Preparation of recombinant peptides with site- and degree-specific lysine (13)C-methylation

Abstract

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 (15)N/(13)C-enriched peptides with a (13)C-methyl-labeled methylated lysine analogue. The method, which relies on the synthesis of (13)C-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.

FIGURE 1

Production of recombinant p53 peptides and installation of MLAs. pGBm/p53p and pGBm/p53pK370C peptides were overexpressed in E. coli as a histidine tag-GBm fusion protein, subjected to reductive alkylation to incorporate methyllysine analogs, cleaved from the fusion, and purified by various chromatographic methods.

FIGURE 2

Syntheses of ¹³C-enriched mono, di and trimethylated 2-chloroethylamine. These reductive alkylations use ¹³C-iodomethane and ¹³C-formaldehyde as sources of ¹³C-methyl. * indicates ¹³C, me stands for methyl. EAme1* and EAme2* have all their methyllysine analog methyls ¹³C-labeled. EAme3* has 3 methyls but only 1 is ¹³C-labeled.

FIGURE 3

¹H-¹³C HSQC titration spectra of ¹³C-methylated p53p peptides with nonlabeled 53BP1-tudor, C20orf104-tudor, and JMJD2A-tudor acquired at pH 7.5. Black peaks represent free p53pK_C370me1* (A), p53pK_C370me2* (B), and p53K_C370me3* (C), and cyan peaks, after addition of excess 53BP1-tudor, C20orf104-tudor, and JMJD2A-tudor, respectively. Intermediate stages of the titrations are shown in other colors.