Chemical approaches to synthesize proteins with site-specific lysine succinylation and in vitro investigation of the effects of histone succinylation on nucleosome dynamics

Abstract

Posttranslational modifications (PTMs) increase the structural and functional diversity of proteins and are involved in the regulation of various biological processes. To date, more than 20 types of PTMs have been identified on histones. Histone PTMs were demonstrated to play vital roles in chromatin-templated cellular processes, including nucleosome conformational dynamics, gene transcription regulation and DNA damage repair. Extensive studies revealed that nucleosome dynamics can be influenced by site-specific histone PTMs, however, most of these studies are confined to a limited range of PTMs (i.e., acetylation and phosphorylation). The potential effects of those newly identified histone PTMs, such as succinylation and glutarylation, on nucleosome dynamics remained unknown. This may partially due to a challenge to generate homogeneous proteins that carry site-specific modification at stoichiometric level. To address this challenge, in this research I have utilized different strategies to prepare homogeneously modified proteins. First, I have developed a chemical approach based on ‘thiol-ene’ chemistry to site-specifically install analogs of PTMs into proteins. In detail, lithium 5-oxo-5-(vinylamino) pentanoate (compound 15) and tert-butyl N-vinyl succinamate (compound 21) were designed and synthesized to react with native cysteine in the protein sequence to generate a glutaryllysine analog (Kcglu) and a succinyllysine analog (Kcsucc) respectively. This synthetic strategy is highly efficient for preparing large quantities of homogeneously modified proteins. Besides, I have utilized expressed protein ligation (EPL) strategy to site-specifically incorporate acetyllysine and succinyllysine into histone H4 at K77 position respectively. Additionally, various posttranslationally modified amino acids have been site-specifically incorporated into proteins via amber codon suppression, including methyllysine, acetyllysine and crotonyllysine. However, there is no successful report on the genetic incorporation of succinyllysine into proteins. This may be because the negatively charged side chain of succinyllysine impairs its cell permeability. To address this issue, I have designed and synthesized compound 4, N6-(4-(2-(4-hydroxy-3,5-dimethoxyphenyl)-2-oxoethoxy)-4-oxobutanoyl)-L-lysine, with a photolabile protecting group caging the negatively charged side chain to enhance its cell permeability. If it can be genetically incorporated, succinyllysine can be effectively released in situ upon UV irradiation. Previous studies indicated that lysine acetylation impaired histone-DNA interaction and influenced nucleosome dynamics by quenching the positively charged lysine side chain (+1 to 0). Comparably, lysine succinylation even changes the positively charged lysine side chain to negatively charged (+1 to -1), which is supposed to have much more significant impacts on histone-DNA interaction and nucleosome dynamics. To test such hypothesis, succinylated histone H2BKc34succ was incorporated into mononucleosomes. Single-pair förster resonance energy transfer (spFRET) based approach indicated that H2BKc34succ influences nucleosome stability by impairing the interaction between H2A-H2B dimers and nucleosomal DNA. Besides, a more comprehensive study was conducted with modified histone H4K77succ and H4K77ac. FRET analysis of the nucleosomes containing the modified histones demonstrated that compared with H4K77ac, H4K77succ exerts a much severer destabilizing effect on nucleosome and promotes the dissociation of H2A-H2B dimer from nucleosome in the salt-induced stepwise nucleosome disassembly process. These studies comprehensively investigated the effects of site-specific PTMs on nucleosome stability and proposed potential regulatory mechanisms of PTMs in the regulation of nucleosome dynamics.