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J. Biol. Chem., Vol. 277, Issue 45, 43443-43453, November 8, 2002
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From the The A and B chains of insulin combine to form
native disulfide bridges without detectable isomers. The fidelity of
chain combination thus recapitulates the folding of proinsulin, a
precursor protein in which the two chains are tethered by a disordered
connecting peptide. We have recently shown that chain combination is
blocked by seemingly conservative substitutions in the C-terminal
Mechanism of Insulin Chain Combination
ASYMMETRIC ROLES OF A-CHAIN 
-HELICES IN DISULFIDE
PAIRING*,
,,,
Department of Biochemistry, Case
Western Reserve School of Medicine, Cleveland, Ohio 44106-4935 and
the § Department of Pharmacology and Biological Chemistry,
Mount Sinai School of Medicine, New York University,
New York, New York 10029
-helix of the A chain. Such analogs, once formed, nevertheless
retain high biological activity. By contrast, we demonstrate here that chain combination is robust to non-conservative substitutions in the
N-terminal -helix. Introduction of multiple glycine substitutions into the N-terminal segment of the A chain (residues A1-A5) yields analogs that are less stable than native insulin and essentially without biological activity. 1H NMR studies of a
representative analog lacking invariant side chains IleA2
and ValA3 (A chain sequence
GGGEQCCTSICSLYQLENYCN;
substitutions are italicized and cysteines are underlined)
demonstrate local unfolding of the A1-A5 segment in an
otherwise native-like structure. That this and related partial folds
retain efficient disulfide pairing suggests that the native N-terminal
-helix does not participate in the transition state of the reaction.
Implications for the hierarchical folding mechanisms of
proinsulin and insulin-like growth factors are discussed.
*
This work was supported in part by a grant from the National
Institutes of Health (to M. A. W. and P. G. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The 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 one figure showing histograms of chemical
shift changes in 3G-DKP-insulin and four tables providing chemical
shift information for 3G-DKP-insulin and DG/RMD restraints.
The atomic coordinates and the structure factors (code 1LKQ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ To whom correspondence may be addressed. Tel.: 212-241-9350; Fax: 212-996-7214; E-mail: panayotis.katsoyannis@mssm.edu.
To whom correspondence may be addressed. Tel.: 216-368-5991;
Fax: 216-368-3419; E-mail: weiss@biochemistry.cwru.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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