Synergistic Inhibitory Effect of PeptideâOrganic Coassemblies on

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Synergistic Inhibitory Effect of PeptideOrganic Co-assemblies on Amyloid Aggregation Lin Niu, Lei Liu, Wenhui Xi, Qiang Li, Qiusen Han, Yue Yu, Qunxing Huang, Fuyang Qu, Meng Xu, Yibao Li, Huiwen Du, Rong Yang, Jacob R Cramer, Kurt V. Gothelf, Mingdong Dong, Flemming Besenbacher, Qingdao Zeng, Chen Wang, Guanghong Wei, and Yanlian Yang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b07396 • Publication Date (Web): 16 Mar 2016 Downloaded from http://pubs.acs.org on March 18, 2016

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Synergistic Inhibitory Effect of Peptide-Organic Coassemblies on Amyloid Aggregation Lin Niu1‡, Lei Liu1,3‡, Wenhui Xi2‡, Qiusen Han1, Qiang Li4, Yue Yu1, Qunxing Huang1, Fuyang Qu1, Meng Xu1, Yibao Li1, Huiwen Du1, Rong Yang1, Jacob Cramer4, Kurt V. Gothelf4, Mingdong Dong4, Flemming Besenbacher4, Qingdao Zeng1, Chen Wang1,*, Guanghong Wei2,*, Yanlian Yang1,* 1

National Center for Nanoscience and Technology, Beijing 100190, China 2

3

4

Department of Physics, Fudan University, Shanghai 200433, China

Institute for Advanced Materials, Jiangsu University, Jiangsu 212013, China

Interdisciplinary Nanoscience Center (iNANO), Center for DNA Nanotechnology (CDNA), Aarhus University, DK-8000 Aarhus C, Denmark

*Corresponding authors ‡

These authors contributed equally to this work.

ABSTRACT: Inhibition of amyloid aggregation is important for developing potential therapeutic strategies of amyloid-related diseases. Herein, we report that the inhibition effect of a pristine peptide motif (KLVFF) can be significantly improved by introducing a terminal regulatory moiety (terpyridine). The molecular level observations by using scanning tunneling microscopy (STM) reveal stoichiometrydependent polymorphism of the co-assembly structures which is originated from the terminal interactions of peptide with organic modulator moieties, and can be attributed to the secondary structures of peptides and conformations of the organic molecules. Furthermore, the polymorphism of the peptide coassemblies is shown to be correlated to distinctively different inhibition effects on amyloid-β 42 (Aβ42) aggregations and cytotoxicity. KEYWORDS: amyloid β (Aβ) peptide, scanning tunnelling microscopy, peptide aggregation, peptide motif, polymorphism effect, amyloid cytotoxicity, inhibitory effect ACS Paragon Plus Environment

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The pathology of some human disorders is likely correlated with the self-assembly of amyloid peptides into various forms of aggregates.1,2 For instance, in Alzheimer’s disease (AD), the amyloid fibrils are elucidated to be composed of β-hairpin structure containing two packed parallel β-sheets of amyloid-β peptides (Aβ),1,3,4 which is investigated by using X-ray diffraction (XRD), solid-state NMR, and scanning probe microscopy.5-12 These assembled aggregates may lead to significantly distinct toxicity in neuronal cells.13 In order to develop effective inhibition strategies to suppress amyloid formation and related cytotoxicity, many efforts have been made to design small molecule inhibitors. Some organic dye molecules (Congo red, thioflavin T (ThT) and their derivatives) and the polyphenol compounds, such as tannic acid and curcumin, are excellent agents for inhibiting the fibrillization of amyloidogenic peptides.14-18 Di-pyridine molecules, for instance, 4, 4’-dipyridine could accelerate the aggregation of peptides as one strategy to rescuing the cytotoxicity of amyloid peptides.9,11,12 Designed short peptide motifs are extensively pursued as potential inhibitors for amyloid peptide aggregation and cytotoxicity based on the peptide-peptide interactions.19-24 A promising peptide motif candidate is one Aβ pentapeptide sequence with the residues 16-20, Lys-Leu-Val-Phe-Phe (KLVFF), which has been identified as not only a key fragment for the formation of β-sheet and amyloid aggregations but also a recognition element for inhibiting the aggregation of full length Aβ peptide (Aβ42). Previous reports illustrated that short pentapeptide motif KLVFF with relatively bulky structures via covalent links to other molecules, oligopeptides, and dendrimers or apocyclen moieties may improve inhibitory effect on Aβ aggregation via specific interaction and the steric hindrance effect at single molecular level.25-31 However, further studies are still keenly needed to unravel the molecular pathways involved in regulating aggregation of amyloid peptides. One of the feasible important strategies is based on synergistic effect of potential co-assembly modulation species that might effectively enhance the inhibitory effect of pristine inhibitors by using non-covalent intermolecular interactions. In comparison with individual inhibitors, for instance, peptide motif for amyloid aggregation in previous study22-24, the 2

