Prion Protein PrP Research Articles

Prolongation of prion disease-associated symptomatic phase relates to CD3+ T cell recruitment into the CNS in murine scrapie-infected mice

Prion diseases are caused by the transconformation of the host cellular prion protein PrP(c) into an infectious neurotoxic isoform called PrP(Sc). While vaccine-induced PrP-specific CD4(+) T cells and antibodies partially protect scrapie-infected mice from disease, the potential autoreactivity of CD8(+) cytotoxic T lymphocytes (CTLs) received little attention. Beneficial or pathogenic influence of PrP(c)-specific CTL was evaluated by stimulating a CD8(+) T-cell-only response against PrP in scrapie-infected C57BL/6 mice. To circumvent immune tolerance to PrP, five PrP-derived nonamer peptides identified using prediction algorithms were anchored-optimized to improve binding affinity for H-2D(b) and immunogenicity (NP-peptides). All of the NP-peptides elicited a significant number of IFNγ secreting CD8(+) T cells that better recognized the NP-peptides than the natives; three of them induced T cells that were lytic in vivo for NP-peptide-loaded target cells. Peptides 168 and 192 were naturally processed and presented by the 1C11 neuronal cell line. Minigenes encoding immunogenic NP-peptides inserted into adenovirus (rAds) vectors enhanced the specific CD8(+) T-cell responses. Immunization with rAd encoding 168NP before scrapie inoculation significantly prolonged the survival of infected mice. This effect was attributable to a significant lengthening of the symptomatic phase and was associated with enhanced CD3(+) T cell recruitment to the CNS. However, immunization with Ad168NP in scrapie-incubating mice induced IFNγ-secreting CD8(+) T cells that were not cytolytic in vivo and did not influence disease progression nor infiltrated the brain. In conclusion, the data suggest that vaccine-induced PrP-specific CD8(+) T cells interact with prions into the CNS during the clinical phase of the disease.

Human prion protein binds Argonaute and promotes accumulation of microRNA effector complexes

Despite intense research in the context of neurodegenerative diseases associated with its misfolding, the endogenous human prion protein PrP(C) (or PRNP) has poorly understood physiological functions. Whereas most PrP(C) is exposed to the extracellular environment, conserved domains result in transmembrane forms of PrP(C) that traffic in the endolysosomal system and are linked to inherited and infectious neuropathologies. One transmembrane PrP(C) variant orients the N-terminal 'octarepeat' domain into the cytoplasm. Here we demonstrate that the octarepeat domain of human PrP(C) contains GW/WG motifs that bind Argonaute (AGO) proteins, the essential components of microRNA (miRNA)-induced silencing complexes (miRISCs). Transmembrane PrP(C) preferentially binds AGO, and PrP(C) promotes formation or stability of miRISC effector complexes containing the trinucleotide repeat-containing gene 6 proteins (TNRC6) and miRNA-repressed mRNA. Accordingly, effective repression of several miRNA targets requires PrP(C). We propose that dynamic interactions between PrP(C)-enriched endosomes and subcellular foci of AGO underpin these effects.

Direct observation of multiple misfolding pathways in a single prion protein molecule

Protein misfolding is a ubiquitous phenomenon associated with a wide range of diseases. Single-molecule approaches offer a powerful tool for deciphering the mechanisms of misfolding by measuring the conformational fluctuations of a protein with high sensitivity. We applied single-molecule force spectroscopy to observe directly the misfolding of the prion protein PrP, a protein notable for having an infectious misfolded state that is able to propagate by recruiting natively folded PrP. By measuring folding trajectories of single PrP molecules held under tension in a high-resolution optical trap, we found that the native folding pathway involves only two states, without evidence for partially folded intermediates that have been proposed to mediate misfolding. Instead, frequent but fleeting transitions were observed into off-pathway intermediates. Three different misfolding pathways were detected, all starting from the unfolded state. Remarkably, the misfolding rate was even higher than the rate for native folding. A mutant PrP with higher aggregation propensity showed increased occupancy of some of the misfolded states, suggesting these states may act as intermediates during aggregation. These measurements of individual misfolding trajectories demonstrate the power of single-molecule approaches for characterizing misfolding directly by mapping out nonnative folding pathways.

