Peptide binding assays using fluorescence anisotropy

For assaying the affinity of the JARID2-K116me3 peptide for EED, various concentrations of EED (amino acids 40–441) were incubated with 40 nM of 5-FAM labelled JARID2-K116me3 peptide in binding buffer (50 mM Tris-HCl pH 7.5 at 25 °C, 100 mM KCl, 2 mM 2-mercaptoethanol, 0.1 mg/mL BSA, 0.05% NP-40, 2.5% glycerol) at 30 °C for 30 min before a fluorescence anisotropy measurement took place using a PHERAstar plate reader. Data processing was carried out as previously described⁶⁶, with some modifications. Specifically, with changing the concentration of EED protein (P), we recorded △Robs: the observable anisotropy of the mixtures after the subtraction of the observable anisotropy of the 5-FAM labelled JARID2-K116me3 peptide ligand. Data were fitted to equation (1), below, by using non-linear least-squares fit (Matlab, MathWorks) to estimate the anisotropy difference △r and the dissociation constant between the protein EED to the ligand JARID2-K116me3 peptide, KL:

where the concentrations of the protein-ligand complex [PL] and the free ligand [L] are calculated from Eqs. (2) and (3) below, respectively, with P₀ and L₀ indicates the total concentration of the protein and the ligand, respectively.

Fluorescence anisotropy displacement titrations were used to assay the dissociation constants of the unlabelled peptides (N) and the protein (P). Assays were carried out as described above, with the exception that 2-fold serial dilutions of unlabelled peptides were combined with EED at a final concentration of 10 µM and 5-FAM labelled JARID2-116me3 peptide at a final concentration of 40 nM before anisotropy data were collected as described above. Data processing was carried out as previously described⁶⁶. Specifically, △Robs were recorded for each peptide [N] concentration point and Eq. (1) used to estimate KN and △r as the fitting parameters. KN is the equilibrium dissociation constant for binding of the unlabelled peptide N to the protein P. N₀ is the total concentration of N. [PL] and [L] are calculated in a different way:

and [L]=L0−PL

where KL are obtained from the measurement above, and P is one of the roots of the cubic equation:

where