Carbon Nanoparticles Inhibit the Aggregation of Prion Protein as

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Computational Chemistry

Carbon Nanoparticles Inhibit the Aggregation of Prion Protein as Revealed by Experiments and Atomistic Simulations Shuangyan Zhou, Yongchang Zhu, Xiaojun Yao, and Huanxiang Liu J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00725 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 24, 2018

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Journal of Chemical Information and Modeling

Carbon Nanoparticles Inhibit the Aggregation of Prion Protein as Revealed by Experiments and Atomistic Simulations Shuangyan Zhoua,d#, Yongchang Zhua,b#, Xiaojun Yaob,c, Huanxiang Liua* a School b State

of Pharmacy, Lanzhou University, Lanzhou 730000, China

Key Laboratory of Applied Organic Chemistry and Department of Chemistry, Lanzhou University, Lanzhou 730000, China

c State

Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for

Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China d

Chongqing Key Laboratory on Big Data for Bio Intelligence, Chongqing university of posts and telecommunications, Chongqing, China

# Authors with equal contribution * Corresponding author Tel.: +86-931-891-5686 Fax: +86-931-891-5686 E-mail address: [email protected]

1

ACS Paragon Plus Environment

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Abstract The specific properties of carbon nanoparticles (NPs) have attracted high attention on the application in biotechnology and biomedicine, e.g. in the field of amyloidosis. To date, it is still indefinable about whether the carbon NPs would promote or inhibit the fibril formation of amyloid proteins. Here, to uncover the effects of carbon nanoparticles (NPs) including graphene and carbon nanotube on the aggregation of prion protein, whose misfolding and aggregation will lead to prion diseases, the ThT fluorescence assay and molecular dynamics (MD) simulation were performed. The ThT fluorescence assay reveals that both graphene and carbon nanotube can inhibit the fibril formation of prion protein, especially graphene. Further MD simulation of PrP127-147 tetramer with or without carbon NPs suggests that the interactions between prion protein and carbon NPs reduce the aggregation tendency of PrP127-147 by decreasing the inter-peptide interactions and thus inhibiting β-sheet formation. Meanwhile, the aromatic residues greatly contribute to the inhibition effects of carbon NPs by π-π stacking interaction. The obtained results can increase our understanding on the interaction between nanoparticles and amyloid-related proteins.

Key words: carbon NPs, PrP aggregation, ThT fluorescence assay, molecular dynamics simulation.

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Introduction Recent advances in nanotechnologies have led to the broad application of nanoparticles (NPs) in drug delivery, molecular imaging and molecular diagnosis1-4. NPs are tiny materials with large surface area and size ranges from 1 to 100 nm. Among variety classes of nanomaterials, carbon nanomaterials such as graphene, carbon nanotubes and fullerenes have attracted particular interests in the field of biomedicine because of their unique architecture and special physical properties5. Carbon nanomaterials are typically characterized by significant adsorption capacity with large hydrophobic surfaces area, which makes it easy to carry other molecules such as drugs, peptides or proteins by covalent bonds or by adsorption6. In addition, carbon NPs can access cells or organs easily due to their ultra-small size, and interact with various biological molecules, particularly proteins like amyloid proteins7, 8. It is well known that the misfolding and aggregation of amyloid proteins would lead to the occurrence of amyloidosis, such as type II diabetes9, Alzheimer’s disease10, prion diseases11. To date, intensive efforts have been devoted to explore the effect of carbon NPs on the amyloid-related protein, including their biological toxicity and potential therapeutic effect. But until now, it still has no consistent conclusion. The obtained results indicate that NPs may promote or inhibit amyloid aggregation and fibril formation depending on the physicochemical properties of NPs and the characteristics of amyloid-related proteins7, 12-15. For example, by thioflavin T (ThT) fluorescence, Linse et al found that depending on the amount of exposed surface area and curvature of nanoparticles, multi-walled carbon nanotubes (MWCNTs) can 3



