Article

Federico Bene

0022-2836/© 2014 Elsevi

Structural Determinants in Prion ProteinFolding and Stability

tti 1, 2, †, Xevi Biarnés3, †, Fr

ancesco Attanasio4, Gabriele Giachin1,Enrico Rizzarelli 4 and Giuseppe Legname1, 2, 5

1 - Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati, Via Bonomea 265, I-34136 Trieste, Italy2 - Italian Institute of Technology, Scuola Internazionale Superiore di Studi Avanzati Unit, Via Bonomea 265, I-34136 Trieste, Italy3 - Department of Physics, Scuola Internazionale Superiore di Studi Avanzati, I-34136 Trieste, Italy4 - National Research Council, Institute of Biostructures and Bioimaging, Viale Andrea Doria 6, I-95125 Catania, Italy5 - 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 StudiAvanzati, Via Bonomea 265, I-34136 Trieste, Italy. legname@sissa.ithttp://dx.doi.org/10.1016/j.jmb.2014.09.017Edited 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, aprototype prion disorder, show a majority of cases being sporadic, while the remaining occurrences are eithergenetic or iatrogenic. The molecular mechanisms by which PrPC is converted into its pathological isoform havenot yet been established. While point mutations and seeds trigger the protein to cross the energy barriers, thuscausing genetic and infectious transmissible spongiform encephalopathies, respectively, the mechanismresponsible for sporadic forms remains unclear. Since prion diseases are protein-misfolding disorders, weinvestigated prion protein folding and stability as functions of different milieus. Using spectroscopic techniquesand atomistic simulations, we dissected the contribution of major structural determinants, also defining theenergy landscape of prion protein. In particular, we elucidated (i) the essential role of the octapeptide region inprion protein folding and stability, (ii) the presence of a very enthalpically stable intermediate in prion-susceptiblespecies, 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 disorderscharacterized by spongiform neurodegeneration ofthe central nervous system caused by themisfoldingof the cellular prion protein (PrPC) into pathogenicconformers (PrPSc) denoted prions. Transmissiblespongiform encephalopathies can manifest assporadic, genetic, or infectious disorders involvingpost-translational modifications of PrPC [1]. Thesemaladies include Creutzfeldt-Jakob disease, Gerst-mann-Straussler-Scheinker syndrome, fatal familialinsomnia, and kuru in humans; bovine spongiformencephalopathy in cattle; scrapie in sheep andgoats; and chronic wasting disease in elk, deer, andmoose [2].

er Ltd. All rights reserved.

The polypeptide PrPC 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]. PrPC is expressedmostly in the central nervous system and periph-eral nervous system, but its precise physiologicalfunction is still unclear [5]. The NMR structures ofseveral species variants of recombinant PrPrevealed a flexibly disordered N-terminal domainencompassing residues 23–124, a globular domainof residues 125–228 with three α-helices, a shorttwo-stranded antiparallel β-sheet, and a shortC-terminal tail [6,7]. The V-shaped arrangementof the two longest helices, the second and the third,forms the scaffold onto which the β-sheet and thefirst α-helix are anchored [7]. The C-terminal

J. Mol. Biol. (2014) 426, 3796–3810

3797Insights into Prion Protein Folding and Stability

domain has a buried disulfide bridge and twoN-linked carbohydrates. The N-terminal domaincontains glycine-rich repeats, each composed ofeight residues. This sequence is termed octarepeatregion (OR) and is highly conserved amongmammals. The OR segment binds copper [8] andother divalent cations such as zinc, nickel, iron, andmanganese [9–13].Prions are characterized by their infectious prop-

erties and intrinsic ability to encipher distinctbiochemical features through their secondary, ter-tiary, and quaternary protein structures [14]. Duringprion disease progression, PrPC is converted into itsabnormal form (PrPSc) through a process wherebymost α-helical motifs are replaced by β-sheetsecondary structures [2,15–17]. The PrPC-to-PrPSc

conversion confers altered biochemical properties,such as resistance to limited proteolysis andinsolubility in non-denaturing detergents [18,19]. Indisease-affected brain homogenates, limited prote-olysis completely hydrolyzes PrPC and produces asmaller, protease-resistant PrPSc molecule of ~142amino acids, designated PrP27-30 [20]. Limitedproteolysis studies on prions have revealed twodomains: one labile, spanning from the N-terminusto residues 78–103, and one protease-resistant[21–23]. The protease-resistant core, formed by theC-terminal domain of PrP, retains infectivity. Thegeneration of synthetic prions from recombinanttruncated mouse PrP has confirmed the importanceof the globular domain in PrP conversion and prionformation [24,25].The molecular pathways leading to PrP misfold-

ing remain unclear. It has been suggested that theβ-rich PrPSc isoform is thermodynamically morestable than PrPC and that folding of PrP to its nativeα-helical conformation is under kinetic control. Theconformational transition from the α-helical to theβ-rich isoform is separated by a large energeticbarrier associated with unfolding and with a high-er-order kinetic process related to oligomerization[26]. Very little is known about the initial stages ofthe conformational transition from PrPC to PrPSc.While point mutations and seeds cause the geneticand infectious forms, respectively, favoring thecrossing of energetic barriers, there is no informa-tion regarding sporadic forms.Here we investigated structural elements and

environmental factors affecting PrP stability andfolding. Combining spectroscopic techniques andatomistic simulations, we identified (i) the centralrole of octarepeats in prion protein folding andstability; (ii) a highly enthalpically stable intermedi-ate state in PrP C-terminal domain unfolding; (iii) thepresence of this enthalpically stable intermediateonly in prion-susceptible species such as human,mouse, cattle, and some sheep variants; and (iv) therole of the disulfide bridge in preserving nativefolding and protein stability.

Results

Prions arise as the result of an aberrant foldingof PrPC. Both the full-length and truncated prionproteins are susceptible to misfolding and prionconversion. Although many studies haveattempted to disclose the molecular mechanismspromoting the conversion, structural determinantsand environmental key factors involved in prionconversion remain unsolved. Here we report theeffects of the major structural determinants (un-folded N-terminal domain, globular domain, anddisulfide bridge) and environmental key factors(pH and dielectric constant) on prion proteinfolding and stability.

