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Colloids and Surfaces B: Biointerfaces 135 (2015) 371–378

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

j o ur nal ho me pa ge: www.elsev ier .com/ locate /co lsur fb

olecular-level insights of early-stage prion protein aggregation onica and gold surface determined by AFM imaging and molecular

imulation

hichao Loua,b, Bin Wanga, Cunlan Guoa, Kun Wanga, Haiqian Zhangb, Bingqian Xua,∗

Single Molecule Study Laboratory, College of Engineering and Nanoscale Science and Engineering Center, University of Georgia, Athens, GA 30605, USACollege of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 210016, PR China

r t i c l e i n f o

rticle history:eceived 29 April 2015eceived in revised form 17 July 2015ccepted 21 July 2015vailable online 26 July 2015

eywords:rion proteinarly-stage aggregationnterfaces

a b s t r a c t

By in situ time-lapse AFM, we investigated early-stage aggregates of PrP formed at low concentration(100 ng/mL) on mica and Au(1 1 1) surfaces in acetate buffer (pH 4.5). Remarkably different PrP assem-blies were observed. Oligomeric structures of PrP aggregates were observed on mica surface, which wasin sharp contrast to the multi-layer PrP aggregates yielding parallel linear patterns observed Au(1 1 1) sur-face. Combining molecular dynamics and docking simulations, PrP monomers, dimers and trimers wererevealed as the basic units of the observed aggregates. Besides, the mechanisms of the observed PrP aggre-gations and the corresponding molecular-substrate and intermolecular interactions were suggested.These interactions involved gold–sulfur interaction, electrostatic interaction, hydrophobic interaction,and hydrogen binding interaction. In contrast, the PrP aggregates observed in pH 7.2 PBS buffer demon-

omputational simulationstomic force microscopy

strated similar large ball-like structures on both mica and Au(1 1 1) surfaces. The results indicate that thepH of a solution and the surface of the system can have strong effects on supramolecular assemblies ofprion proteins. This study provides in-depth understanding on the structural and mechanistic nature ofPrP aggregation, and can be used to study the aggregation mechanisms of other proteins with similarmisfolding properties.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

Prion protein (PrP) has drawn great attention due to its patho-ogical potential to prion diseases, such as Creutzfeldt–Jakobisease and “mad cow disease” [1]. Studies have shown that prioniseases are induced by the formation of extended �-sheet richbrillar PrP aggregations [2–4], as well as various on-pathway

ntermediates prior to the PrP fibrils [5–8]. The detailed under-tanding of these aggregates may help in the search for anti-prionisease treatments. Different types of highly ordered PrP aggrega-ions have been investigated by conventional techniques, such as-ray diffraction, nuclear magnetic resonance and electron diffrac-

ion [9–11]. And compared with these traditional technologies, thetomic force microscopy (AFM) provides additional information

n 3D morphology structures and conformation evolution of PrPggregates with time [12–14]. But most AFM-based studies focusedn imaging the microfibers or large aggregations in extreme

∗ Corresponding author.E-mail address: bxu@engr.uga.edu (B. Xu).

ttp://dx.doi.org/10.1016/j.colsurfb.2015.07.053927-7765/© 2015 Elsevier B.V. All rights reserved.

conditions which are far from the physiological environment ofhuman bodies, such as in high concentrations [15], after long-timeincubation [16], and at high temperature [17]. As a result, detailedinformation on the early-stage PrP aggregation involving the on-pathway intermediates is limited.

For in vitro studies, pH is an important factor among manyexperimental conditions. It may determine whether PrP fibrils andon-pathway intermediates are formed [18]. In 1999, Jackson et al.reported that when GdnHCl-denatured, DTT-reduced recombinanthuman PrP (91–231) was dialyzed against a pH 4.0 buffer, it canrefold to a monomeric �-sheet rich conformer, which shows par-tial protease resistance and is prone to associating into solubleoligomers or fibrils [19]. In addition, the misfolded PrP moleculesare reported to accumulate in the endosomal organelles, wherethe typical pH is around 5.0 but can be as low as 4.3 [20–22].However, most studies have focused on characterizing the acid-induced PrP aggregations [2,19,23] and the PrP molecules in these

studies were mainly truncated forms of PrP, rather than the entireprotein. These truncated forms of PrP lack the flexible portion (N-terminal domain) of the protein while evidence has suggested thatN-terminus can chelate copper and thus acquire some structure