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enhance the appearance of critical nucleus for nucleation of human β2-microglobulin fibril formation by shorting the lag (nucleation) phase16. Combining with the finding of Linse’s, Colven et al hypothesized nanoparticle surfaces can act as platforms for protein association and catalyze fibril formation, which mean the nanoparticles are act as conventional catalysts in promoting fibrillation17. On the other hand, through ThT fluorescence, Kim et al discovered that fullerene can significantly prevent the fibril formation of beta-amyloid (Aβ) protein by specifically binding to the central hydrophobic motif of Aβ18. Later, Xie et al investigated the molecular mechanism of fullerene-inhibited aggregation of Aβ peptide deeply by molecular dynamics (MD) simulation19. They found the strong hydrophobic and aromatic-stacking interactions of fullerene hexagonal rings with Phe ring of Aβ significantly weaken the important peptide-peptide interaction for β-sheet formation. As such, exploring the nanoparticles-mediated aggregation of related amyloid protein is of important value to understand the role of NPs on related amyloidosis. Prion disease, also known as transmissible spongiform encephalopathies (TSEs), is one of the most typical amyloidosis in animals and humans11,

20, 21.

More

significantly, it is also the only known infectious amyloidosis, which can be infected from humans to humans or animals to humans. Generally, the misfolding and conformational transition of cellular prion protein (PrPC) into the abnormal and infectious isoform (PrPSc) which can act as seed to induce the fibril formation of prion protein22, is believed to be the key procedure in prion diseases23, 24. Presently, despite the widely reported studies about the influence of carbon NPs on the aggregation of 4


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many amyloid proteins, e.g. Aβ, β2-microglobulin, islet amyloid polypeptide (IAPP), by either experimental or computational methods16, 19, 25-27, little is known about the effects of carbon NPs on the aggregation of prion protein. Therefore, it is still a mystery for us whether the carbon NPs would promote or inhibit the fibril formation of prion protein. In this work, by combining ThT fluorescence assay and MD simulation, we investigated the potential role of carbon NPs, including graphene and carbon nanotube, on the aggregation of prion protein. The ThT fluorescence assay is an effective approach for amyloid fibril detection28, 29 and has been widely used in the field of peptide-NPs interaction studies. For example, Yousaf et al have used the ThT fluorescence analysis as a method to investigate the inhibiting effect of fluorine functionalized graphene quantum dots or hydroxylated carbon nanotubes on human islet amyloid polypeptide (hIAPP) aggregation30, 31.

As for MD simulation, it can

provide the atomic level understanding on how the carbon material affects amyloid aggregation since it can provide intuitive snapshots of how amyloid aggregates arrange, what interactions contribute most to protein aggregation and which residues are key for interactions, etc. Taken together, our study will provide deep insight into the potential role of carbon NPs on the aggregation of prion protein, which may also provide theory basis for the application of carbon NPs in the field of prion diseases.

Materials and methods The Cloning, Expression and Purification of moPrP117-231 5



In ThT fluorescence assay, the mouse prion protein from 117 to 231 (moPrP117-231) was used. The fully synthetic genes of PrP117-231 tagged by 6×his were cloned into an optimized pET-28b vector with two endonuclease enzyme site NdeI and NotI at 5’ and 3’ terminal, respectively. First of all, the recombinant plasmid was transferred into E.coil BL21(DE3) competent cells by heat-shock at 42℃ for 60 s . Then the cells were cultured overnight at 37℃，220 rpm in 50 ml LB containing 50 μg/ml kanamycin. After overnight culture, the cells were moved into 1L LB resistant medium at 37℃ until an optical density at 600 nm reached 0.6-1.0, and induced with 0.2 mM IPTG at 37℃ for 5 hours. The cultured cells were sonicated and the inclusion bodies were centrifuged at 12000 rpm for 30 min. The whitish pellets were then solubilized in the buffer (10 mM Tris-HCl, 100 mM NaH2PO4, 5 mM reduced glutathione, 8 M urea, pH 8). The solubilized PrP117-231 was refolded with buffers containing a gradient of 8-0 M urea (10 mM Tris-HCl, 100 mM NaH2PO4, 5 mM reduced glutathione, pH 8) by Ni-NTA agarose column. The non-bound proteins were removed by washing buffer (10 mM Tris-HCl ， 100 mM NaH2PO4, 50 mM imidazole), and the refolded PrP117-231 was eluted with an elution buffer (10 mM Tris-HCl 100 mM NaH2PO4, 800 mM imidazole, pH 5.8). The purified protein was concentrated and exchanged into storage buffer containing 10 mM Tirs-HCl, 100 mM NaH2PO4, pH 5.8 using ultrafiltration with a 3 kDa molecular weight cutoff centrifugal filter. The purity and concentration of the refolded protein were determined using a 12% SDS-PAGE and a Nanadrop 2000, respectively. Finally, the protein was stored lyophilized at -80℃. 6