Effect of pH on Prion Protein Foldingand Stability

Like other GPI-anchored proteins, PrPC constitu-tively cycles between plasma membrane and endo-cytic compartments, with subsequent exposure todifferent environmental pH values.Full-length and truncated mouse prion proteins,

MoPrP, fold mostly in α-helical conformation at acidicand neutral pH (SupplementaryFig. 1). Ureaunfoldingmeasurements of full-length MoPrP, MoPrP(23-231),reveal a cooperative two-state unfolding mechanismat both pH conditions (Fig. 1), though it is more stableat neutral pH (Fig. 1, dotted line) than at acidic pH(Fig. 1, continuous line) as also confirmed by [Urea]1/2and ΔGH2O values. [Urea]1/2 values, corresponding tothe urea concentration required to unfold half of theprotein, are 5.7 M and 6.3 M at acidic and neutral pH,respectively (Table 1). These results are corroboratedby free-energy values, ΔGH2O, reported in Table 1. Atneutral pH, ΔGH2O is 5.9 kcal/mol, while at pH 5.5, itdecreases to 4.3 kcal/mol. The variation in slope (m),which reflects the dependence of free-energy ondenaturant concentration and indicates changes inthe solvent-accessible area of hydrophobic residues,is also a function of pH (Table 1).At acidic pH, the truncated form, MoPrP(89-231),

unfolds following a three-state transition mechanism(Fig. 2a). We confirmed this three-state unfoldingprocess by performing equilibrium unfolding mea-surements in 40 mM 4-morpholineethanesulfonicacid buffer (same ionic strength as acetate buffer),pH 5.5, indicating that the intermediate state dependsonly on pH (Supplementary Fig. 2). The transitionmechanism collapses into a two-state process atneutral pH (Fig. 2b). [Urea]1/2 values from the native tothe fully unfolded state of MoPrP(89-231) are pHindependent and correspond to 5.2 M (Table 1).These values are lower than those obtained usingthe full-length protein, suggesting a role for theunfolded N-terminal domain in stabilizing the globulardomain.

Fig. 2. Unfolding curves of MoPrP(89-231) determinedby fluorescence spectroscopy. (a) At pH 5.5 and (b) atneutral pH 7. Inset represents the MoPrP(89-231) unfold-ing process followed by CD. At acidic pH, unfolding curvesare characterized by a three-state transition mechanism.At neutral pH, the unfolding is collapsed into a two-statetransition mechanism. The unfolded fraction is expressedwith standard deviation obtained from three independentexperiments.

Fig. 1. Unfolding curves of MoPrP(23-231) determinedby CD: at acidic pH (black squares and continuous line)and neutral pH (black circles and dotted line). At both pHvalues, the full-length reveals a two-state unfoldingmechanism.

3798 Insights into Prion Protein Folding and Stability

We further investigated MoPrP(23-231) andMoPrP(89-231) stability by thermal denaturation. Atacidic pH, MoPrP(23-231) thermal unfolding stillexhibits a two-state reversible cooperative processwith a melting temperature (Tm) of 68 °C (Supple-mentary Fig. 3a and b), regardless of the scan rate(data not shown). Data analysis using statisticalthermodynamics shows only two states, native andunfolded, confirming the two-state transition mech-anism previously observed (Supplementary Fig. 3c).ΔG25 °C values calculated from differential scanningcalorimetry (DSC) experiments are higher thanthose obtained from circular dichroism (CD)(Table 2). Calorimetry measures the thermallyinduced transitions, taking into account the overallenergetics of protein folding/unfolding reactions.This suggests an interaction between the unfoldedN-terminal domain and the globular part. In contrastto urea denaturation, at neutral pH, MoPrP(23-231)is less stable (Tm, 65 °C) and the unfolding processis irreversible as shown by the decrease in meltingtemperature, reduced ellipticity, and free-energyvalues after cooling (Supplementary Fig. 4a andSupplementary Table 1).Calorimetric experiments on MoPrP(89-231) con-

firm the presence of two thermodynamically distinctdomains with melting temperatures of 52.8 °C (Tm1)and 68.3 °C (Tm2), respectively (Supplementary Fig.

Table 1. Thermodynamic parameters of MoPrP(23-231) anequilibrium experiments

Protein ΔGH2O (kca

MoPrP(23-231), pH 5.5 4.3 ± 0.007MoPrP(23-231), pH 7 5.9 ± 0.081

Fluorescence CMoPrP(89-231), pH 5.5 Native–intermediate 2.6 ± 0.02 2

Intermediate–unfolded 1.65 ± 0.014 1MoPrP(89-231), pH 7 2.1 ± 0.001

5a). A repeated DSC scan after cooling shows thefull irreversibility of the first thermodynamic domainand the partial reversibility of the main endothermictransition. CD experiments reveal that the thermallyinduced transition starts around 40–45 °C and it ischaracterized by an asymmetric denaturing curvereflecting a three-state unfolding mechanism (Sup-plementary Fig. 5b). Changes in calorimetric profiles

d MoPrP(89-231) unfolding processes determined from

l/mol) m (kcal mol−1 M−1) [Urea]1/2 (M)

−0.697 ± 0.002 5.7−0.92 ± 0.016 6.3

D Fluorescence CD.29 ± 0.3 −0.54 ± 0.007 −0.32 ± 0.01 5.2.77 ± 0.013 −0.231 ± 0.002 −0.259 ± 0.002

−0.362 ± 0.0005 5.2

Table 2. Thermodynamic parameters of MoPrP(23-231) and MoPrP(89-231) thermal unfolding processes at acidic pH

Protein Method Tm (°C) ΔG25 °C (kcal/mol) ΔHm (kcal/mol) ΔCp (kcal mol−1 °C−1)

MoPrP(23-231), pH 5.5 DSC Tm, 68.2 8.04 ΔH1, 65.9 ± 0.32 0.171Tm,rev, 68.2 6.91 ΔHrev, 59.3 ± 0.4

MoPrP(23-231), pH 5.5 CD Tm, 68.1 3.94 ΔH1, 27.9 ± 0.68Tm,rev, 67.6 3.99 ΔHrev, 28.1 ± 0.68

MoPrP(89-231), pH 5.5 DSC Tm1, 52.8 0.54 ΔH1, 5.72 ± 0.11 0.232Tm2, 68.3 2.56 ΔH2, 20.8 ± 0.1Tm,rev, 68.4 1.78 ΔHrev, 14.2 ± 0.1

MoPrP(89-231), pH 5.5 CD Tm1, 65.8 2.14 ΔH1, 18.19 ± 0.26Tm,rev, 65.1 1.60 ΔHrev, 13.87 ± 0.15

3799Insights into Prion Protein Folding and Stability

and the reduced ellipticity values after cooling dependonanon-fully reversible thermal unfoldingmechanism.The non-fully reversible transition is confirmed by thereduced free-energy and enthalpy values obtainedanalyzing the second scan (Table 2). ΔG25 °C andenthalpy values from DSC and CD are similar butlower than thoseobtained using full-length protein, andthis strengthens the role of the N-terminal tail instabilizing the folded domain. Similar to full-lengthprotein, at neutral pH, the thermal unfolding process ofthe truncated form is fully irreversible and the proteintends to precipitate (Supplementary Fig. 4b andSupplementary Table 1). The same melting tempera-tures obtained at acidic and neutral pH confirm thatMoPrP(89-231) stability is pH independent.

