Development of novel protein modification strategy by integrating genetic code expansion and chemoselective conjugation

Abstract

Many proteins in living cells undergo numerous post-translational modifications (PTM) including phosphorylation, methylation, acetylation, and so forth. Such modifications play key roles in various cellular processes by regulating protein functions. Hence, an efficient way of installing particular modification site-specifically on protein can give us clues to understand how biological functions are controlled in cells. However, conventional chemical modifications have been limited to installation of PTM mimics. Alternatively, genetic code expansion enabled specific incorporation of modified amino acids, but it requires selective evolution process for each amino acid, and many PTMs are still left unconquered by this technique. Here, we present a novel protein chemical modification strategy integrating the two approaches: Marking-Activation-Coupling (MAC) scheme. In brief, the modification site is marked by site-selective incorporation of precursor amino acid, followed by activation of the marked site and coupling of target PTM moiety on the position via chemoselective conjugation. In this study, we demonstrated our system by selective PTM installation on proteins and biochemical assays using the modified proteins. Using MAC scheme, we produced modified histone H3 variants carrying three different methylation states (Kme1, Kme2, Kme3), respectively. Then, we validated the H3K79 methylation enhances histone acetylation, eventually leading to the increase of transcription level. We expect the MAC scheme may shed new light on disclosing biological meanings of diverse protein modifications occurring in our cells. Protein phosphorylation, the most prevalent PTM in mammalian proteome, is one of the extensively studied research subjects due to its vital regulatory roles. Genetic incorporation of phosphoserine has been successfully applied to produce site-specifically phosphorylated protein in high yield since it was first developed in 2011. Here are the four recent applications using a much improved phosphoserine incorporation system to reveal the roles of phosphorylation in various cellular processes: histone crosstalk, neurodegenerative diseases, NF-κB pathway, and DNA sensing pathway. The diverse applications successfully revealed the generality and utility of our system.