Combining non-canonical amino acid mutagenesis and native chemical ligation for multiply modifying proteins: A case study of a-synuclein post-translational modifications

The Parkinson's disease associated protein & alpha;-synuclein (& alpha;S) has been found to contain numerous post-translational modifications (PTMs), in both physiological and pathological states. One PTM site of particular interest is serine 87, which is subject to both O-linked & beta;-N-acetylglucosamine (gS) modification and phosphorylation (pS), with & alpha;S-pS87 enriched in Parkinson's disease. An often-overlooked aspect of these PTMs is their effect on the membrane-binding properties of & alpha;S, which are important to its role in regulating neurotransmitter release. Here, we show how one can study these effects by synthesizing & alpha;S constructs containing authentic PTMs and labels for single molecule fluorescence correlation spectroscopy measurements. We synthesize & alpha;S-gS87 and & alpha;S-pS87 by combining native chemical ligation with genetic code expansion approaches. We introduce the fluorophore by a click reaction with a non-canonical amino acid. Beyond the specific problem of PTM effects on & alpha;S, our studies highlight the value of this combination of methods for multiply modifying proteins.