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PermaPhos Ser : autonomous synthesis of functional, permanently phosphorylated proteins.

ABSTRACT: Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study protein regulation. Previously, a genetic code expansion (GCE) system was developed to translationally install non-hydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with carbon, but it has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a biosynthetic pathway that produces nhpSer from the central metabolite phosphoenolpyruvate. Using this "PermaPhos Ser " system - an autonomous 21-amino acid E. coli expression system for incorporating nhpSer into target proteins - we show that nhpSer faithfully mimics the effects of phosphoserine in three stringent test cases: promoting 14-3-3/client complexation, disrupting 14-3-3 dimers, and activating GSK3β phosphorylation of the SARS-CoV-2 nucleocapsid protein. This facile access to nhpSer containing proteins should allow nhpSer to replace Asp and Glu as the go-to pSer phosphomimetic for proteins produced in E. coli .

SUBMITTER: Zhu P 

PROVIDER: S-EPMC8687462 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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PermaPhos <sup>Ser</sup> : autonomous synthesis of functional, permanently phosphorylated proteins.

Zhu Phillip P   Franklin Rachel R   Vogel Amber A   Stanisheuski Stanislau S   Reardon Patrick P   Sluchanko Nikolai N NN   Beckman Joseph S JS   Karplus P Andrew PA   Mehl Ryan A RA   Cooley Richard B RB  

bioRxiv : the preprint server for biology 20211214

Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study protein regulation. Previously, a genetic code expansion (GCE) system was developed to translationally install non-hydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with carbon, but it has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into <i>Escherichia coli</i> a biosynthetic pathway that produces nhpSer from the central m  ...[more]

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