Figure 5

Titration of Ki-1/57(122-413) into RACK1 solution monitored by fluorescence. (A) Fluorescence emission spectra titration of purified 6xHis RACK1 protein (1 μM) with titration of Ki-1/57 at 0.1 μM (black, filled square), 0.2 μM (black, filled circle), 0.3 μM (black, filled triangle), 0.5 μM (open, white triangle) and 1 μM (open, white circle). The excitation wavelength was 295 nm and spectra were background corrected. Inset: Area under curve (AUC) of RACK1 fluorescence emission spectrum in function of Ki-1/57(122-413) concentration. The arrow indicates the increase on the RACK1 fluorescence signal at Ki-1/57(122-413) 0.3 uM. (B) The titration curve was represented as difference of fluorescence intensity at 334 nm θ (black, filled square) as function of Ki-1/57(122-413) concentration in the range from 0.3 μM and 1.4 μM and fitted with the modified Hill equation (Equation 5). The dashed line represents the fitting curve. The KD between RACK1 and Ki-1/57(122-413) was of around 0.60 μM which corresponds to the dissociation constant of RACK1\Ki-1/57(122-413) observed in sedimentation equilibrium experiments. The number of cooperative sites n was 8 ± 1.