# Genetic Encoding of Pentafluorophosphato‐Phenylalanine Provides PF5‐Proteins as Phosphoprotein Mimetics

## TL;DR

Scientists genetically encoded a fluorinated amino acid into proteins to create mimics of phosphorylated proteins, enabling new ways to study protein function.

## Contribution

A hyper-fluorinated phosphotyrosine analog was genetically encoded into proteins for the first time, enabling site-specific phosphoprotein mimetics.

## Key findings

- PF5CF2Phe was successfully incorporated into red fluorescent protein, ubiquitin, and GB1 using amber stop codon suppression.
- PF5-GB1 strongly inhibited phosphatases PTP1B and SHP2, unlike native GB1.
- PF5-ubiquitin was converted into a phosphotyrosine mimetic protein with phosphono-difluoromethyl phenylalanine.

## Abstract

Protein phosphorylation is one of the most important posttranslational modifications altering the structure, stability, and activity of more than 13 000 human proteins. In this work, the phosphotyrosine mimetic pentafluorophosphato‐difluoromethyl‐phenylalanine (PF5CF2Phe) was genetically encoded and incorporated into three different proteins. Screening two libraries of orthogonal aminoacyl‐tRNA synthetases identified enzymes enabling the efficient and specific incorporation of PF5CF2Phe into red fluorescent protein (RFP) via amber stop codon suppression. Two model proteins, human ubiquitin (Ubq) and the B1 immunoglobulin‐binding domain of streptococcal protein G (GB1), were prepared with PF5CF2Phe mutations and investigated for potential interaction partners. While native GB1 showed no binding to protein tyrosine phosphatases (PTP), PF5‐GB1, with PF5CF2Phe at position 17, was a strong inhibitor of the phosphatases PTP1B and SHP2. PF5‐Ubq was produced and converted into the first example of a protein carrying the most prominent phosphotyrosine mimetic, phosphono‐difluoromethyl phenylalanine (PO3CF2Phe). With increasing need in the biosciences to delineate the functions of complex phosphorylation patterns, genetic encoding of PF5CF2Phe yielding phosphoprotein mimetics opens unique opportunities for precise functional studies where site‐specific and homogeneous protein modifications are required.

Genetic code expansion with a unique hyper‐fluorinated phosphotyrosine analog. In this work, we successfully incorporated the unnatural amino acid pentafluorophosphato‐difluoromethyl‐phenylalanine, carrying seven fluorine atoms and a permanent negative charge into three different proteins via the use of mutated orthogonal aminoacyl‐tRNA synthetases. Biological testing revealed the great potential of this approach for furnishing functional phosphoprotein mimetics.