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In Vitro Inhibition of Prion Fibrils Formation The ThT fluorescence assay was performed to evaluate the formation level of prion fibrils and the experimental details were described in our previous reported paper32. The carbon nanomaterials used in our work was bought from XFNANO, Inc. Nanjing. The carbon nanotubes are high-purity single-walled carbon nanotubes with the diameter 1-2 nm and the graphene is single layer with high purity (~99%). Both carbon nanotubes and graphene are uncharged without any modification. The purified prion protein was diluted to 20 μM with the dilution buffer (10 mM Tris-HCl, 100 mM NaH2PO4, pH 5.8) and then incubated with 1.5 ml Eppendorf tubes at 37 ℃，220 rpm. Three parallel experiments were performed for each testing. The final concentrations of graphene were 25 μg, 50 μg, 100 μg and the final concentrations of carbon nanotubes were 50 μg, 100 μg, 200 μg. Prion protein without carbon nanoparticles was used as control. At intervals, 100 μl of the samples were taken out and thoroughly mixed with equal volume of 20 μM ThT. As consequence, the final concentrations of ThT and prion protein were 10 μM. In order to monitor the formation of prion fibril, ThT fluorescence intensity was recorded with excitation at 440 nm and emission range of 480-490 nm.

Model preparation in molecular dynamic simulations Presently, it is still a challenge to obtain the amyloid structure of the full-length prion protein by existing techniques, which can be greatly attributed to the aggregation-prone and inherent heterogeneity properties of prion proteins. As thus, 7



many amyloid fragments of prion protein, e.g. PrP106-126, PrP82-146, PrP106-147 etc, are often used as relevant models to explore the aggregation behavior of full-length prion protein33-35. Former studies have shown that the fragment PrP127-147 of prion protein is a central region for amyloid fibril formation in Gerstmann-Straussler-Scheinker (GSS) syndrome and related encephalopathies35-37, which is also reported to exhibit typical characteristics of full-length PrPSc, i. e. fibril formation and cytotoxicity38, 39. Considering this, the fragment PrP127-147, which is included in PrP117-231, was chosen as a simplified model in this work to investigate the aggregation behavior of prion protein as well as the effects of carbon NPs on its aggregation. The sequence of PrP127-147 was shown in Fig. 1a. Three systems were simulated in total, including PrP127-147 tetramer alone, PrP127-147 tetramer in the presence of single-walled carbon nanotube (SWCNT) and in the presence of graphene. For brevity, we denoted the three systems as 4pep, 4pep-SWCTN and 4pep-GRA, respectively. The extracted atomic coordinate of monomeric PrP127-147 (displayed with red color in Fig. 1a) from the NMR structure of human prion protein (PDB ID:1HJM, displayed in Fig. 1a)40 was used as input structure to construct the initial PrP127-147 tetramer with the peptides randomly placed. After that, to obtain reasonable random tetramer structure for simulation with or without carbon NPs, a short replica exchange molecular dynamics (REMD) simulation was performed with 30 replicas by using the modified Generalized Born solvent model41. A web server42 was applied to generate temperature distributions of replicas with temperatures ranging from 270 K to 610 K. After 1 ns equilibrium MD simulations for all replicas 8


