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J. Biol. Chem., Vol. 282, Issue 12, 9090-9097, March 23, 2007

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Site-specific Conformational Studies of Prion Protein (PrP) Amyloid Fibrils Revealed Two Cooperative Folding Domains within Amyloid Structure^*

Ying Sun, Leonid Breydo¹, Natallia Makarava, Qingyuan Yang², Olga V. Bocharova³, and Ilia V. Baskakov⁴

From the Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201 and the Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland 21201

Despite the ability of most proteins to form amyloid, very little is know about amyloid fibril structures and the factors that govern their stability. Using amyloid fibrils produced from full-length prion protein (PrP), we describe a reliable approach for determining both site-specific and global conformational stability of the fibrillar form. To measure site-specific stability, we produced six variants of PrP by replacing the residues at positions 88, 98, 127, 144, 196, and 230 with cysteine, labeled the new cysteines with the fluorescent dye acrylodan, and investigated their conformational status within the amyloid form in guanidine hydrochloride-induced denaturation experiments. We found that the fibrils labeled at positions 127, 144, 196, and 230 displayed cooperative unfolding and showed a very high C value similar to that observed for the global unfolding of the amyloid structure. The unfolding at residue 98 was also cooperative; however, it showed a C value substantially lower than that of global unfolding, whereas the unfolding of fibrils labeled at residue 88 was non-cooperative. These data illustrate that there are at least two independent cooperative folding domains within the amyloid structure of the full-length PrP. In addition, kinetic experiments revealed only a partial overlap between the region that constituted the fibrillar cross- core and the regions that were involved in nucleation. This result illustrates that separate PrP regions accounted for the nucleation and for the formation of the conformationally most stable fibrillar core.

Received for publication, September 6, 2006 , and in revised form, January 16, 2007.

^* This work was supported by the Prion Program at the University of Maryland Biotechnology Institute. 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 three supplemental figures.

¹ Present address: Dept. of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697.

² Present address: Dept. of Pharmacology and Experimental Therapeutics, University of Maryland, Baltimore, MD, 21201.

³ Present address: Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia, 117997.

⁴ To whom correspondence should be addressed: Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 W. Lombard St., Baltimore, MD 21201. Tel.: 410-706-4562; Fax: 410-706-8184; E-mail: Baskakov{at}umbi.umd.edu.

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