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J. Biol. Chem., Vol. 281, Issue 38, 28131-28142, September 22, 2006

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The Folding Nucleus of the Insulin Superfamily

A FLEXIBLE PEPTIDE MODEL FORESHADOWS THE NATIVE STATE^*

Qing-xin Hua, John P. Mayer, Wenhua Jia, Jingwen Zhang^¶, and Michael A. Weiss¹

From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285, and ^¶Amgen, Inc., Thousand Oaks, California 91320

Oxidative folding of insulin-like growth factor I (IGF-I) and single-chain insulin analogs proceeds via one- and two-disulfide intermediates. A predominant one-disulfide intermediate in each case contains the canonical A20–B19 disulfide bridge (cystines 18–61 in IGF-I and 19–85 in human proinsulin). Here, we describe a disulfide-linked peptide model of this on-pathway intermediate. One peptide fragment (19 amino acids) spans IGF-I residues 7–25 (canonical positions B8-B26 in the insulin superfamily); the other (18 amino acids) spans IGF-I residues 53–70 (positions A12–A21 and D1–D8). Containing only half of the IGF-I sequence, the disulfide-linked polypeptide (designated IGF-p) is not well ordered. Nascent helical elements corresponding to native -helices are nonetheless observed at 4 °C. Furthermore, ¹³C-edited nuclear Overhauser effects establish transient formation of a native-like partial core; no non-native nuclear Overhauser effects are observed. Together, these observations suggest that early events in the folding of insulin-related polypeptides are nucleated by a native-like molten subdomain containing Cys^A20 and Cys^B19. We propose that nascent interactions within this subdomain orient the A20 and B19 thiolates for disulfide bond formation and stabilize the one-disulfide intermediate once formed. Substitutions in the corresponding region of insulin are associated with inefficient chain combination and impaired biosynthetic expression. The intrinsic conformational propensities of a flexible disulfide-linked peptide thus define a folding nucleus, foreshadowing the structure of the native state.

Received for publication, March 20, 2006 , and in revised form, July 19, 2006.

^* This work was supported in part by National Institutes of Health Grant DK0697674 (to M. A. W.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5 and Tables S1–S5.

¹ To whom correspondence should be addressed: 10900 Euclid Ave., Cleveland, OH 44106-4935. Tel.: 216-368-5991; Fax: 216-368-3419; E-mail: maw21{at}case.edu.

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