AbstractThe de novo design of porphyrin binding four-helix bundles (maquettes) is a proven strategy to both mimic and expand on the natural functionality of heme-containing proteins, including electron transfer, oxygen transport and catalysis. The rational design of maquettes utilizes simple principles to construct robust and adaptable heme-binding helical structures which can be iteratively engineered towards new functions. However, maquette designs have historically adopted highly dynamic conformations. Although this does not impede or may be integral to their function, enabling the construction of flexible structures capable of accessing diverse oxidoreductase catalysis, the lack of a unique conformation in these designs hinders structural determination at atomic resolution by techniques such as crystallography or NMR. In contrast, computational protein design is advancing to a stage where a range of protein structures can be routinely constructed with atomic accuracy, a level of precision which could be invaluable to engineer maquette designs towards novel properties.
In this project, computational design approaches were integrated into the construction of maquette structures, combining the strengths of functional, rational design strategies with precise computational techniques. The crystal structure of a two-heme binding four-helix bundle maquette was obtained by modification of the previously reported D2 peptide into a single-chain, in vivo expressed design. Computational techniques were employed to construct single and multi-heme binding variants which retain the architecture and stability of this initial structure by the design of precise protein interactions in a hydrophobic core or extending the helical bundle into a four-heme molecular wire, with further structural insights gained by NMR spectroscopy and cryoEM. Furthermore, computational design was harnessed to construct novel c-type cytochromes from scratch, inspired by the recent design of the artificial peroxidase, C45. This work presents novel structures for the continued engineering of oxidoreductase functionality in maquettes and a platform for precise investigation of the fundamental properties of protein-porphyrin complexes.
|Date of Award||24 Jun 2021|
|Supervisor||J L R Anderson (Supervisor) & Adrian J Mulholland (Supervisor)|
- de novo
- Protein Design