Conformational Plasticity of the Gerstmann–Sträussler–Scheinker Disease Peptide as Indicated by Its Multiple Aggregation Pathways

doi:10.1016/j.jmb.2008.06.063 J. Mol. Biol. (2008) 381, 1349–1361

Available online at www.sciencedirect.com

Conformational Plasticity of theGerstmann–Sträussler–Scheinker Disease Peptide asIndicated by Its Multiple Aggregation Pathways

Antonino Natalello1,2, Valery V. Prokorov3, Fabrizio Tagliavini4,Michela Morbin4, Gianluigi Forloni5, Marten Beeg5, Claudia Manzoni5,Laura Colombo5, Marco Gobbi5, Mario Salmona5

and Silvia Maria Doglia1,2⁎
1Department of Biotechnologyand Biosciences, University ofMilano-Bicocca, Piazza dellaScienza 2, 20126 Milan, Italy2Consorzio NazionaleInteruniversitario per le Scienzefisiche della Materia (CNISM)UdR Milano-Bicocca, Via Cozzi53, 20125 Milan, Italy3Probe Microscopy Group,M.M. Shemyakin & Yu.A.Ovchinnikov Institute ofBioorganic Chemistry, RussianAcademy of Sciences, Ul.Miklukho-Maklaya, 16/10,117997 GSP, Moscow V-437,Russia4Fondazione I.R.C.C.S. IstitutoNeurologico Carlo Besta, ViaCeloria 11, 20133 Milan, Italy5Istituto di RicercheFarmacologiche “Mario Negri”,Via G. La Masa, 19, 20156Milan, Italy
Received 28 March 2008;received in revised form17 June 2008;accepted 21 June 2008Available online28 June 2008

*Corresponding author.Department2, 20126 Milan, Italy. E-mail addressAbbreviations used: AFM, atomic

EM, electron microscopy; GSS, Gersconfocal fluorescence microscopy; T

0022-2836/$ - see front matter © 2008 E

The existence of several prion strains and their capacity of overcomingspecies barriers seem to point to a high conformational adaptability of theprion protein. To investigate this structural plasticity, we studied here theaggregation pathways of the human prion peptide PrP82–146, a majorcomponent of the Gerstmann–Sträussler–Scheinker amyloid disease.By Fourier transform infrared (FT-IR) spectroscopy, electron microscopy,

and atomic force microscopy (AFM), we monitored the time course ofPrP82–146 fibril formation. After incubation at 37 °C, the unfolded peptidewas found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands. At a critical oligomer concentration, the emergence ofa new FT-IR band allowed to detect fibril formation. A differentintermolecular β-sheet interaction of the peptides in oligomers and infibrils is, therefore, detected by FT-IR spectroscopy, which, in addition,suggests a parallel orientation of the cross β-sheet structures of PrP82–146fibrils. By AFM, a wide distribution of PrP82–146 oligomer volumes—thesmallest ones containing from 5 to 30 peptides—was observed. Interest-ingly, the statistical analysis of AFM data enabled us to detect a quantizationin the oligomer height values differing by steps of∼0.5 nm that could reflectan orientation of oligomer β-strands parallel with the sample surface.Different morphologies were also detected for fibrils that displayed highheterogeneity in their twisting periodicity and a complex hierarchicalassembly.Thermal aggregation of PrP82–146 was also investigated by FT-IR

spectroscopy, which indicated for these aggregates an intermolecular β-sheet interaction different from that observed for oligomers and fibrils.Unexpectedly, random aggregates, induced by solvent evaporation, werefound to display a significant α-helical structure as well as several β-sheetcomponents.All these results clearly point to a high plasticity of the PrP82–146 peptide,

which was found to be capable of undergoing several aggregationpathways, with end products displaying different secondary structuresand intermolecular interactions.

© 2008 Elsevier Ltd. All rights reserved.

Keywords: amyloid; atomic force microscopy; Fourier transform infraredspectroscopy; prion; protein aggregation
Edited by S. Radford
of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza: [email protected] microscopy; ATR, attenuated total reflection; FT-IR, Fourier transform infrared;tmann–Sträussler–Scheinker; HFIP, hexafluoroisopropanol; LSCFM, laser scanninghT, thioflavin T; TFA, trifluoroacetic acid; PBS, phosphate-buffered saline.

lsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.jmb.2008.06.063

1350 Conformational Plasticity of PrP82–146

Introduction

Prion diseases are neurological disorders found inhumans and animals, which are due to the conversionof the native cellular prion protein (PrPC) into a patho-logical PrPSc conformation. While PrPC is mainly anα-helical protein sensible to protease digestion, PrPSc

displays a high β-sheet content and resistance toprotease.1–4

Among prion diseases, the Gerstmann–Sträussler–Scheinker (GSS) disease is an inherited disordercharacterized by the deposition of amyloid plaquesin the cerebrum and cerebellum.5,6 A major compo-nent of amyloid fibrils purified from brains of GSSpatients was found to be an internal PrP fragment of7 kDa spanning from residues 81–82 to 144–153.5–7