3 s B: Biointerfaces 135 (2015) 371–378

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Fig. 1. (A) The amino acid sequence of the full-length PrP molecule used in theexperiment. The residues involved in the �-sheet structures are labeled. (B) The

72 Z. Lou et al. / Colloids and Surface

24,25], and may also play a role in prion pathogenesis [26]. Inddition, different segments of protein are of different biochem-cal properties, for example, the full-length PrP (23–231) is highlyasic (isoelectric point of 9.3), whereas the isoelectric point of PrP0–231 is about 7.2 [27]. Although some recent aggregation studiestilized the full-length PrP [18,28], its aggregation properties haveot been studied as extensively as those of the truncated forms andhe mechanistic details are missing.

AFM technology is inherently based on the surface. Solid sur-aces, such as mica and gold, have been reported to influenceggregation kinetics and the resulting morphology of PrP aggre-ates [29]. Gold surface has been widely used in biosensors,nd specific proteins can self-assemble on gold surface via theold–sulfur bonds formed by their cysteine residues and goldtoms [30,31]. Our previous investigation has suggested that PrPolecules can form aggregates aligning with the herringbone

tructures of the reconstructed Au(1 1 1) surface via Au S bonds32]. Meanwhile, mica surface is commonly used as substrate foripid bilayers, modeling the native cell membrane with its uniqueroperties of being atomically flat, and negatively charged [33].everal investigations using in situ AFM reported aggregates ofmyloid proteins with varied morphologies on mica surface [34,35].herefore, application of both solid surfaces as model systemsrovides the opportunity to elucidate how specific surface envi-onments influence the architectural features of early-stage PrPggregates.

Herein, we investigate the in situ early-stage architectural fea-ures of PrP aggregates formed at low concentration (100 ng/mL)n mica and Au(1 1 1) surfaces using time-lapse liquid phase AFMmaging at pH 4.5. On mica surface, individual oligomeric PrP aggre-ates, some of which have ring-“head” structures were observed;nd in sharp contrast, parallel linear PrP aggregates were formedn Au(1 1 1) surface. By the aid of molecular dynamics and dock-ng simulations, the formation mechanisms of the observed PrPggregates, as well as the molecular-substrate and intermolecularnteractions happening at the interfaces were determined. Mean-

hile, ball-like structures of PrP aggregates were observed on bothurfaces when the AFM imaging was conducted at pH = 7.2.

. Material and methods

.1. Materials

Frozen full-length human recombinant prion protein (sequence:3–231, theoretical PI/Mw: 9.39/23571.92) was purchased fromalbiochem® in Germany with a concentration of 2 mg/mL, whichas from Escherichia coli expression and purified as previouslyescribed [36]. The purity of >95% was determined by SDS-PAGE.he protein was stored at −20 ◦C in 10 mM sodium acetate buffert pH 4.0 before usage. Before each experiment, purchased prionrotein was centrifuged (20,000 × g) for 30 min at 4 ◦C to removere-existing aggregates. Phosphate buffered saline (PBS: 100 mModium phosphate, 150 mM sodium chloride, pH 7.2, added with.05% sodium azide) was purchased from Pierce (Themo Scien-ific, Waltham, MA, USA). Triple deionized water was provided by aarnstead Nanopure Diamond Laboratory Water System. Mica wasurchased from Ted Pella, Inc. (Product No: 56), and the Au(1 1 1)tructure was attained by annealing a fresh thermal coated goldhip with 2 min hydrogen flame [31].

.2. Single-molecule AFM experimental settings

An Agilent 5500 Controller combined with a 50 �m by 50 �mgilent multipurpose AFM scanner (S/N: 325-00388, with 0.5 AMS of z noise specification) was used to obtain images in an area as

three-dimensional structures of the PrP molecule at pH = 7.2 and 4.5, where three�-helices are in blue and �-sheets in purple. (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of the article.)

large as 1000 nm by 1000 nm. The whole system was shielded fromenvironmental interference by a PicoPlusIsolation Chamber. Siliconcantilevers tip with spring constant of around 0.1 N/m were usedfor experiments. The detailed parameters for the image were as fol-lows: drive is approximately 20%, resonance gain is 2, resonancefrequency is around 6.725 kHz, resonance amplitude is 4.837 V,“Stop At” is set as 0.8, and scan rate is about 1.02 line/s.

