Protein Structure and Folding
Mechanism of Insulin Fibrillation: THE STRUCTURE OF INSULIN UNDER AMYLOIDOGENIC CONDITIONS RESEMBLES A PROTEIN-FOLDING INTERMEDIATE*

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Insulin undergoes aggregation-coupled misfolding to form a cross-β assembly. Such fibrillation has long complicated its manufacture and use in the therapy of diabetes mellitus. Of interest as a model for disease-associated amyloids, insulin fibrillation is proposed to occur via partial unfolding of a monomeric intermediate. Here, we describe the solution structure of human insulin under amyloidogenic conditions (pH 2.4 and 60 °C). Use of an enhanced sensitivity cryogenic probe at high magnetic field avoids onset of fibrillation during spectral acquisition. A novel partial fold is observed in which the N-terminal segments of the A- and B-chains detach from the core. Unfolding of the N-terminal α-helix of the A-chain exposes a hydrophobic surface formed by native-like packing of the remaining α-helices. The C-terminal segment of the B-chain, although not well ordered, remains tethered to this partial helical core. We propose that detachment of N-terminal segments makes possible aberrant protein-protein interactions in an amyloidogenic nucleus. Non-cooperative unfolding of the N-terminal A-chain α-helix resembles that observed in models of proinsulin folding intermediates and foreshadows the extensive α → β transition characteristic of mature fibrils.

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The atomic coordinates and structure factors (code 1SF1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

*

This work was supported in part by National Institutes of Health Grant R01 DK054622 (to M. A. W.) and performed at the Cleveland Center for Structural Biology. 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 Tables S1-S2.