Convergent Chemical Synthesis of Histone H2B Protein for the Site‐Specific Ubiquitination at Lys34

Abstract

Posttranslational modifications (PTMs) play a vital role in regulating the structure and dynamics of chromatin as well as DNA-driven cellular processes by covalently modifying the primary structures of the histone proteins (H2B, H2A, H3, H4) with various chemical entities.[1] One such example is the modification of the histone H2B by ubiquitin (Ub) at multiple lysine residues. Of these, ubiquitination at Lys120 (H2K120Ub) is best studied. This PTM was suggested to modulate gene expression, trigger H3 methylation, and activate DNA damage response pathways.[2] In contrast, little is known about the effects and functions of the other sites of ubiquitination in H2B, K34, K46, K108, and K116. [3] Notably, the preparation of any of these ubiquitinated histone proteins for biochemical analyses has been a challenge in the field and has thus hampered a variety of studies aiming to understand the effect of ubiquitination on chromatin. Only recently, novel nonenzymatic approaches were introduced and enabled the preparation of homogeneously native H2K120Ub on a milligram scale for structural and functional analyses.[4] These approaches proved to be successful for the incorporation of this single modification or any other modification within the short C-terminal synthetic peptide.In order to investigate the role of ubiquitination at other sites and to study the interplay of multiple modifications, a new synthetic strategy is needed. For example, it has been reported recently that ubiquitination of H2B at Lys34 directly regulates H3K4 and K79 methylation through trans-tail crosstalk both in vitro and in cells.[5] Hence, the preparation of such an analogue will enable studies aiming to understand how this modification regulates H3 methylation and potentially affects the structure of chromatin, as well as help to delineate mechanisms of K34 ubiquitination and deubiquitination. Here, we report our endeavors toward the total chemical synthesis of the H2B protein, and the successful sitespecific ubiquitination at Lys34 (H2K34Ub) for initial functional characterization.If one considers the preparation of H2K34Ub semisynthetically through the expression of a large C-terminal H2B fragment that bears an N-terminal Cys or through total chemical synthesis by applying a ligation approach with three fragments, the 57mer H2B(1-57), which bears the K34 modification, has to be prepared synthetically. Because H2B lacks any Cys residue, an Ala to Cys mutation for native chemical ligation (NCL) combined with desulfurization [6] should be performed at Ala58 (Scheme 1). Even if the semisynthetic method were applicable, it would not allow the insertion of different PTMs along the sequence but only at the N-terminal fragment. [7] Our initial studies with a threefragment approach revealed that the synthesis of the 57mer peptide via Fmoc-SPPS was very difficult. We thus concluded that a total chemical synthesis of the H2B protein from four fragments is inevitable. Hence, we divided the H2B sequence, taking into consideration the position of Ala residu...

Aligator: A computational tool for optimizing total chemical synthesis of large proteins

“…2A): Thioester (ligation) kinetics 73 : Segments containing a preferred C-terminal thioester receive +2 points (Ala, Arg, Cys, Gly, His, Met, Phe, Ser, Trp, or Tyr), while other acceptable thioesters (Ile, Leu, Lys 75 , Thr, or Val) result in 0 points. See File S3 for details.…”

An Efficient One‐Pot Four‐Segment Condensation Method for Protein Chemical Synthesis

“…[4] Thes ignificance of this method has been demonstrated in the successful synthesis and studies of important proteins,s uch as hepatocyte growth factor [5] and ubiquitinated histone H2B. [6] It is envisaged that one-pot four-segment ligation will also be an important method for protein chemical synthesis. [7,8] Indeed, in the development of labeled chemokines we encountered the need to establish ao ne-pot four-segment method to prepare these proteins containing multiple cysteine (Cys) residues.…”

Exploiting the MeDbz Linker To Generate Protected or Unprotected C‐Terminally Modified Peptides

“…Upon exposure to various nucleophiles, unprotected peptides 3 would be diversified at the C terminus ( 4 ). The susceptibility of this linker to attack by weaker nucleophiles than thiols and hydrazine 19 remains under-explored. 20 Additionally, the direct nucleophilic cleavage of this linker from resin has not been reported.…”

“…Propargylamine was similarly effective, while the reduced nucleophilicity of aniline resulted in an 8:92 ratio of amide to macrolactam. Peptide hydrazides 19, 32 and hydroxamides were generated with no macrocycle formation. In contrast, treatment with Weinreb amine led to 89% conversion to the macrolactam.…”