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J. Biol. Chem., Vol. 279, Issue 53, 55224-55233, December 31, 2004

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In Vitro Refolding/Unfolding Pathways of Amphioxus Insulin-like Peptide

IMPLICATIONS FOR FOLDING BEHAVIOR OF INSULIN FAMILY PROTEINS^*

Yan Chen, Rui Jin, Hong-Yun Dong, and You-Min Feng

From the Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences Shanghai 200031, China

Amphioxus insulin-like peptide (AILP) belongs to the insulin superfamily and is proposed as the common ancestor of insulin and insulin-like growth factor 1. Herein, the studies on oxidative refolding and reductive unfolding of AILP are reported. During the refolding process, four major intermediates, P1, P2, P3, and P4, were captured, which were almost identical to those intermediates, U1, U2, U3, and U4, captured during the AILP unfolding process. P4 (U4) has the native disulfide A20-B19; P1 (U1), P2 (U2), and P3 (U3) have two disulfide bonds, which include A20-B19. Based on the analysis of the time course distribution and properties of the intermediates, we proposed that fully reduced AILP refolded through 1SS, 2SS, and 3SS intermediate stages to the native form; native AILP unfolded through 2SS and 1SS intermediate stages to the full reduced form. A schematic flow chart of major oxidative refolding and reductive unfolding pathways of AILP was proposed. Implication for the folding behavior of insulin family proteins was discussed. There may be seen three common folding features in the insulin superfamily: 1) A20-B19 disulfide is most important and formed during the initial stage of folding process; 2) the second disulfide is nonspecifically formed, which then rearranged to native disulfide; 3) in vitro refolding and unfolding pathways may share some common folding intermediates but flow in opposite directions. Furthermore, although swap AILP is a thermodynamically stable final product, a refolding study of swap AILP demonstrated that it is also a productive intermediate of native AILP during refolding.

Received for publication, August 6, 2004 , and in revised form, October 12, 2004.

^* This work was supported by National Foundation of Natural Sciences Grant 39670179 and Chinese Academy of Sciences Grant KJ951-B1-606. 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.

These authors contributed equally to this work.

To whom correspondence should be addressed: Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yue Yang Rd., Shanghai 200031, China. Tel.: 86-021-54921133; Fax: 86-021-54921011; E-mail: fengym{at}sunm.shcnc.ac.cn.

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