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: 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:	Physics and Astronomy 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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