Evolvability of a viral protease: experimental evolution of catalysis, robustness and specificity

2020-02-17T03:33:13Z (GMT) by Thomas Shafee
PhD Thesis
2014-02-04

Institution
Department of Biochemistry, University of Cambridge
Antibody Discovery and Protein Engineering, Medimmune

Advisors
Hollfelder, Florian
Minter, Ralph

The aim of this thesis is to investigate aspects of molecular evolution and enzyme engineering using the experimental evolution of Tobacco Etch Virus cysteine protease (TEV) as a model. I map key features of the local fitness landscape and characterise how they affect details of enzyme evolution. In order to investigate the evolution of core active site machinery, I mutated the nucleophile of TEV to serine. The differing chemical properties of oxygen and sulphur force the enzyme into a fitness valley with a >104-fold activity reduction. Nevertheless, directed evolution was able to recover function, resulting in an enzyme able to utilise either nucleophile. High-throughput screening and sequencing revealed how the array of possible beneficial mutations changes as the enzyme evolves. Potential adaptive mutations are abundant at each step along the evolutionary trajectory, enriched around the active site periphery. It is currently unclear how seemingly neutral mutations affect further adaptive evolution. I used high-throughput directed evolution to accumulate neutral variation in large, evolving enzyme populations and deep sequencing to reconstruct the complex evolutionary dynamics within the lineages. Specifically I was able to observe the emergence of robust enzymes with improved mutation tolerance whose descendants overtake later populations. Lastly, I investigate how evolvability towards new substrate specificities changed along these neutral lineages, dissecting the different determinants of immediate and long-term evolvability. Results demonstrate the utility of evolutionary understanding to protease engineering. Together, these experiments forward our understanding of the molecular details of both fundamental evolution and enzyme engineering.