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without replica exchange, 30 ns REMD simulations for each replica were performed subsequently with the exchange interval set to 5 ps and exchange rate about 40%. We then extracted the trajectory at 303 K to choose a conformation as the initial tetramer structures with or without carbon NPs. The selected tetramer structure (4pep in Figure 1b) is mainly random coil and turn structure with four peptide chains well separated. The two termini of each peptide chain were capped with ACE and NH2 as determined experimentally39. For the complex systems of 4pep-SWCTN and 4pep-GRA, the peptide chains were put on the surface of NPs with the minimum distance large than 5 Å. The atomic coordinates of carbon NPs were generated by the Carbon Nanostructure Builder plugin in VMD43 (1.9.2) with the SWCNT 5.82 nm in length and 0.89 nm in diameter, while the dimension of graphene is 7.60 nm×7.60 nm. The initial structures of each system were depicted in Fig. 1b. Each system was then placed in a cubic water box with a minimum distance of 10 Å between the solute and the box boundary. All MD simulations were performed by using the AMBER 16.0 package44. The AMBER ff99SB force field45 which is developed to improve the secondary structure balance and to improve the description of glycine residue was applied, since it has been proved to achieve reasonable agreement with experiment for variety of proteins46,

47.

Besides, previous studies also reported that AMBER ff99SB can

reproduce the secondary structure of adsorbed peptides on graphene sheet comparable to experimental measurements48, 49. For carbon nanotube and graphene, the carbon atoms were modeled as uncharged particles as done by Zuo et al50, 51, corresponding 9



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to sp2 carbon in the AMBER ff99SB force field. The TIP3P solvent model was applied to describe water52. To keep the electroneutrality of systems, 8 Cl- ion was added to each system. Three steps were then performed to minimize each initial configuration by first keeping the peptides constraint, then keeping backbone of peptides constraint and finally free all of the molecules. Later, all systems were warmed up from 0 to 310 K in the NVT ensemble by keeping solute constraint, the temperature was controlled by the Langevin thermostat. After that, 5 ns equilibration MD simulation and 300 ns production MD simulation were performed for each system in the NPT ensemble. To ensure the “inside” position of carbon NPs in the solvent box during the simulation, 1.0 kcal mol-1 Å2 weak force was put on the NPs. During the simulation, a 2 fs time step was used to integrate the equations of motion and the SHAKE algorithm were applied to constrain the hydrogen-involved bonds. The long-range electrostatic interactions were treated with the particle mesh Ewald method. For all simulations, the atomic coordinates were saved every 2 ps for further analysis.

Analysis methods of MD simulation The AMBER 1644 and VMD programs (version 1.9.2)43 were employed to analyze the obtained trajectories. The STRIDE algorithm53 was used to calculate the secondary structure and thus to gain the impacts of NPs on the structural change of PrP127-147 peptides during oligomerization. The average-linkage cluster analysis54 was

used

to

classify

the

sampled

conformations

10


with

a

backbone

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root-mean-square-deviation (RMSD) cutoff of 3 Å. Further, to check the inter-peptide interactions, the residue contacts and backbone hydrogen bond (H-bond) formation between inter-peptides were calculated. We also calculated the total contact number and the binding free energies of each residue between peptides and carbon NPs to characterize the interplay among them. Here, a contact is defined when atoms in peptides or NPs come within 3.5 Å and H-bond is taken to be formed if the Donor…Acceptor distance is less than 3.5 Å and the Donor-H…Acceptor angle is less than 30°.

Meanwhile, the contact surface areas of SWCNT and graphene is

calculated as Scontact area=1/2((SASAtetramer+SASANPs)-SASAcomplex)19, the SASAtetramer, SASANPs and SASAcomplex are represent for the solvent accessible surface area of PrP127-147 tetramer, carbon NPs (SWCNT and graphene), and carbon NPs-tetramer complex, respectively. The binding free energy of NPs with each residue of PrP127-147 is estimated by the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method55-57 implemented in the AMBER 16 package44.

