Abstract
Post-translational methylation of lysine side chains is of great importance for protein regulation, including epigenetic control. Here, we present specific 13CHD2 labeling of dimethylated lysines as a sensitive probe of the structure, interactions, and dynamics of these groups, and outline a theoretical and experimental framework for analyzing their conformational dynamics using 1H and 13C CPMG relaxation dispersion experiments. Dimethylated lysine side chains in calcium-loaded calmodulin show a marked pH dependence of their Carr-Purcell-Meiboom-Gill (CPMG) dispersion profiles, indicating complex exchange behavior. Combined analysis of 1H and 13C CPMG relaxation dispersions requires consideration of 12-state correlated exchange of the two methyl groups due to circular three-state rotamer jumps around the Cε-Nζ axis combined with proton exchange and amine inversion. Taking into account a number of fundamental constraints, the exchange model can be reduced to include only three fitted parameters, namely, the geometric average of the rotamer-jump rate constants, the rate constant of deprotonation of Nζ, and the chemical shift difference between the trans and gauge positions of the 13C or 1H nuclei. The pH dependence indicates that protonation of the end group dramatically slows down rotamer exchange for some lysine residues, whereas deprotonation leads to rapid amine inversion coupled with rotamer scrambling. The observed variation among residues in their exchange behavior appears to depend on the structural environment of the side chain. Understanding this type of exchange process is critical to correctly interpreting NMR spectra of methylated lysine side chains. The exchange model presented here forms the basis for studying the structure and dynamics of epigenetically modified lysine side chains and perturbations caused by changes in pH or interactions with target proteins.
Highlights
Post-translational modifications of the ε-amino group of lysines regulate a number of protein functions, and are notable in histones, where they play essential roles in transcriptional regulation, chromatin regulation, and DNA damage response.[1−3] Lysine methylation of histones signals activation or repression of transcription depending on the number of attached methyl groups and the identity of the methylated lysine.[2]
The present results reveal dramatic pH-dependent changes in 1H and 13C transverse relaxation rates of methylated lysine side chains that cannot be explained by simple exchange models
The model can be fitted to the 1H and 13C CPMG dispersion profiles across a wide pH range by adjusting only three parameters, namely, ⟨krot⟩, kOH, and ΔrotΩ(13C) or ΔrotΩ(1H)
Summary
Post-translational modifications of the ε-amino group of lysines regulate a number of protein functions, and are notable in histones, where they play essential roles in transcriptional regulation, chromatin regulation, and DNA damage response.[1−3] Lysine methylation of histones signals activation or repression of transcription depending on the number of attached methyl groups and the identity of the methylated lysine.[2]. We introduce isotopomer-specific 13CHD2 labeling of dimethylated lysine residues in proteins that enables accurate measurement of their dynamics via 1H and 13C NMR relaxation experiments. Using this approach, we measured pH-dependent 1H and 13C Carr-Purcell-Meiboom-Gill (CPMG) dispersion profiles for Kme[2] groups in calmodulin (CaM). Monoand dimethylation of K94 occurs in the compound eye in Drosophila, while other tissues do not appear to harbor the same modification, indicating a functional role of this methylation pattern.[13] Methylation of lysines in CaM may play a role in protection against ubiquitination and subsequent
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