RVCaB, a calcium-binding protein in radish vacuoles, is predominantly an unstructured protein with a polyproline type II helix.

A unique acidic calcium-binding protein RVCaB, rich in glutamic acid and proline and lacking aromatic amino-acid residues, exists in radish vacuoles, and is thought to be involved in the vacuole Ca(2+)-storage function. In the present study, we focused on the protein physicochemical properties of RVCaB to understand its uniqueness in terms of structure and Ca(2+)-binding function. On differential scanning calorimetry, the protein did not show any sharp transition of heat-denaturation of the folded protein except for a gradual excess of heat capacity when heated up to 99 degrees C from 20 degrees C. The Ca(2+)-binding ability of RVCaB was retained after heat treatment. No alpha-helix or beta-sheet was detected in the far-UV CD spectra of RVCaB as judged by several computer programs for protein structure analysis. However, further analyses with CD spectroscopy suggest that RVCaB has a left-handed polyproline type II (PPII) helix, which is known to be in a collagen chain conformation. The number of Ca(2+) bound to RVCaB was determined to be 21.6, and a 360 M(-1) Ka value for Ca(2+) binding was determined by isothermal titration calorimetry. The analysis also revealed that the binding of Ca(2+) to RVCaB is an entropy-driven phenomenon. We prepared tryptophan-inserted mutants of RVCaB (V136W and V202W) to probe the Ca(2+)-induced structural change by fluorescent spectroscopy. The analysis suggests a small structural rearrangement of RVCaB upon Ca(2+)-binding and that the induced Trp residues at 136 and 202 are exposed to solvent in each mutant. These results suggest that RVCaB does not have a definitive protein fold except for the extended PPII structure and that its structure changes slightly by the binding of Ca(2+) or heat treatment. These findings suggest that the unique structure of RVCaB with its PPII helices is closely related to its high-capacity and low-affinity Ca(2+)-binding properties.