The inhibitory effect of hydroxylated carbon nanotubes on the

Subscriber access provided by University of Winnipeg Library

Article

The inhibitory effect of hydroxylated carbon nanotubes on the aggregation of human islet amyloid polypeptide revealed by a combined computational and experimental study Yuxiang Mo, Sayanti Brahmachari, Jiangtao Lei, Sharon Gilead, Yiming Tang, Ehud Gazit, and Guanghong Wei ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00166 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

The inhibitory effect of hydroxylated carbon nanotubes on the aggregation of human islet amyloid polypeptide revealed by a combined computational and experimental study Yuxiang Mo†a,b, Sayanti Brahmachari†c, Jiangtao Leia, Sharon Gileadc, Yiming Tanga, Ehud Gazit*c and Guanghong Wei*a a

State Key Laboratory of Surface physics, Key Laboratory for Computational Physical Science (Ministry of Education), Collaborative Innovation Center of Advanced Microstructures, and Department of Physics, Fudan University, Shanghai 200433, People's Republic of China b

College of Physical Science and Technology, Guangxi Normal University, 15 Yucai Road, Guilin, 541004, People's Republic of China

c

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

†

These authors contributed equally to this work.

1

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Fibrillar deposits formed by the aggregation of the human islet amyloid polypeptide (hIAPP) are the major pathological hallmark of type 2 diabetes mellitus (T2DM). Inhibiting the aggregation of hIAPP is considered as the primary therapeutic strategy for the treatment of T2DM. Hydroxylated carbon nanoparticles have received great attention in impeding amyloid protein fibrillation owing to their reduced cytotoxicity compared to the pristine ones. In this study we investigated the influence of hydroxylated single-walled carbon nanotubes (SWCNT-OHs) on the first step of hIAPP aggregation-the dimerization by performing explicit solvent replica exchange molecular dynamics (REMD) simulations. Extensive REMD simulations demonstrate that SWCNT-OHs can dramatically inhibit inter-peptide β-sheet formation and completely suppress the previously reported β-hairpin amyloidogenic precursor of hIAPP. Based on our simulation results, we proposed that SWCNT-OH can hinder hIAPP fibrillation. This was further confirmed by our systematic turbidity measurements, thioflavin T fluorescence, circular dichroism (CD), transmission electron microscope (TEM) and atomic force microscopy (AFM) experiments. Detailed analyses of hIAPP-SWCNT-OH interactions reveal that hydrogen bonding, van der Waals, and π-stacking interactions between hIAPP and SWCNT-OH significantly weaken the inter- and intra-peptide interactions that are crucial for β-sheet formation. Our collective computational and experimental data reveal not only the inhibitory effect but also the inhibitory mechanism of SWCNT-OH against hIAPP aggregation, thus providing new clues for the development of future drug candidates against T2DM.

Keywords: Protein aggregation, human islet amyloid polypeptide, hydroxylated carbon nanotubes, inhibitory mechanism, replica exchange molecular dynamics simulations, TEM experiment. Graphical Table of Contents

2


Page 2 of 32

Page 3 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Amyloid fibrillar deposits formed by the aggregation of proteins/peptides in different organs are the hallmark of several diseases. For example, the pathological self-assembly of the human islet amyloid polypeptide (hIAPP), amyloid-β (Aβ) peptide and α-synuclein is associated with type-2 diabetes mellitus (T2DM)1, Alzheimer’s disease2 and Parkinson’s disease3, respectively. Despite different protein compositions, all the amyloid fibrils formed by different proteins display a cross-β structure with β-strands perpendicular and inter-strand hydrogen bonds parallel to the fibril axis, indicating a common aggregation mechanism4. The hIAPP is a 37-residue polypeptide hormone (also known as amylin) which originates in the pancreatic β cells and is co-secreted with insulin5, 6. It was reported that the aggregation of hIAPP leads to the death of islet β cells7. Emerging evidence shows that hIAPP oligomers8 and mature fibrils9 are both toxic and may contribute to β-cell loss in T2DM10. Hence, inhibiting hIAPP aggregation has been considered as one of the primary strategies to treat T2DM. In this respect, finding and designing inhibitor-based drugs against T2DM is a challenge and is of significance. To date, four major classes of inhibitors have been reported. The first class of inhibitors includes proteins11,

peptides12, 13 and peptide analogues14. The second consists of

organic small molecules15

16

(such as EGCG extracted from green tea and resveratrol

often found in the fruits of grape, peanuts, and berries). The third class of inhibitors comprises of a series of nanoparticles including polymer nanoparticles17, gold-sulfur complexes18 and carbon nanoparticles19,

20

. The fourth class of inhibitors is

antibodies21-23. Recently, carbon nanoparticles (including fullerenes, graphenes, carbon nanotubes and their derivatives) have received considerable attention in modulating 3



Page 4 of 32

protein aggregation24-27 due to their ability of crossing biological membranes28-30. For instance, increasing experimental and computational studies show that fullerenes and their derivatives can inhibit the amyloid fibrillation31-37 or disrupt amyloid fibrils38, 39 of both Aβ peptides and Aβ fragments. Graphenes and graphene derivatives can not only retard the fibrillization of Aβ40,

41

and hIAPP19,

20

, but also promote the

fibrillation of other peptides42-44. Also, pristine and hydroxylated single-walled carbon nanotubes (SWCNT-OHs) can inhibit the formation of β-sheet-rich oligomers (from dimers to octamers) and amyloid fibrils of Aβ16-22, Aβ1-40 and Aβ1-4245-48 peptides. In contrast, it was reported that pristine carbon nanotubes enhance the fibrillation of β2-macroglobulin by shortening the lag phase for nucleation49. These contradictory effects of carbon nanoparticles on amyloid fibrillation may depend on the protein-nanomaterial interactions. Nevertheless, in spite of extensive studies, the influence of SWCNT-OH on the aggregation of hIAPP peptide and the underlying molecular mechanisms remain elusive. In this work, we performed 400-ns (per replica) all-atom explicit-solvent replica exchange molecular dynamics (REMD) simulations in the absence or presence of SWCNT-OHs to study the effect of SWCNT-OH on the first step of hIAPP aggregation (i.e. dimerization). Hydroxylated CNTs, due to their enhanced solubility in water, biocompatibility, and reduced cytotoxicity compared to pristine CNTs50, are becoming a more promising candidate for biomedical applications. Our simulations show that SWCNT-OHs can dramatically interfere with the inter- and intra-peptide interactions and inhibit the β-sheet formation of hIAPP via hydrogen bonding, van der Waals and π-π stacking interactions with hIAPP, thus retarding the next step towards aggregation. In addition, we conducted systematic turbidity experiments at different concentrations, ThT fluorescence analysis, CD, TEM and AFM experiments to validate our simulation predictions. Importantly our experimental findings show that SWCNT-OH interacts with the hIAPP and can significantly inhibit hIAPP fibrillation.

RESULTS AND DISCUSSION 4


Page 5 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


We first examined the convergence of the two REMD simulations using the data within two different time intervals (240-320 and 320-400 ns) for the hIAPP dimer and (220-310 and 310-400 ns) for the hIAPP+SWCNT-OH system. Several parmeters were used for the convergence check, including the probability density function (PDF) of radius of gyration (Rg) of hIAPP dimer, the probabilities of each dominant secondary structure over all residues, and the coil, β-sheet, bend, turn and helix probabilities for each residue within the two different time intervals. As shown in Figure S1(A) and Figure S2(A), the Rg distributions within the two independent time intervals overlapped well in the two systems. The time-averaged and the residue-based coil, bend, turn, β-sheet and helix probabilities within the two time intervals were also quite similar (Figure S1(B-G) and Figure S2(B-G)). We also checked the sampling efficiency by comparing the PDF of the number of H-bond for two peptide chains (chain-1 and chain-2) and by following the time evolution of temperature swapping of one representative replica in temperature space. The PDF of the number of H-bond for chain-1 and chain-2 showed similar curves (Figure S1(H) and Figure S2(H)). As shown in Figure S1 (I) and Figure S2(I), the representative replica of the two systems visited the full temperature space several times during the 400 ns simulation, demonstrating that the replica was not trapped in one single temperature. All of these results indicated that our simulations are reasonably converged.