2.3. The liquid phase AFM imaging of the PrP aggregates on micaand Au(1 1 1) surfaces

Centrifugation treated PrPs were re-dissolved in 10 mM pH = 4.5sodium acetate acid buffer or pH = 7.2 PBS buffer to a final concen-tration of 100 ng/mL. And 400 �L of each sample was depositedonto newly caved mica surface or freshly reconstructed Au(1 1 1)surface in a liquid cell. Then, samples were imaged immediately atroom temperature at 20 min intervals, with the AFM tip immersedin the solution. All the images were taken under TopMAC mode witha PicoTREC controller (Agilent Technologies, Santa Clara, CA), whichis a low-force touch method [37]. The detailed description of Top-MAC mode AFM used here is shown in the Supplementary material(in Fig. S1). The AFM images were obtained with 1024 × 1024 pointsand processed using WSxM software [38]. If necessary, only first-order plane-fittings were performed with the analytical software.The height values of the PrP aggregates were determined by indi-vidual cross-section profiles generated by the software.

3. Results

3.1. Dynamics simulation of PrP monomer at pH 4.5 and pH 7.2environment

The human PrP sequence comprising amino acid residues (aa)23–231 is shown in Fig. 1A. The three-dimensional structures ofPrP at pH 4.5 and pH 7.2 are simulated by Amber 11, with 60 nsequilibration and the results are shown in Fig. 1B. The experimen-tal details of the simulations are shown in Supplementary material.Dynamics simulations show that, at pH = 4.5, �-helical-dominant

prion protein (PrPc) conformer converted into the �-sheet richconformer, which is normally called PrPSc. This result is consis-tent with previous studies [39]. The PrPSc monomer has threehydrophilic �-helices and two hydrophobic �-sheets, implying its

Z. Lou et al. / Colloids and Surfaces B: Bi

Fig. 2. Time-lapse in situ AFM images of PrP 23–231 incubated at room temperature,obtained after 20 min, 40 min, 80 min, and 160 min of continuous scanning. The AFMimages of bare mica and gold surfaces are shown in Fig. S3. (A) and (B) show PrPaggregations in pH = 4.5 acetate acid buffer on mica and Au(1 1 1) surfaces, respec-tively. (C) and (D) show PrP aggregations in pH = 7.2 PBS buffer on mica and Au(1 1 1)saa

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urfaces, respectively. The highlighted squared areas are used in later analysis. Thengle marks in Fig. 2B (1) demonstrate the values of 120◦ or 60◦ . 100-nm scale barsre indicated.

mphiprotic property in aqueous solution. These monomers canssemble into dimers or trimers by forming a hydrophobic coreith their �-sheets, and leave the more hydrophilic �-helix regions

acing the aqueous phase (see Supplementary material, Fig. S2).revious studies have shown that these dimers and trimers act ashe nuclei or growth units to form the fibrils [40].

.2. Oligomeric PrP aggregations on mica surface at pH 4.5

Fig. 2A shows the real-time evolution of PrP aggregation on micat pH 4.5, monitored by time-lapse in situ AFM imaging.

.2.1. Determination of the basic units in observed simple PrPligomers

Fig. 3A shows the zoom-in profile of the marked area in Fig. 2A.1.ach aggregation unit has different sizes under the same off-set in

direction of the AFM image (trimer > dimer > monomer, Fig. S4n the Supplementary material), so we can recognize and distin-uish PrP monomer (M), dimer (D), and trimer (T) componentsn these oligomeric PrP aggregates by reading the height of eachomponent from the cross-section profiles (Fig. 3B). To ensurehe reliable height analysis, we generated the distribution of theeometrical height values for these basic oligomeric units usingore than 350 of these oligomers from 10 high-resolution images

