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


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.

SUBMITTER: Pan B 

PROVIDER: S-EPMC8185324 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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

Pan Buyan B   Park Joo Hyung JH   Ramlall Trudy T   Eliezer David D   Rhoades Elizabeth E   Petersson E James EJ  

Chembiochem : a European journal of chemical biology 20210129 8


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 chemo  ...[more]

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