Fluorescence properties of His-ASF/SF2 and quantification of the binding of His-ASF/SF2 to R199G.

(A) Emission spectrum of His-ASF/SF2 (100 nM) at an excitation wavelength of 280 nm. Solid line, purified protein in 20 mM Tris-HCl, 100 mM KCl, pH 7.5; Dashed line, purified protein after a 2 h incubation in 20 mM Tris-HCl, 100 mM KCl, pH 7.5, and 8 M urea; Inset, SDS-PAGE (P) and Western blot (W) of purified His-ASF/SF2. (B) Molar fluorescence of His-ASF/SF2. His-ASF/SF2 was excited at 280 nm and fluorescence intensity at increasing concentration of His-ASF/SF2 was measured at an emission wavelength of 340 nm. (C) Binding curve generated by plotting the observed change in fluorescence of His-ASF/SF2 as a function of increasing concentration of R199G. The curve was fitted to a one-site binding model using non-linear regression and the apparent K_D (appK_D) was calculated. The inset represent typical ASF/SF2 emission spectra generated by the addition of increasing amount of R199G. (D) Variation of the observed change in fluorescence of His-ASF/SF2 as a function of increasing concentration of P11.60, a non-related RNA hairpin competitor. The insert represents the sequence and secondary structure of P11.60.