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Methyllysine sites in proteins are recognized by an array of reader domains that mediate protein–protein interactions for controlling cellular processes. Herein, we engineer a chromodomain, an essential methyllysine reader, to carry 4-azido- l -phenylalanine (AzF) via amber suppressor mutagenesis and demonstrate its potential to bind and crosslink methylated proteins in human cells. We further develop a first-of-its kind chromodomain variant bearing two AzF units with enhanced crosslinking potential suitable for profiling the transient methyllysine interactome. 

Site-specific placement of unnatural amino acids, particularly those responsive to light, offers an elegant approach to control protein function and capture their fleeting ‘interactome’. Herein, we have resurrected 4-(trifluoromethyldiazirinyl)-phenylalanine, an underutilized photo-crosslinker, by introducing several key features including easy synthetic access, site-specific incorporation by ‘privileged’ synthetases and superior crosslinking efficiency, to develop photo-crosslinkable bromodomains suitable for ‘interactome’ profiling. 

Ten-eleven translocation (TET) enzymes oxidize C–H bonds in 5-methylcytosine (5mC) to hydroxyl (5hmC), formyl (5fC) and carboxyl (5caC) intermediates en route to DNA demethylation. It has remained a challenge to study the function of a single oxidized product. We investigate whether alkyl groups other than methyl could be oxidized by TET proteins to generate a specific intermediate. We report here that TET2 oxidizes 5-ethylcytosine (5eC) only to 5-hydroxyethylcytosine (5heC). In biochemical assays, 5heC acts as a docking site for proteins implicated in transcription, imbuing this modification with potential gene regulatory activity. We observe that 5heC is resistant to downstream wild type hydrolases, but not to the engineered enzymes, thus establishing a unique tool to conditionally alter the stability of 5heC on DNA. Furthermore, we devised a chemical approach for orthogonal labeling of 5heC. Our work offers a platform for synthesis of novel 5-alkylcytosines, provides an approach to ‘tame’ TET activity, and identifies 5heC as an unnatural modification with a potential to control chromatin-dependent processes.