Fig. 3C). The detailed method on how we created the histogramn Fig. 3C is described in Supplementary material. Monomer, dimernd trimer units were recognized from Gauss fittings of the heightistograms (Fig. 3C), which clearly show the most probable heightsf 0.80 ± 0.01 nm, 1.01 ± 0.01 nm and 1.19 ± 0.02 nm, respectively.y examining the height values of these oligomers in Fig. 3B, we

ound that the structure labeled as � in Fig. 3A represents a D1.07 nm), � is a 3-unit aggregate including one M (0.78 nm) andwo Ds (1.09 nm and 1.05 nm) laying on the surface side by side,nd � is a 2-unit aggregate containing one D (1.05 nm) and one T

ointerfaces 135 (2015) 371–378 373

(1.2 nm). These observed conformations of oligomers �, �, and �are further confirmed by docking simulations in Fig. 3D. The inter-molecular interactions, which hold together aggregation units (M,D, T) to form the oligomers, are supposed to be hydrogen bond-ing at the interfaces of the aggregation units. This will be furtherdiscussed in Section 4. A list of the predicted residue formingthe intermolecular hydrogen bonds at the interfaces is listed inTable S1 (see Supplementary material). Therefore, combining AFMhigh-resolution images and molecular simulations, we clearly dis-tinguished PrP monomer, dimer and trimer units in each individualoligomer. More representative individual oligomers were selectedfrom the AFM image in Supplementary material Fig. S5. Corre-sponding simulated structures are provided and compared withthe AFM height results in Supplementary material, Fig. S6.

3.2.2. AFM-observed elongation process and “ring-head”oligomers

The oligomers of PrP were observed to continue aggregatingwith time (Fig. 2A (2)–(4)). With the magnified topographic imagesin Fig. 4A, two oligomeric PrP aggregates (highlighted by yellowarrows) are found to gradually combine with each other at 40 minand finally turned into an elongated one at 160 min. The zoom-inview (white square) shows that a trimer (T) unit of one oligomericPrP aggregate combined with a dimer (D) unit of another oligomericPrP aggregate (see Supplementary material, Fig. S7). This impliesthat the growth of PrP aggregates is through the assemblies oftwo or more oligomers. Fig. 4B shows the magnified images ofoligomers selected with red squares in Fig. 4A. Interestingly, theseoligomers appear to have ring-“head” structures formed by stringsof spherical particles and the ring structures have been suggested assoluble oligmeric intermediates which is associated with the toxic-ity of PrP [41]. High-resolution AFM images show these ring-“head”structures are composed with four to six subunits (spherical par-ticles) (Fig. 4B and Supplementary material, Fig. S8B). The heightvalues of each subunit of these ring structures shown in Fig. 4Bare consistent with the ones in the height distribution histogramin Fig. 3C, indicating that the basic units of these ring conforma-tions are also monomer, dimer and trimer. It is also worth noticingthat slight heighter values of corresponding subunits could be dueto the probability that some subunits overlaid on top the others inring conformations, resulting in higher “altitude” than their heightsobtained from the simple PrP oligomers mentioned in Section 3.2.1(marked with red arrows in Fig. 4B). Corresponding moleculardocking simulations revealed the proper combining conformationsfor these ring-like structures in Fig. 4C. More representative “ring-head” oligomers are provided in Supplementary material, Fig. S8.

3.3. Architectural features of PrP aggregations with multiplelayers on Au(1 1 1) surface at pH 4.5

On Au(1 1 1) surface, at pH = 4.5, regular PrP aggregates in themorphology of highly ordered parallel patterns with hundreds ofnanometers in length were observed (Fig. 2B), which is in sharpcontrast to the case on mica surface. Zoom-in views of these aggre-gates and the evolution process are shown in Fig. 5. PrPs wereobserved to form an orderly patterned first layer fairly quicklyafter PrP solution was dropped on the surface (Fig. 5A). Accord-ing to the cross-section profiles of the surface collected at differenttime points, we see that the height of the architecture increasedfrom around 1.0 nm at 40 min (Fig. 5A) to around 2.2 nm at 80 min(Fig. 5B) and further increased up to around 4.3 nm at 160 min(Fig. 5C). This trend implies that more PrP molecules deposited on

top of the first layer from the solution, and then the second layerand multiple layers were formed as time lapsed. For the first layer,the surface height determined by the cross-section profile (Fig. 5A)ranges from 0.8 nm to 1.2 nm, consistent with those of monomer,

374 Z. Lou et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 371–378

Fig. 3. (A) Zoom-in image of the yellow squared area in Fig. 2A (1); (B) Corresponding cross-section profiles for the oligomers in (A); (C) height distribution histogram foreach components of the small oligomers which was fitted to three Gaussian yielding heights of 0.80 ± 0.01 nm, 1.01 ± 0.01 nm and 1.19 ± 0.02 nm (R-squared: 0.98); (D)m dimeri n. (Fort

diutorhifaad

olecular docking simulations for the observed oligomers in (A), monomers (M),

nterfaces squared in the yellow dashed areas along with hydrogen bonds are showo the web version of the article.)

