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

• Published in: Chemical science (Cambridge) 2015-07, Vol.6 (7), p.3712-3717
• Main Authors: Hartley, Andrew M, Zaki, Athraa J, McGarrity, Adam R, Robert-Ansart, Cecile, Moskalenko, Andriy V, Jones, Gareth F, Craciun, Monica F, Russo, Saverio, Elliott, Martin, Macdonald, J Emyr, Jones, D Dafydd
• Format: Article
• Language: English
• ISSN: 2041-6520; 2041-6539
• DOI: 10.1039/c4sc03900a

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. modelling was used to design TEM β-lactamase variants with the non-natural amino acid -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 TEM 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.