HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jia, X.-Y. Articles by Feng, Y.-M. Search for Related Content PubMed PubMed Citation Articles by Jia, X.-Y. Articles by Feng, Y.-M. Social Bookmarking                   What's this?

Peptide models of four possible insulin folding intermediates with two disulfides

¹ Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
² Institute of Hydrobiology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China

Reprint requests to: You-Min Feng, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China; e-mail: fengym{at}sunm.shcnc.ac.cn; fax: 86-021-64338357.

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20–B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20–B19, A7–B7]PIP, [A20–B19, A6–B7]PIP, [A20–B19, A6–A11]PIP, and [A20–B19, A7–A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20–B19, A7–A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20–B19, A7–A11] is much lower than that of forming the other three intermediates.

Keywords: Insulin; folding; intermediate; disulfide bonds; kinetics

Abbreviations: PIP, recombinant single-chain insulin in which the C terminus of porcine insulin B-chain and the N terminus of porcine insulin A-chain were linked together by a dipeptide, Ala-Lys • IGF-1, insulin-like growth factor 1 • BPTI, bovine pancreatic trypsin inhibitor • EGF, epidermal growth factor • GSH, reduced glutathione • GSSG, oxidized glutathione • HPLC, high-performance liquid chromatography • TFA, trifluoroacetic acid • PAGE, polyacrylamide gel electrophoresis • CD, circular dichroism • NMR, nuclear magnetic resonance

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				Y. Chen, R. Jin, H.-Y. Dong, and Y.-M. Feng In Vitro Refolding/Unfolding Pathways of Amphioxus Insulin-like Peptide: IMPLICATIONS FOR FOLDING BEHAVIOR OF INSULIN FAMILY PROTEINS J. Biol. Chem., December 31, 2004; 279(53): 55224 - 55233. [Abstract] [Full Text] [PDF]