The OR Plays a Central Role in PrionProtein Folding and Stability

To identify the key structural element of theN-terminal domain stabilizing the prion protein, wetook advantage of a polymorphism that occurs in thehuman prion protein and results in the absence of anoctapeptide [27,28]. We compared thermal unfoldingcurves at acidic pH of full-length human prion protein[HuPrP(23-231)], full-length human prion protein with-out the octapeptide [HuPrP(23-231, -1OP)], and thetruncated form [HuPrP(90-231)], as reported in Fig. 3.While the thermal unfolding process of HuPrP(23-231)is fully reversible (Fig. 3a), the deletion of only oneoctapeptide—HuPrP(23-231, -1OP)—reduces theα-helical content and determines the loss of reversibil-ity, as is the casewith the truncated form (Fig. 3b andc).

Identification of a Highly EnthalpicallyStable Intermediate State

At acidic pH, the equilibrium unfolding process ofMoPrP(89-231) is characterized by a three-statetransition. To better investigate the thermodynamicparameters of the intermediate state, we performedunfolding curves at three different temperatures(15 °C, 25 °C, and 37 °C). Under all the experimen-tal conditions, the unfolding curves reveal a three-state unfolding mechanism, with the intermediate

state favored at lower temperatures (Fig. 4). At thelowest temperature (15 °C), the intermediate statedominates at 3.6 M urea, whereas at 25 °C and37 °C, it is represented more at 4.4 M and 5.2 Murea, respectively (Supplementary Fig. 6). Theintermediate-state relative population decreasesas a function of temperature, from 0.93 at 15 °C to0.79 at 37 °C. This enthalpically stable intermediatestate is stabilized with respect to the native state byapproximately 20 kcal/mol. Free-energy and [Urea]1/2values confirm that intermediate-state formation isfavored at low temperatures (Table 3).

Computational Search of AlternativePrion Protein Folds

Taking advantage of molecular dynamic simula-tions, we explored PrP folding landscape to identifystructural regions involved in the transition towardhighly enthalpically stable states. To explore alterna-tive conformations of PrP that are accessible from thenative state, we carried out extensive atomisticsimulations exploiting an accurate all-atom descriptionand state-of-the-art enhanced sampling techniques.Simulationswere performed starting fromHuPrP (PDBaccession code 1HJM) [29] at 300 K by classicalmolecular dynamics in explicit water under acidicconditions and at effective protein concentration of6.6 mM. To enhance the probability of observingtransitions toward alternative conformations, we usedmetadynamics [30] and bias-exchangemetadynamics[31] techniques, which have been extensivelyemployed to study folding and aggregation processes.[32–35]

Alternative Folds of PrP

First, we performed bias-exchange metadynamicssimulations specifically aimed at exploring possiblealternative folds of PrP without estimating theirrelative stability. The calculations were extendedfor a total time of 1.5 μs, and the different confor-mations were identified by structural cluster analysis.During this lapse of time, the protein adopts tens ofclearly distinct conformations characterized by sig-nificantly high secondary structure content. Figure

Fig. 3. Thermal unfolding curves of HuPrP(23-231),HuPrP(23-231, -1OP), and HuPrP(90-231) by CD. (a) HuPrP(23-231); (b) HuPrP(23-231, -1OP); (c) HuPrP(90-231). Firstscan (black curve) and second scan after cooling (red curve).

Fig. 4. Unfolding curves of MoPrP(89-231) at pH 5.5 andthree different temperatures: 15 °C (dotted line), 25 °C(short dotted line), and 37 °C (continuous line). Theseunfolding curves are characterized by a three-state mech-anism. The intermediate state is favored at low temperature.The unfolded fraction is expressed with standard deviationobtained from three independent experiments.

3800 Insights into Prion Protein Folding and Stability

5a shows HuPrP native structure, while Fig. 5bdisplays the four structures that appeared morefrequently during the simulations. HuPrP⁎1 structureis very similar to the native fold. The main structuraldifference is the unfolding of the native β-sheet,whereas the three native α-helices are maintained.In HuPrP⁎2, an important secondary structurechange occurs in the first native α-helix. In

HuPrP⁎3 and HuPrP⁎4, different parts of the secondand third native α-helices are refolded into β-sheets.In order to assess the stability of these structures, we

computed their enthalpy. In concordance with aprevious metadynamics study [36], the nativeβ-sheet can be easily disrupted (HuPrP⁎1 structure),with an enthalpy penalty of less than 4 kcal/mol. Theother structures shown in Fig. 5b exhibited differentfolds and were viable candidate metastable states. Bysystematically computing the enthalpy of these struc-tures, we found that altering helices 2 and 3 causesenthalpy penalties exceeding 15 kcal/mol. In line withprevious experimental observations [37–39], theseresults suggest that helices 2 and 3 maintain theirfolding when native HuPrP is refolded into anotherconformation.These results prompted us to restrict the search for

alternative conformations with a second round ofexploratory metadynamics simulations only to theN-terminal part of the C-terminal domain. Indeed,structures with a significant rearrangement in thisregion are often characterized by a relatively lowenthalpy (Fig. 5c).We found thatHuPrP⁎5 is 5 kcal/molmore stable than the native fold. The main structuraldifferences lay in the formation of the β-bridge in adifferent sequence position and a small elongationof the native α-helix. HuPrP⁎6 and HuPrP⁎7 appearenthalpically favored with respect to the native foldby −23 and −31 kcal/mol, respectively. Both struc-tures share essentially the same secondary structure.Structure HuPrP⁎7 is more compact than HuPrP⁎6,with more intramolecular contacts and the lowestenthalpy. The reason for this remarkable enthalpicstability can be traced to two additional salt-bridges inthe tertiary structure that are not present in nativeHuPrP (Supplementary Fig. 7a). Additionally,

Table 3. Thermodynamic parameters of MoPrP(89-231) unfolding processes determined from equilibrium experiments atincreasing temperatures

Protein T (°C) [Urea]1/2 (M) ΔGH2O (kcal/mol)

MoPrP(89-231), pH 5.5 15 Native–intermediate 1.7 1.87Intermediate–unfolded 5.8 1.64

25 Native–intermediate 2.8 2.60Intermediate–unfolded 6.1 1.65

37 Native–intermediate 3.9 3.6Intermediate–unfolded 6.5 2.12

3801Insights into Prion Protein Folding and Stability

HuPrP⁎7 formsonaveragemorehydrogen bondswithsurrounding water (154 compared to 146 in the nativefold).

