Protein kinases govern many cellular pathways and therefore need to be tightly regulated. Kinases can be controlled by changes to their conformation and many require phosphorylation to be fully active. In order to study the activation mechanisms of kinases, and for use in biochemical assays to identify potential inhibitors, a homogeneous protein sample of fixed 'activation state' is often desirable.Attempts to produce homogeneous fully active kinases usually involve mutagenesis to introduce a residue that mimics phosphorylation. For a typical kinase, Aurora-A (AurA), introduction of glutamate or aspartate into the activation loop is not a suitable substitute for serine or threonine phosphorylation. An alternative strategy is to chemically modify cysteine residues via a two-step reaction1 to produce a phospho-cysteine bioisostere of phospho-serine or -threonine. We have applied this approach to selectively determine the relative contributions to kinase activity of two adjacent phospho-threonine residues in AurA, and revealed a novel inhibitory role for activation loop phosphorylation in this kinase.2We now demonstrate that a range of chemical mimics of post-translational modification, and other unnatural amino acids, can be installed by this technique. We have determined reactivity trends for a diverse range of thiol nucleophile addition reactions on AurA. We also show that chemical modification of cysteine residues is possible not only on a flexible surface-exposed loop, but also within a sterically-hindered active site pocket, which reveals the potential use of this method in exploring enzyme function through modification of catalytic site residues.3 In conclusion, we show that chemical modification of cysteine residues is a valuable tool with which to modulate the activity and/or conformation of a recombinant protein. 1) Chalker (2012) Angew. Chem. Int. Ed., 51, 1835�18392) Rowan (2013) ACS Chem. Biol., 8, 2184�21913) Manuscript in preparation

Diverse functionalization of Aurora-A by chemical modification: Insights into kinase activation

248th National Meeting (2014)

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