Abstract
Electron transfer between redox centres is at the core of biochemical processes essential for life, such as respiration and photosynthesis. However, studying these processes can be challenging due to the complex nature of bioenergetic proteins, in which functionality is often obscured by the protein’s evolutionary legacy. This can be addressed by designing proteins de novo, with redox active cofactors, such as hemes or flavins, placed within a simple protein scaffold. Flavins are of particular interest as they are capable of one and two electron transfer reactions, and can be photoactivated, enabling light-induced electron transfer and catalysis.This thesis investigates different computational methods of engineering flavin binding sites into de novo four helix bundles. Whilst the computational workflow did not produce any designs capable of binding flavins noncovalently, similar computational methods were successfully used to engineer a binding site containing a covalently anchored flavin. This approach was used on several de novo proteins, yielding helical and thermostable proteins binding a covalently anchored riboflavin and heme in a desired stoichiometry, or simply the riboflavin alone. These proteins were able to rapidly abstract electrons upon exposure to light, storing the reducing equivalents within the heme or flavin itself. However, the designed proteins were shown to be highly structurally flexible, with in silico simulations highlighting the challenges of incorporating both cofactors in the small helical scaffold. Additionally, natural flavin transferases and their ability to covalently attach flavins to de novo scaffolds were explored. The preliminary results indicated that this is a viable strategy, especially as flavin-bound proteins can be produced in vivo.
The work described here creates a foundation for incorporating covalently bound flavins into designed scaffolds, enabling creation of minimal light activated redox proteins. These proteins can serve as a foundation to develop efficient light harvesting systems for bioinspired solar cells or minimal flavin-based (photo)biocatalysts.
Date of Award | 20 Jun 2023 |
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Original language | English |
Awarding Institution |
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Supervisor | Marc W Van der Kamp (Supervisor) & J L R Anderson (Supervisor) |