Communication: Epistructural thermodynamics of soluble proteins

The epistructural tension of a soluble protein is defined as the reversible work per unit area required to span the interfacial solvent envelope of the protein structure. It includes an entropic penalty term to account for losses in hydrogen-bonding coordination of interfacial water and is determined by a scalar field that indicates the expected coordination of a test water molecule at any given spatial location. An exhaustive analysis of structure-reported monomeric proteins reveals that disulfide bridges required to maintain structural integrity provide the thermodynamic counterbalance to the epistructural tension, yielding a tight linear correlation. Accordingly, deviations from the balance law correlate with the thermal denaturation free energies of proteins under reducing conditions. The picomolar-affinity toxin HsTX1 has the highest epistructural tension, while the metastable cellular form of the human prion protein PrP(C) represents the least tension-balanced protein.

PrionHome: A Database of Prions and Other Sequences Relevant to Prion Phenomena

Prions are units of propagation of an altered state of a protein or proteins; prions can propagate from organism to organism, through cooption of other protein copies. Prions contain no necessary nucleic acids, and are important both as both pathogenic agents, and as a potential force in epigenetic phenomena. The original prions were derived from a misfolded form of the mammalian Prion Protein PrP. Infection by these prions causes neurodegenerative diseases. Other prions cause non-Mendelian inheritance in budding yeast, and sometimes act as diseases of yeast. We report the bioinformatic construction of the PrionHome, a database of >2000 prion-related sequences. The data was collated from various public and private resources and filtered for redundancy. The data was then processed according to a transparent classification system of prionogenic sequences (i.e., sequences that can make prions), prionoids (i.e., proteins that propagate like prions between individual cells), and other prion-related phenomena. There are eight PrionHome classifications for sequences. The first four classifications are derived from experimental observations: prionogenic sequences, prionoids, other prion-related phenomena, and prion interactors. The second four classifications are derived from sequence analysis: orthologs, paralogs, pseudogenes, and candidate-prionogenic sequences. Database entries list: supporting information for PrionHome classifications, prion-determinant areas (where relevant), and disordered and compositionally-biased regions. Also included are literature references for the PrionHome classifications, transcripts and genomic coordinates, and structural data (including comparative models made for the PrionHome from manually curated alignments). We provide database usage examples for both vertebrate and fungal prion contexts. Using the database data, we have performed a detailed analysis of the compositional biases in known budding-yeast prionogenic sequences, showing that the only abundant bias pattern is for asparagine bias with subsidiary serine bias. We anticipate that this database will be a useful experimental aid and reference resource. It is freely available at: http://libaio.biol.mcgill.ca/prion.

Folding Dynamics and Kinetic Schemes from Signal-Pair Correlation Analysis of Single-Molecule Trajectories

Kinetic analysis of single-molecule trajectories is typically done by mapping measured signal trajectories (e.g. molecular extension, FRET, or ion current) onto a discrete set of states and then analyzing the dwell times. State identification can be challenging, however, especially in multi-state systems when signals are noisy or have low amplitude. We present a new method which avoids the need to identify states but can still analyze complex systems, based on correlations between “signal pairs,” two discrete ranges of the measured signal. The concept is related to a recently-introduced method for analyzing single-molecule FRET measurements of diffusing molecules [1]. First, time correlations are calculated between all signal pairs, without assuming any specific kinetic model for the system. Next, kinetic models are tested to determine the correct scheme, by choosing signal ranges associated with different states and fitting all cross-correlations between them to functions which are derived for each model from previously-described matrix methods [2]. We demonstrate the method with three examples measured by single-molecule force spectroscopy: folding of a two-state DNA hairpin, which can also be analyzed with simple standard methods; a folding of a three-state DNA hairpin, for which the kinetic scheme is known; and folding of the prion protein PrP, which forms non-native structures with an unknown kinetic scheme. These examples show that the method can cope with significant overlap of signals from different states due to noise (as high as 70%), states with very low occupancy (as low as 2%), and transition rates which are very similar or differ by several orders of magnitude. We also introduce signal-pair histograms for an unbiased visualization of dynamic processes within the trajectories.[1] Hoffmann et al., PhysChemChemPhys 13:1857 (2011).[2] Gopich & Szabo, J Phys Chem B 113:10965 (2009).

