{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/245207"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/245207","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Evolvability of a viral protease: experimental evolution of catalysis, robustness and specificity","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Shafee, Thomas"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2014,"date_issued":"2014-02-04","date_published":"2014-02-04","updated_at":"2026-07-22T22:24:14Z","subjects":["Directed evolution","Evolution","Evolvability","Promiscuity","Experimental evolution","Robustness","Protease","Enzymology","Nucleophile"],"languages":["en"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/89fc407f-0877-4256-bd35-3965ace9b448/download"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.16528","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.sponsor","label":"Sponsor","values":["This work was supported by the BBSRC in conjunction with MedImmune through a CASE studentship."]},{"key":"dc:creator","label":"Author","values":["Shafee, Thomas"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2014-02-04"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/245207"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Directed evolution","Evolution","Evolvability","Promiscuity","Experimental evolution","Robustness","Protease","Enzymology","Nucleophile"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/89fc407f-0877-4256-bd35-3965ace9b448/download"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.16528"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/42519bb6-8009-49bc-9278-4bc931353c80/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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. 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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. 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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. 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