imer and trimer units determined on mica surface (Fig. 3C), imply-ng no significant difference in the height values of basic combiningnits observed between mica and gold surface system. Meanwhile,he linear patterns are generally oriented with a cross-angle of 60◦

r 120◦ (marked in Fig. 2B), which coincides with the angle of theeconstructed Au(1 1 1) surface [42]. Combining with the fact thatuman prion protein monomer has one disulfide bond connect-

ng Cys 179 and Cys 214 [43], the architectural patterns of PrP

ormed in the first layer were probably induced by the Au S bondst the interface between PrP units (monomer, dimer and trimer)nd the reconstructed Au(1 1 1) surface, though no chemical evi-ences are available for the studied system in this work [32]. For

s (D) and trimers (T) are presented in different colors. The zoom-in images of the interpretation of the references to color in this figure legend, the reader is referred

the second layer, our previous work showed that the fresh PrP unitsalong with their combinations interacted with the first layer not viaAu S bonding, but via the hydrogen bonding, of which the mecha-nism is similar to the interactions between PrP oligomers on micasurface as mentioned in Section 3.2. This mechanism explains thephenomenon that the second and the multiple layers have shownsimilar architectural features to those of the first layer.

3.4. Aggregations on mica and Au(1 1 1) surfaces at pH 7.2

Increasing pH from 4.5 to 7.2 leads to dramatic change in themorphologies of PrP aggregates on both mica and Au(1 1 1) surfaces.

Z. Lou et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 371–378 375

Fig. 4. (A) In situ zoom-in of the squared areas in Fig. 1A (2)–(4). (B) Magnified images of the oligomers squared with red dashed frames in (A). Corresponding cross-section profiles indicating the units for the ring structures are shown below each magnification. (C) Corresponding docking simulations indicating the proper combiningc er unl ctivelr

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onformations for these three ring structures, where each monomer, dimer and trimisted in Table S2. 40-nm and 10-nm scale bars are indicated for (A) and (B), respeeferred to the web version of the article.)

n mica surface, ball-like aggregates of PrP molecules in differentizes were observed instead of any individual oligomers (Fig. 2C).he molecular dynamics simulation shows that monomeric PrP isot able to transform to a stable �-sheet isoform within 60 ns at pH.2 (Fig. 1B). The three �-helices of PrP are more hydrophilic, sug-

esting that monomeric PrP molecule is highly soluble at neutralH [44]. As a result, these �-helix rich PrP monomers can combineith each other freely to form the ball-like aggregates via inter-olecular hydrogen bonding in the solution, and deposit onto the

it is labeled in color. The detailed corresponding bonding sites at the interfaces arey. (For interpretation of the references to color in this figure legend, the reader is

mica surface prior to AFM imaging. Similar ball-like aggregates areobserved on Au(1 1 1) surface at pH 7.2, in contrast to the parallelpatterns observed at pH = 4.5. It implies that no gold–sulfur bond-ing happened in neutral environment. Fig. S9 in the Supplementarymaterial provides the zoom-in top view of the predicated 3D struc-

ture of PrP monomer in the orientation, following which the PrPmonomers are predicted to combine with the Au(1 1 1) surface viagold–sulfur bonds at pH 4.5. Fig. S9B shows the same region includ-ing the disulfide bond at pH 7.2. It shows clearly that at pH 4.5, the

376 Z. Lou et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 371–378

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ig. 5. Time-lapse in situ PrP aggregation on Au surface. Upper panel: magnificatrofile. 50-nm scale bars are indicated. (For interpretation of the references to colo

wo residues (Cys179 and Cys214) are exposed and stabilized byisulfide bond and �-sheet structures, which make them accessi-le to Au(1 1 1) surface and bind via gold–sulfur bond as discussed

n Section 3.3. In contrast, these two residues are partly envelopedy the �-helices at pH 7.2, and this more flexible structure with-ut �-sheets additionally prevent Cys residues from contact to theu(1 1 1) surface. Therefore, the ball-like aggregates in Au(1 1 1)urface system are formed in a similar way as in the mica surfaceystem.