Results and discussion Carbon NPs inhibit the prion fibrils formation in vitro The expressed PrP117-231 was determined by 12% SDS-PAGE and the effect of the carbon nanomaterials on PrP117-231 fibril formation were shown in Fig. 2. As displayed in Fig. 2a and 2b, the incubated prion without carbon nanomaterials converted to amyloid after a short lag phase. After about 40 hours, the ThT fluorescence intensity reached the maximum, showing that the prion fibril reached the 11



maximum amount (black line in Fig. 2a and 2b). Increasing the concentrations of carbon nanomaterials, the fluorescence intensity obviously decreased. Here, to ensure that the decreased fluorescence intensity is mainly caused by the reduced amount of fibril, we further recorded the fluorescence emission spectrum of blank ThT as well as ThT incubated with carbon NPs (graphene and SWCNT) as a control. As can be seen from Fig. 2c, the presence of carbon NPs reduces the fluorescence intensity of ThT to a certain degree. However, it is much weaker than the signal reduction obtained with the co-incubation of carbon NPs and prion protein. Thus, it can be inferred that the reduction of fluorescence emission is indeed caused by the decreased fibril formation, and the presence of carbon NPs can inhibit the amyloid formation. Besides, when the graphene concentration was 100 μg/ml and the carbon nanotubes concentration was 200 μg/ml, the inhibition rate of both graphene and nanotubes were larger than 90% (Fig. 2d). Moreover, when the concentration of carbon nanomaterials (carbon nanotube and graphene) reached 50 μg/ml, the inhibitory effect was more than 50%. It is interesting to note that at the same concentration, the inhibitory rate of graphene was better than that of carbon nanotubes (Fig. 2d). Thereafter, in order to better understand the experimental results and uncover the inhibition mechanism of carbon nanomaterials on prion aggregation at the atomic level, MD simulation was further performed.

SWCNT and GRA reduce the peptide interaction and β-structure formation In order to identify the essence of the inhibition phenomenon observed in the 12


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ThT fluorescence assay, 300 ns conventional MD simulation runs were performed on the three systems, including 4pep, 4pep-SWCTN, 4pep-GRA. To monitor the convergence of each system, we first calculated the root-mean-square deviation (RMSD) of backbone atoms relative to the first conformations as well as the radius of gyration (Rg) for all systems. From Fig. 3a and 3b, it is obvious that the RMSD values and Rg values of all systems are quite stable during the last 50 ns, indicating that all simulated systems were reached equilibrium. Besides, it can be seen that 4pep system has the largest RMSD fluctuations followed by 4pep-SWCNT and 4pep-GRA, which indicates that the tetramer without NPs has the largest structural change and the interaction of tetramer peptides and NPs reduces the structural dynamics of peptides obviously. We further calculated the averaged RMSDs of backbone atoms for each residue of PrP127-147 to exam the flexibility of all residues and identify which portion is influenced most by carbon NPs during the aggregation process. The trajectories of last 50 ns were used and the result was shown in Fig. 3c. As with the RMSD fluctuation in Fig. 3a, the residues in 4pep with large average RMSD values will have the large structural flexibility. It can be seen that the residues in the C-terminus (residue 143-147) for all three systems are quite flexible with large average RMSD values, which consists well with the result of Cʹ-Cα cross-peak characterization of PrP127-147 fibril39 and our previous simulation of PrP127-147 monomer with the AMBER ff99SB force field58. Clearly, the structural flexibility of the N-terminus (residue 127-142) reduced in both carbon NPs systems, especially in system 4pep-GRA, indicating that the N-terminus may serve as the main region to 13



interact with surface of carbon NPs. As discussed above, the structural dynamics of PrP127-147 is largely influenced by the carbon NPs, especially for the N-terminus. Thus, here, to obtain the detailed structural information in three systems, secondary structure analysis was further performed. Fig. 4a shows the average content of different secondary structure of PrP127-147 tetramer with or without NPs. Compared to the initial tetramer structure starting from random coil and turn with all peptide chains well separated, the disordered coil and turn structures are still the main structures for all systems. The percentage of both helix structure and β-structure are relatively low in three systems. However, relative to 4pep-SWCNT and 4pep-GRA systems, the percentage of β-bridge and β-sheet structure in 4pep system is obvious higher. In 4pep, the contents of β-bridge and β-sheet structure are 4.70% and 5.34%, respectively. Whereas, only β-bridge structure is formed in 4pep-SWCNT and 4pep-GRA with contents about 1.40% and 0.64%, respectively. From Fig. 4b-4d, it can be seen more directly that both β-bridge and β-sheet structures in 4pep are formed pretty fast and quite stable. Meanwhile, it can be seen that the β-structures is mainly formed at the N-terminus involving residues 129-131 and residues 136-138 (C2 and C3), which may be largely attributed to the hydrophobic interaction as we reported before58. By contrast, in 4pep-SWCNT and 4pep-GRA, only the β-bridge structure is found to be formed with a few residues in the N-terminus involved (Fig. 4c and 4d). This result indicates that in the presence of carbon NPs, the tendency of β-structure formation is greatly decreased and the N-terminus may serve as an important region for the aggregation of 14