HuPrP⁎7 is a metastable fold of HuPrP

The impressive enthalpic stability of structuresHuPrP⁎6andHuPrP⁎7spurred us to further investigatetheir thermodynamic stability. To this end, we comput-ed free-energy differences among the native state,HuPrP⁎6, and HuPrP⁎7 by bias-exchange metady-namics, using a set of six CVs. Figure 6 shows thefree-energy as a function of two CVs (total RMSDvalues from HuPrP and HuPrP⁎7).The global free-energy minimum (M1) corresponds

to the native conformation. A metastable state (M2)was found very close to M1. In this state, the smallhelix 1 of the native PrP is partially unfolded, while therest of the structure is maintained. The difference infree-energy between the two conformations is almostnegligible (less than 1 kcal/mol). Two further meta-stable states (M3 and M4) are characterized by the

Fig. 5. Cartoon representations of the three-dimensional scode 1HJM); (b) alternative folds of HuPrP identified in the firstHuPrP identified in the second round of metadynamics simulaand loops are in cyan. The differences in enthalpy are also shonot colored and made transparent.

same secondary structure content and distribution asthose observed in HuPrP⁎7, although the tertiarycontacts are different. The structural differencesbetweenM3 andM4 are only due to the compactnessof the global fold, M4 being more compact than M3.Structure HuPrP⁎7 turns out to be only one represen-tative of a relatively large class of structures,belonging to a well-defined free-energy basin (aroundM4) and separated from the folded state by anenergetic barrier. The non-native β-sheet observedin HuPrP⁎7 is not always present in the structures withthe lowest free energy in this basin, although theirtertiary contacts are similar to it. This indicates that theβ-sheet can be formed transiently, at least. The basinincluding M4 and HuPrP⁎7 turns out to be ametastable state, higher in free-energy by approxi-mately 3 kcal/mol with respect to the native fold. Theimportant entropic decompensation (approximately34 kcal/mol) is possibly due to the 50% increase ofnon-polar solvent-accessible surface area (Supple-mentary Fig. 7b). Although the free-energy ofHuPrP⁎7 is marginally higher than that of the native

tructure of HuPrPC. (a) Native fold of HuPrPC (accessionround of metadynamics simulations; (c) alternative folds oftions. α-Helices are in purple/blue, β-sheets are in yellow,wn. Structural regions not activated by metadynamics are

Fig. 6. Free-energy surface of the refolding of HuPrP into HuPrP⁎7 as a function of (i) total RMSD of all protein Cα withrespect to native fold and (ii) total RMSD of all protein Cα with respect to alternative fold. Spheres represent the localminima position. Cartoon representations of the HuPrP structures representative of each metastable state identified in thefree-energy surface. Star indicates the location of HuPrP⁎7 structure.

Table 4. Enthalpy energy differences ofalternative fold (PrP⁎7) with respect to nativefold for different species

Species ΔH (kcal/mol)

Human −31.1 ± 1.4Mouse −23.1 ± 0.5Cattle −26.5 ± 1.8SheepARR −1 ± 0.5SheepARQ −28 ± 0.5

3802 Insights into Prion Protein Folding and Stability

state, an important free-energy barrier is clearly visiblealso in the two-dimensional projection in Fig. 6.Furthermore, unbiased molecular dynamics simula-tions of HuPrP⁎7 indicate that this state is stable for atleast hundreds of nanoseconds, thus suggesting thatit might actually be a metastable fold relevant to thePrP misfolding process.

PrP⁎7 is a Metastable Fold of PrP also in OtherSpecies

We searched for metastable folds in other speciesaffected by sporadic prion diseases, such as mouse,cow, and some sheep variants. As revealed by theiratomic structure, the globular domains of differentmammalian PrPC are very similar to each other [40].The structural superimposition reported in Supple-mentary Fig. 8 shows four significant sequencemismatches (low BLOSUM62 scoring): three ofthem are located at α-helix 1 in the structure, andtheother one is located at theα2-β2 loop.Among thesemismatches, the latter is critical because there is achange of charge in the amino acid between HuPrP(E168) and sheep PrP (R171). Thismismatch actuallycorresponds to a maximum scrapie resistance inOvPrPARR. Interestingly, sheep carrying the polymor-phism ARQ (OvPrPARQ), reverting the charge atposition 171, is generally susceptible to natural andexperimental scrapie [41].

The enthalpy differences between the native andalternative fold structures of PrP for the four mamma-lian species considered in this work were computedfollowing the same procedure as for the humanprotein. Since the PrP sequences of these speciesare highly similar (average sequence identity = 86%),point mutations along the structure were directlyintroduced on the native and alternative fold structuresof the human protein (HuPrP and HuPrP⁎7, respec-tively). Interestingly, the strong enthalpy stability of thealternative fold with respect to the native fold ismaintained in mouse, cow, and the sheep variantARQ, whereas the energetic difference appearsalmost negligible in the scrapie-resistant sheepvariant ARR (Table 4). These results indicate thatstructures such as HuPrP⁎7 may actually correspondto metastable folds of PrP in species susceptible toscrapie.

3803Insights into Prion Protein Folding and Stability

Effect of the Dielectric Constant onPrion Protein Folding and Stability

As mentioned above, PrPC is linked to the plasmamembrane through a GPI anchor. Close to themembranes, the dielectric constant (ε) is lower thanin bulk solutions. We therefore evaluated folding andstability of full-length and truncated MoPrP atdecreasing dielectric constant values.Full-length and truncated MoPrP exposed to

increasing trifluoroethanol (TFE) concentrationsshow significant differences in the 222 nm/208 nmellipticity ratio (Fig. 7). A 222 nm/208 nm ellipticityratio of 1.0 indicates interhelical contacts, such asthose present in coiled-coil or helix-bundle struc-tures. Alternatively, values in the range of 0.90indicate an elongated helix with little or no inter-helical contacts [42].At acidic pH, MoPrP(23-231) has an α-helix-bun-

dle organization that rearranges to an elongated

Fig. 7. Effect of decreasing dielectric constant onfull-length and truncated MoPrP folding. (a) The 222 nm/208 nm ellipticity ratio of MoPrP(23-231) at acidic andneutral pH. (b) The 222 nm/208 nm ellipticity ratio ofMoPrP(89-231) at the two pH values.

helix with helix–TFE contacts in the presence of20% TFE (v/v) (Fig. 7a). The initial ratio of 0.9 isdue to the unfolded N-terminal domain. At neutralpH, the 222 nm/208 nm ellipticity ratio decreasesat lower dielectric constant values (Fig. 7a). Thesedata, together with those obtained with urea,indicate that MoPrP(23-231) in the presence ofchemical denaturants is more stable at neutral pH.Conversely, MoPrP(89-231) is pH insensitive asalso observed when the protein was unfolded withurea (Fig. 7b).

