FIGURE 4.
Quantitative EMSAs on highly purified protein demonstrate the CP2 motif is critical for RNA binding. A, SDS-PAGE gel of equal quantities of WT and R237A RBD protein. B, analytical sizing chromatography demonstrating that both proteins elute identically, consistent with a monomeric protein of ∼40 kDa. C, EMSAs for WT and R237A RBD. The dissociation constant (Kd) was determined for all protein variants as well as the S.D. (error bars) from triplicate measurements and is indicated below the gel as Kd ± S.D. D, Kd quantification for WT and R237A RBD. The Kd was determined by measuring the fraction bound at each concentration and fitting the data to the Hill equation. The Hill coefficients obtained were n = 1.0 and n = 1.9 for WT and R237A constructs, respectively. The higher Hill coefficient for R237A protein is likely the result of the increased contribution of higher molecular mass species (asterisks) skewing the fitting.