Fig. 5

Spectroscopic characterizations of Δ21 and mutant proteins I432V and Q438R/E440G. (A) Differential scanning fluorimetry used to compare protein unfolding rates. Temperature increase was regulated by a Stratagene Mx3005P real-time PCR machine. The first derivative of the change in fluorescence is plotted. (B) Fluorescence anisotropy results revealing the affinities of the proteins for the RNA SL-UC (inserted in the graph to the left). The symbol attached to the 5′ terminus of the RNA represents fluorescein. Purified proteins were titrated into a cuvette fitted with a magnetic stir bar. Background fluorescence of the buffer and protein were small at the wavelengths used (emission: 520 nm; excitation: 560 nm) but were subtracted from the data. The fit to the binding isotherms was performed with a hyperbola equation (y - m1*x/[m2+x]; m2 = Kd) and plotted with KaleidaGraph. The quality of the fit is shown by the R² value.