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J. Biol. Chem., Vol. 281, Issue 22, 15536-15545, June 2, 2006

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Probing the Conformation of the Prion Protein within a Single Amyloid Fibril Using a Novel Immunoconformational Assay^*

Vera Novitskaya, Natallia Makarava, Anne Bellon, Olga V. Bocharova¹, Igor B. Bronstein^¶2, R. Anthony Williamson, and Ilia V. Baskakov^||3

From the Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, ^||Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland 21201, the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, the ^¶Institute for Animal Health, Compton Laboratories, Compton RG20 7NN, United Kingdom

The coexistence of multiple strains or subtypes of the disease-related isoform of prion protein (PrP) in natural isolates, together with the observed conformational heterogeneity of PrP amyloid fibrils generated in vitro, indicates the importance of probing the conformation of single particles within heterogeneous samples. Using an array of PrP-specific antibodies, we report the development of a novel immunoconformational assay. Uniquely, application of this new technology allows the conformation of multimeric PrP within a single fibril or particle to be probed without pretreatment of the sample with proteinase K. Using amyloid fibrils prepared from full-length recombinant PrP, we demonstrated the utility of this assay to define (i) PrP regions that are surface-exposed or buried, (ii) the susceptibility of defined PrP regions to GdnHCl-induced denaturation, and (iii) the conformational heterogeneity of PrP fibrils as measured for either the entire fibrillar population or for individual fibrils. Specifically, PrP regions 159–174 and 224–230 were shown to be buried and were the most resistant to denaturation. The 132–156 segment of PrP was found to be cryptic under native conditions and solvent-exposed under partially denaturing conditions, whereas the region 95–105 was solvent-accessible regardless of the solvent conditions. Remarkably, a subfraction of fibrils showed immunoreactivity to PrP^Sc-specific antibodies designated as IgGs 89–112 and 136–158. The immunoreactivity of the conformational epitopes was reduced upon exposure to partially denaturing conditions. Unexpectedly, PrP^Sc -specific antibodies revealed conformational polymorphisms even within individual fibrils. Our studies provide valuable new insight into fibrillar substructure and offer a new tool for probing the conformation of single PrP fibrils.

Received for publication, February 13, 2006 , and in revised form, March 24, 2006.

^* This work was supported by National Institute of Health Grants NS045585 (to I. V. B.) and AG004243 (to R. A. W.) and Department of Defense (D.A.M.D.) Award 17-03-1-0511 (to R. A. W.). 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.

¹ Present address: Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.

² Supported by the Biotechnology and Biological Sciences Research Council and Department of Health, United Kingdom. Present address: National Institute for Medical Research, Physical Biochemistry Division, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.

³ 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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