FIGURE 4.

Wild-type hCap2_CRD trimer mediated by calcium is unstable. A, native gel analysis showed that wild-type Cap2_CRD exists as a mixture of trimer and monomer, which could be shifted substantially to monomer by EDTA treatment or trimer by calcium. Sample 1 is obtained by loading EDTA-treated wild-type hCap2_CRD on Superdex 75 in a buffer containing 0.5 mm EDTA. Sample 2 is obtained from adding a final concentration of 5 mm CaCl₂ to sample 1. B–D, DAEA mutant, wild-type Cap2_CRD and the latter treated with EDTA were eluted from gel filtration column as trimer, a mixture of trimer and monomer, and monomer, respectively. However, their apparent molecular masses (25, 14, and 9 kDa) calculated from gel filtration standard are obviously lower than expected for unknown reasons. E and F, dynamic light scattering analysis showed that wild-type hCap2_CRD and hCap2_DAEA both exhibit a single peak, and they have a similar average hydrodynamic radius with an estimated molecular mass (MW) of 60 kDa.