Chemoenzymatic Semi-synthesis Enables Efficient Production of Isotopically Labeled α-Synuclein with Site-Specific Tyrosine Phosphorylation

Post-translational modifications (PTMs) can affect the normal function and pathology of alpha-synuclein (alpha S), an amyloid-fibril-forming protein linked to Parkinson's disease. Phosphorylation of alpha S 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 alpha S 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 alpha S 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 N-15-labeled, phosphorylated protein for NMR studies.

Automated summary

This study explores the impact of post-translational modifications on alpha-synuclein and introduces a streamlined method to produce phosphorylated alpha S in large quantities. By co-expressing a kinase with a protein fragment in Escherichia coli and using methyl thioglycolate, the researchers achieved high yields of site-specifically phosphorylated protein through chemoenzymatic methods. This strategy is particularly suitable for producing N-15-labeled, phosphorylated protein for NMR studies.