Journal of Biochemistry Advance Access published online on June 16, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm130
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© 2007 The Japanese Biochemical Society
RVCaB, a calcium-binding protein in radish vacuoles, is predominantly an unstructured protein with a polyproline type II helix
,1,2
* Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, Kouto, Sayo, Hyogo, 679-5148, Japan
Laboratory Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan
Corresponding author: Masashi Miyano: Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan. Tel: +81-791-58-2815, Fax: +81-791-58-2816, e-mail: miyano{at}spring8.or.jp
Corresponding author: Masayoshi Maeshima: Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan. E-mail: maeshima{at}agr.nagoya-u.ac.jp
Received March 8, 2007; Accepted May 21, 2007
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Abstract |
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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 Ca2+-storage function. In the present study we focused on the protein physicochemical properties of RVCaB to understand its uniqueness in terms of structure and Ca2+-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 °C from 20 °C. The Ca2+-binding ability of RVCaB was retained after heat treatment. No 
-helix or ß-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 Ca2+ bound to RVCaB was determined to be 21.6 , and a 360 M–1 Ka value for Ca2+ binding was determined by isothermal titration calorimetry. The analysis also revealed that the binding of Ca2+ to RVCaB is an entropy-driven phenomenon. We prepared tryptophan-inserted mutants of RVCaB (V136W and V202W) to probe the Ca2+-induced structural change by fluorescent spectroscopy. The analysis suggests a small structural rearrangement of RVCaB upon Ca2+-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 Ca2+ 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 Ca2+-binding properties.
Key Words: calcium-binding protein, circular dichroism, unstructured protein, polyproline type II helix
1 The authors J.I. and N.N. equally contributed to this work.
2 Correspondence may be addressed to either of these authors (miyano{at}spring8.or.jp or maeshima{at}agr.nagoya-u.ac.jp)
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