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dc.contributor.authorHartley, AM
dc.contributor.authorZaki, AJ
dc.contributor.authorMcGarrity, AR
dc.contributor.authorRobert-Ansart, C
dc.contributor.authorMoskalenko, AV
dc.contributor.authorJones, GF
dc.contributor.authorCraciun, MF
dc.contributor.authorRusso, S
dc.contributor.authorElliott, M
dc.contributor.authorMacdonald, JE
dc.contributor.authorJones, DD
dc.date.accessioned2016-04-25T11:08:42Z
dc.date.issued2015-03-31
dc.description.abstractPost-translational modification (PTM) modulates and supplements protein functionality. In nature this high precision event requires specific motifs and/or associated modification machinery. To overcome the inherent complexity that hinders PTM's wider use, we have utilized a non-native biocompatible Click chemistry approach to site-specifically modify TEM β-lactamase that adds new functionality. In silico modelling was used to design TEM β-lactamase variants with the non-natural amino acid p-azido-L-phenylalanine (azF) placed at functionally strategic positions permitting residue-specific modification with alkyne adducts by exploiting strain-promoted azide–alkyne cycloaddition. Three designs were implemented so that the modification would: (i) inhibit TEM activity (Y105azF); (ii) restore activity compromised by the initial mutation (P174azF); (iii) facilitate assembly on pristine graphene (W165azF). A dibenzylcyclooctyne (DBCO) with amine functionality was enough to modulate enzymatic activity. Modification of TEMW165azF with a DBCO–pyrene adduct had little effect on activity despite the modification site being close to a key catalytic residue but allowed directed assembly of the enzyme on graphene, potentially facilitating the construction of protein-gated carbon transistor systems.en_GB
dc.description.sponsorshipDDJ and ARM would like to thank the Advanced Research Computing @ Cardiff facility, especially Thomas Green for help with access and usage of the Raven cluster. DDJ, JEM and ME would like to thank the BBSRC (BB/H003746/1 and BB/ M000249/1), EPSRC (EP/J015318/1) and Cardiff SynBio Initiative/SynBioCite for supporting this work. SR and MFC acknowledge financial support from EPSRC (EP/J000396/1, EP/ K017160/1, EP/K010050/1, EP/G036101/1, EP/M001024/1, EPM002438/1). AMH was supported by a BBSRC studentship, AJZ by a Ministry of Higher Education in Kurdistan Region and Kurdistan Regional Government studentship and ARM by a Cardiff School of Biosciences personal studentship.en_GB
dc.identifier.citationChemical Science, 2015, 6, 3712en_GB
dc.identifier.doi10.1039/C4SC03900A
dc.identifier.urihttp://hdl.handle.net/10871/21217
dc.language.isoenen_GB
dc.publisherRoyal Society of Chemistryen_GB
dc.rightsThis is the final version of the article. Available from the Royal Society of Chemistry via the DOI in this record.en_GB
dc.titleFunctional modulation and directed assembly of an enzyme through designed non-natural post-translation modificationen_GB
dc.typeArticleen_GB
dc.date.available2016-04-25T11:08:42Z
dc.identifier.issn2041-6539
dc.identifier.journalChemical Scienceen_GB


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