Hydroxylated SWCNTs significantly inhibit β-sheet formation and induce disordered coil conformations of hIAPP dimer. We first examined the influences of SWCNT-OH on the secondary structure of hIAPP dimer by calculating the probability of each dominant secondary structure. As shown in Table 1, after the SWCNT-OH were added into the hIAPP dimer system, the β-sheet content decreased from 10.6 ± 1.1 (%) to 3.8 ± 0.3 (%), while the coil content increased from 40.5 ± 0.8 (%) to 48.8 ± 0.2 (%). The helix, bend and turn contents changed very slightly. We also calculated the residue-based secondary structure of hIAPP in the absence or presence of SWCNT-OHs. From Figure 1(A), one can see that the SWCNT-OHs significantly 5



Page 6 of 32

reduced the β-sheet content of residues C7~L16 and S19~T36. Strikingly, the β-sheets of residues C7~Q10, F15 and T30~G33 were completely suppressed. The coil content increased in the region of T6~F15, A25~L28 and N31~S34 (Figure 1 (B)), and the helix content was reduced dramatically in the region of A24~S29 and S34~T36 (Figure 1(C)). Overall our simulations results demonstrate that SWCNT-OHs significantly prevented β-sheet formation and induced coil conformations of hIAPP dimer. Secondary structure System

β-sheet (%)

Helix

Coil

Bend

Turn

(%)

(%)

(%)

(%)

hIAPP

10.6±1.1

7.5±0.1

40.5±0.8

27.0±0.1

11.7±0.1

hIAPP+SWCNT-OH

3.8±0.3

5.2±0.2

48.8±0.4

28.7±0.1

10.3±0.3

Table 1 The probability of each dominant secondary structure of hIAPP dimer in hIAPP and hIAPP+SWCNT-OH systems.

Figure 1. Propensity of (A) β-sheet, (B) coil and (C) helix as a function of amino acid residue of hIAPP dimer in the two systems at 310 K. We then classified hIAPP dimers into different type of conformations by performing cluster analysis using a chain-independent main-chain RMSD cutoff of 0.35 nm. The hIAPP dimer conformations in hIAPP and hIAPP+SWCNT-OH systems were separated into 84 and 72 clusters, respectively. The top six most-populated clusters and their populations are shown in Figure 2(A, B), which represent 45% and 47% of all conformations of hIAPP dimer in hIAPP and hIAPP+SWCNT-OH systems respectively. These representative conformations show that hIAPP dimers have 6


Page 7 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


different structure propensities in the two systems. In the absence of SWCNT-OHs, hIAPP dimers have a preference to adopt a three-stranded antiparallel β-sheet structure consisting of a β-hairpin structure (two β-strands: A8-L16 and A25-G33 and a loop: V17-G24) formed by one chain and a β-strand by the other chain (Cluster_1), and disordered conformations containing short β-sheets (Cluster_2, Cluster_6) and a mixture of β-sheet and helix structures (Cluster_3, Cluster_4, Cluster_5). The β-hairpin structure in Cluster-1 is of particular interest as the positions of its β-strand regions strongly resemble those of the solid-sate NMR-derived hIAPP fibrils (two β-strands: A8-V17 and S28-Y37, and a loop H18-L27)51. This β-hairpin was reported to be the amyloidogenic precursor of hIAPP52, 53. In the presence of SWCNT-OH, the β-hairpin-containing three-stranded antiparallel β-sheet structure disappeared (Figure 2(B)). The vanishing of the β-hairpin-containing β-sheet conformation in the presence of SWCNT-OH indicate that SWCNT-OH can prevent hIAPP from forming prefibrillar aggregate, thus inhibit hIAPP fibrillation. The hIAPP dimers are mostly in disordered coil-rich states with much shorter β-sheets. These results demonstrate that the SWCNT-OHs shift the conformations of hIAPP dimer toward more disordered states. This finding is further supported by the probability of β-sheet length (Figure 2(C)). In the isolated hIAPP system, we found that the two-, nine-, three-, six-, four-, and eight-residue β-sheets have the top six highest probabilities of 3.6%, 1.9%, 1.4%, 1.4%, 0.7% and 0.7% respectively. However, in the hIAPP+SWCNT-OH complex, the probabilities of two-, three-, six- and four-residue β-sheet decrease to 1.0%, 1.0%, 0.5% and 0.4% respectively, while eight- and nine-residue β-sheets almost vanish. These data indicate that SWCNT-OH greatly prevented hIAPP peptides from forming long β-sheet conformations that are important for hIAPP fibrillization54. In Figure 2(D), one can see that hIAPP dimers in the presence of SWCNT-OH exhibit a decreased number of H-bonds, indicating that the SWCNT-OH block the inter- and intra-peptide interactions. The increase of Rg observed in Figure 2(E) suggests that SWCNT-OHs induce hIAPP to form loosely packed conformations.

7



Figure 2. Conformational analyses of hIAPP dimers in the absence or presence of SWCNT-OHs. Representative conformations of the first six most-populated clusters of hIAPP dimers in the absence (A) or presence (B) of SWCNT-OHs. The corresponding population of each cluster is given in parentheses. The two hIAPP chains are colored in cyan and purple, and SWCNT-OH are in bond representation. The green sphere refers to the Cα atom of K1 residue of each chain. (C) Probability of β-sheet length in hIAPP dimers without (green) or with (wine) SWCNT-OH. (D) PDFs of the total number of intra- and inter-molecular H-bonds, (E) the radius of gyration of the hIAPP dimer in the two different systems.

Van der Waals, aromatic stacking and hydrogen bonding interactions between SWCNT-OH and hIAPP play important roles in inhibiting β-sheet formation of hIAPP dimer. To understand the molecular mechanism of SWCNT-OH in inhibiting β-sheet formation of hIAPP dimer, we calculated the inter- and intra-peptide main chain-main chain (MC-MC) and side chain-side chain (SC-SC) contact probabilities between all pairs of residues of hIAPP in the absence or presence of SWCNT-OH (Figure 3). The inter-peptide MC-MC contact pattern between residues R11~L16 and 8


Page 8 of 32

Page 9 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H18~F23 (marked by a rectangle in the upper left of the contact probability matrix (Figure 3(A)) and the intra-peptide contact pattern between residues A8~V17 and G24~V32 (marked by a rectangle in the lower right of the contact probability matrix (Figure 3(A)) both disappeared in the presence of SWCNT-OH (Figure 3(C)). The disappearance of the two MC-MC contact patterns further indicate that the SWCNT-OH completely suppressed the formation of the above-mentioned β-hairpin-containing three-stranded β-sheets. An increase of intra-peptide contact probabilities between residues T30~S34 and V17~S20 is also observed in Figure 3(C) (marked by a rectangle in the lower right of the contact probability matrix), indicating the formation of other patterns of short β-sheets which were induced by SWCNT-OH. Quantitatively similar results are seen for the inter- and intra-peptide SC-SC interactions (Figure 3(B, D)). The intra-peptide SC-SC contact patterns were changed in the presence of SWCNT-OH in Figure 3(B, D) (marked by rectangles in the contact probability matrix). These data demonstrate that SWCNT-OHs dramatically weakened both inter- and intra-hIAPP interactions that are important for the amyloidogenic precursor formation, thus shifting the hIAPP dimer from fibril-competent β-sheet rich conformations to fibril-incompetent coil-rich conformations.

9



Figure 3. Inter- and intra-chain MC–MC and SC–SC contact probability maps for hIAPP dimer in the absence (A and B) or presence (C and D) of SWCNT-OHs.

To identify the key residues and dominant interactions that contributed to the binding of hIAPP to the SWCNT-OH, we calculated the residue-based relative binding free energy, the vacuum potential energy components (the van der Waals and electrostatic interaction energies) and the H-bond formation probability between hIAPP and the SWCNT-OH. As shown in Figure 4(A), aromatic residues F15 (-5.8 kcal/mol) and Y37 (-9.7 kcal/mol), hydrophobic residue L12 (-5.6 kcal/mol), and polar residues Q10 (-6.1 kcal/mol), H18 (-5.0 kcal/mol), N31 (-5.0 kcal/mol) and N35 (-6.8 kcal/mol) interacted most strongly with SWCNT-OHs than other residues. The contact probability between hIAPP and SWCNT-OH in Figure S3(A) showed quantitatively similar results. Structural visualization shows that the nonpolar aliphatic groups of polar residues interacted mostly strongly with the carbon atoms of SWCNT-OH. Our observation of strong interactions between polar residues and carbon nanotubes is also in consistent with previous experimental55 and computational56,57 studies of the adsorption of proteins/peptides to the surfaces of carbon nanotubes and fullerenes. Figure S3(B) shows that the solvation energy of each residue was unfavorable for the binding, especially for charged residues K1 and R11. By comparing Figure 4(B) with Figure 4(A), we found that residue-based van der Waals interaction energy (Figure 4(B)) displayed similar trends with the relative binding free energy (except for charged residues K1 and R11) and van der Waals interaction energy dominated over the electrostatic interaction energy (Figure 4 (C)). This finding implies that the van der waals interaction made a dominant contribution to the binding of hIAPP to SWCNT-OH. Figure 4(D) shows that polar residues Q10 (5.1%), N3 (4.2%), N31 (4.4%) and N35 (5.1%), and charged residue R11 (5.5%) have relatively higher probability to form H-bonds with hydroxyl groups of SWCNT-OH (Figure 4(D)). These results indicate that aromatic stacking and hydrogen binding interactions played critical roles on the binding of hIAPP with SWCNT-OH, consistent with our previous studies showing that π-π stacking and 10


Page 10 of 32

Page 11 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hydrogen bonding interactions are the dominant forces that inhibit the aggregation of Aβ fragments by carbon nanoparticles 34, 45, 58.