Effect of the Disulfide Bridge on PrionProtein

Prion protein contains a disulfide bridge connect-ing α2-α3 helices. The role of the disulfide bond inprion conversion is controversial [43–47]. In vitrostudies have shown that its reduction under dena-turing conditions causes the loss of secondarystructure and disrupts native tertiary interactions[48]. To elucidate the role of the disulfide bridge inprion protein folding and stability, we investigatedthe best conditions to expose the disulfide bridge toreducing agents without dramatically altering sec-ondary and tertiary structures. To this end, weestablished that 4 M urea is the minimal concentra-tion required for exposing MoPrP to reducing agentDTT, as confirmed by unfolding experiments andbiotin-switch assay [49]. Free thiol groups wereblocked by alkylating with iodoacetamide, andreduced MoPrP(23-231) and MoPrP(89-231) sec-ondary structures were analyzed by CD. While boththe oxidized and reduced forms of the full-lengthprotein foldmainly in α-helices (Fig. 8a), the reducedform of MoPrP(89-231) has higher β-content thanthe oxidized one (Fig. 8b) with the subsequentexposure of hydrophobic regions (SupplementaryFig. 9). This finding agrees with recently publishedresults showing the detachment of α-helix 1 fromα-helices 2 and 3, as well as the increase inhydrophobic residues exposed to solvent in thereduced PrP form [50].We studied the effect of the disulfide bridge on the

thermodynamic stability of MoPrP by thermal unfold-ing. At acidic pH, thermal unfolding of reducedMoPrP(23-231) is a non-reversible two-state transi-tion mechanism. Indeed, melting temperature andfree-energy values decrease from 69 °C and4.1 kcal/mol to 66.8 °C and 2.7 kcal/mol. It isnoteworthy that the thermodynamic parameters ofthe first thermal unfolding transition correspond tothose obtained for the fully reversible oxidized form.Under similar experimental conditions, reducedMoPrP(89-231) unfolds over a very broad tempera-ture range, showing no well-defined cooperativetransition. Also in this case, the unfolding process isnot reversible.

Fig. 8. CD spectra of oxidized and reduced MoPrP atpH 5.5. (a) CD spectra of oxidized MoPrP(23-231) (contin-uous line) and reduced MoPrP(23-231) (dotted line); (b) CDspectra of oxidized MoPrP(89-231) (continuous line) andreduced MoPrP(89-231) (dotted line).

3804 Insights into Prion Protein Folding and Stability

Discussion

Epidemiological studies on Creutzfeldt-Jakob dis-ease have revealed that close to 90% of cases aresporadic, 8–10% are genetic, and less than 1% areiatrogenic [51]. Structural insights into PrP mutantshave provided some hints on the early events of theconversion and generation of genetic forms, such asthe loss of π-stacking interactions in the α2-β2 loop.Infectious forms of prion diseases are due to theintrinsic ability of prions to convert the physiologicalPrPC into its pathological form by acting as atemplate [14,52]. Limited information on sporadicforms is available. In this work, we combinedspectroscopic analyses and computer simulationsto identify structural elements and environmentalfactors affecting PrP stability and folding. To thisend, we used both full-length and truncated forms,since PrPC can be cleaved in vivo in the centralregion generating a soluble N-terminal domain andan anchored C-terminal globular domain [53,54].

Chemical denaturation studies show that full-lengthMoPrP is more stable than the truncated form and itsstability is pH dependent because of the histidineresidues in the OR. The pH dependency of full-lengthMoPrP has been confirmed performing folding andstability studies at decreasing dielectric constantvalues to mimic plasma membrane proximity. Also inthis case, full-length MoPrP is more stable at neutralpH than at acidic pH. The higher stability ofMoPrP(23-231) compared to MoPrP(89-231) hasbeen also confirmed by thermal unfolding. Full-lengthfree-energy values from DSC measurements arehigher than those obtained analyzing CD unfoldingcurves, suggesting an interaction between the unfold-ed N-terminal domain and the globular one. Calorim-etry, indeed, measures the thermally inducedtransitions taking into account the overall energeticsof protein folding/unfolding reactions. The N-terminaldomain confers reversibility to unfolding, as thermaldenaturation of the truncated form is not fully revers-ible. This confirms the interaction of the unfoldedN-terminal domain with the globular part and itsessential role in PrP folding, stability, and reversibility.The role of theN-terminal domain in stabilizingPrPandin conferring reversibility to the unfolding process isconfirmed also for HuPrP. Taking advantage of apolymorphism occurring in HuPrP, we show that theN-terminal domain exerts its effect throughoctapeptiderepeats. The absence of only one octapeptide in theN-terminal region prevents the fully reversible processafter thermal unfolding, resembling the truncated form.At acidic conditions, similar to those present in

endocytic compartments, denaturation of the truncat-ed form is characterized by a three-state transitionwith the intermediate state enthalpically stabilized byapproximately 20 kcal/mol. We investigated theenthalpically stable intermediate state and its confor-mational rearrangement also by molecular dynamicsin combination with metadynamics. This approachallowed us to explore in typical microsecond timesprocesses that usually take place over longertimescales (e.g., protein folding). First, we exploredthe conformational neighborhood of HuPrP globulardomain. Most identified conformers are less compactthan the native structure and strongly unfavored interms of enthalpy. Surprisingly, we observed a set ofconformations with a remarkable enthalpic stabilitycompared to the native state. These highly stableconformations are characterized by a repositioning ofα1-helix to residues 139–149 and a new β-sheetbetween residues 124–128 and residues 151–155.These conformations belong to a well-defined free-energy basin that is separated from the folded state byan energetic barrier. They are characterized by alarger number of intramolecular salt-bridges andintermolecular hydrogen bonds with water, not acces-sible from the native conformation. The tertiarytopology of the metastable state is compatible withan extension of this β-sheet, which could grow to a

