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Pressure Equilibrium and Jump Study on Unfolding of 23-kDa Protein from Spinach Photosystem II

Cui-Yan Tan ^*, Chun-He Xu , Jun Wong , Jian-Ren Shen  , Shinsuke Sakuma , Yasusi Yamamoto , Reinhard Lange ^||, Claude Balny ^|| and Kang-Cheng Ruan ^*

^* Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai, China; Institute of Plant Physiology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai, China; Graduate School of Natural Science and Technology, Department of Biology, Faculty of Science, Okayama University, Okayama, Japan; and ^|| Institut National de la Santé et de la Recherche Médicale Unit 710, Institut Fédératif de Recherche 122, Université Montpellier 2, Montpellier, France

Correspondence: Address reprint requests to K. C. Ruan, Shanghai Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China. Tel.: 86-21-5492-1168; Fax: 86-21-5492-1011; E-mail: kcruan{at}sibs.ac.cn. C. Balny, INSERM U 710, Université Montpellier 2, Place E. Bataillon, CC105, 34095 Montpellier Cedex 5, France. Tel.: 33-46-714-9347; Fax: 33-46-713-3386; E-mail: balny{at}monpt.inserm.fr.

Pressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20°C and pH 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (G_u) and volume change (V_u) for the unfolding are 5.9 kcal/mol and –160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in G_u but also with a decrease in V_u. The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (–66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.

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