This fragment was found to be the smallest segmentthat can lead to amyloidogenesis.7 Low molecularweight fragments have also been detected even inareas not interested by amyloid deposition.8–10 How-ever, all these fragments contained the PrP spanningregion 89–140, which is known to be fundamentalfor the conformational transition of PrPC intoPrPSc.11,12 It has been seen that the human syntheticPrP peptide from residue 82 to 146 (PrP82–146)forms amyloid fibrils, which bind thioflavin S andare birefringent under polarized light after Congored staining.13 Interestingly, PrP82–146 was foundto interact with biological membranes13 and to formion channels in lipid bilayers.14 For these reasons,the human PrP82–146 peptide has been used as amodel system to investigate the peculiar features ofamyloid polymerization,15 the anti-prionic proper-ties of tetracyclines,16 and the role of metal ions infibrillogenesis.17 Interestingly, PrP82–146 oligomerswere found to have biological activities triggeringneuron cell death and activating a gliotrophicresponse.18 To highlight the conformational plasti-city of the PrP82–146 prion peptide, we studied itskinetics of aggregation in different experimentalconditions and the structural properties of theaggregation end products by Fourier transforminfrared (FT-IR) spectroscopy. This technique hasbeen proven to be a powerful tool for the structuralcharacterization at the molecular level of amyloidand prion proteins.19–21 Laser scanning confocalfluorescence microscopy (LSCFM), electron micro-scopy (EM), and high-resolution atomic forcemicroscopy (AFM) were also employed to highlightthe ultrastructures of PrP82–146 fibrils. The PrP82–146 aggregation induced by thermal treatment andby solvent evaporation is also reported.Evidence is provided for the high plasticity of the

PrP82–146 peptide, which was found to aggregatealong several pathways, each leading to assembliescharacterized by different peptide secondary struc-tures and intermolecular interactions.We noted that the PrP82–146 peptide contains the

residue sequence 90–106, which was recently foundto participate in the dimer interactions during theearly aggregation of Syrian hamster PrP90–231.22

Moreover, the peptide studied here contains also the

88–98 sequence that was seen to be involved in thenucleation process of the mouse PrP23–230 fibrilformation.23

Results

Kinetics of PrP82–146 aggregation: FT-IRspectroscopy of unordered peptides, low-orderoligomers, and fibrils

The assembly of the PrP82–146 peptide was invest-igated by FT-IR spectroscopy. Since this process hasbeen found to depend critically on the startingconditions of the peptide solution, namely, on thepresence of preformed aggregates, PrP82–146 wasfirstly solubilized in hexafluoroisopropanol (HFIP).The solvent was then removed by evaporation toobtain a protein film that was subsequently dissolvedin D2O at a concentration of 5 mg/ml. The infraredabsorption spectra, collected immediately and afterincubation at 37 °C at different times up to 2 months,are presented in Fig. 1a. The second derivatives of themeasured spectra are reported in Fig. 1b in the AmideI region (1700–1600 cm−1), in order to identify thepeptide secondary structure and the intermolecularβ-sheet bands characteristic of aggregation.24,25 Allthe spectra in Fig. 1a and b display an intense band at1673 cm−1, due to a residual trace of trifluoroaceticacid (TFA)26 that, in spite of its very low concentra-tion, gives rise to a strong infrared absorption due toits high extinction coefficient. At the beginning ofincubation at 37 °C, in addition to this band, only abroad component was observed around 1645 cm−1,which can be assigned to the peptide's random coilstructure;27,28 no intermolecular β-sheet bands—indicative of aggregation—were detected at thisstage. The observed random coil structure is actuallyan expected result. Indeed, the NMR study of the full-length human PrP protein indicated that the 23–144sequence is unordered with the exception of the short128–131 β-strand.4 At later times of incubation, up toapproximately 7 days, two components of very lowintensity appeared at 1623 cm−1 and—as a weakshoulder—around 1690 cm−1, as can be seen in Fig.1b and c. These bands can be assigned to theintermolecular β-sheet interactions of the peptide inthe early aggregates that are forming in solution, asclearly indicated by the lowwavenumber componentaround 1623 cm−1 that is a marker of aggrega-tion.19,28–30 It is interesting to note that the spectra ofthe early stages of aggregation (up to 7 days) displayan isosbestic point around 1627 cm−1, which is astrong indication that the unfolded peptides wereconverting into β-sheet-rich aggregates. After a lagtime of approximately 3–7 days of incubation, whichwas found to vary in different experiments, a newpeak component around 1626 cm− 1 appearedabruptly, as shown in Fig. 1d, where the intensity ofthis band versus incubation time is reported. After alag phase, a sudden increase of this band wasobserved fromday 7 to day 10, followed by a constant

Fig. 1. Kinetics of fibril formation of the human PrP82–146 peptide in D2O. (a) FT-IR absorption spectra in the Amide Iregion of samples taken during the time course of aggregation: immediately after sample preparation and after 3,7–11 days of incubation at 37 °C. Arrows point toward increasing time. (b) FT-IR second derivatives of the absorptionspectra in (a). (c) FT-IR second-derivative spectra at time 0 and 3, 7, and 8 days from (b), reported on an enlarged scale inthe β-sheet absorption region from 1635 to 1613 cm−1. (d) Kinetics of aggregation monitored by the β-sheet bandintensities at 1626 and 1623 cm−1 taken from (b). The reported data refer to a single experiment. The same trend wasalways observed (four independent experiments), with a variability in the lag time length.

