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Chemoenzymatic Semi‐synthesis Enables Efficient Production of Isotopically Labeled α‐Synuclein with Site‐Specific Tyrosine Phosphorylation
Author(s) -
Pan Buyan,
Park Joo Hyung,
Ramlall Trudy,
Eliezer David,
Rhoades Elizabeth,
Petersson E. James
Publication year - 2021
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000742
Subject(s) - phosphorylation , chemistry , biochemistry , native chemical ligation , tyrosine , escherichia coli , protein phosphorylation , fibril , biophysics , protein kinase a , chemical synthesis , biology , in vitro , gene
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.