Ru Catalyst Facilitates Total Chemical Protein Synthesis to Investigate the Effect of Epigenetic Modifications Decorated on Linker Histone H1.2 and Heterochromatin Protein 1α (HP1α)

For epigenetics research, preparing homogeneous proteins bearing site-specific posttranslational modifications (PTMs) is essential to understand the behavior of chromatin. Total chemical protein synthesis is a very powerful method to obtain target proteins with various modifications at site-specific positions. To produce large proteins efficiently, one-pot ligation of multiple peptide fragments was previously reported through repetitive deprotection of protecting groups for N-terminal Cys with palladium complexes. However, this method demanded more than a catalytic amount of metal complexes, and, in general, it had been challenging to achieve catalytic cycles of metal complexes especially for reactions on proteins. Here, we report an efficient and facile method of chemical protein synthesis using Ru catalyst. The use of 10–20 mol% of Ru complexes enabled us to remove the protecting groups on peptides or proteins under peptide ligation conditions, and this complex showed more than 50-fold activity compared to the previous palladium complexes due to the great stability toward thiol moieties. By using this Ru catalyst, we accomplished total chemical synthesis of linker histone H1.2 (212 amino acids) and heterochromatin protein 1a (HP1a) (191 amino acids), which are important components of heterochromatin, through one-pot multiple peptide ligation. This method prompted the preparation of H1.2 and HP1a bearing various patterns of PTMs. Moreover, we found that R53Cit at H1.2 reduced its binding affinity toward nucleosomes and four consecutive phosphorylations at N-terminus HP1a controlled its binding ability against DNA. We envisage that homogeneously modified proteins prepared by our method would facilitate epigenetics research and be applied for the elucidation of various biological phenomena.