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J. Biol. Chem., Vol. 283, Issue 14, 9012-9022, April 4, 2008

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Dissociation from the Oligomeric State Is the Rate-limiting Step in Fibril Formation by -Casein^*

Heath Ecroyd, Supported by an NHMRC Peter Doherty Fellowship¹, Tomas Koudelka, David C. Thorn, Danielle M. Williams, Glyn Devlin^¶2, Peter Hoffmann, and John A. Carver

From the School of Chemistry and the Physics and the School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia and the ^¶Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom

Amyloid fibrils are aggregated and precipitated forms of protein in which the protein exists in highly ordered, long, unbranching threadlike formations that are stable and resistant to degradation by proteases. Fibril formation is an ordered process that typically involves the unfolding of a protein to partially folded states that subsequently interact and aggregate through a nucleation-dependent mechanism. Here we report on studies investigating the molecular basis of the inherent propensity of the milk protein, -casein, to form amyloid fibrils. Using reduced and carboxymethylated -casein (RCM-CN), we show that fibril formation is accompanied by a characteristic increase in thioflavin T fluorescence intensity, solution turbidity, and β-sheet content of the protein. However, the lag phase of RCM-CN fibril formation is independent of protein concentration, and the rate of fibril formation does not increase upon the addition of seeds (preformed fibrils). Therefore, its mechanism of fibril formation differs from the archetypal nucleation-dependent aggregation mechanism. By digestion with trypsin or proteinase K and identification by mass spectrometry, we have determined that the region from Tyr²⁵ to Lys⁸⁶ is incorporated into the core of the fibrils. We suggest that this region, which is predicted to be aggregation-prone, accounts for the amyloidogenic nature of -casein. Based on these data, we propose that fibril formation by RCM-CN occurs through a novel mechanism whereby the rate-limiting step is the dissociation of an amyloidogenic precursor from an oligomeric state rather than the formation of stable nuclei, as has been described for most other fibril-forming systems.

Received for publication, December 5, 2007 , and in revised form, January 28, 2008.

^* This work was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia and the Australian Research Council (to J. A. C. and P. H.), postgraduate scholarships from Dairy Australia (to D. C. T.), an Australian Postgraduate Award (to D. M. W.), and the University of Adelaide (to T. K.). 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.

² Supported by an NHMRC C. J. Martin Fellowship.

¹ To whom correspondence should be addressed. Tel.: 61-8-830-35505; Fax: 61-8-830-34358; E-mail: heath.ecroyd{at}adelaide.edu.au.

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