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PrP127-147. Additionally, to intuitively observe the structural property for each system, the average-linkage cluster analysis54 with a backbone RMSD cutoff of 3 Å was further performed to extract representative conformations from the last 50 ns trajectories. The representative structures and their corresponding percentage of the most populated five clusters are displayed in Fig. 5. Obviously, typical β-structures including β-sheet and β-bridge are observed in all clusters at system 4pep. Besides, the percentages of the first two clusters are 24.7% and 18.2%, respectively, suggesting the conformations in these two clusters are relative stable. In comparison, only short β-bridges are found in 4pep-SWCNT and no β-structure is observed in 4pep-GRA and their cluster percentages are all less than 10%. Meanwhile, in both 4pep-SWCNT and 4pep-GRA, the peptide chains are partially or completely adsorbed in the carbon NPs, especially in system 4pep-GRA, which directly prove the absorptive property of carbon NPs at molecular level. We thus speculate that the strong absorptive property of carbon NPs may reduce the inter-peptide interactions and thus reduce the tendency of β-structure formation. Further residue contact map for all contacts (side chain contacts and main chain contacts) and backbone H-bond formation map between inter-peptide chains prove our above speculation very well. As depicted in Fig. 6, peptides in 4pep have the most residue contacts and higher backbone H-bond formation probability. For 4pep without SWCNT or graphene, the residue contacts between different peptide chains are mainly found at the N-terminus from residue 127 to residue 141, indicating that 15



residues 127-141 play an important role in the aggregation process of PrP127-147, which is consistent with the above secondary structure analysis. This result is also in accordance with the previous experiment report that residues from 127 to 142 constitute the β-sheet structure of human PrP127-147 fibril39. In comparison, similarity tendency of residue contact is observed in both 4pep-SWCNT and 4pep-GRA but with the corresponding residue contacts significantly decreased or even disappeared, suggesting the reduced inter-peptide interactions. As with the residue contact map, backbone H-bond formation between inter-peptides is also largely influenced. In system 4pep, more than 10 stable backbone H-bonds can be found between the peptides, 3 stable H-bonds are formed in 4pep-SWCNT, and no H-bond is observed in 4pep-GRA. This observation indicates that the interactions between peptides and NPs prevent the backbone H-bond formation which further prevents the β-sheet formation since the backbone H-bond formation between inter-peptide chains is the prerequisites of amyloid aggregation. Moreover, the inhibitory effect of GRA on PrP127-147 aggregation is more effective than SWCNT as reflected by the less backbone H-bond formation in Fig. 6, which is consistent well with the above inhibition rate shown in Fig. 2d.

The aromatic amino acids contribute significantly to the carbon NPs-peptide interaction The above MD simulations of PrP127-147 tetramer suggest that peptides can interact with carbon NPs and adsorb on their surface, which directly decrease the 16


