Structure, Orientation, and Surface Interaction of Alzheimer Amyloid-β

Apr 2, 2012 - Xiaofeng Wang , Jeffrey K. Weber , Lei Liu , Mingdong Dong , Ruhong ... Mrigya Agarwal , BongKeun Kim , Igor V. Pivkin , Joan-Emma Shea...
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Structure, Orientation, and Surface Interaction of Alzheimer Amyloid-β Peptides on the Graphite Xiang Yu,†,§ Qiuming Wang,†,§ Yinan Lin,‡ Jun Zhao,† Chao Zhao,† and Jie Zheng*,† †

Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, Ohio 44325, United States Department of Polymer Science, The University of Akron, Akron, Ohio 44325, United States

‡

S Supporting Information *

ABSTRACT: The misfolding and aggregation of amyloid-β (Aβ) peptides into amyloid fibrils in solution and on the cell membrane has been linked to the pathogenesis of Alzheimer’s disease. Although it is well-known that the presence of different surfaces can accelerate the aggregation of Aβ peptides into fibrils, surface-induced conformation, orientation, aggregation, and adsorption of Aβ peptides have not been well understood at the atomic level. Here, we perform all-atom explicit-water molecular dynamics (MD) simulations to study the orientation change, conformational dynamics, surface interaction of small Aβ aggregates with different sizes (monomer to tetramer), and conformations (α-helix and β-hairpin) upon adsorption on the graphite surface, in comparison with Aβ structures in bulk solution. Simulation results show that hydrophobic graphite induces the quick adsorption of Aβ peptides regardless of their initial conformations and sizes. Upon the adsorption, Aβ prefers to adopt random structure for monomers and to remain β-rich-structure for small oligomers, but not helical structures. More importantly, due to the amphiphilic sequence of Aβ and the hydrophobic nature of graphite, hydrophobic C-terminal residues of higher-order Aβ oligomers appear to have preferential interactions with the graphite surface for facilitating Aβ fibril formation and fibril growth. In combination of atomic force microscopy (AFM) images and MD simulation results, a postulated mechanism is proposed to describe the structure and kinetics of Aβ aggregation from aqueous solution to the graphite surface, providing parallel insights into Aβ aggregation on biological cell membranes.

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INTRODUCTION Interaction of biomolecules (i.e., peptides, proteins, cells, DNA, RNA, and phospholipids) with many solid surfaces (i.e., metals, oxides, minerals, semiconductors, and nanoparticles) is a fundamental phenomenon in a wide range of practical applications for biomaterials, biosensor, nano/biotechnology, and medicine.1−3 Physical binding or chemical immobilization of proteins/peptides on the surface enables to develop new biomaterials with desirable functionalities for sensing, imaging, antifouling, catalysis, and tissue engineering. On the other hand, the physicochemical properties of the surface can also regulate the function and activity of surface-bound proteins/peptides by controlling their conformations, folding pathways, and selfassembly process. Thus, understanding of biophysicochemical interactions between proteins/peptides and surface is fundamentally essential for establishing not only the relationship between protein/peptide sequences and their binding affinities and specificities but also the rational design of novel peptides with desirable properties of interest (e.g., binding, synthesis, catalysis, antimicrobial, biocompatibility, and assembly properties) in natural and engineering systems.4 It is more biologically important to study the disease-related proteins/peptides (e.g., amyloid peptides associated with neurodegenerative diseases including Alzheimer’s, Parkinson’s, © 2012 American Chemical Society

and diabetes type II) interacting with different biological or artificial surfaces, which could provide general insights into the mechanism of protein (mis)folding, aggregation, and its relation to pathological process. Amyloid-β (Aβ) peptide is a short 39−42 residue amphiphilic peptide (∼4 kDa), which is produced by a proteolytic cleavage of the transmembrane amyloid precursor protein by β and γ secretases. A number of in vitro and in vivo studies have shown that the self-aggregation of Aβ peptides from soluble and unstructured monomers to insoluble and β-sheet-rich amyloid fibrils is strongly linked to the pathogenesis of Alzheimer’s disease.5,6 Aggregation kinetics and structural characterization of Aβ in solution and on surfaces have been intensively studied by both experiments7−10 and simulations.11−16 Many studies have focused on the Aβ-cell membrane interactions and their effects on membranedisrupting mechanisms, which are associated with Aβ toxicity.6,17 Membrane disruption could be induced by the selective Aβ ion channel18−20 or by the unselective membrane thinning.21,22 Both scenarios of selective ion channel and nonselective membrane thinning enable ions and small Received: January 16, 2012 Revised: March 2, 2012 Published: April 2, 2012 6595

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actions can greatly promote the adsorption of Aβ and the structural conversion of Aβ to β-sheet structure on the graphite, and such similar hydrophobic interactions between Aβ peptides could also facility fibril seeds formation on the surface and fibril prolongation in vitro.