. Discussion

In this study, we investigated the early-stage architectural fea-ures of PrP aggregations at molecular level on both mica andu(1 1 1) surfaces using in situ liquid phase AFM imaging. AFMrovides a capability to image PrP aggregates in a hydrated state,ithout the need for complex pretreatments involved in the tradi-

ional technologies mentioned in Section 1 [9–11]. Only low forceontacts with the samples are made, minimizing disruption due tompact of the AFM tip (see the detailed description of TopMAC modeFM in the Supplementary material). Though measured dimen-ions of samples in AFM images display larger values due to thenavoidable tip broadening effect, their dimensions can still bestimated based on spherical [45] and half-spherical [46] molec-lar shapes models. But in our work, most of PrP basic units attacho each other, forming oligomeric aggregates. As a result, the restndividual basic units are not enough for us to do the statisticsf their dimensions. Here, the high-resolution AFM data provideshe height distribution histogram for each component of the smallligomers in liquid that are used to distinguish and recognize therP basic units monomer, dimer, and trimer. The obtained meaneight of monomer (0.80 ± 0.01 nm, Fig. 3C) is very close to previousork [47].

Previous structural and biochemical analysis of the prion pro-eins indicate that the assembly of misfolded protein conformersnto fibrils is a complex and multistep process involving theopulation of transient or metastable intermediates prior to theormation of fibrils [48]. Recent studies have suggested that

hese intermediates exhibit greater cytotoxicity than either the

onomeric molecules or fibrils [5–8]. One important advantage ofur study is that, a lower concentration (100 ng/mL) was inducedo slow down the aggregation process so as to directly follow

f the squared areas in Fig. 2B (2)–(4); bottom panel: corresponding cross-sectione text, the reader is referred to the web version of the article.)

these intermediates. This low concentration prevents the veryfast aggregations by too many PrP molecules, providing oppor-tunities to follow more individual early-stage PrP aggregates(Figs. 3A and 4B and Supplementary material, Figs. S5, S6 and S8)and follow the aggregation processes in both mica and gold surfacesystems with time-lapse in situ AFM imaging (Figs. 4A and 5). Espe-cially due to the hydrophilicity of mica, the formed PrP aggregatescan move freely and further combine with each other into morecomplex oligomeric aggregates on mica surface (see Section 3.2.2).

Both hydrophilic �-helix structures and hydrophobic �-sheetstructures of the three basic units (M, D, and T) at pH 4.5 are essen-tial for the intermolecular interactions to occur at their interfaces.The intermolecular interactions can be classified into two non-covalent types: (1) �-helix induced hydrogen bonding interactionsand (2) �-sheet interactions. Based on the results of the dockingsimulations for PrP dimers and trimers (see Supplementary mate-rial, Fig. S2), we determined the number of the solvent-exposedresidues in �-helix structures and �-sheet structures of each basicunit in Supplementary material, Table S3. As shown in Table S3,the residues from the �-helix structures dominate the surfaces ofthese units (91.2% in dimers and 88.9% in trimers), suggesting thatthe �-helix induced intermolecular hydrogen bonding happensmore frequently than the �-sheet interactions. This mechanismleads to the formation of the oligomers observed on mica surface(Figs. 3A and 4B, and Supplementary material, Figs. S6E–G and S8).Similarly, the interaction between the first and second PrP layersin Au(1 1 1) surface system is also caused by hydrogen bonding[32], which is much weaker than the Au S bonding at the inter-faces between first PrP layer and the Au(1 1 1) surface. Thus, theoligomeric PrP units in the higher layers could change their orien-tations freely after they deposited on the lower layer, resulting inthe phenomenon that the observed number of the linear patternscrossed the red dashed line decreases from 8 (Fig. 5B) to 7 (Fig. 5C).

Gathering all the information of high-resolution single-molecule AFM images and the molecular simulations discussedabove, we can construct a schematic illustration for PrP aggre-gations on mica and Au(1 1 1) surface at pH 4.5 as shown inFig. 6. Firstly, acid-induced PrP basic units are formed rapidly in

solution in both mica and Au(1 1 1) surfaces systems (Fig. 6A).Then individual ring-like, rod-like oligomers, and their combina-tions (“ring-head” oligomers) are formed in the solution, by thesebasic units via intermolecular hydrogen bonding at their interfaces

