Total Chemical Synthesis Of Histones With Post-translational Modifications

Histone post-translational modifications(PTMs) including methylation, acetylation, phosphorylation, glycosylation, ubiquitylation and so on, play important roles in regulating the structures and functions of nucleosome, and are closely related to many dieases. Studies for its regulatory mechanism need homogenously modified histones as molecular tools. Now, four metnods including Cys-directed protein modification, amber suppression, protein semi-synthesis and protein total chemical synthesis are used for preparing modified histones. In these methods, total chemical synthesis that is not limited to the sites, numbers and types of modification can handle the molecular structure exactly, so ti is the most flexible method. But its applications in studies of histone PTMs are limited due to its low synthetic efficiency that usually low than milligram.For this purpose, the author is aimed at developing practical and efficient chemical synthesis methods for histone PTMs. For simple modifications(such as methylation, acetylation), the author developed a one-pot method for the ligation of peptide hydrazides which could be used for preparing modified histones in multi-milligram-scale. For ligation of peptide hydrazides that is practical and efficient in protein total chemical synthesis, the first one-pot method by using Dobz as the protecting group of Cys in the N-terminal is established.For the synthesis of ubiquitinated histones, the author developed trifluoroacetic acid sensitive auxiliary-mediated ligation of peptide hydrazides, achiving the ligation in Cys-free sits by the first time. Compare to the previous methods, the new method simplified the organic synthesis largely. Then, with the help of new strategy, the author prepared ubiquitinated histone H2 B including H2B-K34 ub and H2B-K120 ub in tens of milligram-scale. Also, the first total chemical synthesis of modified histones including ubiquitinated H2 A and H3-K56ac-K122 ub in tens of milligram-scale were finished. Especially, other three methods could not be able to prepare the double modified histone H3-K56ac-K122 ub, due to acetylation happens in the middle of the sequence and ubiquitylation in the C-terminal.In summary, this work established efficient total chemical synthesis methods for making modified histones including H2 A, H2 B, H3 and H4 in tens of milligram-scale. The synthetic proteins were reconsituted successfully into nucleosomes or histone octamers. And these could meet the escalating needs in the functional and stuctural studies of modified histones. Meanwhile, these methods are also useful for synthesis of other proteins, just like poly-ubiquitin chains.