Figure 4. Analyses of residue-based interactions between hIAPP and SWCNT-OH. (A) Binding free energy, (B) van der Waals (VDW) and (C) electrostatic (Elec) contribution to the binding free energy, and (D) H-bond probability. The unit of the energy is in kcal/mol. To better understand the stacking patterns between the aromatic residues of hIAPP and the carbon rings of SWCNT-OH, we plotted in Figure 5(A) the free energy surface as a function of two reaction coordinates: the centroid distance and the angle between the rings of aromatic residues (F15, F23 and Y37) of hIAPP and the carbon rings of SWCNT-OH. The free energy surface in Figure 5(A) exhibited three minimum-energy basins centered at (distance, angle) values of (0.4 nm, 10.0º), (C) (0.6 nm, 60.0º), and (D) (0.6 nm, 89.1º), which correspond to parallel-, herringbone-, and perpendicular stacking patterns respectively between the rings of aromatic residues and the carbon rings of SWCNT-OH. These data imply that the stacking patterns between hIAPP aromatic rings and SWCNT-OH carbon rings are distance-dependent, and at a shorter distance, a parallel-aligned π-π stacking geometry is likely to be the most favorable. Of interest, a parallel-displaced aromatic stacking was found to be the major organization of π-π interactions in proteins59. Three 11



representative snapshots in Figure 5(B-D) show the parallel-, herringbone-, and perpendicular stackings between the aromatic rings of residues Y37 and the carbon rings of SWCNT-OH. We also calculated the parallel-, herringbone-, and perpendicular stacking probability between the rings of each aromatic residues (F15, F23 and Y37) and the carbon rings of SWCNT-OH (Figure 5 (E-G)). It can be seen that the contributions of aromatic residues to the three different stacking patterns have the order of Y37 > F23 > F15, indicating that residue Y37 makes a dominant contribution to the π–π stacking interaction between hIAPP and SWCNT-OH.

Figure 5. Analysis of aromatic-stacking interactions between the rings of three aromatic residues (F15, F23 and Y37) of hIAPP and the carbon rings of SWCNT-OH. (A) The free energy landscape as a function of the centroid distance and angle between the aromatic rings of hIAPP and the carbon rings of SWCNT-OH. Representative snapshots showing the stacking patterns of Y37 with the carbon rings of SWCNT-OH at a (Distance, Angle) value of (B) (0.4 nm, 10.0º), (C) (0.6 nm, 60.0º), and (D) (0.6 nm, 89.1º). (E) Parallel, (F) herringbone and (G) perpendicular stacking probability between the rings of each aromatic residue (F15, F23 and Y37) and carbon rings of SWCNT-OH for their centroid distance < 0.5 nm and angle < 30º,

12


Page 12 of 32

Page 13 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.5 nm < distance < 0.7 nm and 40º< angle < 60º , and 0.5 nm < distance < 0.7 nm and 60º < angle < 90º, respectively.

Turbidity measurements, ThT fluorescence, CD and microscopy experiments demonstrate that hydroxylated SWCNT can significantly inhibit the hIAPP fibrillation. Our REMD simulations showed that SWCNT-OHs can dramatically inhibit inter-peptide β-sheet formation and completely suppress the previously reported β-hairpin amyloidogenic precursor of hIAPP by blocking inter- and intra-peptide interactions. Based on our simulation results, we proposed that SWCNT-OH could impede the next step of hIAPP aggregation and the formation of hIAPP mature fibrils. To validate our speculation, we probed the system using turbidity measurements, ThT fluorescence measurement, CD, TEM and AFM imaging experiments. To investigate the effect of the SWCNT-OH on the process of hIAPP aggregation, firstly the turbidity of the SWCNT-OH in the presence of hIAPP in PB and that of a control system of SWCNT-OH alone in PB were studied. 1 mg of SWCNT-OHs was weighed and sonicated in ice with 0.1 mg hIAPP in 2 mL PB for 10 min. The solution was allowed to settle overnight and the supernatant was collected and compared with the control dispersion of 1 mg of SWCNT-OH similarly treated but in the absence of the protein (Figure 6(A, B)). The absorbance of the solution was measured at 550 nm and the dispersion remained stable for 24h and after which aggregation was observed. The system containing SWCNT-OH and hIAPP showed an absorbance of 0.47±0.02 while the absorbance value of the control system containing SWCNT-OH alone was found to be 0.12±0.02. Next, a series of hIAPP solutions were prepared in PB with varying concentration of hIAPP from 0.0125 mg/mL to 0.125 mg/mL. These solutions were incubated with fixed amount of SWCNT-OH (0.5 mg/mL) and the samples were processed as described in the methods section. The absorbance was measured and plotted as a function of varying hIAPP concentration. The obtained plot (Figure 6(C)) clearly shows that initially the amount of SWCNT-OH interacting with the hIAPP 13



increased with increasing amount of hIAPP but after a concentration of 0.05 mg/mL the system reached saturation and there was no noticeable change in the absorbance value. Similarly, the effect of the varying concentration of SWCNT-OH on fixed concentration of hIAPP was studied. The concentration of SWCNT-OH was varied from 0.25 mg/mL to 1 mg/mL while the concentration of the hIAPP was fixed at 0.05 mg/mL. The samples were treated as previously and the obtained absorbance was plotted as a function of varying SWCNT-OH concentration (Figure 6(D)). Interestingly there was no observable change in the absorbance upon increasing concentration of SWCNT-OH at a fixed concentration of hIAPP of 0.05 mg/mL. Pristine SWCNTs are intrinsically amphiphobic in nature, i.e. they are not soluble or dispersed in any kind of solvent60. However, the dimension, nature and the functional groups play an instrumental role in the dissolution process of these materials in solvent27. Also, the protein hIAPP has a high aggregation propensity and it can form amyloid fibrils under physiological conditions14. However, the turbidity experiments of the first control system indicate that SWCNT-OH by itself still has low solubility in PB in spite of the presence of the -OH functional groups. Subsequently, the turbidity analysis of the SWCNT-OH in the presence of hIAPP indicates that the protein interacts and assists in the dissolution of the SWCNT-OH in the aqueous buffer. Also from the varying concentration ratio analysis we inferred that the optimum mass ratio of nanomaterial to the protein was 0.5 mg/mL of SWCNT-OH to 0.05 mg/mL of hIAPP since other combinations do not substantially affect the absorbance. This provides the first and most important experimental evidence that the hIAPP protein and the SWCNT-OH interacts in solution as suggested by the simulation results. It is noted that the mass ratio of hIAPP to SWCNT-OH in our simulation is about 1:4, which is of the same order of magnitude as that used in experiments (1:10). However, the concentrations of SWCNT-OH and hIAPP in our simulations are respectively 4.3 and 16.5 mg/mL, much higher than those in experiments. The use of high hIAPP and SWCNT-OH concentrations enables us to characterize the relatively complete conformational ensembles within bearable simulation time using current computer facilities, as done in previous REMD simulation studies45, 61, 62. 14


Page 14 of 32

Page 15 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Images showing the dispersions of SWCNT-OH with hIAPP in PB (A) and SWCNT-OH alone in PB (B). (C) Plot of absorbance of SWCNT-OH at 550 nm with varying hIAPP concentrations. (D) Plot of absorbance of varying concentrations of SWCNT-OH in hIAPP solution. All the experiments were repeated three times.

