PermaPhosPeptide Technology: Recombinant Production of Permanently Phosphorylated Oligopeptides for 14–3–3 Protein Interactomic Studies

Phosphorylation is a prevalent post-translational modification that controls many signaling pathways by regulating protein-protein interactions. Traditionally, these interactions are studied with chemically synthesized phosphopeptides, which are often expensive and prone to dephosphorylation, or <i>in vitro</i> kinase reactions, which often give incomplete or off-target phosphorylation. Here, using genetic code expansion with amber-codon-directed incorporation of a nonhydrolyzable phosphoserine analog (nhpS) that is autonomously produced in <i>E. coli</i>, we developed a technology to produce PermaPhosPeptides and validated its utility by obtaining functional 12-mer fragments of the SARS-CoV-2 nucleocapsid protein (N) from Wuhan and later coronavirus variants. PermaPhosPeptides are phosphatase-proof and accurately mimic authentic phosphopeptides in being recognized by pS/pT-binding 14-3-3 proteins, exhibiting an average <i>K</i>_D difference at pH 7-8 with respect to the Wuhan phosphopeptide of only 9%, as measured by fluorescence polarization. At pH 5.5, <i>K</i>_D for the 14-3-3 complex with PermaPhosPeptide increases by 68% compared with the phosphopeptide but remains in the low micromolar range despite the charge -1 of the nhpS-group, indicating that stereochemistry of the target group is a more critical driver for 14-3-3 recognition than its precise charge. Furthermore, PermaPhosPeptides revealed consistent effects of N mutations on binding affinities for the seven human 14-3-3 isoforms, indicating specificity and sensitivity of the interaction. Given the modular genetic encoding system used, PermaPhosPeptide technology is scalable and adaptable, in principle enabling production of almost any phosphopeptide in a permanently phosphorylated form for studies by low- and high-throughput methods.