Partially Folded Intermediates in Insulin Fibrillation
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- 作者：Atta Ahmad ; Ian S. Millett ; Sebastian Doniach ; Vladimir N. Uversky ; and Anthony L. Fink
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：October 7, 2003
- 年：2003
- 卷：42
- 期：39
- 页码：11404 - 11416
- 全文大小：379K
- 年卷期：v.42,no.39(October 7, 2003)
- ISSN：1520-4995
文摘
Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions,the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical propertiesof amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulinsolutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn·HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the additionof as little as 0.25 M Gdn·HCl leads to dissociation of insulin hexamers into dimers. Moderateconcentrations of Gdn·HCl lead to formation of a novel partially unfolded dimer state, which dissociatesinto a partially unfolded monomer state. High concentrations of Gdn·HCl resulted in unfolded monomerswith some residual structure. The addition of even very low concentrations of Gdn·HCl resulted insubstantially accelerated fibrillation, although the yield of fibrils decreased at high concentrations.Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, whichexisted up to >6 M Gdn·HCl, accounting for the formation of substantial amounts of fibrils under suchconditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was alsoaccelerated by Gdn·HCl. The enhanced fibrillation of the monomer was due to the increased ionic strengthat low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformationat Gdn·HCl concentrations above 1 M. The data suggest that under physiological conditions, the fibrillationof insulin involves both changes in the association state (with rate-limiting hexamer dissociation) andconformational changes, leading to formation of the amyloidogenic expanded monomer intermediate.