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peptide motifs and organic molecules in the non-covalent co-assembled structures are integrated together to interact with amyloid peptides, giving rise to greatly improved capability of tuning inhibition effects of pristine peptide motifs. Furthermore, the introduction of organic moieties linked to peptide motifs via non-covalent interactions maybe generate hierarchical architectures which may affect the binding strength with biological entities and also resulting in varied inhibition effect of amyloid aggregation and cytotoxicity in the current study. In the strategy, the promising peptide motif with good inhibitory effect on amyloid aggregation can be a candidate to construct the co-assembled structures with functional organic molecules by non-covalent interactions. In the previous studies, we have investigated the modulation effect of pyridine derivatives on Aβ42 peptide assembly due to the strong hydrogen bonds formed between pyridine derivatives and C-terminus of amyloid peptides.9,11,12 Herein, we expand this approach to construction of KLVFF-molecule complex by interaction between terpyridine molecule and C-terminus of peptide motif KLVFF, resulting in significant inhibition effect on amyloid aggregation. The terpyridine molecules have multiple aromatic N atoms which could lead to versatile interaction configurations for tunable complex. Thus, the bulky molecular species and the resulted bulky complex could give rise to increased steric hindrance for neighboring amyloid peptide contact resulting in increased inhibitory effect. In this work, short peptide KLVFF and small organic terpyridine molecules were utilized to construct the co-assembled inhibitors. The polymorphic co-assembled structures were explored to be related to the stoichiometry of peptides and terpyridine molecules by using scanning tunneling microscopy (STM) and supported by simulation analysis. Distinctively different molecular arrangements and secondary structures of peptides were further revealed in different polymorphic structures by STM, theoretical calculation and simulation, and Fourier transform infrared (FT-IR) spectroscopy. The obtained submolecular structural information facilitated understanding of the mechanism of peptide assembly and the interaction between the peptide and modulator molecule.9,11,12,32,33 Furthermore, the inhibitory effect 3


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of the peptide-organic molecule co-assemblies on Aβ42 aggregation and cytotoxicity were investigated, and The correlation between co-assembly polymorphism and the inhibitory effects were reported, which provides a new strategy for exploring synergistic effect of peptide-molecule co-assembly in amyloid diseases. RESULTS AND DISCUSSION Aβ16-20 KLVFF self-assembled structure on surface is observed to be typical continuous lamella on highly oriented pyrolytic graphite (HOPG) by using STM (Figure S1, supporting information). Separations between two neighboring peptides is approximately 0.47 ± 0.05 nm, which is in agreement with the previously reported inter-stand distances in Aβ and other amyloid peptides.9-12,33 In order to construct the peptide-organic molecular co-assembly, we introduced pyridine-containing species, 4'-chloro2,2':6',2''-terpyridine (hereafter refer as Ter in this work), that are expected to interact preferentially with the C-termini of the peptides33. The Ter molecules have inherent structural allotropy due to different conformations of pyridine moieties. When co-assembled with KLVFF, the polymorphism effect on the assemblies of KLVFF/Ter complexes could be examined from high resolution STM images. At a molar ratio of KLVFF vs. Ter 5:1 (representative of relatively low Ter content), typical STM images reveal the heterogeneous assembly of Ter co-adsorbed with KLVFF on the HOPG surface, in which Ter and KLVFF form a sandwich-like striped assembly structure (Figure 1a). The features exhibiting a brighter contrast in the linear array are attributed to Ter molecules, while the two stripes of reduced contrast are associated with KLVFF. In this molar ratio, the angle between peptide backbone and the stacking direction is 30 ± 2° (Table1). Peptides in one lamella structure assembled in a side-by-side form as their C-termini are anchored by Ter in the same direction (Figure 1d). At the same time, it also interacts with the molecule in the neighboring KLVFF lamella by tail-to-tail interactions. Neighboring two peptides within one stripe have a position offset of around 0.7 ± 0.1 nm, equivalent to the length of

4


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two amino acids. The side chain pairing could be K16-V18, L17-F19, V18-F20, in the case of KLVFF vs. Ter 5:1 (Table 1). The illustrative structure of the co-assembly is shown in Figure 1d. By adjusting the molar ratio of KLVFF and Ter to 1:1, a ladder-like peptide assemblies can be constructed (Figure 1e). Dimers of Ter molecules are formed by N…H hydrogen bonding intermolecular interactions and the distance between dimers of Ter is about 1.0 nm (Figure 1h). The hydrogen bonds between N atoms of Ter and C-terminus of KLVFF peptide lead to the formation of the alternating lamella structure with Ter dimer array insertion (Figure 1h). The angle between peptide backbone and the stacking direction of this ladder-like co-assembly is 85 ± 2°. Considering the alternating head-to-tail configuration discussed above, the inter-strand alignment of KLVFF peptides in this case can be deduced as Figure 1h. The peptides with head-to-tail form without residue offset were observed and differentiated. K16-F20; L17-F19; V18-V18; F19-L17 and F20-K16 as the side chain pairing are shown in Table 1. In addition, other forms of KLVFF/Ter co-assembly structures and KLVFF lamellae can be observed originating from local concentration fluctuations: coexistence of zig-zag type lamellar structures (Figure S2a), Ter tetramer (Figure S2b), Ter monomer impaction into KLVFF lamellae (Figure S2c) and KLVFF/Ter network structures (Figure S2d). Significant differences between KLVFF/Ter network structures (Figure 1i) and the lamellar ones discussed above (Figure 1a and e) should be noticed. When the molar ratio of KLVFF to Ter is adjusted to 1:5 (representative of relatively low KLVFF content), this network structure become predominant as shown in Figure 1i in which four peptides in rhomboidal βsheet pattern are surrounded by 4 tetramers of Ter. The tetramer unit is consisted of anti-parallel KLVFF molecules. In the neighboring network cavities, the peptides are oriented almost perpendicularly to each other. Four network cavities comprise one unit cell and the C-H…N hydrogen bond between the four Ter molecules comprises the crossover point of two kinds of lattices in perpendicular directions. A tentative structural model for the network of KLVFF/Ter is presented in Figure 1l. In each unit, the measured angle between peptide backbone and the stacking direction is 60 ± 2°, and each peptide has an offset of 5


