A chemical biology route to site-specific authentic protein modifications

Abstract

Many essential biological processes are controlled by posttranslational protein modifications. The inability to synthetically attain the diversity enabled by these modifications limits functional studies of many proteins. We designed a three-step approach for installing authentic posttranslational modifications in recombinant proteins. We first use the established O-phosphoserine (Sep) orthogonal translation system to create a Sep-containing recombinant protein. The Sep residue is then dephosphorylated to dehydroalanine (Dha). Last, conjugate addition of alkyl iodides to Dha, promoted by zinc and copper, enables chemoselective carbon-carbon bond formation. To validate our approach, we produced histone H3, ubiquitin, and green fluorescent protein variants with site-specific modifications, including different methylations of H3K79. The methylated histones stimulate transcription through histone acetylation. This approach offers a powerful tool to engineer diverse designer proteins.

Fig. 1. Approach for protein chemical modifications

(A) Schematic representation of the three-step synthesis of proteins with authentic PTMs. (B) Our coupling scheme enables chemoselective carbon-carbon bond formation in proteins. R, PTM moiety.

Fig. 2. Synthesis of proteins with selective differential methylations

(A) MALDI-TOF MS analysis of proteins after trypsin digestion. Formation of H3Sep79 is evidenced by the Sep-containing peptide (Sep79, blue).Synthesis of H3Dha79 generates a new Dha-containing peptide (Dha79, green) but eliminates the Sep-containing peptide. Coupling of the methyl iodides onto H3Dha79 produces new methylated lysine-containing peptides (K79me1, K79me2, and K79me3, red). m/z, mass/charge ratio; [M+H]⁺, monoisotopic mass; a.u., arbitrary units. (B) Western blot analysis of the modified proteins, using antibodies (α) against H3K79me1, H3K79me2, and H3K79me3. CB, Coomassie blue. (C) Chemical structures of lysine and differentially methylated lysine residues.

Fig. 3. Effects of H3K79 methylation on chromatin transcription

(A) Schematic of the standard in vitro transcription assay. AcCoA, acetyl coenzyme A; NTP, nucleoside triphosphate. (B) Effect of methylated H3K79 on chromatin transcription (x axis, lane number; y axis, relative transcription activity. (C) Effect of methylated H3K79 on p300-mediated chromatin acetylation. Histone acetylation status was monitored by fluorography. (D) Schematic representation of transcriptional activation by H3K79 methylation. Pol, polymerase; GTF, general transcription factor; TBP, TATA-binding protein.