly in E. coli cells. These should prove useful as a new de novo designed CC module for other applications, and in high throughput characterisation of other CC designs in the future.
| Date of Award | 24 Jan 2023 |
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| Original language | English |
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| Supervisor | Dek N Woolfson (Supervisor) & Nigel J Savery (Supervisor) |