After having established that the SWCNT-OH and hIAPP interacts in a PB solution in a way that it led to observable dissolution of the nanomaterial, we then probed the phenomenon using the ThT binding assay. The ThT binding assay is an established method widely used for the detection of amyloid fibrils.55 The dispersed solution of SWCNT-OH with hIAPP was incubated with ThT and the spectrum was recorded after 3 h of incubation (Figure 7(A iv)). The spectra of blank ThT (Figure 7(A v)) and ThT in the presence of hIAPP (Figure 7(A ii)) were also recorded as control systems. Additionally, as a control we recorded the fluorescence emission spectrum of ThT incubated with SWCNT-OH (Figure S4). Compared to blank ThT solution, a significant increase in the fluorescence emission intensity of ThT was observed when incubated with hIAPP, a feature of ThT usually observed in the presence of amyloids. However, a significant decrease of this emission was observed when SWCNT-OH was co-incubated with hIAPP. Importantly, quenching in the fluorescence emission intensity of ThT was also observed in the presence of SWCNT-OH only (without hIAPP in Figure S4), but it is much weaker than the signal reduction obtained with the co-incubation of SWCNT-OH and hIAPP. Hence it can be inferred that the reduction of the fluorescence emission is cumulative due to the significant decrease in the amount of amyloid fibrils in the presence of the 15



SWCNT-OH and the quenching effect of SWCNT-OH itself. Subsequently the fluorescence was recorded after 12 h of incubation and a significant increase in the emission intensity was detected in the hIAPP control system which corresponds to an increase in the amyloid content with increasing time (Figure 7(A i)). However, after 12 h the emission intensity of the co-incubation system of SWCNT-OH with hIAPP did not increase substantially, which highlights the efficiency of the nanotubes towards inhibiting the formation of the amyloidogenic structures over time (Figure 7(A iii)). This investigation indicates that the interaction of the nanomaterial with the protein eventually lead to a decrease in the amyloid formation and the process of aggregation is arrested by the nanomaterial over time. Furthermore, the process of hIAPP protein aggregation in the presence of SWCNT-OH was also monitored using CD. Specifically, two different kinds of experiments were conducted, in order to understand first the effect of SWCNT-OH on already formed hIAPP structures and second the effect on co-incubation of SWCNT-OH and the protein. For the first analysis, hIAPP was sonicated and dissolved in PB at a concentration of 0.05 mg/mL and the spectrum was recorded after 2 h (Figure 7(B i)). The spectra of the blank hIAPP clearly showed a minimum at 223 nm which corresponds to the formation of β-sheet secondary structure (Figure 7(B i)). Next, SWCNT-OHs were sonicated (in 20 µL of PB) and added to these pre-formed protein structures (i.e. the 2h-incubated hIAPP solution), the solution was shaken for 2 h and the spectra was recorded after 30 min and 2 h incubation (Figure 7B ii, iii). As shown in Figure 7(B ii) a significant decrease in the β-sheet content is observed after 30 min. Subsequently, the feature of the spectrum after 2 h (Figure 7(B (iii))) indicates the formation of the α-helical structure with double minima at 208 and 220 nm, which is consistent with the simulation results suggesting that there is a decrease in the β-sheet content when the protein interacts with the nanomaterial. Similarly, for the second analysis, 0.05 mg/mL hIAPP was co-incubated with 0.5 mg/mL SWCNT-OH and the spectra was recorded after 2 h (Figure 7(B iv)). However, a complete loss of this secondary structure was observed in case when the SWCNT-OH and hIAPP was co-incubated (i.e., added at time zero) (Figure 7(B iv)). The complete disappearance 16


Page 16 of 32

Page 17 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of any kind of secondary structure when the SWCNT-OHs and hIAPP were co-incubated from the beginning might be because the nanomaterial inhibited the amyloid structure formation of the protein at the initial steps of aggregation. This experiment indicates that the SWCNT-OH hindered the formation of β-sheet like structures of hIAPP, and reduced the β-sheet content of already formed aggregates hence inhibiting the fibrillization of hIAPP. The influence of the SWCNT-OH on hIAPP aggregation was further investigated by TEM experiments. Samples were prepared and imaged after 3 h of incubating hIAPP in PB or hIAPP in the presence of SWCNT-OH in PB (Figure. 7(C, D)). The blank hIAPP clearly showed the formation of short fibrillar aggregates. In the presence of SWCNT-OHs a clear decrease in the small fibrillar structures was observed, instead globular aggregates on very long fibers were noted. The TEM image of a blank SWCNT-OH was also recorded (SI, Figure S5) to distinguish the protein and the SWCNT-OH and it showed the presence of long fibril-like structures. Hence from these control images it can be inferred that the short thin fibrils denote the proteins, the long thick fibrils are the nanomaterials. Also Figure 7(D) clearly shows the interaction of the hIAPP with the SWCNT-OH walls. The process was further studied by the prolonged incubation of hIAPP with the SWCNT-OH for 12 h and 24 h. The images recorded after 12 h and 24 h clearly showed an increase in the density and changes in the morphology of the hIAPP fibrils (Figure 7(E and G)), noticeably the protein fibrils were longer instead of the small ones previously observed after 3 h of incubation. However, in the presence of the SWCNT-OH after 12 and 24 h, most of the proteins are seen on the sides of the nanotubes, whereas the area devoid of the nanotubes appears to be clean from any fibrillar structures (Figure 7(F and H)). The obvious decrease of short thin protein fibrils indicates that SWCNT-OH suppressed the fibrillization of hIAPP. The TEM images also indicate that the two heteromaterials stick and interact with each other, supporting our hypotheses that the SWCNT-OH interacts with hIAPP. It is noted that the inhibitory effect of SWCNT-OH is not specific for hIAPP as a previous study reported that SWCNT-OH can also inhibit the aggregation of Aβ(16-22) peptide45. 17



Figure 7. (A) Fluorescence of ThT in (i) hIAPP solution after 12 h incubation, (ii) hIAPP solution after 3 h incubation, (iii) hIAPP with SWCNT-OH after 12 h co-incubation, (iv) hIAPP with SWCNT-OH after 3 h, and (v) blank solution of ThT upon excitation at 460 nm. (B) CD spectra of i) hIAPP solution after 2 h incubation, ii) 30 min after SWCNT-OH added to the 2h-incubated hIAPP solution, iii) 2 h after SWCNT-OH were added to the 2h-incubated hIAPP solution, iv) CD spectra of hIAPP co-incubated with SWCNT-OH. TEM images of hIAPP after (C) 3 h, (E) 12 h and (G) 24 h incubation. TEM images of hIAPP co-incubated with SWCNT-OH after (D) 3 h, (F) 12 h and (H) 24 h incubation.

The inhibition of SWCNT-OH on hIAPP fibrillization was further probed using AFM experiments. The AFM images were collected for SWCNT-OH, hIAPP and a co-incubated system containing the nanomaterial and the protein (Figure 8(A-C)). SWCNT-OH, hIAPP alone and the SWCNT-OH with hIAPP was incubated for 24 h and cast onto freshly cleaved mica surface, the dried samples were subsequently imaged. The height analysis of the nanostructures was done for all observed structures 18


Page 18 of 32

Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and histograms were prepared to understand the exact dimension of the two materials (Figure 8(D-E)). The analysis revealed that the diameters of the SWCNT-OH and hIAPP are about 1 nm and 2-3 nm respectively. Interestingly the height analysis in the co-incubated system indicates the predominant presence of globular aggregates of size greater than 5 nm (Figure 8(F)). Also, the image of the co-incubated SWCNT-OH and hIAPP system vividly showed the presence of globular structures attached on the walls of the SWCNT-OH and the absence of long fibrillar aggregates. This further delineates that the two nanomaterials mutually interacted, which eventually led to the fibril inhibition as indicated by the Fluorescence and CD studies.

Figure 8. AFM images of (A) SWCNT-OH, (B) hIAPP, (C) Co-incubated hIAPP and SWCNT-OH after 24 h. The histogram of the height profiles of (D) SWCNT-OH, (E) hIAPP, and (F) co-incubated hIAPP and SWCNT-OH.

