Polymorphism and Ultrastructural Organization of Prion Protein Amyloid Fibrils: An Insight from High Resolution Atomic Force Microscopy Maighdlin Anderson 1 , Olga V. Bocharova 1 , Natallia Makarava 1 Leonid Breydo 1 , Vadim V. Salnikov 1 and Ilia V. Baskakov 1,2 * 1 Medical Biotechnology Center University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA 2 Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD 21201, USA Amyloid fibrils were produced from the full-length mouse prion protein (PrP) under solvent conditions similar to those used for the generation of synthetic prions from PrP 89-230. Analysis of the ultrastructure by atomic force microscopy revealed extremely broad polymorphism in fibrils formed under a single growth condition. Fibrils varied with respect to the number of constitutive filaments and the manner in which the filaments were assembled. PrP polymerization was found to show several peculiar features: (i) the higher-order fibrils/ribbons were formed through a highly hierarchical mechanism of assembly of lower-order fibrils/ribbons; (ii) the lateral assembly proceeded stepwise; at each step, a semi-stable fibrillar species were generated, which were then able to enter the next level of assembly; (iii) the assembly of lower into higher-order fibrils occurred predominantly in a vertical dimension via stacking of ribbons on top of each other; (iv) alternative modes of lateral association co-existed under a single growth condition; (iv) the fibrillar morphology changed even within individual fibrils, illustrating that alternative modes of filament assembly are inter-convertible and thermodynamically equivalent. The most predominant fibrillar types were classified into five groups according to their height, each of which was divided in up to three subgroups according to their width. Detailed analysis of ultrastructure revealed that the fibrils of the major subtype (height 3.61(G0.28) nm, width 31.1(G2.0) nm) were composed of two ribbons, each of which was composed of two filaments. The molecular volume calculations indicated that a single PrP molecule occupied a distance of w1.2 nm within a single filament. High polymorphism in fibrils generated in vitro is reminiscent of high morphological diversity of scrapie-associated fibrils isolated from scrapie brains, suggesting that polymorphism is peculiar for polymerization of PrP regardless of whether fibrils are formed in vitro or under pathological conditions in vivo. q 2006 Elsevier Ltd. All rights reserved. Keywords: prion protein; amyloid fibrils; atomic force microscopy; lateral association; polymorphism *Corresponding author 0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. Present addresses: O. V. Bocharova, Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia, 117997; V. V. Salnikov, Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, Kazan, Russia, 420111. Abbreviations used: PrP, recombinant full-length PrP; a-PrP, a-helical isoform of PrP; PrP C , cellular isoform of the prion protein; PrP Sc , disease-associated isoform of the prion protein; PK, proteinase K; AFM, atomic force microscopy; SAF, scrapie-associated fibrils; EM, electron microscopy; GndHCl, guanidine hydrochloride. E-mail address of the corresponding author: baskakov@umbi.umd.edu doi:10.1016/j.jmb.2006.02.007 J. Mol. Biol. (2006) 358, 580–596