Förster Resonance Energy Transfer Assay for Investigating the Reactivity of Thioesters in Biochemistry and Native Chemical Ligation

Abstract

Thioesters are considered to be “energy-rich” functional groups that are susceptible to attack by thiolate and amine nucleophiles while remaining hydrolytically stable at neutral pH, which enables thioester chemistry to take place in an aqueous medium. Thus, the inherent reactivity of thioesters enables their fundamental roles in biology and unique applications in chemical synthesis. Here, we investigate the reactivity of thioesters that mimic acyl-coenzyme A (CoA) species and S-acylcysteine modifications as well as aryl thioesters applied in chemical protein synthesis by native chemical ligation (NCL). We developed a fluorogenic assay format for the direct and continuous investigation of the rate of reaction between thioesters and nucleophiles (hydroxide, thiolate, and amines) under various conditions and were able to recapitulate previously reported reactivity of thioesters. Further, chromatography-based analyses of acetyl- and succinyl-CoA mimics revealed striking differences in their ability to acylate lysine side chains, providing insight into nonenzymatic protein acylation. Finally, we investigated key aspects of native chemical ligation reaction conditions. Our data revealed a profound effect of the tris-(2-carboxyethyl)phosphine (TCEP) commonly used in systems where thiol–thioester exchange occurs, including a potentially harmful hydrolysis side reaction. These data provide insight into the potential optimization of native chemical ligation chemistry.

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

“…In the presence of 5 mM tris(2-carboxyethyl)phosphine (TCEP), concentrations as low as 10 mM BME at pH 7 were effective for quantitative on-protein hydrolysis within a few hours (Supplementary Fig. 3f ) 32 .…”

Lactoylglutathione promotes inflammatory signaling in macrophages

“…24 This provides a pH-dependent balance of acyltransfer from acylGSH and/or acylCoA species in biological systems. 24,25 . We thus hypothesized that LGSH may serve as a metabolic source for lactoyl-CoA through this non-enzymatic mechanism, transferring the lactate moiety to CoASH ( Figure 2e ).…”

“…Although lactoylLys has been proposed to be derived from lactoyl-CoA in an enzyme catalyzed reaction involving p300, this metabolite has not been quantified in macrophages, and its existence was only later found in cultured HepG2 cells. 3,21 As S-to-S acyl transfers are well-established in biology, 24 we hypothesized that lactoyl-CoA may be derived from LGSH, rather than through lactate and an unknown acyl-CoA synthetase. Indeed, this reaction takes place in vitro, with acyl transfer from LGSH to CoASH occurring more rapidly than its acetyl counterparts.…”

CysteineS-acetylation is a post-translational modification involved in metabolic regulation

“…The thioester bond, which connects acyl modifications to the cysteine sidechain, is sensitive to temperature, pH, and reducing agents and is susceptible to nucleophilic attack, which degrades short-chain cysteine acyl PTMs over time. 6 To address this, we developed a system for MS sample preparation that preserves the cysteine acetylome. Incubation with tris(2-carboxyethyl)phosphine (TCEP), which is minimally reactive towards the thioester bond, allows for the preservation of the cysteine acetylome compared to other common reducing agents such as dithiothreitol (DTT).…”