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0.35 nm (size of one amino acid) in Figure 1j. In one quadrilateral cavity, each KLVFF molecule forms backbone and side chain binds to its two neighboring molecules into anti-parallel direction in the same KLVFF tetramer. Thus, the residue pairs are supposed to be F20-L17; F19-V18; V18-F19 and L17-F20 (Table 1). To understand the concentration-dependent polymorphic conformation of the co-assemblies, molecular simulations were performed to study the conformations and energies of Ter, peptides and the complex, based on STM observations. A single Ter molecule has three pyridine moieties, which means two degree of freedom on rotations of pyridines on both sides: trans-trans(C00), trans-cis(C01), cis-cis(C11) (Figure 2a).32,34 We performed quantum-chemical computation to investigate these three different conformations. Compared to the C00, the other two isomers are disfavored in energy (7.3 kcal/mol for C01 and 16.9 kcal/mol for C11 higher than that of C00 as shown in Figure 2b, and the histogram should be reversed for normal looking). Furthermore, with the monomer conformation C00, three pyridine moieties of a single Ter molecule are in the same plane, but not with C01 and C11 conformations. The bending angles are 19.5° and 28.3° for C01 and C11 respectively (As shown in lateral view in Figure 2a). Therefore, the following models are based on the C00 type of Ter due to its predominant energy stability. Conformations in various KLVFF assemblies, including amino acid side chain and backbone, are also investigated by molecular dynamics. All the possible peptide conformations and assembly structures in STM observations are enumerated. The lamellar structures are analyzed and categorized as parallel or antiparallel β-sheets (represented by P-/AP-). The directions of side chain on neighboring peptides could be the same or opposite (represented by Cis-/Trans-). Finally, two neighboring peptides having two residues offset, one residue offset and no offset are represented by (2), (1) and (0). There are six suggested β-sheet patterns of peptides in all: P-Cis(0), AP-Cis(0); P-Trans(1), AP-Trans(1) and P-Cis(2), AP-Cis(2) (Figure 2b and Figure S3). The calculated angles between peptide backbone and the stacking direction are listed in Figure 2b. According to the angles of peptides in co-assemblies, there are two alternative 6


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patterns for each molar ratio: P-Cis(0) and AP-Cis(0) for 1:1 ratio ( ~90°), P-Cis(1) and AP-Cis(1) for 5:1 ratio (~35°) and P-Trans(2) and AP-Trans(2) for 1:5 ratio (~60°). The average binding energies between adjacent peptides calculated by MMGBSA methods (Figure 2d) reveal that the AP-Cis(0) (-22.1 kcal/mol), AP-Trans(1) (-20.2 kcal/mol) and P-Cis(2) (-9.0 kcal/mol) are the most possible conformations for each corresponding patterns. After obtaining the above simulation results, quantum chemical (QC) calculations and molecular dynamics (MD) simulations were performed for the optimal conformations to explore the interactions between Ter (C00) and peptides in the co-assemblies. The stable structures of tetramers of Ter (C00) were obtained by QC calculations. Two conformations are found with four N…H-C hydrogen bonds in Ter tetramers (Figure 3a). Conformation A can be attributed to the observed pattern in 1:5 ratio assemblies and the Conformation B can be attributed to the pattern in 1:1 ratio assemblies. In the case of 5:1 ratio, the interaction between Ter molecules can be ignored. The binding energies of these two tetramers are calculated to be -17.9 kcal/mol for Conformation A and -14.0 kcal/mol for Conformation B (Figure 3b). Furthermore, the interaction between amino acid C/N-termini and Ter molecules is calculated by QC calculation. The binding energy between the C-terminus and Ter is calculated to be -23.1 kcal/mol, which is much lower than the one between the N-terminus and Ter (-5.8 kcal/mol) (Figure 3b & 3c). This calculation result is also consistent with the previous report that Ter molecule would prefer to bind to C-terminus rather than N-terminus of peptide.31, 32. At the 5:1 molar ratio of KLVFF to Ter, representative of high KLVFF peptide content in the coassemblies, C-terminus of two peptide molecules interact with the Ter molecules in C00 conformation. Based on this hydrogen-bond interaction configuration, peptide molecules are arranged into tworesidue-position-offset lamellar with the P-Cis(2) conformation, the Ter molecules are located in the center of the sandwich-like striped assembling structure. The Ter-Ter interaction is weak and negligible in this assembly configuration. The optimal co-assembly of peptide/Ter in this ratio was calculated and 7