It is interesting to investigate whether SWCNT-OH can disassemble the pre-formed hIAPP amyloid fibrils. To this aim, we performed additional 200 ns MD simulations of a pre-formed hIAPP protofibril (pentamer) in the absence or presence of one/four SWCNT-OH (corresponding to low and high SWCNT-OH concentration). The pentameric hIAPP protofibril was constructed on the basis of a previous solid-state NMR study by Tycko et al63. The use of two different number of SWCNT-OHs is to simulate the influence of nanoparticles at low and high concentrations on the 19



structural stability of the protofibril. The three protofibril systems are denoted as protofibril, protofibril+1SWCNT-OH, and protofibril+4SWCNT-OH. A 200-ns MD run was carried out for each of the three systems. The initial states of the three systems are shown in Figure S6. Simulation details are given in SI. The time evolution of the all-atom RMSD with respect to the initial state in Figure 9(A) shows that the RMSD increases rapidly within the first few nanoseconds and gradually reaches a plateau after 125 ns. In the presence of one SWCNT-OH, the hIAPP protofibrils have similar RMSD (~0.50 nm) as the isolated hIAPP protofibril. With the increase of SWCNT-OH concentration, the RMSD (~0.57 nm) of the hIAPP protofibril markedly increases. From the RMSF of each residue based on the 125~200 ns data in Figure 9(B), we can see that the N-terminal residues of hIAPP protofibril in the presence of four SWCNT-OH have an increased RMSF compared to those in the isolated hIAPP protofibril. These data indicate that SWCNT-OH at high concentration can potentially disassemble the pre-formed hIAPP amyloid fibrils. This is supported by the CD spectra in (Figure 7(B ii and iii)), where a significant decrease in the β-sheet content is observed at 30 min and 2 h after SWCNT-OH were added to the 2h-incubated hIAPP solution.

Figure 9 Influence of SWCNT-OHs on the pre-formed hIAPP amyloid protofibril. (A) Time evolution of all-atom RMSD of hIAPP with respect to the initial pre-formed protofibril, (B) RMSF of each residue based on the 125-200 ns simulation data of hIAPP protofibril, protofibril+1SWCNT-OH and protofibril+4SWCNT-OH systems.

CONCLUSIONS

20


Page 20 of 32

Page 21 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In summary, we have studied the influence of hydroxylated SWCNT on the dimers – the smallest oligomers formed in the early stage of hIAPP aggregation by performing 400-ns all-atom REMD simulations in the absence or presence of SWCNT-OH. Secondary structure calculation of hIAPP dimer demonstrates that SWCNT-OH can reduce the β-sheet content of residues in the region of C7~L16 and S19~T36, and increase the coil content of residues in the region of T6~F15, A25~L28 and N31~S34. Strikingly, we found that SWCNT-OH suppressed completely the three-stranded antiparallel β-sheet structure which contains the previously reported amyloidogenic precursor - the β-hairpin, and shifted the hIAPP dimer from fibril-competent

β-sheet

rich

conformations

to

fibril-incompetent

coil-rich

conformations. This leads to the retardation of hIAPP aggregation and fibrillation. hIAPP-hIAPP and hIAPP--SWCNT-OH interaction analyses reveal that the interactions between aromatic residues of F15 and Y37, polar residues of N3, Q10, H18, N31 and N35 and charged residue R11 and SWCNT-OH play important roles in blocking peptide-peptide interactions and inhibiting β-sheet formation. We note that further experiments, such as point mutations and NMR-based investigations on interaction sites can provide evidence on the critical residues inferred from our simulations. This clearly remains to be determined in future study. We found that H-bonding, van der Waals and π-π stacking interactions between hIAPP and SWCNT-OH are the dominant driving forces in impeding hIAPP aggregation. To further investigate the influence of SWCNT-OH on the aggregation and fibril formation, we probed the hIAPP aggregation process in the absence or presence of SWCNT-OH using spectroscopic and microscopic techniques. Our turbidity analyses, ThT fluorescence studies, CD experiments, TEM and AFM images provide direct evidence showing that the SWCNT-OHs interact with hIAPP in solution and this leads to the inhibition of hIAPP amyloid formation. In addition, our MD simulations and CD experiments suggest that SWCNT-OHs can disassemble the pre-formed hIAPP amyloid. This study will be beneficial for the design of therapeutic agents against T2DM. 21



METHODS Computational section hIAPP and hIAPP+SWCNT-OH systems. The two systems, hIAPP dimer in the absence or presence of SWCNT-OH, are denoted as hIAPP and hIAPP+SWCNT-OH. The amino acid sequence of full-length hIAPP is NH3+-KCNTATCATQ10RLANFLV HSS20 NNFGAILSST30NVGSNTY-CONH2. There is a disulfide bond between Cys2 and Cys7. The C-terminus is amidated. To mimic the experimental neutral pH condition (around pH 7.364), the N-terminus, the side chains of residues Lys and Arg are protonated (NH3+, Lys+ and Arg+). Thus the net charge of hIAPP is +3. As done in our previous work65, we placed twenty-four/sixteen extended coil hIAPP monomers in turn in a perpendicular, parallel or antiparallel orientation to obtain twelve/eight initial hIAPP dimer states for hIAPP and hIAPP+SWCNT-OH systems (Figure S7(A) and (B)). The minimum distance between the two hIAPP monomers is 1.0 nm. In the initial states of hIAPP+SWCNT-OH system, the minimum distance between SWCNT-OH and hIAPP dimer (Figure S7(B)) is 0.8 nm. The SWCNT-OH used in our simulations consists of 216 carbon atoms and 30 hydroxyl groups. It has a diameter of 0.407 nm and a length of 4.25 nm, in agreement with our previous study45. It is noted that short and thin carbon nanotubes have very low cytotoxicity as they can be cleared through lymphatic system66. Oxygen and hydrogen atoms in hydroxyl groups and carbon atoms bonded with hydroxyl groups in SWCNT-OH have a partial charge of -0.8, +0.3 and +0.5 |e|, respectively. Other carbon atoms in the SWCNT-OH are uncharged45. In accordance with our previous computational study65, we used the OPLS-AA force field67 for hIAPP and TIP4P for water molecules. Each initial state of hIAPP and hIAPP+SWCNT-OH systems was placed in the center of a cubic box (6.7 × 6.7 × 6.7 nm3) and then solvated with TIP4P water molecules. Counterions (Cl-) were added to neutralize the two systems. The total numbers of atoms for the two systems are 39122 and 39328, respectively. REMD simulations. The REMD simulation, an enhanced sampling method68-71, was used to sample the conformational space of hIAPP dimer in the absence or presence 22


Page 22 of 32

Page 23 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of SWCNT-OH. A 400-ns REMD simulation was performed for both hIAPP and hIAPP+SWCNT-OH systems in the isothermal-isobaric (NPT) ensemble using the GROMACS-4.5.3 soft package72. There are 48 replicas (400 ns for each replica), distributed in the temperature range of 306-409 K for hIAPP dimer system and 305-445 K for hIAPP+SWCNT-OH system. The distribution of the 48 temperatures was generated using an approach reported previously73. The temperature lists were given in Table S1. The aim of using different temperature range for the two different systems was to obtain similar acceptance ratio and the same number of replicas. The exchange between two adjacent replicas was attempted every 1000 integration steps. The acceptance ratios for hIAPP and hIAPP+SWCNT-OH systems are 16% and 19% respectively. The simulation of the hIAPP dimer without SWCNT-OH is an extension of our previous REMD simulations from 36065 to 400 ns. The bond lengths of hIAPP and water molecules were constrained respectively using the LINCS74 and SETTLE75 algorithms. The pressure was maintained at 1 bar controlled by the Parrinello-Rahman method76 with a coupling time constant of 1.0 ps. The temperature was kept constant using a velocity rescaling coupling method77 with a coupling constant of 0.1 ps. The solute and solvent were separately coupled to external temperature and pressure baths. Electrostatic interactions were treated with the particle mesh Ewald method with a real space cutoff of 1.0 nm. The van der Waals interactions were calculated using a cutoff of 1.4 nm. Analysis methods. All the analyses were performed using our in-house-developed codes and the tools implemented in GROMACS software. All the results reported in this study are based on the REMD trajectories at 310 K and the data generated in the last 160 ns (from 240 to 400 ns) for the hIAPP dimer system and 180 ns (from 220 to 400 ns) for hIAPP+SWCNT-OH system. The secondary structures of hIAPP dimer were calculated using DSSP algorithm78. The cluster analysis was performed using the Daura algorithm based on the main chain root-mean-square deviation (RMSD) with a cutoff of 0.35 nm for hIAPP dimer. The chain-independent main chain RMSD58 was calculated by completely neglecting the chain identifier in the coordinate file of 23



