The lysine-specific demethylase 1 (LSD1) removes methyl groups from modified lysines as they are typically found in histones. LSD1 acts in the histone modifying complex CoREST that plays an important role in epigenetic modification of chromatin. Association of a substrate with complex-bound LSD1 can be exploited to measure protein-ligand binding. The tumbling of free fluorescent-labelled substrate-peptides gives a lower fluorescence polarization value than the LSD1-CoREST-bound peptide.
In order to measure the binding of H3-derived peptides to the LSD1-protein complex, the peptides were labelled with TAMRA. These were titrated with the LSD1-CoREST-complex and FP was measured using a CLARIOstar®. The binding of the chosen peptides to LSD1 was confirmed as indicated by an increase in FP. Similarly, competitive binding of peptides was determined by titration of a saturated (TAMRA-)peptide-LSD1 mixture with an unlabeled competitive peptide which led to a decrease in FP due to the release of the labelled peptide.