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presented in Figure 3d-e (5:1, P-Cis(2)/Ter), which is consistent with STM investigation. By adjusting the molar ratio of KLVFF and Ter to 1:1, in the ladder-like peptide co-assemblies, KLVFF peptides molecules arrange into the aligned lamellar with AP-Cis(0) conformation with the lowest binding energy and most stable co-assembly structure. This conformation enables the peptide molecules to interact with Ter, with an angle 89.8° ± 1.9° between peptide backbone and Ter dimer occlusion array. The hydrogen bond interaction between Ter molecule and peptide in antiparallel form is much lower than the peptide in parallel form. In addition, in the Ter dimer occlusion array, neighboring two Ter molecules also interact with each other by C-H…N hydrogen bond, with an average binding energy of -3.5 kcal/mol per monomer or -14.0 kcal/mol for Ter tetramer (Figure 3d and 3e). When the molar ratio of KLVFF to Ter is adjusted to 1:5, the quadrangular network structure becomes the predominant co-assembly configuration. With the high Ter content in the assemblies, four pairs of C-H…N hydrogen bond in every Ter tetramer are formed with the average binding energy -4.5 kcal/mol per monomer or -17.9 kcal/mol for Ter tetramer. The quadrangular network formed by these Ter tetramers could encapsulate KLVFF peptide tetramers in the cavity with AP-Trans(1) conformation. This assembly structure is stabilized by the terminus interaction between the peptide and Ter molecule, as well as the optimal conformations of peptides (AP-Trans(1)) and Ter molecules (Conformation A), respectively. The MD simulations (methods details is SI) were performed for the above three optimal conformations of peptide co-assemblies, and the structures are shown in Figure 3d, which are in excellent agreement with STM observations. The above analysis of the modulation of secondary structure of KLVFF in different assemblies was further verified by FT-IR spectroscopy. Amide I perpendicular band (around 1632 cm-1) and amide I parallel band (around 1695 cm-1) are sensitive to the hydrogen bond between the peptides. Only amide I perpendicular band represents parallel β-sheet, while both amide I perpendicular band and weak parallel band stand for anti-parallel β-sheet.35-37 Individual KLVFF in the self-assembly was identified to be parallel β-sheet structures (1636 cm-1 ± 2 cm-1)38-42 (Figure 4a). When Ter molecules modulate the peptide 8


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assembly at the molar ratio 5:1 (KLVFF : Ter), the β-sheet characteristic of KLVFF is retained in the similar region only with slight change from 1636 cm-1 ± 2 cm-1 to 1631 cm-1 ± 2 cm-1 and a peak disappearance at 1537 cm-1 (Figure 4b), which implied the molecular modulation of peptides by Ter. STM study (Figure 1a) also supported the modulation of the assembly structures of KLVFF by Ter molecules into the side-by-side parallel β-sheet conformation in the same KLVFF lamella. Due to the two amino acid offset to the neighboring peptide in the parallel assemblies, the inter-molecular hydrogen bond density is relatively lower than the one in the individual KLVFF continuous lamella structures. Consequently the peak at 1631 cm-1 ± 2 cm-1 becomes a little weaker compared to the individual KLVFF (Figure 4e). At the molar ratio of 1:1, we observed a new weak band at 1708 cm-1 ± 2 cm-1 under the jurisdiction of amide I parallel band. The presence of two major bands at 1633 cm-1 ± 2 cm-1 (strong) and 1708 cm-1 ± 2 cm-1 (weak) indicated the presence of an anti-parallel β-sheet conformation of the peptide assemblies, consistent with the conjecture of a new alternating lamella of KLVFF structure from the STM investigation (Figure 1e). It seems that the weak peak 1708 cm-1 in this work is out of the commonly accepted range of anti-parallel β–sheet (1695 cm-1). However, it is reported that this peak is resulted from vibration of each amide bonds within the peptide and protein, which could be influenced by the hydrogen bonding patterns, and the surrounding environment such as the pH, temperature and interaction with other molecules.43 In this work, the temperature and pH were maintained constant, while introduction of chaperone-like molecular modulator could change the hydrogen bonding configurations (see Figure 1, STM investigation, different hydrogen bonding configurations were observed for the peptide assembly and peptide/Ter complex) and further affect the amide bond vibration of peptide. Therefore, it is rationalized that FT-IR peak representing the anti-parallel β–sheet secondary structure will slightly blue shifted from 1695 cm-1 to 1708 cm-1 after introduction of molecular modulator. Furthermore; along with the increase of Ter to the molar ratio of 1:5 (KLVFF : Ter), the anti-parallel β-sheet can also be observed with amide I perpendicular band at 1632 cm-1 ± 2 cm-1 (strong) and amide I parallel band at 1710 cm-1 ± 9