1351Conformational Plasticity of PrP82–146

plateau reached around days 10 and 11. The1626 cm−1 band can be assigned to PrP82–146 fibrilsfrom the LSCFM, EM, and AFM results reported inthe next paragraphs. Figure 1d also reports the timecourse of the 1623 cm−1 band intensity, which differsfrom that of the 1626 cm−1 fibril band. The 1626 cm−1

component—as expected—is absent up to the suddenfibril formation, while the 1623 cm−1 band displays aminor but significant intensity increase with timeduring the lag phase. This temporal evolution and theEM andAFM studies reported in the next paragraphssuggest that the 1623 cm−1 band can be taken as anoligomer signature. At the end of the lag phase, in Fig.1d, the two 1626 cm−1 and 1623 cm−1 band intensitiesseem to merge together, due to the rapid increase ofthe 1626 cm−1 fibril component that covers the1623 cm−1 oligomer response.The spectra of PrP82–146 fibrils are characterized

by a single sharp cross β-sheet band, whose peakposition around 1626 cm−1 was found to be constantover a long span of time (2 months). It is worthnoting that during fibril formation, the growth of the1626 cm−1 band is not accompanied by the growth ofa second component around 1690 cm−1, as observedduring oligomer formation. The single aggregationpeak at 1626 cm−1 indicates that a parallel inter-

molecular β-sheet interaction characterizes thePrP82–146 fibrils.25,31

In Fig. 2, the FT-IR spectra of PrP82–146 fibrils inD2O solution are reported at two spectral resolutions(2 and 1 cm−1) to verify that the 1626 cm−1 peak isdue to a single component. The fibril spectrumdisplays also a broad component around 1648 cm−1

that can be assigned to random coil structures20,27,28

of the PrP82–146 polypeptide chain. Thermal treat-ment up to 100 °C of the fibrils was found to induceonly aminor increase of the 1626 cm−1 band intensity(data not shown).

FT-IR spectra of thermal and random PrP82–146aggregates

The spectrum of thermal aggregates, obtained byheating the peptide solution (previously treated byHFIP) up to 100 °C and then cooling it down to37 °C, is reported in Fig. 2. Two components at1690 cm−1 and at 1622 cm−1 were observed, whichare typical of aggregates with antiparallel β-sheetinteraction.25,29,30,32–34 It should be noted that, aftera thermal cycle of 37 °C→100 °C→37 °C, the aggre-gate spectrum was similar to that measured at

Fig. 2. FT-IR spectra of PrP82–146 fibrils and thermalaggregates. (a) FT-IR absorption spectra of fibrils at aresolution of 1 cm−1 (····) and at 2 cm−1 (—). Thespectrum of thermal aggregates at a resolution of 2 cm−1

(- - -) is also reported to compare the response of the twoaggregates. (b) Second derivatives of absorption spectrareported in (a).

Fig. 3. ATR/FT-IR spectra of PrP82–146 random aggre-gates. (a) ATR/FT-IR absorption spectra of PrP82–146random aggregates obtained by water evaporation of thepeptide solution. Spectra from two independent samplepreparations are reported. (b) Second derivatives of spectrain (a), presented after normalization for protein content atthe tyrosine 1517 cm−1 band. α-Helical secondary struc-tures are evident around 1659 cm−1, in addition to severalβ-sheet components around 1698 cm−1 and in the range1633–1623 cm−1.


100 °C (data not shown), indicating that thermalaggregation is an irreversible process.In order to explore other aggregation pathways of

PrP82–146, we investigated by FT-IR spectroscopythe formation of random aggregates35 obtained byputting a small amount of PrP82–146 water solutionon an attenuated total reflection (ATR) diamondplate and then letting the bulk solvent evaporate atroom temperature. The random aggregate spectra oftwo independent samples—taken as examples—arereported in Fig. 3. Even if a high spectral hetero-geneity characterizes the response of these aggre-gates, specific band components were usuallyobserved in the Amide I region. In particular, anα-helical structure,35 whose intensity was found tovary from sample to sample, was always seenaround 1659 cm−1. In addition, several β-sheet com-ponents were present in the region 1633–1623 cm−1

together with the high wavenumber β-sheet bandaround 1698 cm−1 (Fig. 3). This result, therefore,indicates that when random aggregates are induced,the PrP82–146 peptide undergoes a conformationaltransition from an unordered structure into α-helixandβ-sheet structures. Instead,whenmature PrP82–146 fibrils were measured in the form of protein film

on the ATR plate, their absorption spectrum wasfound to be identical with that measured in trans-mission mode for the sample in solution (data notshown), as expected for a rigid structure.

Overview of PrP82–146 fibril formation obtainedby EM and LSCFM

An overview of fibril formation and of the struc-tural properties of PrP82–146 oligomers and fibrilswas first obtained by EM and LSCFM. A detailedmorphological analysis of oligomers and fibrils,performed by high-resolution AFM, will be reportedin the next paragraph.