Reconstruction of the Energy Landscape Profile for Native Folding of Thepprion Protein from Single-Molecule Force Spectroscopy

Free energy landscapes drive protein folding phenomena but are difficult to measure directly. We measured the folding landscape of the prion protein PrP, a protein notable for its ability to form infectious non-native structures, using single molecule force spectroscopy with optical tweezers. Folding/unfolding trajectories of a single PrP molecule were observed directly by measuring the extension of the molecule under an applied tension, under both equilibrium constant-force and non-equilibrium force-ramp conditions. Native folding/unfolding was found to be a two-state process. The height and location of the energy barrier between the two states were determined from the distribution of unfolding forces in force-extension curves (FECs), and independently from the force-dependent lifetimes measured at constant force. The full profile of the landscape for native folding was then reconstructed from the FECs using the Hummer-Szabo method (Hummer and Szabo, 2001; Gupta A.N., et al., 2011). Because the landscape profile reconstructed this way is smoothed by the elastic compliance of the measurement set-up, we recovered the actual PrP folding landscape by using the measured point-spread function of the instrument to deconvolve the Hummer-Szabo result. The height and position of the barrier in the deconvolved landscape profile agreed well with the single reference point provided by the unfolding-force and force-dependent lifetime analyses, but the profile includes additional information about the width and shape of the potential wells and barriers. We found that the native folding pathway of PrP has an extended transition state, in agreement with mutational phi-analysis (Hart et al., 2009). These results show how protein folding landscapes can be recovered from non-equilibrium pulling experiments.

Prionemia and Leukocyte-Platelet-Associated Infectivity in Sheep Transmissible Spongiform Encephalopathy Models

The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.

Functional mechanisms of the cellular prion protein (PrP(C)) associated anti-HIV-1 properties.

The cellular prion protein PrP(C)/CD230 is a GPI-anchor protein highly expressed in cells from the nervous and immune systems and well conserved among vertebrates. In the last decade, several studies suggested that PrP(C) displays antiviral properties by restricting the replication of different viruses, and in particular retroviruses such as murine leukemia virus (MuLV) and the human immunodeficiency virus type 1 (HIV-1). In this context, we previously showed that PrP(C) displays important similarities with the HIV-1 nucleocapsid protein and found that PrP(C) expression in a human cell line strongly reduced HIV-1 expression and virus production. Using different PrP(C) mutants, we report here that the anti-HIV-1 properties are mostly associated with the amino-terminal 24-KRPKP-28 basic domain. In agreement with its reported RNA chaperone activity, we found that PrP(C) binds to the viral genomic RNA of HIV-1 and negatively affects its translation. Using a combination of biochemical and cell imaging strategies, we found that PrP(C) colocalizes with the virus assembly machinery at the plasma membrane and at the virological synapse in infected T cells. Depletion of PrP(C) in infected T cells and microglial cells favors HIV-1 replication, confirming its negative impact on the HIV-1 life cycle.

Crystallization and preliminary X-ray diffraction analysis of prion protein bound to the Fab fragment of the POM1 antibody.

Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrP(c) to the pathogenic isoform PrP(sc). Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120-232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°.