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2 cm-1 (weak) (Figure 4d). In comparison, 1635 cm-1 ± 2 cm-1 peak is obviously weakened in this case (Figure 4e) though the band 1710 cm-1 ± 2 cm-1 is observed to be a little stronger instead (Figure 4f). This is ascribed to the transformation of peptide assemblies from ladder-like lamellar structure to quadrilateral cavities. In the interdigitated and compact ladder-like lamellar structure at the molar ratio 1:1, peptides can form a strong backbone hydrogen bonding network and side chain packing, which could enhance the intensity of amide I perpendicular band at 1633 cm-1 ± 2 cm-1 (Figure 4e). However, once increasing the small organic molecule Ter to the molar ratio of 1:5 (KLVFF : Ter), the scattered peptide oligomers embedded in the quadrilateral cavity dramatically block and intercept β-sheet formation in peptide self-assembly into long lamellae (Figure 1i). To further explore the polymorphic structures of peptide assembly on nanoscale, co-assembled nanostructure of KLVFF and Ter was characterized by atomic force microscopy (AFM). The polymorphism of assembled nanostructures of KLVFF/Ter was obtained, which was mainly due to the molecular modulation of Ter agent binding at C-terminus of peptides (Figure 5). Typical amyloid fibril assembled from KLVFF was observed by AFM with 2 days incubation (Figure 5a). The height was determined to be ~ 6 nm (Figure 5e). Morphological change from short fibrils (Figure 5b), granular particles and oligomers (Figure 5c), to small flocky particles (Figure 5d) as the molar ratios are varied from 5:1 to 1:1 to 1:5 for the KLVFF/Ter co-assemblies with same incubation, respectively. They were determined to be 7 nm for short fibril (Figure 5f), 4 nm for oligomers and 20 nm for granular particles (Figure 5g) and 3 nm for flocky particles (Figure 5h) presented in the height histograms. The polymorphism of peptide coassembly on nanoscale can be clearly recognized. Nanoparticles appear as the molar ratio of peptide and Ter is changed to 5:1, which is characterized both by height analysis and area coverage analysis (Figures 5i and 5j). After the threshold, the granular particles (1:1) and flocky particles (1:5) dominated the morphology of co-assemblies of peptide and Ter, rather than the fibrils. The different stoichiometry of peptide and Ter will lead to distinct peptide packing, geometrical arrangement and molecular interaction 10


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between the peptide and Ter at the terminus, which is the essence of the peptide-organic molecular coassembly polymorphism. The capability of the peptide co-assemblies in inhibiting Aβ42 aggregation and cytotoxicity were demonstrated and the polymorphism effect on the inhibitory ability was also observed. The process involved in aggregation of Aβ42 was monitored by ThT fluorescent assay. Binding of ThT to amyloid fibrils leads to increased fluorescence emission at 482 nm upon excitation at 450 nm, which has been widely used for screening inhibitors.44 In Figure 6a, typical sigmoidal curve of Aβ42 peptide (50 µM) aggregation process was observed, and the aggregation behaviors of Aβ42 peptide with a series of KLVFF/Ter polymorphic assembly were further explored. The general inhibiting effect was concluded that although the individual KLVFF marginally slows down the aggregation of Aβ42, the co-assemblies of KLVFF/Ter with different stoichiometry presented the pronounced inhibiting effect on Aβ42 aggregation (Figure 6a). In comparison, when the molar ratio of Aβ42/KLVFF/Ter is 1:1:1, the optimized inhibiting effect was obtained which completely suppressed the aggregation of Aβ42. In addition, the inhibitory effect of different peptide co-assembly on the morphologies of Aβ42 was investigated, from mature amyloid fibrils (Aβ42), decreased amount of amyloid fibrils (Aβ42/KLVFF), short fibrils (Aβ42/KLVFF/Ter=5:5:1), to nanoparticles (Aβ42/KLVFF/Ter = 1:1:1, 1:1:5) (Figure. 6b, Figure S5). The height of these modulated nanostructures was generally decreasing, which was consistent with the results obtained from the ThT assay that the maximum fluorescence intensity also decreased approximately in the presence of different peptide/Ter co-assemblies (Figure 6b). It is obviously displayed that the optimized inhibiting effect of peptide co-assembly on Aβ42 aggregation was at the molar ratio of Aβ42/KLVFF/Ter = 1:1:1. To explore polymorphism effect of peptide assemblies on inhibiting the Aβ42 cytotoxicity, the dependence of cytotoxicity on amyloid peptide aggregation was examined on the neuroblastoma cells. All the SH-SY5Y cells were incubated for 48 h at 37℃ in Aβ42 solutions with and without KLVFF and KLVFF/Ter complexes. The cell viability was monitored by using MTT (3-(4, 5dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide) assay. Aβ42 alone is detected to have no-