Electron microscopy

EMmicrographs of PrP82–146 after different timesof incubation at 37 °C are reported in Fig. 4. Imme-diately after dissolution, a low degree of aggregationwas observed (Fig. 4a). To verify that the gray areasin Fig. 4a were due to PrP82–146 and not to artifacts,

Fig. 4. Electron micrographs of PrP82–146 at different times of incubation at 37 °C. (a) Immediately after samplepreparation; (b) immunogold staining performedwith 3F4 antibody of sample as in (a); (c) 18 h after incubation; (d) 7 daysafter incubation; (e) 15 days after incubation. Magnification of (c–e) are reported, respectively, in (c1), (c2), (d1), (d2), (e1),and (e2) where 25-nm scale bars are indicated.


we have performed immunogold labeling with themonoclonal antiboby 3F4. As shown in Fig. 4b, thepresence of PrP82–146 deposits, evenly distributed onthe grid, was confirmed. After 18 h of incubation, anincrease of morphologically defined peptide assem-bly was observed. In particular, EM micrographshowed round-shaped particles, with an averagediameter ranging from 20 to 60 nm, organized in a“rosary like” structure (Fig. 4c, c1, and c2). Seven daysafter incubation at 37 °C, a spongiform network ofPrP82–146 aggregateswas observed (Fig. 4d), indicat-ing (Fig. 4d1 and d2) the presence of small structureswith round or rod-like shape at higher magnification.Fifteen days after incubation, PrP82–146 formed fullyassembled amyloid fibrils (Fig. 4e) with a diameterranging from 5 to 8 nm (Fig. 4e1 and e2).

Laser scanning confocal fluorescence microscopy

Even if detailed structural information cannot beobtained by LSCFM, due to the low spatial resolutionof this technique (≥0.1 μm), fibril formation can stillbe monitored in a rapid way through the binding ofthioflavin T (ThT),36 a well-known fluorescent probeof amyloids.37 Indeed, LSCFM, an easy-to-use tech-nique that enables studying large sample areas, is auseful complementary technique to monitor peptideassembly on the same samples examined by FT-IRspectroscopy. LSCFM images of PrP82–146 fibrils inthe presence of ThT show the presence of dispersedfibrils or fibril bundles (Fig. 5a) and of large fibrilsupramolecular assemblies38 (Fig. 5b and c).

High-resolution AFM of PrP82–146 oligomers andfibrils

To characterize the morphology of PrP82–146oligomer and fibril structures, we report here theresults of high-resolution AFM measurements

undertaken on the very same samples studied byFT-IR spectroscopy.AFM images of PrP82–146 in the early stage of

aggregation (3–7 days) are presented in Figs. 6 and 7,where the coexistence of globular oligomers andfibrillar structures, over a dense grainy background,is evident.

AFM of oligomers

Globular oligomers in Figs. 6 and 7 display a largedispersion in heights and lateral dimensions asindicated by height image analysis. The smallestoligomers (particles 1 and 2 in Fig. 7a) have roundshapes, while larger oligomers have often smoothedrectangular shapes. Rectangular shapes with acentral depression (Fig. 7b) are evident for largeroligomers, suggesting a toroid topology similar tothat reported for α-synuclein.39 Actually, oligomershapes seem to be smoothed as a consequence of thebroadening effect due to the probe curvature radius.The oligomer lateral dimension and volume areaffected by the AFM probe curvature and by theheight of the structure (see Supplemental Fig. S1).Therefore, to evaluate oligomer volumes, we per-formed AFM measurements with the use of ultra-sharp whiskers (as described in Materials andMethods) in order to obtain reliable height valuesand correct the volume data by the broadeningcoefficient. The value of this coefficient (typicallybetween 2 and 4) has been determined from theheight profile of the structure as illustrated inSupplemental Fig. S1.The statistical analysis of the measured oligomer

heights and volumes is presented in Fig. 7e, f, and g.As it can be seen, the oligomer height value rangesfrom 1.5 to 10 nm, whereas their volumes vary fromseveral hundreds to several thousands of cubicnanometers. A rough estimate of the number of

Fig. 5. LSCFM images of PrP82–146 fibrils stained by ThT, after incubation at 37 °C for 20 days. Images displaying (a)dispersed fibrils or fibril bundles and (b and c) supramolecular assemblies of fibrils. Scale bars of 20 μm are reported ineach image.


molecules inside each oligomer can be, therefore,obtained from the volume values (appropriatelycorrected for the broadening effect), considering thata volume of 1 nm3 can be associated to a globularprotein with amolecular mass of about 800 Da.40 Forinstance, considering the PrP82–146 molecular massof 6.550 kDa, the number of peptides within a singleoligomerwithmeasured volume ranging from 250 to600 nm3 is found to vary from 5–10 to 15–30.

Fig. 6. AFM images of PrP82–146 oligomers and fibrils.(a) Height image of oligomers and fibrils. (b and c) Theheight profile analysis of fibrils F1 and F2 from (a), betweenthe positions marked by triangles. In fibril F2, a gap ofreduced height can be seen, as indicated by the dottedarrows (a and c), suggesting the presence of two or moreprotofilaments outside the gap. Sample areas marked bydashed rectangles in (a) are reported with higher magni-fication in Fig. 7a and b.

