Figure 8.

ITC results for SF1mut-U2AF⁶⁵_R12U (striped) or SF1pa-U2AF⁶⁵_R12U (stippled) complexes titrated with B3P3_SUBOPT RNA. (A) Schematic diagram of the ITC experiment. (B and C) Representative isotherms fit with identical site models for (B) 50 μM B3P3 RNA into 5 μM SF1pa-U2AF⁶⁵_R12U complex, c = 59, and (C) 50 μM B3P3 RNA into 5 μM SF1mut-U2AF⁶⁵_R12U complex, c = 61. Nonidentical-site models were discarded based on 1) fitting errors greater than the resulting thermodynamic values; 2) stoichiometry values of nearly zero for one class of nonidentical sites; and 3) thermodynamic values for the remaining class of sites that matched the results of the identical-site fit. (D) Bar graph of apparent affinities (K_A) of the interactions, colored as in (A). The apparent equilibrium dissociation constants (K_D = K_A⁻¹) and standard deviations of at least two replicates are given. For comparison, the nonidentical-site affinities of the parent SF1_1–255-U2AF⁶⁵_R12U complex titrated with B3P3 RNA (details in Fig. 6) are plotted at left. The inset shows an expanded view of the B3P3 RNA affinities for the SF1mut- or SF1pa-U2AF⁶⁵_R12U complexes. A control titration of B3P3_SUBOPT RNA into buffer is shown in Fig. S1 C and a titration of B3P3_SUBOPT RNA into SF1mut-U2AF⁶⁵_R12U in phosphate buffer (as opposed to HEPES) is shown in Fig. S6.