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table cytotoxicity at a concentration of 10 µM and the cytotoxicity is slightly reduced in the presence of the inhibitor peptide KLVFF (Figure 6c), in consistent with previous reports.23,29 If different KLVFF/Ter stoichiometries are introduced into the medium, the cytotoxicity is neutralized to varying degrees at Ter concentrations of 2, 10 and 50 µM (Figure 6c). The introduction of KLVFF/Ter at the molar ratio 5:1 and 1:1 can enhance the inhibiting effect of the peptide motif KLVFF on Aβ42 cytotoxicity. It is proposed that the inhibiting effect on both the amyloid aggregation and the cytotoxicity of Aβ42 are ascribed to the synergistic effect of co-assembled structure of short peptide and Ter molecule. Various forms of amyloid aggregates have been identified including nanometer scale oligomers, fibrils, fibers and eventually senile plaques. Soluble oligomers of amyloid peptides have been shown to have primary toxic effects in cell cultures. Modulation of the assembly behaviors of Aβ42 is considered as an important approach to explore the possible diagnosis and treatment of AD. Possible mechanisms could be associated with the retained recognition of crucial peptide motifs to the target Aβ42 peptides and the enlarged steric hindrance between neighboring Aβ42 peptides because of the peptide-molecule coassemblies. Different inhibitory effects are possibly associated with the KLVFF/Ter co-assembled structures. From STM results, KLVFF/Ter tunable complexes could be formed depending on peptide:Ter stoichiometry. At a molar ratio of KLVFF vs. Ter 5:1 (representative of relatively low Ter content), Ter and KLVFF form a sparse sandwich-like striped assembly structure, in which two KLVFF peptide molecules interact with one Ter. By adjusting the molar ratio of KLVFF and Ter to 1:1, a tighter ladder-like peptide assembly can be constructed. The hydrogen bonds between N atoms of Ter and C-terminus of KLVFF peptide lead to the formation of the alternating lamella structure with Ter dimer array, in which one KLVFF peptide molecule interacts with one Ter. The bulky Ter dimer array and the resulted more bulky complex could give rise to increased steric hindrance for neighboring Aβ42 amyloid peptide contact. Such compact co-assembly structure may enhance the inhibitor effect on Aβ42 aggregation. In this case, with the stoichiometry increase to 1:5, the tight lamellar assembly structure is re-arranged into looser network. Such variations in the co-assembly structures could inevitably influence the interaction


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sites, side chain interactions and interaction between Aβ42 and its inhibitor motif KLVFF. Lower inhibitory effects of cytotoxicity are observed at molar ratio of 1:1 and 1:5 due to the toxicity of Ter molecule at higher concentration (Figure S6). The polymorphism effect suggests that only the co-assemblies at optimized molar ratio of peptide to molecule are more suitable to interfere with the Aβ42 aggregation, modulate Aβ42 assembly structures at different stages including oligomers and fibrils and reduce the amyloid cytotoxicity. The investigations could be expanded into general strategy for versatile combinations between peptide inhibitors and small molecules for broad range of amyloid peptides related to degenerative diseases. CONCLUSIONS To sum up, the polymorphic effect of the amyloid peptide assemblies can be clearly recognized in the assembly characteristics, aggregation morphology and cellular functionalities. In this paper, we introduced terperidine molecules which can form binary complex with KLVFF by hydrogen bonding to enhance the modulating effect of KLVFF on Aβ42 aggregation. The molecular mechanistic studies of the peptide-organic molecular co-assembly revealed the contributions from multiple interactions in polymorphic peptide assemblies with distinct geometrical arrangement. The enhanced inhibitory effect of the peptide-molecule co-assembly demonstrated the improved rescue effect on Aβ42 induced cytotoxicity. It could be a feasible strategy to utilize peptide-small molecule complex inhibitors to interact with amyloid peptide at the initial stage, and modulate amyloid assembly structures at different stages including oligomers and fibrils, and further regulate the aggregation dynamics of amyloid peptide ultimately reduce the cytotoxicity of amyloid peptides.

MATERIALS AND METHODS Sample Preparation. Aβ42 (purity, 96.3%) was obtained from American Peptide Inc. KLVFF (purity, 98%) was obtained from Shanghai Science Peptide Biological Technology Co. Ltd. 4’-chloro-2,2’:6’,2”terpyridine was purchased from Acros. KLVFF and Ter were dissolved to 1 mM concentrations in MilACS Paragon Plus Environment

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liQ water and ethanol (AR grade), respectively. The final concentrations and the molar ratio of KLVFF and Ter molecules were obtained by mixing the stock solutions with different volumes. STM Experiments. Assembly of KLVFF peptide and co-assemblies of KLVFF/Ter at different molar ratios were prepared by placing approximately 5 µL solutions on freshly cleaved atomically flat HOPG surface (quality ZYB). After the solvent was evaporated, the experiments were performed with a Nanoscope IIIA system (Veeco Inc. USA) operating under ambient conditions. The STM tips were mechanically formed Pt/Ir wire (80/20). All STM images were recorded using the constant current mode. The specific tunneling conditions are given in the corresponding figure captions. QC Calculation. The monomeric and tetrameric Ter molecules and alanine-Ter complex were built and first optimized by VegaZZ45. For the monomeric Ter, three conformations were compared (Figure 2a). For the tetramers, various initial conformations were tried and only two plane conformations were stable (Figure 3a). Furthermore, all systems were then optimized in Gaussian 0346 at the wB97XD/6-31G* level. Comparing to the other DFT methods, such as B3LYP, the wB97XD methods have better performance in representing the long-range interactions47. The basis set superposition error (BSSE) was taken into account in calculating the interactions between molecules. In the tetrameric Ter systems, the binding energy of four molecules was treated as the sum of pairwise interaction and the three-body interaction were neglected. Atomistic MD Simulations. According to our experimental results, six initial β-sheet structures were built. They were different in three aspects: the parallel/anti-parallel β-sheet (P/AP), the direction of side chain (Cis-/Trans-) and the residue offset between neighboring peptides (0/1/2). Six systems were named as follow: P-Cis(0), AP-Cis(0), P-Trans(1), AP-Trans(1), P-Cis(2) and AP-Cis(2) (shown in Figure 2c). In the simulations of co-assemblies, the Ter molecules were placed near the C-terminal of peptides according to the experimental and quantum-chemical results (Shown in Figure 3d and Figure S3). The peptides were placed on a large flat graphene surface. Atomistic MD simulations were performed using the AMBER12 software package48 with Amber ff12SB force field. The system was solvated with


