Figure 3.
Binding of mant-AMPPNP to PKR measured by reverse fluorescence anisotropy. mant-AMPPNP was titrated against 40 μM PKR and the fluorescence anisotropy was measured at an excitation wavelength of 360 nm and emission wavelength of 443 nm with spectral bandwiths of 2 nm and 4 nm for excitation and emission respectively. Fluorescence intensity at each polarizer orientation were integrated for 1 s and measurements were averaged until the maximum standard deviation drops below 0.1%. Each data point represents the average of 3–4 measurements. The solid line represents a fit of the data to a 1:1 binding model to obtain Kd = 34.8± 3.6 μM, rL = 0.0407± 0.0016 and rEL = 0.2356 ± 0.0096.