FIGURE 5.
The affinity of the EN/RR UBA domain mutant for ubiquitin is significantly reduced. A, binding of ubiquitin to WT, E/R, N/R, or EN/RR cIAP1 UBA domain (as indicated) was measured using ITC. Because the ITC curve generated from injections of ubiquitin into UBA-EN/RR did not yield an accurate Kd estimation, SPR analyses were carried out (B). Equilibrium binding analyses was carried out. The sensorgrams resulting from injections of 0–450 μm ubiquitin onto chip-immobilized WT or EN/RR GST-UBA are shown to the right. Dissociation constants and stoichiometry determined from ITC and SPR measurements are shown in Table 1.