Structural and physicochemical analysis of a novel Ca²⁺-binding protein of radish (RVCaB)

Abstract

A Ca²⁺-binding protein found in radish (RVCaB) has no similarity to the other known Ca²⁺-binding proteins (Plant Physiol. 2000). A high content of glutamic acid and absence of aromatic residues and cysteine are characteristics of RVCaB. Expression of RVCaB gene was enhanced under Ca²⁺-deficient condition and suppressed by high Ca²⁺ concentrations. In the present study, we expressed RVCaB with a His₆ tag in E. coli to determine its physicochemical properties. The CD spectrum analysis revealed that RVCaB is a unique protein without α-helix or β-structure. Although Ca²⁺ did not give a marked difference in the structure of RVCaB, a slight difference in CD spectrum was observed. It may be a reflection of binding of Ca²⁺ to RVCaB. Binding analysis of Ca²⁺ by isothermal titration calorimetry indicated that RVCaB has four high-affinity Ca²⁺-binidng sites. We will report more detailed information and discuss its physiological roles.