In addition, from the histogram of Fig. 7e, it ispossible to see that oligomer heights that are morefrequent, in the range from 1.5 to 4 nm, differ onefrom the other by increments of ∼0.5 nm, occurringat 2.00, 2.56, 3.13, and 3.64 nm. This quantizationindicates that the oligomer structure is periodicallyordered in the direction normal to the surface.Interestingly, the ∼0.5 nm difference in the heightpeaks could reflect the spacing between strands ofintermolecular β-sheets, which were observed inPrP82–146 oligomers by FT-IR spectroscopy (seeFig. 1). Indeed, a strand spacing of about 0.48 nmwas typically found in β-sheet structures.41

The observation of this periodicity in the normaldirection implies that in small oligomers, β-strandsare parallel with the sample surface and, conse-quently, intermolecular β-sheets are normal to it(opposite towhat occurs in fibrils whereβ-sheets runparallel with the sample surface).A similar pattern could be also present for larger

oligomers, but the statistical analysis of their datadoes not clearly confirm a height quantization.In addition,we shouldnote that in Figs. 6 and 7a–d,

oligomers with height less than 1.5 nm are notevident, as they seem to be embedded in a grainybackground. However, by using ultrasharp probeswith very high lateral resolution, the backgroundstructure can be identified (Fig. 7h). The backgroundappears to be made by linear and annular structures.Linear structures (one is selected by a rectangle inFig. 7h) have lengths ranging from 5 to 10 nm and anapparent width of about 2–3 nm. Interestingly, theannular objects (see the dotted circle in Fig. 7h) aresimilar to annular protofibrils already reported forother proteins such as α-synuclein.42

AFM of fibrils

The PrP82–146 fibrils (see Figs. 6, 8, and 9) displaylengths that reach severalmicrometers, heights in therange of∼3–10 nm, and twisting periodicity varyingfrom 30 to 130 nm, depending on their height. Fibrilswithout apparent periodicity were very rarelyobserved. The largest periodicity (∼100–130 nm)

Fig. 7. AFM images of PrP82–146 oligomers. (a–d) Height images of oligomers with different dimensions, markedfrom 1 to 6 for comparison with the data reported in (g). In (b), the two triangles mark the line along which the heightprofile is reported in Supplemental Fig. S1a. (e and f) Statistical distribution of height (e) and volume (f) of oligomers. Peakpositions in the height histogram, marked by vertical lines in the top of (e), display a separation of∼0.5 nm. (g) Oligomers'height–volume cross-correlation plot. The arrows with numbers 1 to 6 point to the height–volume coordinates of thecorresponding oligomers in (a), (c), and (d). The presence of two different branches is due to the use of cantilevers withdifferent curvatures, which induce a different broadening of lateral dimensions. (h) High-resolution AFM height image ofthe background layer in Fig. 6a obtained by ultrasharp probe operating in the “soft-repulsion” regime. Dashed circle andrectangle select annular and linear structures, respectively.


was found for thinner fibrils (height of 2–4 nm) andthe smallest periodicity (∼30–40 nm) was found forthicker fibrils with a height from 4 to 8 nm. It isnoteworthy to remark that the majority of fibrilsdisplay a clear periodicity along their axes that indi-cates a twist of two ormore subfilaments. In Fig. 8a, along isolated fibril is reported, with an enlargementin Fig. 8b, where two protofilament subunits can beseen to split and, subsequently, to merge together, aspointed by the arrows. Also, the fibril F2 in Fig. 6seems to be composed of two or more protofilamentsthat break along the fiber between the two dottedarrows,where only one protofilamentwith a height of∼2 nm (the lowest found in fibrils) is observed. Thisstructural feature is evident when fibril longitudinalcross sections are examined (Fig. 6c).The presence of more intertwining protofilament

subunits in isolated fibrils suggests that a hierarchicalmechanism of assembly43–45 is at work in PrP82–146fibril formation. Additional examples supporting acomplex hierarchical assembly are presented inFig. 9 in which triangles and an arrowmark the sites

where fibrils split, respectively, into three and twosubunits, as also indicated by the height cross sectionalong the broken lines.

Discussion

A conformational transition of the cellular prionprotein PrPC into the PrPSc aggregated form, foundwithin amyloid plaques in vivo, is responsible forprion diseases in animals and humans. The mechan-ism of this transition at the molecular level is not yetcompletely understood. A number of crucial issuesare still open, such as the existence of several prionstrains for the same animal species and their abilityto overcome species barriers. This behavior seems torequire a high conformational plasticity of the prionproteins and of their internal fragments involved inamyloidogenesis.20,46 To highlight these peculiardynamic features of the prion protein, we investi-gated the conformational transitions of a humanprion peptide PrP82–146,which has been found to be

Fig. 8. AFM height image of an isolated PrP82–146fibril. A long isolated fibril obtained after 15 days of incu-bation at 37 °C is presented in (a). In (b), an enlargement isreported where two protofilament subunits can be seen.The splitting and the subsequent merging of the two sub-units are indicated by the arrows.