3805Insights into Prion Protein Folding and Stability

platform involving the N-terminal part of the globulardomain. We speculate that this could happen uponbinding to another PrP molecule. A favorable interac-tion with a partner PrP is also suggested by thepresence, on the surface of the intermediate state, ofseveral hydrophobic residues, which are hidden in thecore of the native PrPC structure. A large body ofexperimental evidence [37–39,55] has supported therefolding of α1-helix in the conversion [40], as apromoter of PrPC aggregation. In particular, theidentified structural rearrangements of the enthalpicallystable intermediate state agree with the PrPSc modelproposed by Govaerts et al. [56]: this left-handedβ-helix model points to the rearrangement of PrPresidues from 90 to 170 into β-strands and subse-quently into β-helices [56].Our investigations reveal that the highly enthalpi-

cally stable states in PrPC folding landscape arepeculiar to prion-susceptible species such as mouse,cattle, and some sheep variants. Sheep PrP variantcarrying residues A136, R154, and R171, namelyOvPrPARR, is scrapie resistant and does not show theenthalpically stable state. On the contrary, OvPrPARQ

is susceptible to scrapie and shows the highlyenthalpically stable state. Charge reversion at position171 and the subsequent intermediate formationindicate that a basic amino acid in this region of theprotein may stabilize the native PrP structure. Thepresence of enthalpically stable states in prion-sus-ceptible species suggests a common path in thepathological conversion of PrPC.Under normal conditions, PrPC contains a disulfide

bridge connecting C178 and C213 in α2-α3 helices. Itsrole in prion conversion is still elusive, though it hasbeen previously reported that disulfide bridge reduc-tion under denaturing conditions causes the loss ofsecondary structure and disrupts native tertiary inter-actions [48]. Recently, molecular dynamics studies onreduced PrP showed structural changes similar tothose occurring at low pH, under denaturing condi-tions, and with pathological conditions, suggesting arole for disulfidebond in preventingPrPmisfolding [50].To disclose the role of disulfide bond in PrP folding andstability, we first investigated the minimal denaturantconcentration required to expose and reduce thedisulfide bond preserving the secondary structure. Inthe presence of 4 M urea, the disulfide bridge isreduced and full-length and truncatedMoPrP preservethe secondary structure. The reduced form of the full-length protein has lower α-helical content than theoxidized one, while reduced MoPrP(89-231) presentsa larger β-sheet content, as shown by the decrease inmolar ellipticity to 208 nm and 222 nm. Moreover,compared to the oxidized form, the reduced truncatedone has a larger hydrophobic region exposed to thesolvent, as indicated by higher 8-anilino-1-naphthale-nesulfonic acid (ANS) fluorescence values. Thermalunfolding of both forms is characterized by a non-re-versible process. However, the first thermal unfolding

transition of full-length protein generates parameters,such as melting temperature and free-energy, similarto those obtained for the fully reversible oxidized form.The reduced β-sheet-enriched truncated form unfoldsover a very broad range of temperature and aggre-gates. Therefore, the disulfide bridge has a key role inpreserving the native secondary structure, stabilizingboth full-length and truncated forms and preventinghydrophobic surface exposure that leads to misfoldingand aggregation.In conclusion, our study describes the higher stability

of full-length PrP compared to the truncated form. Thisis due to the presence of octapeptide repeats in theunfolded N-terminal region, as the polymorphismHuPrP(23-231, -1OP) behaves similarly toHuPrP(90-231). The unfolding process of the globulardomain is characterized by a three-state mechanismwith a highly enthalpically stable intermediate state.Enthalpically stable states are present in the foldinglandscape of prion-susceptible species, suggesting acommon path in the pathological prion protein conver-sion. Our study also describes the role of the disulfidebridge in preserving native folding. Indeed, reducingthe disulfide bridge under quasi-native conditionscauses major changes in MoPrP folding. In particular,the reduced truncated form of PrP shows an increasein β-sheet content confirmed by a higher hydrophobicsurface exposed to the solvent, causing aggregationduring the unfolding process.

Materials and Methods

Protein production

The plasmid pET-11a (Novagen) encoding for the full-length MoPrP(23-231) was kindly provided by Dr. J. R.Requena (University of Santiago de Compostela, Santiagode Compostela, Spain). MoPrP(89-231) was cloned in apET-11a vector using as PCR template the murine DNAgenome with NdeI-GGA ATT CCA TAT G GG C CA AGGAGG GGG TAC CCA T and BamHI-CGG GAT CCC TAGCTGGATCTTCTCCCGTCGTAA primers. The full-lengthHuPrP(23-231) was cloned according to Kosmač et al. [57],while the truncated form, HuPrP(90-231), was cloned in apET-11a vector using as PCR template the human DNAgenome with NdeI-GGA ATT CCA TAT GGG TCA AGGAGG TGG CAC CCA C and BamHI-CGG GAT CCC TAGCTC GAT CCT CTC TGG TAA TAG GCC obtained usingrestriction-free cloning. The HuPrP(23-231, -1OP) constructwas generated by using the restriction-free method [58,59].Human and murine constructs were expressed in Escher-ichia coli BL21 (DE3) cells (Stratagene). Freshly trans-formed overnight culture was inoculated into 10 LZYM-5052 medium [60] and 100 μg/mL ampicillin using aBioStat-B Plus fermentor. Cells were harvested after 24 h,lysed by French press (EmulsiFlex-C3), and repeatedlyrinsed in buffer containing 25 mM Tris–HCl, 5 mM ethyl-enediaminetetraacetic acid, 0.8% TritonX-100 (pH 8), andthen water. Inclusion bodies containing MoPrP(89-231) andHuPrP(90-231) were dissolved in 5 volumes of 8 M

3806 Insights into Prion Protein Folding and Stability

guanidine hydrochloride (GndHCl), loaded onto pre-equili-bratedHiLoad26/60Superdex200-pg column, andeluted in25 mM Tris–HCl (pH 8), 5 mM ethylenediaminetetraaceticacid, and 6 M GndHCl at a flow/rate of 2 mL/min.Subsequently, the protein was purified by reverse-phase(Jupiter C4, 250 mm × 21.2 mm, 300 Å pore size; Phenom-enex) and separated using a gradient of 0–95% acetonitrileand 0.1% tri f luoroacetic acid. MoPrP(23-231),HuPrP(23-231), and HuPrP(23-231, -1OP) were purifiedusing a 5-mL HisTrap column (GE Healthcare) pre-equili-brated in binding buffer [2 MGndHCl, 500 mMNaCl, 20 mMTris–HCl, and 20 mM imidazole (pH 8)] and eluted with500 mM imidazole. The protein was further purified inreverse-phase. Purified proteins were analyzed by SDS-po-lyacrylamide gel electrophoresis under reducing conditions,Western blot, and electrospray mass spectroscopy andwere lyophilized and stored at −80 °C. Refolding wasperformed by dialysis against refolding buffer [20 mMsodium acetate and 0.005% NaN3 (pH 5.5)] using aSpectrapor membrane (molecular weight, 3000).