Z. Lou et al. / Colloids and Surfaces B: Biointerfaces 135 (2015) 371–378 377

Fig. 6. (A) Schematic illustration of the formation of PrP dimer and trimer units, (a) is �-sheet interaction, (b) is hydrogen bonding interaction. The acorn-like particle is themodel for PrP monomer, dumbbell-like particles are the models for PrP dimers, and the truncated three pyramids are the models for PrP trimers. D-1, 2 and T-1, 2, 3 are theirdifferent orientations, where �-helix and �-sheet structural surfaces are colored as blue and fuchsia, respectively. The surface ratios of the blue areas for dimer and trimer are0.912 and 0.889, corresponding to the simulation (Tables S3 and S4). (B) Schematic illustration of the observation of individual oligomeric PrP aggregations in mica surfaces eposito ractior

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ystem at pH = 4.5, where oligomeric PrP aggregates formed in solution, and then df the formation of PrP aggregations in gold surface system, (c) is gold–sulfur inteeferred to the web version of the article.)

Fig. 6A). The formation of such “ring-head” structures was sup-osed to involve the spatial rearrangement of the units to provide

more compact conformation minimizing the surface exposure ofydrophobic areas to water [13]. The isoelectric point of human

ull-length PrP is 9.39, implying the obtained PrP aggregates areositively charged in pH = 4.5 buffer. Meanwhile, fresh mica sur-ace is negatively charged [49], making the oligomers easily depositnto the surface via weak electrostatic interactions and can bebserved immediately after PrP samples were dropped on the sub-trate (Fig. 6B). In contrast, the reconstructed Au surface is rich inu(1 1 1) lattice planes [50], which are able to align PrP aggregatesia a surface assisted coupling reaction between the disulfide bondnd gold atoms (Fig. 6C). Thus, the length of each linear pattern isnly restricted by the area of the Au terrace. As the green solid linen Fig. 5A exemplifies one of linear aggregates with the length of30 nm.

. Conclusions

The early-stage architectural features of full-length PrP aggre-ations were investigated at molecular level using in situ liquidhase AFM imaging in both mica and Au(1 1 1) surface systems.ligomeric PrP aggregates, some of which have “ring-head” struc-

ures with strings of spherical particles, were observed on micaurface at pH 4.5. Two individual oligomers were observed to com-ine with each other into an elongated one via time-lapse AFM

maging. In contrast, highly-ordered parallel linear patterns follow-ng the underlying Au(1 1 1) herringbone geometry are formed onu(1 1 1) surface at pH = 4.5. And multiple layers yielding similaratterns were formed with time going on. The results indicate atrong surface effect on the supramolecular assemblies of prionroteins. Based on the results from molecular dynamics, dockingimulations, and high-resolution AFM images, we propose that PrPonomers, dimers and trimers perform as basic units for the forma-

ion of the observed aggregates. Intermolecular hydrogen bonding

nteractions happen in both surface systems, but play differentoles. For mica surface, the basic units assembled into individ-al oligomers with unique structures (“ring-head” oligomers) viaydrogen bonding in the solution, then these oligomers deposited

ed onto the mica surface via (d) electrostatic interaction. (C) Schematic illustrationn. (For interpretation of the references to color in this figure legend, the reader is

onto mica surface via weak electrostatic interaction. However, forAu(1 1 1) surface, the hydrogen bonding interactions happen at theinterfaces between the two PrP layers upon the surface. The firstPrP layer yielding linear patterns was formed via a surface assistedcoupling reaction between the disulfide bond and gold atoms.Meanwhile, ball-like structures of PrP aggregates were observedon both surfaces when the AFM imaging was conducted at pH = 7.2,suggesting the strong influence of environmental pH. These resultscould help us gain deep insight into structural and mechanisticunderstanding on PrP aggregation related to prion diseases, andprovide a way to identify specific inhibitors against the aggrega-tion interaction of PrPs. We also suggest that this TopMAC mode,liquid phase AFM methodology is applicable to study the effects ofthe substrate and pH on architectural features of aggregations ofother proteins with similar misfolding properties.

Acknowledgements

We thank Mr. Weijie Huang for guidance in drawing the 3Dmodels of the PrP units using 3Ds Max software. The research wassupported in part by the U.S. National Science Foundation (CBET1139057, ECCS 1231967), the Chinese National Defense Basic Sci-entific Research Project (Grant No. B2520133007) and Fundingof Jiangsu Innovation Program for Graduate Education (CXLX12-0145).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.07.053

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