Preparation of Recombinant Peptides with Site- and Degree-Specific Lysine 13C-Methylation

Lysine methylation is an important post-translational modification that affects protein function; for example.. the transcriptional activity of the p53 tumor suppressor protein. To facilitate structural characterization of complexes involving proteins and methylated targets by nuclear magnetic resonance spectroscopy, we devised a simple method for preparing recombinant N-15/C-13-enriched peptides with a C-13-methyl-labeled methylated lysine analogue. The method, which relies on the synthesis of C-13-enriched alkylating agents, was applied to the production of 15-residue p53 peptides variously methylated at lysine analogue 370. The peptides were used to probe the methylation state-dependent interactions of mono, di, and trimethylated p53 with three different proteins.