Lysine methylation is an important post-translational modification of histone proteins that defines epigenetic status and controls heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair, and transcriptional regulation. Despite considerable efforts by chemical biologists to synthesize modified histones for use in deciphering the molecular role of methylation in these phenomena, no general method exists to synthesize proteins bearing quantitative site-specific methylation. Here we demonstrate a general method for the quantitative installation of N(epsilon)-methyl-L-lysine at defined positions in recombinant histones and demonstrate the use of this method for investigating the methylation dependent binding of HP1 to full length histone H3 monomethylated on K9 (H3K9me1). This strategy will find wide application in defining the molecular mechanisms by which histone methylation orchestrates cellular phenomena.
A molecular understanding of the biological phenomena orchestrated by lysine N(ɛ)-methylation is impeded by the challenge of producing site-specifically and quantitatively methylated histones. Here, we report a general method that combines genetic code expansion and chemoselective reactions, for the quantitative, site-specific installation of dimethyl-lysine in recombinant histones. We demonstrate the utility of our method by preparing H3K9me2 and show that this modified histone is specifically recognized by heterochromatin protein 1 beta. Extensions of the strategy reported here will allow a range of chemoselective reactions (which have been used for residue-selective, but not site-selective protein modification) to be leveraged for site-specific protein modification.
All biological processes rely on the formation of protein-ligand, protein-peptide and proteinprotein complexes. Studying the affinity, kinetics and thermodynamics of binding between these pairs is critical for understanding basic cellular mechanisms. There are many different technologies designed for probing interactions between biomolecules, each based on measuring different signals (fluorescence, heat, thermophoresis, scattering and interference; among others). Evaluation of the data from the binding experiments and its fitting is an essential step towards the quantification of binding affinities. Here, we present user-friendly online tools to analyze biophysical data from steady-state fluorescence spectroscopy, microscale thermophoresis and differential scanning fluorimetry experiments. The modules from our data analysis platform (spc.embl-hamburg.de) contain classical thermodynamic models and clear user guidelines for the determination of equilibrium dissociation constants (Kds) and thermal unfolding parameters such as melting temperatures (Tms).
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