RESUMEN
We have created modified protein variants by introducing a non-canonical amino acid p-azido-l-phenylalanine (azF) into defined positions for photochemically-induced covalent attachment to graphene. Attachment of GFP, TEM and cyt b 562 proteins was verified through a combination of atomic force and scanning tunnelling microscopy, resistance measurements, Raman data and fluorescence measurements. This method can in principle be extended to any protein which can be engineered in this way without adversely affecting its structural stability.
RESUMEN
[This corrects the article DOI: 10.1039/C4SC03900A.].
RESUMEN
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 ß-lactamase that adds new functionality. In silico modelling was used to design TEM ß-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.