Post-translational modifications (PTMs) are reversible chemical modifications that can modulate protein structure and function. Methylation and acetylation are two such PTMs with integral and well-characterized biological 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 enables pursuit of molecular mechanisms. Here, we present a protocol for implementing nuclear magnetic resonance (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.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)