Deciphering functional roles of protein succinylation and glutarylation using genetic code expansion

Abstract

Post-translational modifications (PTMs) dynamically regulate cellular processes. Lysine undergoes a range of acylations, including malonylation, succinylation (SucK) and glutarylation (GluK). These PTMs increase the size of the lysine side chain and reverse its charge from +1 to −1 under physiological conditions, probably impacting protein structure and function. To understand the functional roles of these PTMs, homogeneously modified proteins are required for biochemical studies. While the site-specific encoding of PTMs and their mimics via genetic code expansion has facilitated the characterization of the functional roles of many PTMs, negatively charged lysine acylations have defied this approach. Here we describe site-specific incorporation of SucK and GluK into proteins via temporarily masking their negative charge through thioester derivatives. We prepare succinylated and glutarylated bacterial and mammalian target proteins, including non-refoldable multidomain proteins. This allows us to study how succinylation and glutarylation impact enzymatic activity of metabolic enzymes and regulate protein–DNA and protein–protein interactions in biological processes from replication to ubiquitin signalling.

Cellular and Biophysical Applications of Genetic Code Expansion

“…366 Recently, ncAAs 78 and 79, which are thioester-masked to neutralize the negative charge of the carboxylate group, were incorporated into proteins and then hydrolyzed to introduce succinylated and glutarylated Lys indirectly. 367 Citrullination has also been introduced into proteins only through indirect methods. Mondal et al introduced the photocaged precursor ncAA 80 site-specifically into target proteins and subsequently converted it to citrulline upon exposure to light.…”

“…The Liu group introduced the precursor ncAA 77 site-specifically into target proteins and subsequently introduced succinylated Lys indirectly using the Staudinger ligation reaction . Recently, ncAAs 78 and 79 , which are thioester-masked to neutralize the negative charge of the carboxylate group, were incorporated into proteins and then hydrolyzed to introduce succinylated and glutarylated Lys indirectly . Citrullination has also been introduced into proteins only through indirect methods.…”