Journal of Biological Chemistry
Volume 280, Issue 51, 23 December 2005, Pages 42345-42355
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Protein Structure and Folding
Proinsulin Is Refractory to Protein Fibrillation: TOPOLOGICAL PROTECTION OF A PRECURSOR PROTEIN FROM CROSS-β ASSEMBLY*

https://doi.org/10.1074/jbc.M507110200Get rights and content
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Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-β assembly. Protection from fibrillation in β cells is provided by sequestration of the susceptible monomer within zinc hexamers. We demonstrate that proinsulin is refractory to fibrillation under conditions that promote the rapid fibrillation of zinc-free insulin. Proinsulin fibrils, as probed by Raman microscopy, are nonetheless similar in structure to insulin fibrils. The connecting peptide, although not well ordered in native proinsulin, participates in a fibril-specific β-sheet. Native insulin and proinsulin exhibit similar free energies of unfolding as inferred from guanidine denaturation studies: relative amyloidogenicities are thus not correlated with global stability. Strikingly, the susceptibility of proinsulin to fibrillation is increased by scission of the connecting peptide at single sites. We thus propose that the connecting peptide constrains a large scale conformational change in the misfolded protein. A tethering mechanism is proposed based on a model of an insulin protofilament derived from electron-microscopic image reconstruction. The proposed relationship between cross-β assembly and protein topology is supported by studies of single-chain analogs (mini-proinsulin and insulin-like growth factor I) in which foreshortened connecting peptides further retard fibrillation. In addition to its classic function to facilitate disulfide pairing, the connecting peptide may protect β cells from toxic protein misfolding in the endoplasmic reticulum.

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*

This work was supported by National Institutes of Health Grant DK0697674 (to M. A. W.). This article is a contribution from 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 five figures providing high-performance liquid chromatography purification profile of split proinsulin analogs, CD spectra of insulin, proinsulin, and insulin analogs at fibril-promoting conditions (pH 2.0 and 65 °C), CD difference spectra, kinetic profile of B-chain fibrillation, and control studies of the kinetics of ThT fluorescence enhancement in relation to optical scattering and EM morphology.

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