Figure 3. Conformational stability of the BIR3 domain of XIAP.

The constructs shown in (A) were expressed as His₆-tagged proteins in E. coli and purified (B). Expression levels of the most extreme truncations (from 262 at the N-terminus or at 336 in the C-terminus) were much lower than the other constructs, but sufficient quantities could be obtained for unfolding and kinetic analyses, and each mutant was normalized to the same concentration prior to analysis. (C) Proteins were incubated for 1 h at room temperature in the presence of the indicated GdmCl concentrations. Unfolding was measured by fluorescence at 350 nm following excitation at 295 nm. For all experiments the protein concentration was 200 nM.