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inter-peptide interactions and prevent the β-sheet formation. But what is the essence for the strong interaction between peptide and carbon NPs? Why does graphene show larger inhibiting effect on PrP127-147 aggregation than SWCNT? To answer these questions, we further analysis the detailed interaction of PrP127-147 peptides and carbon NPs deeply based on the last 50 ns MD trajectories. The atom contact numbers between peptide and carbon NPs was firstly calculated since atom contact is the first step for the interaction between peptides and carbon NPs. Here, atom contact is considered to be existent if the distance between two atoms was less than 3.5 Å. As shown in Fig. 7a, the peaks of atom contact number for 4pep-SWCNT and 4pep-GRA are about 650 and 900, respectively, indicating the stronger peptide-GRA interaction than that of peptide-SWCNT. We speculate that the strong peptide-GRA interaction may be attributed to the large contact areas as reported by Xie et al19. Considering this, the contact surface areas of 4pep-SWCNT and 4pep-GRA were further calculated. Expectedly, as shown in Fig. 7b, larger peptide-GRA contact surface areas are found in 4pep-GRA system with the peak at around 1300 Å2, while the contact surface areas peak in 4pep-SWCNT is about 950 Å2. Consequently, it can be concluded that the larger contact surface area of GRA makes the stronger peptide-GRA interaction and thus interferes the inter-peptide interactions of PrP127-147 tetramer more effectively. This observation explains well with the stronger inhibiting effect of graphene on PrP127-147 aggregation. To further probe which residues and which region of PrP127-147 are important for the interactions between carbon NPs and PrP127-147, the interaction energy 17



between each residue of PrP127-147 and carbon NPs (SWCNT and graphene) was further plotted in Fig. 8. The interaction energy was calculated by the MM-GBSA method. It can be seen that most residues of PrP127-147 show larger binding energy with graphene, which is consistent with the stronger peptide-GRA interaction shown in Fig. 7. Besides, it is clear that the residues from 128 to 141 in the N-terminus of PrP127-147 show larger binding energies with carbon NPs, consistent well with our above speculation that the N-terminus of PrP127-147 may serve as the main region for carbon NPs-peptide interaction as reflected by the reduced averaged RMSD values of residue in the N-terminus. The strong interaction between carbon NPs and the N-terminus of PrP127-147 is also the main reason for the reduced inter-peptide residue contacts from 127 to 141, which further inhibits the aggregation of PrP127-147. Among these residues, hydrophobic residues such as M129, L130, M134, I138, I139, F141, are showing large interaction energies due to the hydrophobic property of carbon NPs. In addition, aromatic amino acids including Y128, F141 and Y145 have relative strong interaction energies with carbon NPs, especially residues Y128 and Y145 in 4pep-GRA, indicating that aromatic amino acids play an important role in the carbon NPs-peptide interaction. As the above result reveals that aromatic amino acids are significant for the carbon NPs-peptide interaction, we thus speculate that π-π stacking between the aromatic ring of aromatic amino acids and the hexagonal rings of carbon NPs is the main reason for the strong interaction energies of aromatic amino acids. As shown in Fig. 9a, most of aromatic amino acids in PrP127-147 tetramer can act as “anchor” to 18


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adsorb themselves onto the surface of carbon NPs, especially in 4pep-GRA. We also examined the packing interaction between aromatic side chain of aromatic residues and the carbon rings of NPs by calculating the probability distribution of the average distance between heavy atoms of aromatic residues sidechain and carbon NPs. Consistent with the above interaction energy of aromatic amino acids including Y128, F141 and Y145, the average distances of F141 in 4pep-SWCNT and Y128, Y145 in 4pep-GRA are relative small with their main peaks all center at 3.6 Å, and probability of 35.29%, 45.13% and 39.42%, respectively (Fig. 9b and Fig. 9c), indicating the π-π stacking of the three residue is strong. In comparison, two peaks and three peaks are observed for Y128 (main peak at 4.7 Å and probability of 20.66%) and Y145 (main peak at 3.6 and probability of 15.51%) in 4pep-SWCNT (Fig. 9b), respectively, suggesting the relative weaker π-π stacking. Although, for F141 in 4pep-GRA, there also exist two obvious peaks (Fig. 9c), the main peak is center at 3.6 Å, and the probability is 30.78%. As thus, it can be concluded that relative to the SWCNT, the overall π-π staking between the aromatic residues of PrP127-147 and graphene is stronger. This is also supported by the larger contact surface areas of graphene (Fig. 7b), since large contact areas is usually beneficial to the staking interaction. Besides, similar result about the π-π staking ability of SWCNT and graphene was exactly reported by previous MD simulation27.