hIAPP as the two chains are topologically identical. The inter- and intra-peptide interactions were estimated by calculating the pairwise residue-residue contact probabilities. The centroid distance and angle between the rings of aromatic residues of hIAPP and the carbon rings of SWCNT-OH were used to analyze the aromatic stacking interaction between hIAPP and SWCNT-OH. The binding free energy was estimated using molecular-mechanics/Poisson-Boltzmans surface area (MM/PBSA) approach (g_mmpbsa script)79. The calculation of binding free energy (∆Gbinding) between hIAPP and SWCNT-OH was performed using ∆Gbinding = ∆Gcomplex - (∆Gprotein + ∆Gligand), where ∆Gcomplex, ∆Gprotein and ∆Gligand are free energies of hIAPP+SWCNT-OH complex, hIAPP and SWCNT-OH respectively. The free energy for each individual component in given by ∆G = EMM + Gsolvation - T∆S. The vacuum potential energy EMM includes the energy of both bonded and nonbonded interactions, EMM = Ebonded + Enonbonded = Ebonded + (EvdW + Eelec), where Ebonded consists of bond, angle, dihedral and improper interactions, and Enonbonded consists of van der Waals (EvdW) and electrostatic (Eelec) interactions. The solvation free energy is expressed as two terms: ∆Gsolvation = ∆Gpolar + ∆Gnonpolar, where ∆Gpolar and ∆Gnonpolar are the electrostatic and non-electrostatic contributions to the solvation free energy respectively. The two-dimensional (2D) free energy surface was calculated using -RTlnP(x, y), where P(x, y) is the probability of a conformation having a certain value of two selected reaction coordinates x and y. In this work, the two selected reaction coordinates x and y are respectively the centroid distance and angle between the rings of aromatic residues of hIAPP and the carbon rings of SWCNT-OH. The VMD program80 was used for structure visualization. Experimental section Materials: Single-walled carbon nanotubes (-OH) +90% Pure, OD: 1-2 nm, Length: 5-30µm -OH Content: ~ 4wt% was purchased from MK nano, human IAPP amide of 95% purity was purchased from Anaspec. Turbidity: 1 mg of SWCNT-OH was weighed and to it 2 mL of Phosphate Buffer (10 mM, pH 7.0) (PB) was added and 0.1 mg hIAPP was added when experiments were 24


Page 24 of 32

Page 25 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


done in the presence of the protein. The mixture was tip sonicated on ice for 10 min, 20% amplitude using a Vibra-Cell Sonics. The solutions were allowed to settle overnight and the turbidity at 550 nm was measured using an ELISA reader (Synergy HT, Biotek Instruments, Winooski, VT, USA). For the study involving the concentration variation of hIAPP the concentration was varied from 0.0125 mg/mL-0.125 mg/mL where the SWCNT-OH concentration was fixed at 0.5 mg/mL. In case of SWCNT-OH variation, the concentration was varied from 0.25 mg/mL-1 mg/mL where hIAPP concentration was fixed at 0.05 mg/mL. ThT Fluorescence: The suspension of protein and SWCNT-OH was incubated with 20 mM ThT solution in PB and the ThT fluorescence emission at 490 nm (excitation at 450 nm) was collected via the BMG Labtech Clariostar plate reader. CD spectrum: Sample solutions were placed in1 mm path length cuvette at 25⁰C and the range of 190–260 nm was recorded on a Chirascan spectrometer. Background (blank PB) was subtracted from the CD spectra.

The samples were equilibrated for

10 min at the desired temperature before carrying out the CD measurement. TEM: The suspension samples were cast on copper grids and stained with Uranyl Acetate for 1 min. TEM micrographs were recorded under a JEOL 1200EX electron microscope (JEOL, Tokyo, Japan) operated at 80 kV. AFM: For atomic force microscopy (AFM), 10 µ L of solution was dropped onto a freshly cleaved mica surface and adsorbed. A topographic image was recorded under a NanoWizard 3 BioScience AFM (JPK, Berlin, Germany) in the tapping mode at ambient temperature, with 512 × 512 pixel resolution and a scanning speed of 1.0 Hz. Using reported protocols, the diameter of the nanotubes was calculated by the height analysis of the nanostructure observed in the AFM images81.

ASSOCIATED CONTENT Supporting Information

25



Page 26 of 32

The Supporting Information is available free of charge on ACS Publications website http://pubs.acs.org. The

temperature

lists

used

in

the

REMD

simulations

of

hIAPP

and

hIAPP+SWCNT-OH systems; The twelve initial conformations of hIAPP dimer and the eight initial conformations hIAPP+SWCNT-OH system; Simulation convergence assesments

for

hIAPP

system;

Simulation

convergence

assesments

for

hIAPP+SWCNY-OH system; Interaction analysis of hIAPP and SWCNT-OH; The image of unstained SWCNT-OH and hIAPP; The initial states of (A) hIAPP protofibril, (B) protofibril+1SWCNT-OH, and (C) protofibril+4SWCNT-OH. AUTHOR INFORMATION Corresponding Authors *Email: [email protected] *Email: [email protected] ORCID Guanghong Wei: 0000-0001-5814-3328 Author Contributions G.W., E.G. and Y.M. conceived the project. E.G., S.B. and S.G. designed and performed the experiments. Y.M., J.L., Y.T. and G.W. performed simulations and analyzed all of the simulation data. Y.M. and G.W. drafted the manuscript. The manuscript was written through contributions of all authors and all authors have given approval to the ﬁnal version of the manuscript. Funding This work is supported by the financial support from the National Key R&D Program of China (Grant No. 2016YFA0501702) and National Natural Science Foundation of China (Grant No. 11674065 and 11274075). All simulations were performed using the high-performance computational facilities at Fudan University and National Supercomputer Center in Guangzhou. Notes The authors declare no competing ﬁnancial interest. 26


Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1)

Hull, R. L., Westermark, G. T., Westermark, P., and Kahn, S. E. (2004) Islet amyloid: a critical entity in the pathogenesis of type 2 diabetes, J Clin Endocrinol Metab 89, 3629-3643.

(2)

Hardy, J., and Selkoe, D. J. (2002) The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics, Science 297, 353-356.

(3)

Yang, S. A., Yoon, J., Kim, K., and Park, Y. (2017) Measurements of morphological and biophysical alterations in individual neuron cells associated with early neurotoxic effects in Parkinson's disease, Cytometry A 91, 510-518.

(4)

Luhrs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Dobeli, H., Schubert, D., and Riek, R. (2005) 3D structure of Alzheimer's amyloid-beta(1-42) fibrils, Proc Natl Acad Sci U S A 102, 17342-17347.

(5)

Cooper, G. J., Willis, A. C., Clark, A. ,Turner, R. C., Sim, R. B., and Reid, K. B. (1987) Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients, Proc Natl Acad Sci U S A 84, 8628-8632.

(6)

Westermark, P., Wernstedt, C., Wilander, E., Hayden, D. W., O'Brien, T. D., and Johnson, K. H. (1987) Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells, Proc Natl Acad Sci U S A 84, 3881-3885.

(7)

Kapurniotu, A., Bernhagen, J., Greenfield, N., Al-Abed, Y., Teichberg, S., Frank, R. W., Voelter, W., and Bucala, R. (1998) Contribution of advanced glycosylation to the amyloidogenicity of islet amyloid polypeptide, Eur J Biochem 251, 208-216.

(8)

Kim, J., Cheon, H., Jeong, Y. T., Quan, W., Kim, K. H., Cho, J. M., Lim, Y. M., Oh, S. H., Jin, S. M., Kim, J. H., Lee, M. K., Kim, S., Komatsu, M., Kang, S. W., and Lee, M. S. (2014) Amyloidogenic peptide oligomer accumulation in autophagy-deficient beta cells induces diabetes, J Clin Invest 124, 3311-3324.

(9)

Lorenzo, A., Razzaboni, B., Weir, G. C., and Yankner, B. A. (1994) Pancreatic islet cell toxicity of amylin associated with type-2 diabetes mellitus, Nature 368, 756-760.