PrionOme: A database of prions and other sequences relevant to prion phenomena

AbstractPrions are units of propagation of an altered state of a protein or proteins. Prions can propagate from cell to cell, and from organism to organism, through cooption of other protein copies. Prions contain no necessary nucleic acids, and are important both as both pathogenic agents, and as a potential force in epigenetic phenomena. The original prions were derived from a misfolded form of the mammalian Prion Protein PrP. Infection by these prions causes neurodegenerative diseases. Other prions cause non-Mendelian inheritance in budding yeast, and sometimes act as diseases of yeast. We have compiled a database of >2000 prion-related sequences, called the PrionOme. The database comprises seven PrionOme classification categories: prionogenic sequences (i.e., sequences that can make prions), ‘prionoids’ (i.e., phenomena that have some prion characteristics), orthologs, paralogs, pseudogenes, prion interactors, and prion-like molecules. Database entries list: supporting information for PrionOme classifications, prion-determinant areas (where relevant), and disordered and compositionally-biased regions. Also included are original references for the PrionOme classifications, transcripts and genomic coordinates, and structural data (including comparative models). We provide database usage examples for both vertebrate and fungal prion contexts. As development of this resource is on-going, we will be very happy to receive and act on any constructive comments from peer scientists in the areas of prion biology and protein misfolding, either by email or using the feedback form provided on the PrionOme website. We hope that this database will be a valuable experimental aid and reference resource. It is freely available at: http://libaio.biol.mcgill.ca/prion.

Calreticulin Inhibits Prion Protein PrP-(23–98) Aggregationin Vitro

Because prion protein PrP-(23-98) was recently found to polymerize into amyloid-like and proteinase K-resistant spherical aggregates in the presence of NADPH plus copper ions, we tested to determine whether calreticulin (CRT) inhibits PrP-(23-98) aggregation in vitro. The results indicated that CRT suppressed PrP-(23-98) aggregation, and that CRT-mediated solubilization occurred in the aggregates.

Quantification of surviving cerebellar granule neurones and abnormal prion protein (PrPSc) deposition in sporadic Creutzfeldt–Jakob disease supports a pathogenic role for small PrPSc deposits common to the various molecular subtypes

Neuronal death is a major neuropathological hallmark in prion diseases. The association between the accumulation of the disease-related prion protein (PrP(Sc) ) and neuronal loss varies within the wide spectrum of prion diseases and their experimental models. In this study, we investigated the relationships between neuronal loss and PrP(Sc) deposition in the cerebellum from cases of the six subtypes of sporadic Creutzfeldt-Jakob disease (sCJD; n=100) that can be determined according to the M129V polymorphism of the human prion protein gene (PRNP) and PrP(Sc) molecular types. The numerical density of neurones was estimated with a computer-assisted image analysis system and the accumulation of PrP(Sc) deposits was scored. The scores of PrP(Sc) immunoreactive deposits of the punctate type (synaptic type) were correlated with neurone counts - the higher the score the higher the neuronal loss - in all sCJD subtypes. Large 5- to 50-µm-wide deposits (focal type) were found in sCJD-MV2 and sCJD-VV2 subtypes, and occasionally in a few cases of the other studied groups. By contrast, the highest scores for 5- to 50-µm-wide deposits observed in sCJD-MV2 subtype were not associated with higher neuronal loss. In addition, these scores were inversely correlated with neuronal counts in the sCJD-VV2 subtype. These results support a putative pathogenic role for small PrP(Sc) deposits common to the various sCJD subtypes. Furthermore, the observation of a lower loss of neurones associated with PrP(Sc) type-2 large deposits is consistent with a possible 'protective' role of aggregated deposits in both sCJD-MV2 and sCJD-VV2 subtypes.

PSI+] Maintenance Is Dependent on the Composition, Not Primary Sequence, of the Oligopeptide Repeat Domain

[PSI+], the prion form of the yeast Sup35 protein, results from the structural conversion of Sup35 from a soluble form into an infectious amyloid form. The infectivity of prions is thought to result from chaperone-dependent fiber cleavage that breaks large prion fibers into smaller, inheritable propagons. Like the mammalian prion protein PrP, Sup35 contains an oligopeptide repeat domain. Deletion analysis indicates that the oligopeptide repeat domain is critical for [PSI+] propagation, while a distinct region of the prion domain is responsible for prion nucleation. The PrP oligopeptide repeat domain can substitute for the Sup35 oligopeptide repeat domain in supporting [PSI+] propagation, suggesting a common role for repeats in supporting prion maintenance. However, randomizing the order of the amino acids in the Sup35 prion domain does not block prion formation or propagation, suggesting that amino acid composition is the primary determinant of Sup35's prion propensity. Thus, it is unclear what role the oligopeptide repeats play in [PSI+] propagation: the repeats could simply act as a non-specific spacer separating the prion nucleation domain from the rest of the protein; the repeats could contain specific compositional elements that promote prion propagation; or the repeats, while not essential for prion propagation, might explain some unique features of [PSI+]. Here, we test these three hypotheses and show that the ability of the Sup35 and PrP repeats to support [PSI+] propagation stems from their amino acid composition, not their primary sequences. Furthermore, we demonstrate that compositional requirements for the repeat domain are distinct from those of the nucleation domain, indicating that prion nucleation and propagation are driven by distinct compositional features.