Conclusion In this work, we investigated the potential role of carbon NPs on the aggregation 19



of PrP by combing ThT fluorescence assay and MD simulations. Our obtained results consistently demonstrate that carbon NPs including graphene and carbon nanotube can inhibit the fibril formation as shown in the decreased fluorescence intensity of PrP117-231 in ThT fluorescence assay and the reduced β-sheet content of PrP127-147 tetramer in MD simulations. The MD simulations of PrP127-147 tetramer with or without the carbon NPs uncover the molecular mechanism of the carbon NPs inhibiting the prion aggregation, which should attribute to the reduced inter-peptide interactions due to the strong carbon NPs-peptide interactions. Meanwhile, the N-terminus of PrP127-147 is the main region for interactions between peptides and carbon NPs. The inhibition effect of graphene is more obvious than carbon nanotube due to its larger contact surface areas. The interaction energy analysis indicates aromatic amino acids including Y128, F141 and Y145 contribute most to the interactions between peptide residues and carbon NPs, which is largely attributed to the π-π stacking interaction between aromatic ring of PrP127-147 peptide and hexatomic ring of carbon NPs. Overall, our results uncover the effect and the corresponding molecular mechanism of carbon NPs on the PrP aggregation, which can increase our understanding on the interaction between nanoparticles and amyloid-related proteins as well as is also valuable to the further applications of graphene and SWCNT in the biological medicine.

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Acknowledgements This work is supported by the National Nature Science Foundation of China (Grant No. 21675070 to H. L.) and the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2017-k24 to H. L.).

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Figure Caption Fig. 1 Structure display of (a) human prion protein (PDB ID: 1HJM) with fragment PrP127-147 colored in red. The sequence of PrP127-147 is shown below, the residues 128-131 are β-sheet structure and the residues 144-147 are α-helix structure. (b) The start simulation structure of PrP127-147 tetramer for each system. 4pep, 4pep-SWCNT, 4pep-GRA represent for tetramer, tetramer with SWCNT and tetramer with graphene, respectively. Fig. 2 ThT fluorescence assay of (a) SWCNT and (b) graphene on the fibril formation of PrP117-231. (c) Fluorescence of 10 μM ThT and ThT incubated with graphene and SWCNT upon excitation at 440 nm. (d) The inhibition rate of SWCNT and graphene after 40 hours. Fig. 3 Structural characteristics of PrP127-147 tetramer. (a) Structural stability of PrP127-147 tetramer with or without carbon NPs calculated by the RMSDs of backbone atoms relative to the start structures. (b) Radius of gyration as a function of simulation time. (c) Averaged RMSD values of backbone atoms for each residue in fragment PrP127-147. Fig. 4 Secondary structure analysis for PrP127-147 tetramer. (a) The average contents for each system to adopt different secondary structures. (b-d) Time series of the 29



secondary structure change for residues in PrP127-147 tetramer (b) without carbon NPs, (c) with SWCNT, and (d) with graphene. C1, C2, C3 and C4 in figures are representing for each peptide chain in tetramer. Fig. 5 The representative structures of the most five populated clusters for each system with their corresponding probability. Fig. 6 Inspection of the inter-peptide interactions of PrP127-147 tetramer. (a) Residues contact map including backbone contacts and side chain contacts, (b) backbone H-bond map between different peptide chains. The largest probability is considered as 1 for reference. Fig. 7 Inspection of the interactions between peptides and carbon NPs. (a) Probability distribution of contact numbers between peptides and carbon NPs and (b) Probability distribution of contact surface areas for SWCNT and graphene. Fig. 8 The interaction energy of each residue in fragment PrP127-147 with SWCNT and graphene. Fig. 9 Inspection of π-π staking interaction between aromatic amino acids and carbon NPs. (a) Representative structures of SWCNT and graphene interacting with PrP127-147 tetramer, respectively, all aromatic amino acids are displayed as sticks. (b)-(c) Probability distribution of the average distance between non-hydrogen atoms of aromatic amino acids side chain and carbon NPs.

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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Fig. 7

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Fig. 8

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Fig. 9

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Carbon Nanoparticles Inhibit the Aggregation of Prion Protein as Revealed by Experiments and Atomistic Simulation Shuangyan Zhou, Yongchang Zhu, Xiaojun Yao, Huanxiang Liu

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Carbon Nanoparticles Inhibit the Aggregation of Prion Protein as

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