Fluorescence spectroscopy and far-UV CDspectroscopy

Urea-induced denaturation was studied by fluorescencespectroscopy using LS 50B Perkin Elmer spectrofluorimetercoupledwithPTP-1FluorescencePeltier System. Theproteinwasexcitedat 280 nmand the fluorescenceemission spectrawere recorded in the wavelength range between 300 nm and450 nm. Slits were set at 5 nm for excitation and at 7.5 nm foremission. The path length of the sample was 1 cm and thescanspeedwas150 nm/min.Measurementswereperformedat protein concentration of 2 μM and pH values 5.5 and 7using acetate buffer and phosphate buffer with the same ionicstrength. Before measurements, the protein was incubated inthe presence of increasing urea concentrations for 2 h atworking temperature. Appropriate controls containing thedenaturant for the study were run. Each spectrum was theaverage of at least three scans.Hydrophobic regions were detected using ANS fluores-

cence. The protein was incubated at room temperature inthe presence of 100 μM ANS and emission spectra werecollected exciting at 380 nm and recording in thewavelength range 400–600 nm.The fraction of the unfolded protein, FU, was calculated

as follows:

FU ¼ fN − fð Þ= fN − fUð Þwhere f is the observed fluorescence at a given ureaconcentration, fU is the fluorescence of the completelyunfolded protein, and fN is the fluorescence of the nativeprotein.Folding/unfolding processes were also studied by

far-UV CD on JASCO model J-810 spectropolarimetercoupled with a Peltier System. All spectra were recordedby averaging two scans in the 185–260 nm range, using abandwidth of 2 nm and a time constant of 1 s at a scanspeed of 50 nm/min. Spectra were acquired at proteinconcentration of 4–6 μM, using HELLMA quartz cells withSuprasil windows and an optical path length of 0.1 cm.Samples in the presence of urea or the lipid mimetic

co-solvent TFE were incubated for 2 h at room tempera-ture, and CD spectra were collected with the same setup

described above. Thermal unfolding was investigated byCD in the 20–85 °C temperature range, using a scanspeed of 60 °C/h.The fraction of unfolded protein FU at various urea

concentrations was calculated using the following equa-tion:

FU ¼ θN − θð Þ= θN − θUð Þwhere θ is the observed molar ellipticity at a given ureaconcentration, θU is the molar ellipticity of the completelyunfolded state, and θN is the molar ellipticity of the nativeprotein. The free energy of the protein unfolding, ΔGU, canbe calculated by

ΔGU ¼ −RT ln FU

This ΔGU is linearly related to the concentration ofdenaturant, urea, as

ΔGU ¼ ΔGH2O − m Urea½ �where ΔGH2O is the apparent free energy of unfolding in theabsence of denaturant, and m depends on the denaturantbinding surface newly exposed in the unfolding reaction.Thermal denaturation curves were analyzed using the

Gibbs–Helmholtz equation [61]:

ΔG Tð Þ ¼ ΔHm 1−T =Tmð Þ þ ΔCp T−Tm−T ln T =Tmð Þð Þwhere Tm and ΔHm is the temperature and the enthalpy atthe midpoint of the thermal denaturation curve, respec-tively, and ΔCp is the heat capacity change.

Differential scanning calorimetry

Thermodynamic parameters underlying the folding/unfolding mechanisms of murine prion proteins weredetermined by DSC. Thermal denaturation experimentswere carried out on a VP-DSC microcalorimeter (MicroCal,Northampton, MA). Protein solutions, in a concentrationrange of 20–30 μM,were degassed for 8 min under vacuumat 20 °C for calorimetric measurements. The DSC scanswere run between 5 and 90 °C at a rate of 60 °C/h.Reversibility of the unfolding transition was estimated byrescanning the sample after cooling. To obtain the excessheat capacity (Cp,exc) curves, we recorded buffer–bufferbaseline scans at the same scanning rate and subtractedthem from the sample curve before further analysis. Thedata were analyzed using Origin 7.0 software (Origin Lab,Northampton, MA) to obtain thermodynamic parameters.

Disulfide bridge reduction

MoPrP(23-231) and MoPrP(89-231) were reduced in thepresence of 100 μM DTT, 100 μM iodoacetamide, and 4 Murea. After reduction, the proteins were dialyzed against20 mM acetate buffer (pH 5.5).

Molecular dynamics simulation

The initial set of atomic coordinates for human PrP(HuPrP)was obtained from thePDB (accession code1HJM)[29]. The ionization state of amino acid side chains was set

3807Insights into Prion Protein Folding and Stability

accordingly to acidic pH (between 4 and 5). The protein wasinserted into a cubic box of water molecules, ensuring thatthe solvent shell extended for at least 8 Å around thesystem.One chloride counterionwas added to neutralize thetotal charge of the system. Protein interactions wereparameterized with the AMBER99 force field [62] andwater molecules were described using the TIP3P model[63]. Periodic boundary conditionswere applied. Long-rangeelectrostatic interactions were treated with the particle meshEwald method [64], using a grid spacing of 1.2 Å combinedwith a fourth-order B-spline interpolation. The cutoff radiusfor the Lenard-Jones interactions and for the real part ofparticlemesh Ewald calculationswas set to 8 Å. The pair listwas updated every 10 steps, and the LINCS algorithm [65]was used to constrain all bond lengths. The integration timestep was set to 2 fs.The system was initially equilibrated imposing harmonic

position restraints of 1000 kJ mol−1 nm−2 on solute atoms,allowing the equilibration of the solvent without distortingthe solute structure. After an energy minimization of thesolvent and the solute without harmonic restraints, the totaltemperature of the system was gradually increased from 0to 298 K in 12 steps of 8 ps each. Constant temperature–pressure (T = 298 K, P = 1 bar) 20-ns dynamics was thenperformed through the Nosé–Hoover [66,67] and Ander-sen–Parrinello–Rahman [68] coupling schemes. The finalsimulation box was equilibrated at a volume of approxi-mately 64.8 nm × 64.7 nm × 64.2 nm. After this setup,the protein kept its native folding. This configuration wasthe starting point of all the metadynamics simulationsperformed in this work. All the simulations presented herewere performed under the same conditions employing theGROMACS4 simulation package [69].

