Multiple Site‐Specific Installations of N^ε‐Monomethyl‐L‐Lysine into Histone Proteins by Cell‐Based and Cell‐Free Protein Synthesis

Abstract

Lysine methylation is one of the important post-translational modifications of histones, and produces an N(ε) -mono-, di-, or trimethyllysine residues. Multiple and site-specific lysine methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A method was previously developed to build an analogue of N(ε)-monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple N(ε)-monomethyllysine residues at specified positions. Release factor 1-knockout (RFzero) Escherichia coli cells or a cell-free system based on the RFzero cell lysate was used for protein synthesis, as in RFzero cells UAG is redefined as a sense codon for non-canonical amino acids. During protein synthesis, a tert-butyloxycarbonyl-protected N(ε)-monomethyllysine analogue is ligated to Methanosarcina mazei pyrrolysine tRNA (tRNA(Pyl)) by M. mazei pyrrolysyl-tRNA synthetase mutants, and is translationally incorporated into one or more positions specified by the UAG codon. Protecting groups on the protein are then removed with trifluoroacetic acid to generate N(ε)-monomethyllysine residues. We installed N(ε)-monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the N(ε)-monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic N(ε)-monomethyllysine residue at position 27 better than the N(ε)-monomethyllysine analogue built with cysteine. Mass spectrometry analyses also confirmed the lysine modifications on the produced histone H3. Thus, our method enables the installation of authentic N(ε)-monomethyllysines at multiple positions within a protein for large-scale production.

Chemical biology approaches for studying posttranslational modifications

“…22,28 But the resulting methyllysine analogs exhibit different properties and binding abilities toward reader molecules; thus, their biological relevance is often questionable. 85 Very recently, a facile chemical biology route that enables attachment of PTM moieties at a selected position of a target protein through chemoselective C-C bond formation was developed. 30 Using this method, which applies a clever combination of genetic code expansion and selective chemical conjugation, these researchers generated recombinant histones containing selective methylations in three modification states.…”

Chemical and Genetic Approaches to Study Histone Modifications

“…Therefore, in RF-1-knockout (RFzero) cells, or a cellfree system based on the RFzero cell lysate, the UAG codon may be entirely repurposed as a sense codon for non-natural amino acids. Sakamoto and co-workers demonstrated the incorporation of a Boc-protected N-ε-monomethyllysine analog in H3 by a mutant PylRS-tRNA CUA pair from M. mazei at multiple positions specified by the UAG codon [70]. The Boc-groups were subsequently removed with strong acid to generate N-ε-monomethyllysine residues at positions 4, 9, 27, 36, and/ or 79 of human histone H3.…”

Pyrrolysine Amber Stop‐Codon Suppression: Development and Applications

“…Indeed, incorporation and post‐translational deprotection yielded modified myoglobin and histone H3K9me1, with K9 methylation shown to be essential for the binding of H3 to heterochromatin protein 1. Further work demonstrated how this route could be used to install multiple monomethylated lysine residues in histone H3 through the use of E. coli RF1‐knockout strain RF0 . A later strategy achieved the same overall incorporation of 3 into a protein, but by using an alternative protecting group.…”

“…Further work demonstrated how this route could be used to install multiple monomethylated lysine residues in histone H3 through the use of E. coli RF1-knockout strain RF0. [30] Al ater strategy achieved the same overall incorporation of 3 into ap rotein, butb yu sing an alternative protecting group. As Alloc-Lys (11)i sasuitable substrate for PylRS, the N-methylated derivative 15 could serve as an analogous "caged" precursor to 3 if the Alloc group could be cleaved in ab ioorthogonal mannera kin to aB oc group.…”