Advances in direct detection of lysine methylation and acetylation by nuclear magnetic resonance using 13C-enriched cofactors

Post-translational modifications (PTMs) are reversible chemical modifications that can modulate protein struc-ture and function. Methylation and acetylation are two such PTMs with integral and well-characterized bio-logical roles, including modulation of chromatin structure; and unknown or poorly understood roles, exemplified by the influence of these PTMs on transcription factor structure and function. The need for biological insights into the function of these PTMs motivates the development of a nondestructive and label-free method that en-ables pursuit of molecular mechanisms. Here, we present a protocol for implementing nuclear magnetic reso-nance (NMR) methods that allow for unambiguous detection of methylation and acetylation events and demonstrate their utility by observing these marks on histone H3 tail as a model system. We leverage strategic isotopic enrichment of cofactor and peptide for visualization by [1H, 13C]-HSQC and 13C direct-detect NMR measurements. Finally, we present 13C-labeling schemes that facilitate one-dimensional NMR experiments, which combine reduced measurement time relative to two-dimensional spectroscopy with robust filtering of background signals that would otherwise create spectral crowding or limit detection of low-abundance analytes.

Automated summary

Post-translational modifications (PTMs) are reversible chemical modifications that can affect protein structure and function. Methylation and acetylation are two well-known PTMs with important biological roles, but their specific effects on transcription factor structure and function are not well understood. This study presents a nondestructive and label-free method using nuclear magnetic resonance (NMR) to detect methylation and acetylation events on histone H3 tail. The authors introduce isotopic enrichment and 13C labeling schemes that enable efficient NMR experiments for detecting low-abundance analytes.