Chemoenzymatic Semi‐synthesis Enables Efficient Production of Isotopically Labeled α‐Synuclein with Site‐Specific Tyrosine Phosphorylation
Buyan Pan, Joo Hyung Park, Trudy F. Ramlall, David Eliezer, Elizabeth Rhoades, E. James Petersson
Abstract
Abstract Post‐translational modifications (PTMs) can affect the normal function and pathology of α‐synuclein (αS), an amyloid‐fibril‐forming protein linked to Parkinson's disease. Phosphorylation of αS Tyr39 has recently been found to display a dose‐dependent effect on fibril formation kinetics and to alter the morphology of the fibrils. Existing methods to access site‐specifically phosphorylated αS for biochemical studies include total or semi‐synthesis by native chemical ligation (NCL) as well as chemoenzymatic methods to phosphorylate peptides, followed by NCL. Here, we investigated a streamlined method to produce large quantities of phosphorylated αS by co‐expressing a kinase with a protein fragment in Escherichia coli . We also introduced the use of methyl thioglycolate (MTG) to enable one‐pot NCL and desulfurization. We compare our optimized methods to previous reports and show that we can achieve the highest yields of site‐specifically phosphorylated protein through chemoenzymatic methods using MTG, and that our strategy is uniquely well suited to producing 15 N‐labeled, phosphorylated protein for NMR studies.