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J. Biol. Chem., Vol. 282, Issue 47, 34204-34212, November 23, 2007

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Probing the Role of PrP Repeats in Conformational Conversion and Amyloid Assembly of Chimeric Yeast Prions^*

Jijun Dong, Jesse D. Bloom^¶1, Vladimir Goncharov², Madhuri Chattopadhyay^||, Glenn L. Millhauser^||, David G. Lynn, Thomas Scheibel^**, and Susan Lindquist, An investigator of the Howard Hughes Medical Institute³

From the Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, the Department of Chemistry and Biology, Emory University, Atlanta, Georgia 30322, the ^¶Department of Molecular Genetics and Cell Biology, Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637, the ^||Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, and the ^**Institut für Organische Chemie und Biochemie, Technische Universität München, D-85747 Garching, Germany

Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. Whereas the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. To provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI⁺] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation. It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials.

Received for publication, June 15, 2007 , and in revised form, September 11, 2007.

^* This work was supported in part by a Howard Hughes Medical Institute undergraduate research fellowship (to J. D. B.), National Institutes of Health Grant GM065790 (to G. M. and M. C.), National Institutes of Health Grant GM25874 (to S. L., J. D., and V. G.), the DuPont-MIT alliance, and DOE Grant ER15377 (to J. D. and D. G. L.). 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 supplemental Figs. S1-S5.

¹ Present address: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA.

² Present address: Francis Bitter Magnet Laboratory/MIT, Cambridge, MA.

³ To whom correspondence should be addressed: Whitehead Institute for Biomedical Research, Cambridge, MA 02142-1479. Tel.: 617-258-5184; Fax: 617-258-7226; E-mail: lindquist_admin{at}wi.mit.edu.

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