the smallest fragment required for amyloidogenesisand themajor amyloid plaque component in the GSSdisease.5–7 Indeed, we disclosed by FT-IR spectro-scopy several aggregation pathways, each leading toend products with a different molecular structure.FT-IR spectroscopy has been recently successfullyapplied to the characterization at the molecular levelof amyloid aggregates, since their infrared absorp-tion in the Amide I region displays a specific signa-ture of β-sheet intermolecular structures.19,47,48 Inparticular, the potential of FT-IR spectroscopy forprion strain discrimination and for understandingtransmission barriers has been recently demon-strated through the strain-specific infrared responsein the Amide I region.20 Biomedical applications of

FT-IR spectroscopy for strain identification have alsobeen recently proposed.47,49

In the present study, we characterized by FT-IRspectroscopy different aggregation pathways ofPrP82–146. The starting peptide in solution displayedmainly a random coil structure, in agreement withwhatwas observed for similar prionpeptides. Indeed,the humanPrP fragment 23–144was found to have anunordered structure using infrared spectroscopy20

and circular dichroism,50 like themouse prion peptide89–143 by NMR spectroscopy.35

Incubation at 37 °C leads to the formation ofoligomers with an intermolecular β-sheet interactionindicated by the two Amide I components around1623 and 1690 cm−1. At a critical oligomer concen-tration, fibril formation starts to take place, asdetected by the appearance of the 1626 cm−1 bandand confirmed by EM, AFM, and LSCFM. We recallthat, β-sheet structures typically display two bandsof different intensity in the Amide I absorptionregion. For antiparallel structures in aggregates, alow-frequency band occurs around 1630–1611 cm−1

and a high-frequency band of lower intensity occursaround 1695–1680 cm−1. For parallel β-sheets, onlythe low-frequency band is expected,31,51–53 usuallyupshifted of a few wavenumbers. The single band ofaggregation and its spectral position at 1626 cm−1,therefore, indicate that parallel intermolecular β-sheet interactions characterize PrP82–146 fibrils. Inoligomers, instead, the two β-sheet components—and their band positions—could suggest the pre-sence of distorted parallel or of antiparallel β-sheetstructures.25 Indeed, a reliable assignment of parallelβ-sheet structure can be proposed when only thelow-frequency β-sheet band is observed.31,51–53 Adifferent conformation of the peptide in oligomersand fibrils is, therefore, demonstrated by the infraredabsorption of these structures, which indicates adifferent β-sheet interaction in the two cases.We should note that it is still an open question

whether globular oligomers belong to the on- or off-pathways of fibril formation.54 In themodel presented

Fig. 9. AFM height image ofPrP82–146 fibrils. Fibrils with differ-ent morphology, periodicity, andheight can be appreciated. Trianglesand arrow mark the sites wherefibrils split, respectively, into threeand twosubunits. The asteriskmarksthe fibril with the lowest twistingperiodicity (∼27 nm). Height crosssection along the directions indi-cated by the three broken lines isalso reported.


here, we propose that globular PrP82–146 oligomersare on the pathway of fibril formation from thefollowing considerations. The time course of fibrilformation monitored by FT-IR spectroscopy enabledus to identify first a nucleation phasewhere oligomersaccumulate, followed by a steep fibril growth. Inaddition, by AFM and EM, it has been possible toidentify the alignment of oligomers into short linearaggregates. Furthermore, as recently reported inliterature,15 PrP82–146 globular oligomers (with adiameter of about 31±11 nm as seen by EM and withRH=12 nm determined by light scattering) werefound to bind prefibrillar aggregates previouslyimmobilized on the sensor chip of a surface plasmonresonance spectroscopy device.15 However, fromthese results, we cannot exclude that PrP82–146oligomers, obtained during 37 °C incubation, areoff-pathway of fibril formation.Nevertheless, the “dock and lock”model proposed

in the surface plasmon resonance spectroscopystudy15 is in agreement with the peptide conforma-tional transition from oligomers into fibrils suggestedhere by FT-IR spectroscopy. In thismodel, the bindingof PrP82–146 oligomers to prefibrillar aggregates(docking) is followed by a conformational rearrange-ment that stabilizes (locking) the new structure.15

The morphology of PrP82–146 oligomers andfibrils, obtained under the same experimental condi-tions that were used in the FT-IR study, was investi-gated by AFM.Globular oligomers were embedded in a grainy

background whose structure had been resolved byhigh-resolution AFM. Linear and annular aggregates(Fig. 7h), resembling the annular protofibrils detectedby EM for α-synuclein,42 were observed. Over thisstructured background, PrP82–146 oligomers withheight ranging from 1 to 10 nm displayed a globularshape—round and rectangular. Our interpretation isthat theymight also result from the assembly of edge-on oriented annular protofibrils, withβ-sheets normalto the sample surface, as suggested by the ∼0.5-nmheight quantization observed for oligomers withheight in the range 1.5–4 nm.Concerning PrP82–146 fibrils, the AFM images

