Monitoring Nucleosome Binding Kinetics

Histone H4(A15C) was labeled with Cy3 maleimide and reconstituted into nucleosomes (26 (link)). Changes in Cy3 fluorescence intensity were monitored in a Horiba Jobin Yvon Fluorolog fluorometer with the sample chamber set at 25°C, excitation monochromator at 510 nm (5 nm slit width), and emission monochromator at 565 nm (5 nm slit width). Increasing amounts of Chd1 were titrated into solutions containing 5 nM nucleosome in 20 mM HEPES, pH 7.5; 5 mM MgCl₂; 0.1 mM EDTA; 5% (w/v) sucrose; 1 mM DTT; 0.02% Nonidet P40 Substitute (Roche); 0.1 mg/ml BSA; 1 mM adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) and 100 mM KCl. Titrations with LacI contained 500 nM LacI. The binding data was fit in KaleidaGraph (Synergy) to the following binding isotherm using nonlinear least squares regression,

where Y is the signal intensity, X is the concentration of Chd1, A_X are the signal amplitudes, K_X are the dissociation constants, N is the nucleosome concentration and C is the signal from nucleosome alone. Titrations for each experimental condition were performed three or more times, with the average 1/K_1/2 values and standard deviations reported in Figure 3.