|Ph.D Student||Jbara Muhammad|
|Subject||Chemical Protein Synthesis of Modified Histone Proteins for|
|Department||Department of Chemistry||Supervisor||Professor Ashraf Brik|
|Full Thesis text|
The eukaryotic genome is stored in a polymeric structure called chromatin, which is composed of a fundamental repeating unit known as the nucleosome. The nucleosome complex has an octameric structure of four histone proteins H2A, H2B, H3, and H4, and DNA coil. Nucleosome plays highly important role in organization and compaction of the genome, which is regulated via different enzymes that covalently modify it by various post-translation modifications (PTMs) such as methylation, phosphorylation, and ubiquitination. Each one of these modifications can affect chromatin structure and stability, therefore, play a critical role in gene transcription and DNA damage repair and is linked to various diseases such as cancer. The chemical preparation of histone analogues is critical to facilitate important biochemical, biophysical and structural studies.
To develop new chemical strategies for the synthesis of inaccessible modified histones targets, there is an intrinsic requirement for incorporating orthogonal protecting groups (PGs) of the thiolated amino acids mainly by using thiazolidine (Thz), acetamidomethyl (Acm), and t-butyl. Despite the utility of the current toolbox for Cys protection/deprotection, there are still limitations to these strategies. The need for harsh removal conditions, prolonged reaction times and additional HPLC-purifications steps all limit the application of these approaches to more challenging systems. In course of my Ph.D. studies we have developed novel chemical tools for accelerating the synthesis and manipulation of synthetic proteins (i.e. histones proteins). We reported that palladium complexes can remove multiple Cys PGs within minutes in a fully aqueous medium to provide excellent yields of the desired products.
Using the new chemistry we were able to synthesize H2AY57p and H2BK120Ub analogues in high efficiency, which were assembled into nucleosomal complex. This complex enabled us to study the effect of phosphorylated H2A in the regulation of deubiquitination of H2B by the SAGA deubiquitinase module (DUBm). By performing enzymatic studies, we provided direct evidence for the cross-talk between phosphorylation and ubiquitination marks on the nucleosome, which found to inhibit the SAGA DUBm activity. In addition, molecular modeling showed that a phosphate group at H2AY57 is in a position to clash with Sgf11 residues Arg84 or Arg91, which are required for efficient H2B DUB activity.
The utility of the new removal conditions for Acm and Thz protecting groups was exemplified in the rapid and efficient synthesis of other modified histone proteins including, monoubiquitination histone H2B at Lys 34 (H2BK34Ub) and chemical synthesis of different analogues of trimethylated H3 at Lys79 (H3K79me3) for studying its regulation via the demethylases enzymes.
In addition, we have recently demonstrated an unprecedented tuning of palladium chemoselectivity in a fully aqueous medium for on-demand orthogonal deprotection of several Cys-protecting groups that are useful in protein synthesis and modification. Based on this chemistry we reported the first total chemical synthesis of an activity-based probe comprising ubiquitinated histone protein (H2AUb), which was incorporated into nucleosomes context and reacted with a Calypso/ASX DUB to yield a stable, covalent nucleosome-enzyme complex. This should assist in understanding the structural and functional mechanisms of Calypso/ASX DUB in chromatin context.