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J. Biol. Chem., Vol. 281, Issue 14, 9058-9065, April 7, 2006

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Polyvinylpyrrolidone 40 Assists the Refolding of Bovine Carbonic Anhydrase B by Accelerating the Refolding of the First Molten Globule Intermediate^*

Yan Jiang, Yong-Bin Yan¹, and Hai-Meng Zhou^¶2

From the Department of Biological Sciences and Biotechnology, the State Key Laboratory of Biomembrane and Membrane Biotechnology, and the ^¶Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084, China

Protecting proteins from aggregation is one of the most important issues in both protein science and protein engineering. In this research, the mechanism of enhancing the refolding of guanidine hydrochloride-denatured carbonic anhydrase B by polyvinylpyrrolidone 40 (PVP40) was studied by both kinetic and equilibrium refolding experiments. The reactivation and refolding kinetics indicated that the rate constant of refolding the first refolding intermediate (I₁) to the second one (I₂) is promoted by the addition of PVP. Fluorescence quenching studies further indicated that PVP could bind to the aggregation-prone species I₁, resulting in the protection of the exposed hydrophobic surface, a minimization of the protein surface, and more importantly, an increase of the refolding rate of I₁. These properties were quite different from those of poly(ethylene glycol) (PEG), which has been shown to have a strong and stoichiometric binding to I₁ and does not interfere with the refolding pathway. Unlike PEG, the binding of PVP to I₁ does not block the aggregation pathway directly but decreases the energy barrier for I₁ to refold to I₂ and thus reduces the accumulation of I₁. These results suggested that PVP works by a quite different mechanism from those well established ones in chaperones and chemical promoters. PVP is more like a folding catalyst rather than a chemical chaperone. The distinct mechanism of enhancing protein aggregation by PVP is expected to facilitate the attempt to develop new chemical compounds as well as new strategies to protect proteins from aggregation.

Received for publication, July 20, 2005 , and in revised form, January 9, 2006.

^* This investigation was supported by Grants 30500084 and 30221003 from the National Natural Science Foundation, Grant G1999075607 from the National Key Basic Research Special Foundation of China, and the 985 Fund from Tsinghua University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: Dept. of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China. Tel.: 86-10-6278-3477; Fax: 86-10-6277-1597; E-mail: ybyan{at}tsinghua.edu.cn. ² To whom correspondence may be addressed: Dept. of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China. Tel.: 86-10-6278-4700; Fax: 86-10-6277-2245; E-mail: zhm-dbs{at}tsinghua.edu.cn.

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