(10) Butler, A. E., Janson, J., Soeller, W. C., and Butler, P. C. (2003) Increased beta-cell apoptosis prevents adaptive increase in beta-cell mass in mouse model of type 2 diabetes: evidence for role of islet amyloid formation rather than direct action of amyloid, Diabetes 52, 2304-2314. (11) Sellin, D., Yan, L. M., Kapurniotu, A., and Winter, R. (2010) Suppression of IAPP fibrillation at anionic lipid membranes via IAPP-derived amyloid inhibitors and insulin, Biophys Chem 150, 73-79. (12) Scrocchi, L. A., Chen, Y., Waschuk, S., Wang, F., Cheung, S., Darabie, A. A., McLaurin, J., and Fraser, P. E. (2002) Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis, J Mol Biol 318, 697-706. (13) Porat, Y., Mazor, Y., Efrat, S., and Gazit, E. (2004) Inhibition of islet amyloid polypeptide fibril formation: a potential role for heteroaromatic interactions, Biochemistry 43, 14454-14462. (14) Gilead, S., and Gazit, E. (2004) Inhibition of amyloid fibril formation by peptide analogues modified with alpha-aminoisobutyric acid, Angew Chem Int Ed Engl 43, 4041-4044.

27



(15) Porat, Y., Abramowitz, A., and Gazit, E. (2006) Inhibition of amyloid fibril formation by polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism, Chem Biol Drug Des 67, 27-37. (16) Mishra, R., Sellin, D., Radovan, D., Gohlke, A., and Winter, R. (2009) Inhibiting islet amyloid polypeptide fibril formation by the red wine compound resveratrol, Chembiochem 10, 445-449. (17) Cabaleiro-Lago, C., Lynch, I., Dawson, K. A., and Linse, S. (2010) Inhibition of IAPP and IAPP(20-29) fibrillation by polymeric nanoparticles, Langmuir 26, 3453-3461. (18) Wang, W., Zhao, C., Zhu, D., Gong, G., and Du, W. (2017) Inhibition of amyloid peptide fibril formation by gold-sulfur complexes, J Inorg Biochem 171, 1-9. (19) Yousaf, M., Huang, H., Li, P., Wang, C., and Yang, Y. (2017) Fluorine Functionalized Graphene Quantum Dots as Inhibitor against hIAPP Amyloid Aggregation, ACS Chem Neurosci 8, 1368-1377. (20) Nedumpully-Govindan, P., Gurzov, E. N., Chen, P., Pilkington, E. H., Stanley, W. J., Litwak, S. A., Davis, T. P., Ke, P. C., and Ding, F. (2016) Graphene oxide inhibits hIAPP amyloid fibrillation and toxicity in insulin-producing NIT-1 cells, Phys Chem Chem Phys 18, 94-100. (21) Sheridan, C. (2009) J&J's billion dollar punt on anti-amyloid antibody, Nat Biotechnol 27, 679-681. (22) Cheng, B., Gong, H., Xiao, H., Petersen, R. B., Zheng, L., and Huang, K. (2013) Inhibiting toxic aggregation of amyloidogenic proteins: a therapeutic strategy for protein misfolding diseases, Biochim Biophys Acta 1830, 4860-4871. (23) Lee, C. C., Julian, M. C., Tiller, K. E., Meng, F., DuConge, S. E., Akter, R., Raleigh, D. P., and Tessier, P. M. (2016) Design and Optimization of Anti-amyloid Domain Antibodies Specific for beta-Amyloid and Islet Amyloid Polypeptide, J Biol Chem 291, 2858-2873. (24) Li, C., and Mezzenga, R. (2013) The interplay between carbon nanomaterials and amyloid fibrils in bio-nanotechnology, Nanoscale 5, 6207-6218. (25) Mahmoudi, M., Kalhor, H. R., Laurent, S., and Lynch, I. (2013) Protein fibrillation and nanoparticle interactions: opportunities and challenges, Nanoscale 5, 2570-2588. (26) Zaman, M., Ahmad, E., Qadeer, A., Rabbani, G., and Khan, R. H. (2014) Nanoparticles in relation to peptide and protein aggregation, Int J Nanomedicine 9, 899-912. (27) De Leo, F., Magistrato, A., and Bonifazi, D. (2015) Interfacing proteins with graphitic nanomaterials: from spontaneous attraction to tailored assemblies, Chem Soc Rev 44, 6916-6953. (28) Andreev, I., Petrukhina, A., Garmanova, A., Babakhin, A., Andreev, S., Romanova, V., Troshin, P., Troshina, O., and Buske. L. D. (2008) Penetration of Fullerene C60 Derivatives Through Biological Membranes, Fullerene Science & Technology 16, 89-102. (29) Wang, X. Y., Lei, R., Huang, H. D., Wang, N., Yuan, L., Xiao, R. Y., Bai, L. D., Li, X., Li, L. M., and Yang, X. D. (2015) The permeability and transport mechanism of graphene quantum dots (GQDs) across the biological barrier, Nanoscale 7, 2034-2041. (30) Shi, X., von dem Bussche, A., Hurt, R. H., Kane, A. B., and Gao, H. (2011) Cell entry of one-dimensional nanomaterials occurs by tip recognition and rotation, Nat Nanotechnol 6, 714-719. (31) Dugan, L. L., Turetsky, D. M., Du, C., Lobner, D., Wheeler, M., Almli, C. R., Shen, C. K., Luh, T. Y., Choi, D. W., and Lin, T. S. (1997) Carboxyfullerenes as neuroprotective agents, Proc Natl Acad Sci U S A 94, 9434-9439.

28


Page 28 of 32

Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(32) Kim, J. E., and Lee, M. (2003) Fullerene inhibits beta-amyloid peptide aggregation, Biochem Biophys Res Commun 303, 576-579. (33) Bobylev, A. G., Kornev, A. B., Bobyleva, L. G., Shpagina, M. D., Fadeeva, I. S., Fadeev, R. S., Deryabin, D. G., Balzarini, J., Troshin, P. A., and Podlubnaya, Z. A. (2011) Fullerenolates: metallated polyhydroxylated fullerenes with potent anti-amyloid activity, Org Biomol Chem 9, 5714-5719. (34) Xie, L., Luo, Y., Lin, D., Xi, W., Yang, X., and Wei, G. (2014) The molecular mechanism of fullerene-inhibited aggregation of Alzheimer's beta-amyloid peptide fragment, Nanoscale 6, 9752-9762. (35) Radic, S., Nedumpully-Govindan, P., Chen, R., Salonen, E., Brown, J. M., Ke, P. C., and Ding, F. (2014) Effect of fullerenol surface chemistry on nanoparticle binding-induced protein misfolding, Nanoscale 6, 8340-8349. (36) Bednarikova, Z., Huy, P. D., Mocanu, M. M., Fedunova, D., Li, M. S., and Gazova, Z. (2016) Fullerenol C60(OH)16 prevents amyloid fibrillization of Abeta40-in vitro and in silico approach, Phys Chem Chem Phys 18, 18855-18867. (37) Sun, Y., Qian, Z., and Wei, G. (2016) The inhibitory mechanism of a fullerene derivative against amyloid-beta peptide aggregation: an atomistic simulation study, Phys Chem Chem Phys 18, 12582-12591. (38) Andujar, S. A., Lugli, F., Hofinger, S., Enriz, R. D., and Zerbetto, F. (2012) Amyloid-beta fibril disruption by C60-molecular guidance for rational drug design, Phys Chem Chem Phys 14, 8599-8607. (39) Zhou, X., Xi, W., Luo, Y., Cao, S., and Wei, G. (2014) Interactions of a water-soluble fullerene derivative with amyloid-beta protofibrils: dynamics, binding mechanism, and the resulting salt-bridge disruption, J Phys Chem B 118, 6733-6741. (40) Mahmoudi, M., Akhavan, O., Ghavami, M., Rezaee, F., and Ghiasi, S. M. (2012) Graphene oxide strongly inhibits amyloid beta fibrillation, Nanoscale 4, 7322-7325. (41) Yang, Z., Ge, C., Liu, J., Chong, Y., Gu, Z., Jimenez-Cruz, C. A., Chai, Z., and Zhou, R. (2015) Destruction of amyloid fibrils by graphene through penetration and extraction of peptides, Nanoscale 7, 18725-18737. (42) Mao, X., Wang, Y., Liu, L., Niu, L., Yang, Y., and Wang, C. (2009) Molecular-level evidence of the surface-induced transformation of peptide structures revealed by scanning tunneling microscopy, Langmuir 25, 8849-8853. (43) Ou, L., Luo, Y., and Wei, G. (2011) Atomic-level study of adsorption, conformational change, and dimerization of an alpha-helical peptide at graphene surface, J Phys Chem B 115, 9813-9822. (44) Yu, X., Wang, Q., Lin, Y., Zhao, J., Zhao, C., and Zheng, J. (2012) Structure, orientation, and surface interaction of Alzheimer amyloid-beta peptides on the graphite, Langmuir 28, 6595-6605. (45) Xie, L., Lin, D., Luo, Y., Li, H., Yang, X., and Wei, G. (2014) Effects of hydroxylated carbon nanotubes on the aggregation of Abeta16-22 peptides: a combined simulation and experimental study, Biophys J 107, 1930-1938. (46) Luo, J., Warmlander, S. K., Yu, C. H., Muhammad, K., Graslund, A., and Pieter Abrahams, J. (2014) The Abeta peptide forms non-amyloid fibrils in the presence of carbon nanotubes, Nanoscale 6, 6720-6726.

