Amyloidogenic Unfolding Intermediates Differentiate Sheep Prion Protein Variants Human Rezaei 1 *, Yvan Choiset 2 , Frederic Eghiaian 3 , Eric Treguer 1 Pascale Mentre 3 , Pascale Debey 3 , Jeanne Grosclaude 1 and Thomas Haertle 2 1 Virologie et Immunologie Mole ´culaires (VIM), INRA F-78352 Jouy-en-Josas, France 2 Laboratoire d’Etude des Interactions des Mole ´cules Alimentaires (LEIMA), INRA F-44316 Nantes, France 3 Institut National de la Recherche Agronomique (INRA) UC 806/EA 2703 Muse ´um National d’Histoire Naturelle, Institut de Biologie Physico-Chimique, 13, rue P. et M. Curie, F-75005 Paris France Sheep is a unique example among mammalian species to present a strong correlation between genotype and prion disease susceptibility phenotype. Indeed a well-defined set of PrP polymorphisms at positions 136, 154 and 171 (sheep numbering) govern scrapie susceptibility, ranging from very high susceptibility for V136-R154-Q171 variant (VRQ) to resistance for A136-R154-R171 variant (ARR). To get better insight into the molecular mechanisms of scrapie suscepti- bility/resistance, the unfolding pathways of the different full-length recombinant sheep prion protein variants were analysed by differential scanning calorimetry in a wide range of pH. In the pH range 4.5–6.0, thermal unfolding occurs through a reversible one-step process while at pH , 4.5 and . 6.0 unfolding intermediates are formed, which are stable in the temperature range 65–80 8C. While these general behaviours are shared by all variants, VRQ and ARQ (susceptibility variants) show higher thermal stability than AHQ and ARR (resistance variants) and the formation of their unfolding intermediates requires higher activation energy than in the case of AHQ and ARR. Furthermore, secondary struc- tures of the unfolding intermediates differentiate variants: ARR unfolding intermediate exhibits random coil structure, contrasting with the b-sheet structure of VRQ and ARQ unfolding intermediates. The rate of the unfolding intermediate formation allows us to classify genetic variants along increasing scrapie susceptibility at pH 4.0, VRQ and ARQ rates being the highest. Rather poor correlation is observed at pH 7.2. Upon cooling, these intermediates refold into stable species, which are rich in b-type secondary structures and, as revealed by thioflavin T fluorescence and electron microscopy, share amyloid characteristics. These results highlight the prion protein plasticity genetically modulated in sheep, and might provide a molecular basis for sheep predisposition to scrapie taking into account both thermodynamic stability and transconformation rate of prion protein. q 2002 Elsevier Science Ltd. All rights reserved Keywords: sheep prion protein; PrP genetic polymorphism; unfolding pathway; amyloidogenic intermediate; scrapie susceptibility *Corresponding author Introduction The transmissible spongiform encephalopathies (TSE), or “prion diseases”, are neurodegenerative pathologies affecting humans as well as animals. Sheep scrapie, bovine encephalopathy, Kuru, Gerstmann–Stra ¨ussler–Scheinker syndrome and Creutzfeldt – Jakob disease (CJD) belong to these diseases. They can be sporadic or transmitted by a not yet identified infectious agent. Several hypotheses have been advanced concerning the nature of the infectious agents, but the most widely accepted, the “protein only” hypothesis, designates protein as the infectious agent. 1,2 According to this hypothesis, the key event in the pathogenesis is the conversion of the cellular form of the prion 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved E-mail address of the corresponding author: rezaei@jouy.inra.fr Abbreviations used: ANS, 8-anilino-1-naphtalene- sulphonate; CD, circular dichroism; DSC, differential scanning calorimetry; ThT, thioflavine T. doi:10.1016/S0022-2836(02)00856-2 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 322, 799–814