Structural Determinants in Prion Protein Folding and Stability Federico Benetti 1,2, † , Xevi Biarnés 3, † , Francesco Attanasio 4 , Gabriele Giachin 1 , Enrico Rizzarelli 4 and Giuseppe Legname 1,2,5 1 - Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati, Via Bonomea 265, I-34136 Trieste, Italy 2 - Italian Institute of Technology, Scuola Internazionale Superiore di Studi Avanzati Unit, Via Bonomea 265, I-34136 Trieste, Italy 3 - Department of Physics, Scuola Internazionale Superiore di Studi Avanzati, I-34136 Trieste, Italy 4 - National Research Council, Institute of Biostructures and Bioimaging, Viale Andrea Doria 6, I-95125 Catania, Italy 5 - Elettra - Sincrotrone Trieste S.C.p.A., AREA Science Park, I-34149 Basovizza Trieste, Italy Correspondence to Giuseppe Legname: Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati, Via Bonomea 265, I-34136 Trieste, Italy. legname@sissa.it http://dx.doi.org/10.1016/j.jmb.2014.09.017 Edited by D. P. Raleigh Abstract Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases, involving post-trans- lational modifications of the cellular prion protein. Epidemiological studies on Creutzfeldt-Jakob disease, a prototype prion disorder, show a majority of cases being sporadic, while the remaining occurrences are either genetic or iatrogenic. The molecular mechanisms by which PrP C is converted into its pathological isoform have not yet been established. While point mutations and seeds trigger the protein to cross the energy barriers, thus causing genetic and infectious transmissible spongiform encephalopathies, respectively, the mechanism responsible for sporadic forms remains unclear. Since prion diseases are protein-misfolding disorders, we investigated prion protein folding and stability as functions of different milieus. Using spectroscopic techniques and atomistic simulations, we dissected the contribution of major structural determinants, also defining the energy landscape of prion protein. In particular, we elucidated (i) the essential role of the octapeptide region in prion protein folding and stability, (ii) the presence of a very enthalpically stable intermediate in prion-susceptible species, and (iii) the role of the disulfide bridge in prion protein folding. © 2014 Elsevier Ltd. All rights reserved. Introduction Prion diseases or transmissible spongiform en- cephalopathies are a group of rare disorders characterized by spongiform neurodegeneration of the central nervous system caused by the misfolding of the cellular prion protein (PrP C ) into pathogenic conformers (PrP Sc ) denoted prions. Transmissible spongiform encephalopathies can manifest as sporadic, genetic, or infectious disorders involving post-translational modifications of PrP C [1]. These maladies include Creutzfeldt-Jakob disease, Gerst- mann-Straussler-Scheinker syndrome, fatal familial insomnia, and kuru in humans; bovine spongiform encephalopathy in cattle; scrapie in sheep and goats; and chronic wasting disease in elk, deer, and moose [2]. The polypeptide PrP C is a sialoglycoprotein, tethered to the outer leaflet of the plasma mem- brane by a glycosylphosphatidylinositol (GPI) anchor, and its primary structure is highly con- served among mammals [3,4]. PrP C is expressed mostly in the central nervous system and periph- eral nervous system, but its precise physiological function is still unclear [5]. The NMR structures of several species variants of recombinant PrP revealed a flexibly disordered N-terminal domain encompassing residues 23–124, a globular domain of residues 125–228 with three α-helices, a short two-stranded antiparallel β-sheet, and a short C-terminal tail [6,7]. The V-shaped arrangement of the two longest helices, the second and the third, forms the scaffold onto which the β-sheet and the first α-helix are anchored [7] . The C-terminal 0022-2836/© 2014 Elsevier Ltd. All rights reserved. J. Mol. Biol. (2014) 426, 3796–3810 Article