Constructing catalytically proficient and functionally diverse enzymes from simple, de novo designed proteins

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Bespoke protein catalysts compatible with the natural biomolecular components of living cells are keyto realising the ambitious goals of synthetic biology and to the provision of cheap, green catalysts forindustrial biotechnology. To this end, the manmade protein maquettes designed in our lab haveproved particularly adaptable to a tractable de novo enzyme design process, as highlighted throughour successful design of a hyperthermostable and catalytically proficient de novo peroxidase, recentlypublished in Nature Communications. Since then, we have atomistically designed a new series ofheme-containing de novo proteins that allow for precision engineering of the protein structure andactive site. We have done so using a powerful combination of Rosetta protein design and MolecularDynamics simulations, allowing for both computational design and rapid structural assessment insilicon prior to protein expression biophysical characterisation.The aims of this project are to implement a powerful synthesis of computational and experimentalmethods in the de novo design of functional, catalytically active heme-containing proteins andenzymes. These proteins will be designed with particular emphasis on addressing challengingchemistries pertinent to industry, including catalytic monooxygenation and carbene transfer activities.Initially, they will be designed and assessed using computational methods (e.g. Rosetta protein designsuite & Molecular Dynamics software), and proteins that are subsequently selected for expression andpurification will be subjected to a comprehensive biophysical analysis (e.g. circular dichroism, EPR andNMR spectroscopies, redox potentiometry, X-ray crystallography). Catalytic activity will bedetermined through a variety of steady-state and pre-steady-state kinetic methods (e.g. plate readerassays, stopped-flow spectrophotometry), and products will be indentured using a variety oftechniques (e.g. NMR spectroscopy, HPLC, LC-MS). QM/MM calculations will be employed to examinethe catalytic cycles in detail, informing future protein designs. Directed evolution will also beemployed to improve and hone nascent activity.