dc.contributor.author | Hartley, AM | |
dc.contributor.author | Zaki, AJ | |
dc.contributor.author | McGarrity, AR | |
dc.contributor.author | Robert-Ansart, C | |
dc.contributor.author | Moskalenko, AV | |
dc.contributor.author | Jones, GF | |
dc.contributor.author | Craciun, MF | |
dc.contributor.author | Russo, S | |
dc.contributor.author | Elliott, M | |
dc.contributor.author | Macdonald, JE | |
dc.contributor.author | Jones, DD | |
dc.date.accessioned | 2016-04-25T11:08:42Z | |
dc.date.issued | 2015-03-31 | |
dc.description.abstract | Post-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.sponsorship | DDJ 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.citation | Chemical Science, 2015, 6, 3712 | en_GB |
dc.identifier.doi | 10.1039/C4SC03900A | |
dc.identifier.uri | http://hdl.handle.net/10871/21217 | |
dc.language.iso | en | en_GB |
dc.publisher | Royal Society of Chemistry | en_GB |
dc.rights | This is the final version of the article. Available from the Royal Society of Chemistry via the DOI in this record. | en_GB |
dc.title | Functional modulation and directed assembly of an enzyme through designed non-natural post-translation modification | en_GB |
dc.type | Article | en_GB |
dc.date.available | 2016-04-25T11:08:42Z | |
dc.identifier.issn | 2041-6539 | |
dc.identifier.journal | Chemical Science | en_GB |