Metadynamics simulations

Metadynamics [30,70] simulations were performed com-bining GROMACS4 with PLUMED plugin [71]. Bias-ex-change metadynamics [31] simulations were distributed in agrid-like architecture using in-house scripts that combineindependent and short normal metadynamics executions ofGROMACS4 + PLUMED. The collective variables (CVs)used in this work are based on RMSD values and areinspired by the variables introduced by Pietrucci and Laio[72]. These variables basically describe the total content ofα-helices and β-sheets in different regions of PrP structure.The first bias-exchange metadynamics simulation pre-

sented in this work used 10 such variables: (i) total α-helixcontent in amino acids 124–168; (ii) total α-helix content inamino acids 179–212; (iii) total α-helix content in aminoacids 169–178 and 213–227; (iv) total antiparallel β-sheetscontent in amino acids 124–168; (v) total antiparallelβ-sheets content in amino acids 124–133, 159–168, and179–212; (vi) total antiparallel β-sheets content in aminoacids 124–133, 159–178, and213–227; (vii) total antiparallelβ-sheets content in amino acids 134–158 and 179–212; (viii)total antiparallel β-sheets content in amino acids 134–158,169–178, and 213–227; (ix) total antiparallel β-sheetscontent in amino acids 179–212; and (x) total antiparallelβ-sheets content in amino acids 169–178 and 213–227.Twelve independent simulation replicas were executed,

each biased on one of these 10 CVs plus two free replicasnot biased on any CV. The following parameters of thetime-dependent bias potential terms were the same for

every replica: height = 4.0 kJ/mol; width = 0.15; deposi-tion time = 10 ps. Exchanges of the bias among replicaswere attempted every 50 ps and only accepted accordingto the Metropolis criterion introduced in Piana et al. [31].The simulation was extended up to 125 ns per replica(total simulation time of 1.5 μs).In the second metadynamics simulation, only half of

the N-terminal portion of the PrP globular domain wasexplored (amino acids 124–171). Two CVs were used:(i) total α-helix content and (ii) total antiparallelβ-sheet content. The simulation was biased on bothCVs in a single replica metadynamics execution. Theparameters of the time-dependent bias potential termswere as follows: height = 4.0 kJ/mol; width = [0.1–0.4];deposition time = 10 ps. The simulation was extended up to290 ns.The last bias-exchange metadynamics simulation pre-

sented in this work focused on refolding PrPC into analternative fold (named HuPrP⁎7 in Results). The followingsix CVs used here describe the total RMSD of proteinbackbone atoms from fragments of both native PrPC

structure and alternative fold: (i) total RMSD with respect tonative α-helix in amino acids 142–156; (ii) native β-sheet inamino acids 127–130 and 160–163; (iii) alternative α-helixin amino acids 138–149; (iv) alternative β-sheet in aminoacids 124–128 and 151–155; (v) total RMSD of all proteinCα with respect to native fold; and (vi) total RMSD of allprotein Cα with respect to alternative fold. Six replicas wereused, each biasing one of these CVs. The followingparameters of the time-dependent bias potential termswere the same for every replica: height = 2.0 kJ/mol;width = 0.12 Å; deposition time = 10 ps. Exchanges of thebias among replicas were attempted every 50 ps. Thesimulation was extended up to 250 ns per replica (totalsimulation time of 1.5 μs). In order to avoid systematicerrors at the boundaries of the CVs, we applied theinversion condition [73] together with a soft wall potential oforder 2. The lower limit was set at 0.0 Å for all the CVs andthe upper limit is set to 7 Å, 16 Å, 6 Å, 16 Å, 17 Å, and14 Å for each CV, respectively. The inversion scalingfactor was set to 6, the reflection distance was set at 1.5 Å,and the maximum hills height was set at 2.0 kJ/mol.

Structure clustering

The large number of structures sampled during all thesimulations were grouped in structurally similar clustersfollowing the method introduced in Daura et al. [74]. Thismethod is based on the RMSD values between all thestructures sampled during the simulation. Approximately25 structurally different relevant clusters were identified ineach simulation using an RMSD cutoff of 2.0 Å. Themiddle structure of each cluster was selected as therepresentative for further characterization.

Enthalpic stability calculation

The relative enthalpies between all the different con-formers identified in this work were determined fromaverages in total potential energy. The averages werecomputed over the structures sampled during longindependent molecular dynamics simulations (200 ns) foreach conformer.

3808 Insights into Prion Protein Folding and Stability

Multidimensional free-energy landscape

The data gathered in the last bias-exchange metady-namics simulation formed the basis to determine thethermodynamics of the relevant conformational states ofPrPC refolding between the two conformations. Theanalysis was performed using METAGUI [75], a visualanalysis tool implemented for VMD‡ [76]. The conforma-tional space explored was discretized as a function of twocollective variables: (i) total RMSD of all protein Cα withrespect to native fold and (ii) total RMSD of all protein Cα

with respect to alternative fold. These variables made itpossible to assign unambiguously all the structuressampled during the simulation to different microstates.The bidimensional CV space was discretized in bins of0.2 Å in each direction. Structures explored duringsimulations were assigned to different microstates if theircorresponding CV values fell within the limits of a givenbin. The relative free energies between the differentmicrostates were then obtained by a weighted-histogramanalysis procedure, as described in Marinelli et al. [77].This procedure corrects the (non-equilibrium) population ofevery microstate observed during the biased simulations,with the average metadynamics biases acting on them. Inthis simulation, the bias potentials start growing evenlyafter 140 ns (Supplementary Fig. 10). The convergence ofthe bias potentials used for the re-weighting procedurewas further assessed by taking the average at two differenttime intervals after 140 ns (Supplementary Fig. 11). Thefree-energy estimator was obtained only from the con-nected segment of bias that deviates less than few kBTfrom the average of both time intervals (further details aregiven in Biarnés et al. [75]). Finally, the microstates wereclustered into kinetic basins, defining the different “free-energy wells”. This was accomplished by constructing anapproximate rate matrix among these microstates andanalyzing its spectrum [78].

Acknowledgments

E.R. and G.L. gratefully acknowledge MIUR(Italian Ministry of Education, University and Re-search) for partially supporting this work through thePRIN 2010-2011 Program (2010M2JART_001).X.B. acknowledges financial support from theGovernment of Catalonia (AGAUR) through aBeatriu de Pinós fellowship (BP-A-2007). Thiswork was supported by the European Union'sSeventh Framework Programme (FP7/2007–2013)under grant agreement number 222887—the PRI-ORITY project to G.L. The authors wish to thankAlessandro Laio for extensive discussions on themanuscript.

Appendix A. Supplementary data

Supplementary data to this article can be foundonline at http://dx.doi.org/10.1016/j.jmb.2014.09.017.

Received 3 April 2014;Received in revised form 30 July 2014;

Accepted 15 September 2014Available online 2 October 2014

Keywords:N-terminal domain;

disulfide bond;intermediate state;

prion-susceptible species;energy landscape

Present address: F. Benetti, European Center for theSustainable Impact of Nanotechnology, Veneto Nanotech

S.C.p.A., I-45100 Rovigo, Italy.

Present address: X. Biarnés, Laboratory of Biochemistry,Institut Químic de Sarrià, Universitat Ramon Llull, Via

Augusta 390, 08017 Barcelona, Spain.

†F.B. and X.B. contributed equally to this work.‡This plugin can be downloaded from http://www.plumed-

code.org/contributions.

Abbreviations used:PrP, prion protein; OR, octarepeat region; DSC, differ-

ential scanning calorimetry; TFE, trifluoroethanol; ANS, 8-anilino-1-naphthalenesulfonic acid; GndHCl, guanidine

hydrochloride.

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