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bioRxiv · 10.1101/2021.10.22.465468

PermaPhosSer: autonomous synthesis of functional, permanently phosphorylated proteins

Zhu, P.·Franklin, R.·Vogel, A.·Stanisheuski, S.·Reardon, P.·Beckman, J. S.·Karplus, P. A.·Mehl, R. A.·Cooley, R. B.
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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 {gamma}-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 "PermaPhosSer" 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 GSK3{beta} 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.

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Zhu, P., Franklin, R., Vogel, A., Stanisheuski, S., Reardon, P., Beckman, J. S., Karplus, P. A., Mehl, R. A., Cooley, R. B.. 2021-10-23. PermaPhosSer: autonomous synthesis of functional, permanently phosphorylated proteins. https://doi.org/10.1101/2021.10.22.465468

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