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Functional modulation and directed assembly of an enzyme through designed non-natural post-translation modification

Hartley, Andrew M., Zaki, Athraa J., McGarrity, Adam R., Robert-Ansart, Cecile, Moskalenko, Andriy V., Jones, Gareth F. ORCID:, Craciun, Monica F., Russo, Saverio, Elliott, Martin ORCID:, MacDonald, J. Emyr ORCID: and Jones, D. Daffydd ORCID: 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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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: Physics and Astronomy
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
Date of First Compliant Deposit: 30 March 2016
Date of Acceptance: 31 March 2015
Last Modified: 11 Oct 2023 17:15

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