Hybrid phase ligation for efficient synthesis of histone proteins.

We introduce a hybrid solid-solution phase ligation approach that combines the efficiency of solid phase ligation with solution phase ligation in the total synthesis of modified histone proteins. A two linker strategy allows analysis throughout work on the solid phase and maximizes yields through cleavage at an external Rink, while an internal HMBA linker allows the native carboxyl terminus for any protein sequence. We demonstrate this approach for two histone proteins: triple-acetylated H4-K5ac, K12ac, K91ac and CENP-A-K124ac.

Chemical and biological tools for the preparation of modified histone proteins.

Eukaryotic chromatin is a complex and dynamic system in which the DNA double helix is organized and protected by interactions with histone proteins. This system is regulated through a large network of dynamic post-translational modifications (PTMs) which ensure proper gene transcription, DNA repair, and other processes involving DNA. Homogenous protein samples with precisely characterized modification sites are necessary to understand better the functions of modified histone proteins. Here, we discuss sets of chemical and biological tools developed for the preparation of modified histones, with a focus on the appropriate choice of tool for a given target. We start with genetic approaches for the creation of modified histones, including the incorporation of genetic mimics of histone modifications, chemical installation of modification analogs, and the use of the expanded genetic code to incorporate modified amino acids. We also cover the chemical ligation techniques which have been invaluable in the generation of complex modified histones indistinguishable from their natural counterparts. We end with a prospectus on future directions.

Development of Convergent Hybrid Phase Ligation for Efficient and Convenient Total Synthesis of Proteins.

Simple and efficient total synthesis of homogeneous and chemically modified protein samples remains a significant challenge. Here, we report development of a convergent hybrid phase native chemical ligation (CHP-NCL) strategy for facile preparation of proteins. In this strategy, proteins are split into ~100-residue blocks, and each block is assembled on solid support from synthetically accessible peptide fragments before ligated together into full-length protein in solution. With the new method, we increase the yield of CENP-A synthesis by 2.5-fold compared to the previous hybrid phase ligation approach. We further extend the new strategy to the total chemical synthesis of 212-residue linker histone H1.2 in unmodified, phosphorylated, and citrullinated forms, each from eight peptide segments with only one single purification. We demonstrate that fully synthetic H1.2 replicates the binding interactions of linker histones to intact mononucleosomes, as a proxy for the essential function of linker histones in the formation and regulation of higher order chromatin structure.