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TIP3P water. The bond length were constrained using SHAKE algorithms49 with integration time step of 2 fs. Simulations were performed in the isothermal-isobaric (NPT) ensemble at 300 K. Particle mesh Ewald (PME) method were employed to treat electrostatic interactions50. The initial conformation underwent 5-ns MD simulation in which the backbone of peptides and Ter molecules were loose restrained. Then for each system, two 30-ns MD simulations were performed. The distance was constrained to 0.5 nm between the mass centers of carboxyl groups at C-terminal of peptide and nitrogen atoms at Ter during the simulations. Besides, the graphene surface was restrained to the initial coordinate. In all systems, most part of the co-assembly maintained well except that peptides at the edge were separated gradually. The analysis of MD simulations based on average of two trajectories with the first 15-ns omitted. The binding energy of neighboring peptides were calculated by MMPBSA.py51 with generalized Born (GB) methods develop by J. Mongan et al.52. AFM Experiments. For KLVFF/Ter complexes: The KLVFF/Ter complex solutions were prepared by mixing KLVFF solution in phosphate buffer (10 mM sodium phosphate, 140 mM NaCl, pH 7.4) with Ter in ethanol and stored at 37℃ for 2 days for the formation of different aggregation structures. The final KLVFF/Ter complex concentrations for incubation were 1 mM for AFM experiments. For Aβ42 inhibited by KLVFF/Ter complex: The KLVFF/Ter complex solutions were prepared by mixing KLVFF solution in phosphate buffer with Ter in ethanol and stored at room temperature for 1 h for complex formation. The pure Aβ42 peptides were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma) and then lyophilized. The lyophilized Aβ42 peptides were dissolved in phosphate buffer and mixed with the KLVFF/Ter complex solutions and incubated up to 2 days at 37℃. The final Aβ42 peptide concentration for incubation was 50 µM for AFM experiments. These solution preparation procedures are also applied in fluorescence experiments. 2 µL of mixed solutions are dropped onto the freshly cleaved mica surface followed by air drying. All the AFM experiments were performed in tapping mode under ambient conditions (Nanoscope IIIA SPM system, Bruker, USA). Commercial silicon tips with a


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nominal spring constant of 40 N/m and resonant frequency of 300 kHz were used in all the AFM experiments. ThT Fluorescence Assay. The ThT fluorescence assay was performed using a PerkinElmer LS55 fluorescence spectrophotometer with excitation and emission wavelengths of 450 and 482 nm, respectively. For each measurement, the incubated Aβ42 solutions with KLVFF/Ter complexes were mixed with ThT solutions in phosphate buffer to final concentration of Aβ42 peptide at 2.5 µM and ThT at 50 µM. The ThT fluorescence intensity as a function of incubation time was fitted using a sigmoidal curve. The solutions were incubated in shaker at 37°C and 100 rpm. The ThT fluorescence assay was repeated for 3 times under the same experimental conditions and all the fluorescence intensity values were the average value of every sample at each time point. MTT Assay. MTT assay was used to assess the cytotoxicity. SH-SY5Y cells were initially seeded on 96-well plates at a concentration of 15000 cells/well (100 µL/well) in full medium under certain incubation conditions overnight. Then SH-SY5Y cells were treated with or without various concentrations of Aβ42 in the presence or absence of modulator molecules for another 48 h. After that, 10 µL MTT (5 mg/ml, CCK8 kit, Dojindo, Japan) were added into each well and the plates were incubated at 37°C for 4 h. Medium was then removed and 200 µL of dimethyl sulfoxide (DMSO) were added to dissolve the formazan crystals. Absorbance was immediately determined on a microplate auto reader at wavelengths of 492 nm. Blank controls without cells were subtracted from sample absorbance values. Cell viability was expressed as the percentage of viable cell relative to untreated cells. Experiments are means of triplicates, and each experiment was performed three times. Data are expressed as mean ± SD. Statistical analyses were performed with SPSS (SPSS, Chicago) by using a paired t–test. Differences were considered statistically significant when P＜0.05.


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ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected].

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS Financial support from National Natural Science Foundation of China (91127043, 21261130090, 21273051) is gratefully acknowledged. R. Yang, C. Wang, M. D. Dong, and F. Besenbacher wish to acknowledge the financial support from SinoDanish Center. L. Liu also acknowledges National Natural Science Foundation of China (21573097) and Youth Natural Science Foundation of Jiangsu Province (BK20130493). MD simulations were performed at the National High Performance Computing Center of Fudan University. We thank Wei Wang of Nanjing University to perform QC calculation using Gaussian03. Supporting Information Characterization data about KLVFF self-assembly structure and KLVFF/Ter co-assembly structures and aggregation nanostructures and height analysis of Aβ42 in bulk solution before and after molecular binding. This material is available free of charge via the Internet at http://pubs.acs.org.