29



(47) Lin, D., Qi, R., Li, S., He, R., Li, P., Wei, G., and Yang, X. (2016) Interaction Dynamics in Inhibiting the Aggregation of Abeta Peptides by SWCNTs: A Combined Experimental and Coarse-Grained Molecular Dynamic Simulation Study, ACS Chem Neurosci 7, 1232-1240. (48) Fu, Z., Luo, Y., Derreumaux, P., and Wei, G. (2009) Induced beta-barrel formation of the Alzheimer's Abeta25-35 oligomers on carbon nanotube surfaces: implication for amyloid fibril inhibition, Biophys J 97, 1795-1803. (49) Linse, S., Cabaleiro-Lago, C., Xue, W. F., Lynch, I., Lindman, S., Thulin, E., Radford, S. E., and Dawson, K. A. (2007) Nucleation of protein fibrillation by nanoparticles, Proc Natl Acad Sci U S A 104, 8691-8696. (50) Liu, Z., Liu, Y., and Peng, D. (2014) Hydroxylation of multi-walled carbon nanotubes reduces their cytotoxicity by limiting the activation of mitochondrial mediated apoptotic pathway, J Mater Sci Mater Med 25, 1033-1044. (51) Luca, S., Yau, W.-M., Leapman, R., and Tycko, R. (2007) Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State NMR, Biochemistry 46, 13505-13522. (52) Dupuis, N. F., Wu, C., Shea, J. E., and Bowers, M. T. (2009) Human islet amyloid polypeptide monomers form ordered beta-hairpins: a possible direct amyloidogenic precursor, J Am Chem Soc 131, 18283-18292. (53) Qiao, Q., Bowman, G. R., and Huang, X. (2013) Dynamics of an intrinsically disordered protein reveal metastable conformations that potentially seed aggregation, J Am Chem Soc 135, 16092-16101. (54) Dupuis, N. F., Wu, C., Shea, J. E., and Bowers, M. T. (2011) The amyloid formation mechanism in human IAPP: dimers have beta-strand monomer-monomer interfaces, J Am Chem Soc 133, 7240-7243. (55) Brown, S., Jespersen, T. S., and Nygard, J. (2008) A genetic analysis of carbon-nanotube-binding proteins, Small 4, 416-420. (56) Zuo, G., Kang, S. G., Xiu, P., Zhao, Y., and Zhou, R. (2013) Interactions between proteins and carbon-based nanoparticles: exploring the origin of nanotoxicity at the molecular level, Small 9, 1546-1556. (57) Lei, J., Qi, R., Xie, L., Xi, W., and Wei, G. (2017) Inhibitory effect of hydrophobic fullerenes on the beta-sheet-rich oligomers of a hydrophilic GNNQQNY peptide revealed by atomistic simulations, RSC Advances 7, 13947-13956. (58) Li, H., Luo, Y., Derreumaux, P., and Wei, G. (2011) Carbon nanotube inhibits the formation of beta-sheet-rich oligomers of the Alzheimer's amyloid-beta(16-22) peptide, Biophys J 101, 2267-2276. (59) McGaughey, G. B., Gagne, M., and Rappe, A. K. (1998) pi-Stacking interactions. Alive and well in proteins, J Biol Chem 273, 15458-15463. (60) Bianco, A., Kostarelos, K., and Prato, M. (2005) Applications of carbon nanotubes in drug delivery, Curr Opin Chem Biol 9, 674-679. (61) Matthes, D., Gapsys, V., and de Groot, B. L. (2012) Driving forces and structural determinants of steric zipper peptide oligomer formation elucidated by atomistic simulations, J Mol Biol 421, 390-416.

30


Page 30 of 32

Page 31 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(62) Sun, Y., Qian, Z., Guo, C., and Wei, G. (2015) Amphiphilic Peptides A6K and V6K Display Distinct Oligomeric Structures and Self-Assembly Dynamics: A Combined All-Atom and Coarse-Grained Simulation Study, Biomacromolecules 16, 2940-2949. (63) Luca, S., Yau, W. M., Leapman, R., and Tycko, R. (2007) Peptide conformation and supramolecular organization in amylin fibrils: constraints from solid-state NMR, Biochemistry 46, 13505-13522. (64) Nanga, R. P., Brender, J. R., Vivekanandan, S., and Ramamoorthy, A. (2011) Structure and membrane orientation of IAPP in its natively amidated form at physiological pH in a membrane environment, Biochim Biophys Acta 1808, 2337-2342. (65) Mo, Y., Lei, J., Sun, Y., Zhang, Q., and Wei, G. (2016) Conformational Ensemble of hIAPP Dimer: Insight into the Molecular Mechanism by which a Green Tea Extract inhibits hIAPP Aggregation, Scientific Reports 6, 33076. (66) Kostarelos, K. (2008) The long and short of carbon nanotube toxicity, Nat Biotechnol 26, 774-776. (67) Jorgensen, W. L., and Tirado-Rives, J. (1988) The OPLS [optimized potentials for liquid simulations] potential functions for proteins, energy minimizations for crystals of cyclic peptides and crambin, J Am Chem Soc 110, 1657-1666. (68) Zhou, R. (2007) Replica exchange molecular dynamics method for protein folding simulation, Methods Mol Biol 350, 205-223. (69) Sugita, Y., and Okamoto, Y. (1999) Replica-exchange molecular dynamics method for protein folding, Chem Phys Lett 314, 141-151. (70) Okamoto, Y. (2004) Generalized-ensemble algorithms: enhanced sampling techniques for Monte Carlo and molecular dynamics simulations, J Mol Graph Model 22, 425-439. (71) Nadler, W., and Hansmann, U. H. (2008) Optimized explicit-solvent replica exchange molecular dynamics from scratch, J Phys Chem B 112, 10386-10387. (72) Pronk, S., Pall, S., Schulz, R., Larsson, P., Bjelkmar, P., Apostolov, R., Shirts, M. R., Smith, J. C., Kasson, P. M., van der Spoel, D., Hess, B., and Lindahl, E. (2013) GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit, Bioinformatics 29, 845-854. (73) Patriksson, A., and van der Spoel, D. (2008) A temperature predictor for parallel tempering simulations, Phys Chem Chem Phys 10, 2073-2077. (74) Hess, B., Bekker, H., Berendsen, H. J. C., and Fraaije, J. G. E. M. (1997) LINCS: A linear constraint solver for molecular simulations, J. Comput. Chem 18, 1463-1472. (75) Miyamoto, S., and Kollman, P. A. (1992) Settle - an Analytical Version of the Shake and Rattle Algorithm for Rigid Water Models, J. Comput. Chem 13, 952-962. (76) Nosé, S., and Klein, M. (1983) Constant pressure molecular dynamics for molecular systems, Molecular Physics 50, 1055-1076. (77) Bussi, G., Donadio, D., and Parrinello, M. (2007) Canonical sampling through velocity rescaling, J Chem Phys 126, 014101. (78) Kabsch, W., and Sander, C. (1983) Dictionary of protein secondary structure: Pattern recognition of hydrogen-bonded and geometrical features, Biopolymers 22, 2577-2637. (79) Kumari, R., Kumar, R., and Lynn, A. (2014) g_mmpbsa—A GROMACS Tool for High-Throughput MM-PBSA Calculations, J. Chem. Inf. Model 54, 1951-1962. (80) Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: visual molecular dynamics, J Mol Graph 14, 33-38, 27-38. 31



(81) Backes, C., Schmidt, C. D., Hauke, F., Bottcher, C., and Hirsch, A. (2009) High population of individualized SWCNTs through the adsorption of water-soluble perylenes, J Am Chem Soc 131, 2172-2184.

32


Page 32 of 32

The inhibitory effect of hydroxylated carbon nanotubes on the

Recommend Documents