“Preclinical” MSA in definite Creutzfeldt‐Jakob disease

Multiple system atrophy (MSA) is a sporadic alpha-synucleinopathy clinically characterized by variable degrees of parkinsonism, cerebellar ataxia and autonomic dysfunction. The histopathological hallmark of MSA is glial cytoplasmic inclusion (GCI). It is considered to represent the earliest stage of the degenerative process in MSA and to precede neuronal degeneration. Sporadic Creutzfeldt-Jakob disease (sCJD) is a fatal, rapidly progressive dementia generally associated with ataxia, pyramidal and extrapyramidal symptoms and myoclonus. Definite diagnosis needs neuropathological demonstration of variable degrees of spongiform degeneration of neuropil, neuronal loss, astro- and microgliosis, and the presence of abnormal deposits of the misfolded prion protein PrP(res) . Both diseases, CJD and MSA are infrequent among neurodegenerative diseases. In the present report we describe clinical and neuropathological findings of a previously healthy 64-year-old woman who developed symptoms of classical CJD. At post mortem examination, the brain showed in addition to classical methionine/methionine PrP(res) type 1 (MM1) sCJD changes and moderate Alzheimer-type pathology, features of "preclinical" MSA with minimal histopathological changes. These were characterized by discrete amounts of alpha-synuclein immunoreacive glial cytoplasmic inclusions in the striato-nigral system, isolated intraneuronal inclusions in pigmented neurons of the substantia nigra, as well as some vermiform intranuclear inclusions. To our knowledge, this is the first report on the coexistence of definite sCJD and "minimal changes" MSA in the same patient.

Copper(II)-Induced Secondary Structure Changes and Reduced Folding Stability of the Prion Protein

The cellular isoform of the prion protein PrP C is a Cu 2 +-binding cell surface glycoprotein that, when misfolded, is responsible for a range of transmissible spongiform encephalopathies. As changes in PrP C conformation are intimately linked with disease pathogenesis, the effect of Cu 2+ ions on the structure and stability of the protein has been investigated. Urea unfolding studies indicate that Cu 2+ ions destabilise the native fold of PrP C. The midpoint of the unfolding transition is reduced by 0.73 ± 0.07 M urea in the presence of 1 mol equiv of Cu 2+. This equates to an appreciable difference in free energy of unfolding (2.02 ± 0.05 kJ mol − 1 at the midpoint of unfolding). We relate Cu 2 +-induced changes in secondary structure for full-length PrP(23–231) to smaller Cu 2 + binding fragments. In particular, Cu 2+-induced structural changes can directly be attributed to Cu 2+ binding to the octarepeat region of PrP C. Furthermore, a β-sheet-like transition that is observed when Cu ions are bound to the amyloidogenic fragment of PrP (residues 90–126) is due only to local Cu 2+ coordination to the individual binding sites centred at His95 and His110. Cu 2+ binding does not directly generate a β-sheet conformation within PrP C; however, Cu 2+ ions do destabilise the native fold of PrP C and may make the transition to a misfolded state more favourable.