indicate that they are composed by twisted protofila-ment subunits. Furthermore, the different morpholo-gies with fibril heterogeneity in height distributioncould reflect the presence of more constituent sub-filaments.43 Moreover, the correlation observedbetween PrP82–146 fibril height and periodicitycould result from the different tightness of thesubfilament twist.55 All these results suggest that ahierarchical fibrils assembly43–45 seems to be at workfor PrP86–142, even if the underlyingmechanisms44,45

of this process cannot be identified.The assembly pathway from the PrP82–146 unor-

dered peptide into mature fibrils is schematized inFig. 10, where other aggregation pathways are alsoreported.In particular, when the unfolded PrP82–146 pep-

tides are heated from 37 to 100 °C, thermal aggre-gates are formed, displaying an intermolecularβ-sheet infrared absorption similar to that observed

for thermal amorphous aggregates of several pro-teins.25,29,30,32–34 These results clearly indicated thatPrP82–146 oligomers, fibrils, and thermal aggregatesare characterized by different intermolecular β-sheetconformations (Figs. 1 and 2).Furthermore, random aggregates,35 formed by

solvent evaporation on a diamond ATR plate, werealso studied. Unexpectedly, these assemblies werecharacterized not only by the presence of severalβ-sheet components but also by an intense α-helixstructure, which is not usually induced by aggrega-tion. The high intensity of this last component, indeed,suggests that a large peptide region can be induced toform α-helix structures. It should be noted that, ingeneral, when films are formed from native foldedproteins, no major changes in protein secondarystructure are found by ATR/FT-IR spectroscopy.26Interestingly, when unordered proteins are measuredin form of films, the formation of β-sheet structureswas typically observed,56 with the rising of α-helicalstructures being an unusual behavior.With the aim of investigating the aggregation of

PrP82–146 under different conditions, we, therefore,observed peptide assemblies with distinct infraredresponse: oligomers, fibrils, thermal aggregates, andrandom aggregates.All these results, therefore, demonstrate the high

plasticity of the PrP82–146 peptide, which is capableof undergoing distinct aggregation pathways, whoseend products are assemblies with different secondarystructures and intermolecular interactions. This con-formational plasticity is crucial to determine prionstrains and to allow species barrier overcoming.20,57 Itis therefore clear that structural characterizations ofprion proteins and peptides are of paramountimportance for the understanding of prion diseases.

Materials and Methods

Synthesis and purification of the prion fragmentPrP82–146

The human prion fragment PrP82–146 was synthesizedand purified as previously reported13,16 and brieflydescribed here. The prion fragment PrP82–146 (GQPHGG-WGQGGTHSQWNKPSKPKTNMKHMAGAAAAGAV-VGGLGGYMLGSAMSRPIIHFGSDYE) was synthesizedin SSPS on a 433A synthesizer (Applied Biosystems, FosterCity, CA) at 0.1 mM scale with 4-hydromethylphenoxya-cetic acid resin using N-(9-fluoroenyl)methoxycarbonyl-protected L-amino acid derivatives. Amino acids wereactivated by reactionwith 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/1-hydroxyben-zotriazole and N,N,-diisopropylethylamine. Peptideswere cleaved from the resin with a mixture of phenol/thioanisole/ethandiol/TFA, precipitated with cold diethylether, andwashed several timeswith the same solvent. Thepurification was performed by reverse-phase HPLC on asemipreparative C18 column (190–300 mm, 300 Å poresize, 15 μm particle size; Delta Pack, Nihon Waters,Tokyo, Japan) with a mobile phase of 0.1% TFA/water(eluent A) and 0.08% TFA/acetonitrile (eluent B) using alinear gradient of 0–80% eluent B in 60 min with a flowrate of 3 ml/min. The fractions containing PrP peptides

Fig. 10. Model for multiple aggregation pathways of the PrP82–146 peptide.


were separately collected, lyophilized, and kept at−80 °C. Their purity and composition were determinedby amino acid sequencing (46600 Prosequencer; Milligen,Bedford, MA) and electrospray mass spectrometry(model 5989 A; Hewlett-Packard, Palo Alto, CA) aspreviously described.13,16

Since fibril formation is strongly affected by preformedaggregates, 1 mg of lyophilized peptide was dissolved in1 ml of HFIP, to eliminate every preformed assembly.After HFIP evaporation, the peptide was solubilized in thebuffers specified below.For fibril formation, the above solution was incubated at

37 °C up to 2 months. Thermal aggregates were formed byheating the PrP82–146 sample solution from 20 to 100 °C,at a constant rate of 0.2 °C/min. Random aggregates35

were also obtained by drying a freshly prepared PrP82–146 water solution on the ATR plate.

FT-IR spectroscopy

For FT-IR measurements of PrP82–146 in solution, theHFIP-treated peptide was solubilized in heavy water(D2O) to a final concentration of 5 μg/μl. The time courseof the aggregation process was studied by keeping thesolution at 37 °C up to 2 months.The infrared absorption spectrum of the peptide solution

was measured in transmission in a thermostated BaF2transmission cell, with 50 μm optical path. The FT-IRspectrometer FTS-40A (Bio-Rad, Digilab Division, Cam-bridge, MA)was employed under the following conditions:1–2 cm−1 spectral resolution, 256–500 scan coaddition,5 kHz scan speed, and triangular apodization. Second-derivative analysis of absorption spectra was performedby the Savitsky–Golay (5 points) procedure, after bino-mial smoothing of the measured spectra (11 points), inorder to identify the secondary structures and aggregatebands.ATR/FT-IRmeasurements were recorded using a single-

reflection diamond element (Golden Gate, Specac, USA)under the same conditions employed for transmissionspectra. Five microliters of PrP82–146 sample was depos-ited on the ATR plate, and spectra were recorded aftersolvent evaporation to allow the formation of a hydratedprotein film.

