Unfolding intermediate of a multidomain protein, calmodulin, in urea as revealed by small-angle X-ray scattering

AbstractThe denaturation of calmodulin (CaM) induced by urea has been studied by small-angle X-ray scattering, which is a direct way to evaluate the shape changes in a protein molecule. In the absence of Ca2+, the radii of gyration (Rg) of CaM are 20.8±0.3 Å in the native state and about 34±1.0 Å in the unfolded state. The transition curve derived from Kratky plots indicates a bimodal transition via a stable unfolding intermediate around 2.5 M urea. In the presence of Ca2+ and in the presence of both Ca2+ and a target peptide, the Rg values are 21.5±0.3 and 18.1±0.3 Å in the native state and 26.7±0.4 and 24.9±0.4 Å at 9 M urea, respectively. The results indicate that a stable unfolding intermediate still persists in 9 M urea. The present results suggest that the shape of unfolding intermediates is an asymmetric dumbbell-like structure, one in the folded and one in the unfolded state