Molecular Simulation Study of Prion Peptide Self-Aggregation in the Presence of Lipid Membranes

Neurodegenerative pathologies such as Alzheimer's disease affect millions of people worldwide and are a major cause of morbidity and mortality. One such cause of dementia, Creutzfeld-Jakob disease, is thought to be caused by the human prion protein PrP. Interactions with membranes have been shown to affect the behaviour of amyloid-forming peptides, including prion peptides, and have been implicated in their toxicity. To gain insight into the molecular basis of these effects, we use atomistic molecular dynamics simulations in explicit solvent to examine the interaction of several blocked PrP and yeast-prion protein fragments with zwitterionic and anionic lipid bilayers.

Understanding the neurospecificity of Prion protein signaling

The cellular prion protein PrP(C) is the normal counterpart of the scrapie prion protein PrP(Sc), the main component of the infectious agent of transmissible spongiform encephalopathies (TSEs). It is a ubiquitous cell-surface glycoprotein, abundantly expressed in neurons, which constitute the targets of TSE pathogenesis. The presence of PrP(C) at the surface of neurons is an absolute requirement for the development of prion diseases and corruption of PrP(C) function(s) within an infectious context emerges as a proximal cause for PrP(Sc)-induced neurodegeneration. Experimental evidence gained over the past decade indicates that PrP(C) has the capacity to mobilize promiscuous signal transduction cascades that, notably, contribute to cell homeostasis. Beyond ubiquitous effectors, much data converge onto a neurospecificity of PrP(C) signaling, which may be the clue to neuronal cell demise in prion disorders. In this article, we highlight the requirement of PrP(C) for TSEs-associated neurodegeneration and review the current knowledge of PrP(C)-dependent signal transduction in neuronal cells and its implications for PrP(Sc)-mediated neurotoxicity.

The α-Helical C-Terminal Domain of Full-Length Recombinant PrP Converts to an In-Register Parallel β-Sheet Structure in PrP Fibrils: Evidence from Solid State Nuclear Magnetic Resonance

We report the results of solid state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the full-length prion protein PrP (residues 23−231, Syrian hamster sequence). Measurements of intermolecular 13C−13C dipole−dipole couplings in selectively carbonyl-labeled samples indicate that β-sheets in these fibrils have an in-register parallel structure, as previously observed in amyloid fibrils associated with Alzheimer’s disease and type 2 diabetes and in yeast prion fibrils. Two-dimensional 13C−13C and 15N−13C solid state NMR spectra of a uniformly 15N- and 13C-labeled sample indicate that a relatively small fraction of the full sequence, localized to the C-terminal end, forms the structurally ordered, immobilized core. Although unique site-specific assignments of the solid state NMR signals cannot be obtained from these spectra, analysis with a Monte Carlo/simulated annealing algorithm suggests that the core is comprised primarily of residues in the 173−224 range. These results are consistent with earlier electron paramagnetic resonance studies of fibrils formed by residues 90−231 of the human PrP sequence, formed under somewhat different conditions [Cobb, N. J., Sonnichsen, F. D., McHaourab, H., and Surewicz, W. K. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 18946−18951], suggesting that an in-register parallel β-sheet structure formed by the C-terminal end may be a general feature of PrP fibrils prepared in vitro.

Pathogenic Mutations in the Hydrophobic Core of the Human Prion Protein Can Promote Structural Instability and Misfolding

Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. These diseases can be hereditary in humans and four of the many disease-associated missense mutants of PrP are in the hydrophobic core: V180I, F198S, V203I and V210I. The T183A mutation is related to the hydrophobic core mutants as it is close to the hydrophobic core and known to cause instability. We used extensive molecular dynamics simulations of these five PrP mutants to compare their dynamics and conformations to those of the wild type PrP. The simulations highlight the changes that occur upon introduction of mutations and help to rationalize experimental findings. Changes can occur around the mutation site, but they can also be propagated over long distances. In particular, the F198S and T183A mutations lead to increased flexibility in parts of the structure that are normally stable, and the short β-sheet moves away from the rest of the protein. Mutations V180I, V210I and, to a lesser extent, V203I cause changes similar to those observed upon lowering the pH, which has been linked to misfolding. Early misfolding is observed in one V180I simulation. Overall, mutations in the hydrophobic core have a significant effect on the dynamics and stability of PrP, including the propensity to misfold, which helps to explain their role in the development of familial prion diseases.