Electron microscopy

Tenmicroliters of peptide solution (in 100mMTris–HCl,pH 7.4) was deposited onto a 200-mesh formvar/carbon-coated nickel grids (Electron Microscopy Science) for6min; the drop of solutionwas then absorbed onWhatmanNo. 1 paper. Grids were air dried and stained with 10 μl ofuranil acetate (saturated solution inwater) for 5min beforeremoval by Whatman No. 1 paper. Grids were air driedand examined with a Tecnai 12 electron microscope ope-rating at 120 kV, equipped with an ULTRA VIEW CCDdigital camera (Philips Eindhoven, The Netherlands).Immunogold labeling was performed using the mono-

clonal antibody 3F4 to confirm the presence of PrP82–146peptide. Peptide material deposited onto the nickel gridswas fixed with 5 μl of phosphate-buffered saline (PBS)containing 4% paraformaldehyde and 0.05% glutaralde-hyde. The fixing buffer was replaced by 5 μl of incubationbuffer (PBS with 2% bovine serum albumin) containing 5%horse serum for 20 min. Grids were then incubated for 2 hwith 3F4 antibody (DakoCytomation, Glostrup, Denmark)diluted 1:40 in incubation buffer. After washing in PBS,grids were incubated for 2 h, in 12-nm Colloidal Gold-AffiniPure Donkey Anti-Mouse IgG (H+L) (1:30) (JacksonImmunoResearch Labs, Inc., Baltimore, PA). The stainingwith uranyl acetate was performed after water washing.

Laser scanning confocal fluorescence microscopy

For LSCFM, an aliquot of the PrP82–146 sample solutionwas stained with ThT at a final concentration of 100 μM.The fluorescence images of these sampleswere obtained bya laser scanning confocal microscope, Leica TCS SP2,equipped with an oil immersion objective (PL APO 63×)with numerical aperture of 1.4. The ThT fluorescence wasexcited by the Argon laser line at 458 nm, and the emissionwas collected in the region 470–700 nm.

Atomic force microscopy

For AFM measurements, ∼5 μl of 5 μg/ml PrP82–146solutionwas deposited on freshly cleavedmica andwashedafter a few seconds with distilled water. Ultrasharp


whiskers were used in the tapping mode to reach a highmolecular resolution.58 On the tip of standard AFMprobes(with spring constant∼10N/m and resonance frequencieswithin 200–350 kHz), whiskers were grown with ananometer curvature radius, in order to reduce adhesionand broadening effect. AFM images were collected atambient conditions in air by an AFM Solver-Bio (NT-MDT,Zelenograd, Moscow, Russia) and using both standardAFM probes and ultrasharp whiskers.58 The lateral andvertical calibration of the AFM device was done with theuse of calibration gratings TGZ1 (NT-MDT, Zelenograd,Russia) with an 18-nm step height.Special attention was paid to the choice of the optimal

probe–surface interaction regime.59 It was found that withthe use of ultrasharp whiskers, the best lateral resolutionwas achieved in the repulsion regime with low amplitudesof probe oscillations, typically in the range of 3–10 nm.Indeed, the repulsion regime was chosen for a bettersurface profiling, thanks to its direct probe–surface contact.The very small curvature radius of the ultrasharpwhiskersincreases the resolution and reduces the adhesion force(which is proportional to the tip curvature radius) and,consequently, the net interaction probe–surface force. Thedominant force in the probe–surface interaction in air wasmainly the capillary force,60 which was further reduced bythe fact that carbon nano-whiskers of the ultrasharp probeswere highly hydrophobic. All these prerequisites provide a“very soft” scanning mode, with small elastic forces andsample deformations.The image analysis was performed using the software

Femtoscan†. For automatic analysis of oligomer heights,radius, and volumes in AFM images, the specializedsoftware SPM Image Magic‡ was used. The evaluation offibril height maxima and minima was done manually.

Acknowledgements

We are grateful to Dimitry V. Klinov for his kindsupply of ultrasharp cantilevers. This work wassupported by a Fondo di Ateneo per la Ricerca grantof the University of Milano-Bicocca to S.M.D., theEuropean Union within the frame of Neuroprion,Fondazione Cariplo (Grant NOBEL-Guard), theItalian Ministry of University and Research (FIRB,RBNE03PX83), and the Negri-Weizmann Founda-tion. A.N. acknowledges a postdoctoral fellowshipof the University of Milano-Bicocca.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.06.063

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Conformational Plasticity of the Gerstmann–Sträussler–Scheinker Disease Peptide as Indicated by Its Multiple Aggregation Pathways

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