Hartley, Andrew M., Zaki, Athraa J., McGarrity, Adam R., Robert-Ansart, Cecile, Moskalenko, Andriy V., Jones, Gareth F.  ORCID: https://orcid.org/0000-0001-7709-3995, Craciun, Monica F., Russo, Saverio, Elliott, Martin ORCID: https://orcid.org/0000-0002-9254-9898, MacDonald, J. Emyr ORCID: https://orcid.org/0000-0001-5504-1692 and Jones, D. Daffydd ORCID: https://orcid.org/0000-0001-7709-3995
2015.
Functional modulation and directed assembly of an enzyme through designed non-natural post-translation modification.
Chemical Science
6
(7)
, pp. 3712-3717.
10.1039/C4SC03900A
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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
| Item Type: | Article |
|---|---|
| Date Type: | Publication |
| Status: | Published |
| Schools: | Schools > Physics and Astronomy Schools > Biosciences |
| Subjects: | Q Science > QC Physics Q Science > QH Natural history > QH301 Biology |
| Additional Information: | First published online 31 Mar 2015 This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. |
| Publisher: | Royal Society of Chemistry |
| ISSN: | 2041-6520 |
| Funders: | EPSRC, BBSRC |
| Date of First Compliant Deposit: | 30 March 2016 |
| Date of Acceptance: | 31 March 2015 |
| Last Modified: | 11 Oct 2023 17:15 |
| URI: | https://orca.cardiff.ac.uk/id/eprint/81057 |
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