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

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.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