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Figure captions Figure 1. High-resolution STM images and the corresponding tentative models of KLVFF/Ter co-assemblies at different molar ratios: 5:1 (a, b, c, d), 1:1 (e, f, g, h), and 1:5 (I, j, k, l). The corresponding tunneling conditions: (a) I = 321.4 pA, V = 699.8 mV; (b) I = 433.3 pA, V = 630.2 mV; (c) I = 532.3 pA, V = 689.0 mV. (b, f, j) Periodicity of single KLVFF peptide molecule. Five peaks represent five residues of KLVFF, which is consistent with the observation from high-resolution STM images. (c, g, k) Separation between two neighboring KLVFF peptide strands. The separation is approximately 4.8 ± 0.1 Å, attributed to the β-sheet formation according to the similar inter-stand distances in Aβ with β-sheet secondary structures. (d, h, l) Detailed chemical structures illustrating the terminal H-bonding interactions. Only backbone atoms of KLVFF peptide are shown. The O-H…N hydrogen bonds between the carboxyl groups of KLVFF peptides and the nitrogen atoms of Ter modulators are illustrated with red dotted lines. Figure 2. (a) Three isomer of Ter in both front view and lateral view. The energy of molecular and the dihedral between pyridines are listed. (b) Enumerating all possible β-sheet structures. The C-termini of peptides are color in red to identify the A/AP of β-sheet. The sidechain of Val18 is color in blue to distinguish the Cis-/ Trans- pattern. (c) The energy of Ter molecules in different conformations. The energy of C00 is set as zero point. The positive value of energy represent disfavor. (d) The binding energy between adjacent peptides. Figure 3. (a) The conformation of interaction between C/N-terminus of peptide and Ter. (b) The binding energy between C/N-terminus and Ter is colored in red. The binding energy between tetramer of Ter is colored in green (tetramer) and purple (averaged on monomer of Ter). (c) Two stable conformations of tetramer of Ter. (d) The coassemblies structures for three molar ratio. The oxygen atoms (red beads) and nitrogen atoms in pyridine (blue beads) formed strong interactions. Figure 4. FT-IR spectra indicate the modulation of β-sheet secondary structure with introduction of pyridine molecules: (a) KLVFF(parallel), (b) KLVFF/Ter at a 5:1 molar ratio of KLVFF to Ter (parallel), (c) KLVFF/Ter at a 1:1 molar ratio of KLVFF to Ter (anti-parallel), (d) KLVFF/Ter at a 1:5 molar ratio of KLVFF to Ter (antiparallel). The characteristic bands have been labeled in the spectra. (e) and (f) The difference of Amide I perpendicular band and Amide I parallel band for β-sheet secondary structures in peptide assembly tuned by Ter molecule, respectively.


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Figure 5. Polymorphism of assembled nanostructures of KLVFF/Ter: KLVFF self-assembled nanostructure (a, e) and KLVFF/Ter co-assembled nanostructures at the KLVFF/Ter molar ratio of 5:1 (b, f), 1:1 (c, g) and 1:5 (d, h) characterized by AFM. (i, j) The height and coverage analyses of different peptide co-assembled nanostructures, respectively. The scale bar is 2 µm. Figure 6.The polymorphism effect of peptide KLVFF assembly on the aggregation behaviors and the cytotoxicity of Aβ42. (a) The ThT assay of the self-assembly process of Aβ42 (50 µM), Aβ42 with individual KLVFF (50 µM : 50 µM), Aβ42 with co-assemblies of KLVFF/Ter (50 µM : 50 µM : 10 µM, 50 µM : 50 µM : 50 µM, 50 µM : 50 µM : 250 µM). (b) Maximum fluorescence intensity and height distributions of Aβ42 decreases with stoichiometry variation of peptide vs. Ter molecule in the co-assemblies, and the morphology of Aβ42 changes from fibrils, short fibrils to particles. (c) Cytotoxicity of SH-SY5Y cells induced by Aβ42, Aβ42/KLVFF and Aβ42/KLVFF/Ter solutions at different molar ratios. Table 1. The assembly behaviors of KLVFF peptide self-assembly at different molar ratios of peptide to Ter.

ToC Molecular level observations by using scanning tunneling microscopy (STM) reveal stoichiometry-dependent polymorphism of the KLVFF/terperidine co-assemblies. The polymorphism of the peptide-organic co-assemblies is shown to be correlated to distinctively different inhibition effects on amyloid-β 42 (Aβ42) aggregations and cytotoxicity.


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Table 1

KLVFF & Ter Co-assembly Structure Molar Ratio

5:1

1:1

1:5

Secondary structure

Parallel

Anti-Parallel

Anti-Parallel

Angle between peptide to strip axis

35 ± 2º

85 ± 2º

60 ± 2º

K16V18

K16F20

F20L17

L17F19

L17F19

F19V18

V18F20

V18V18

V18F19

F19L17

L17F20

Side chain pairing

F20K16


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Figure 1. 243x303mm (150 x 150 DPI)


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Figure 2. 564x493mm (72 x 72 DPI)


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Figure 3. 219x219mm (300 x 300 DPI)


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Figure 4. 684x394mm (72 x 72 DPI)


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Figure 5. 543x423mm (72 x 72 DPI)


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Figure 6 846x317mm (72 x 72 DPI)


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ToC 317x141mm (72 x 72 DPI)


Synergistic Inhibitory Effect of PeptideâOrganic Coassemblies on

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