molecules to pass through the membrane, disrupting cellular homeostasis. But, exact membrane-disruption mechanisms are still hotly debated with many controversial data and explanations, presumably due to the complex nature of cell membranes and notorious environmental sensitivity of Aβ aggregation. In parallel to Aβ toxicity studies, many studies focused on the structure and dynamics of Aβ and their aggregation mechanisms on some molecularly designed model surfaces including mica,23−25 graphite,23,24,26 and self-assembled monolayer (SAM).27,28 These model surfaces usually have simple surface chemistry and large, uniform atomic-resolution domain, allowing to decoupling surface physicochemical properties to examine their effects separately on Aβ aggregation, structure, and kinetics. Kowalewski et al.24 reported the first in situ atomic force microscopy (AFM) study of Aβ aggregation on hydrophobic graphite and hydrophilic mica surfaces. They observed that Aβ formed uniform, elongated β-sheets on hydrophobic graphite but micelle-like aggregates on hydrophilic mica. Dusan et al.26 showed that highly oriented pyrolytic graphite (HOPG) acted as template to promote the assembly of Aβ into fibrils with a distinctive helical morphology depending on incubation times. We recently studied the adsorption kinetics, aggregation behavior, and conformational change of Aβ42 on different SAM surface terminated CH3, OH, COOH, and NH2 groups using surface plasmon resonance (SPR), atomic force microscopy (AFM), and circular dichroism (CD).27 The CD, AFM, and SPR data showed that all of these SAMs greatly accelerated the formation of β-sheets and amyloid fibrils through surface-enhanced interactions, but Aβ1−42 peptides preferentially adsorbed on hydrophobic CH3−SAM and positively charged NH2−SAM with much stronger interactions than on hydrophilic OH−SAM and negatively charged COOH−SAM. Although these experimental studies have enhanced the fundamental understanding of surface impacts on the folding kinetics and aggregation morphologies of Aβ, atomic details of adsorption behavior, structural dynamics, and surface interactions of Aβ at surfaces still remain largely unknown. Complementary to experiments, molecular dynamics (MD) simulations are able to provide nanosecond and nanometer scale information on the structure, dynamics, and orientation of proteins/peptides on different surfaces.23,29−33 In this work, we employ all-atom explicit-solvent MD simulations to study the peptide adsorption, conformational dynamics, and surface interaction of full-length Aβ monomers and small oligomers on the graphite surface. Two distinct conformations of α-helix and β-hairpin are used to present the initial structures of Aβ monomers and oligomers. Experimental and simulation studies of Aβ have revealed that Aβ adopts α-helical structure in cell membrane before releasing to extracelluar media for aggregation but β-hairpin structure in high-ordered oligomers, protofibrils, and mature fibrils after nuclei formation. The use of two representative but different Aβ conformations which appear at initial and final Aβ aggregation states enables not only to examine the effect of initial Aβ conformations on Aβ adsorption, structure, and dynamics on the graphite but also to determine the preferential conformation of Aβ upon adsorption on the graphite, given that conventional MD simulations are not likely to capture a complete structural transition and spontaneous aggregation of Aβ from α-helical to β-hairpin within nanoseconds. Combined MD simulations and AFM images demonstrate that strong surface hydrophobic inter-

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MATERIALS AND METHODS

Molecular Models of Aβ1−42 Aggregates. Aβ Monomers. Two distinct conformations of Aβ1−42 monomers starting from α-helical and β-hairpin structures were simulated in solution and on the graphite surface or used as building blocks to construct Aβ oligomers. The α-helical Aβ1−42 conformation mimics the native structure of the amyloid precursor peptide (APP) embedded in the membrane, while the β-hairpin conformation is a general structural motif in high-ordered Aβ oligomers, protofibrils, and fibrils. The α-helical structure of Aβ1−42 obtained from NMR data (PDB: 1Z0Q).34 The βhairpin structure of Aβ9−40 was obtained from Dr. Tycko’s lab by averaging NMR-derived Aβ 18-mer.35,36 Since the residues 1−8 are structurally disordered and residues 41−42 are missing, the structural coordinates of residues 1−8 and 41−42 were copied from α-helix structure (PDB: 1Z0Q) and reassembled into the β-hairpin structure of Aβ9−40, yielding a full-length Aβ monomer with the β-hairpin structure. The β-hairpin Aβ1−42 monomer was composed of two antiparallel β-strands (residues 10−22 and 30−42) connecting by a bend (residues 23−29).35 For both the α-helical and β-hairpin Aβ monomers, the N- and C-termini of Aβ1−42 were capped with charged −NH3+ and −COO− groups, respectively, yielding a net charge of −3 at pH 7. α-Helical Aβ Dimers. To obtain initial configurations of α-helical Aβ dimers, the Patchdock program37 was first used to coarsely search Aβ dimeric complexes, and the top 200 dimeric complexes at the lower free energy states were subject to the Firedock program38 to optimize the side-chain arrangement at the residue-overlapped interface. Top two α-helical Aβ dimers ranked by the lowest free energy were selected for simulations both in solution and on the grapheme surface. β-Hairpin Aβ Oligomers. Since the experimental crystal structures of Aβ oligomers are currently not available, we used β-hairpin Aβ monomer as a building block to construct a series of β-hairpin Aβ oligomers from dimer to tetramer by stacking Aβ monomers on top of each other in a parallel and register way with an initial peptide− peptide separation distance of ∼4.7 Å, corresponding to the experimental data. Initial structures of α-helical Aβ monomer/dimer and β-hairpin monomer to tetramer are given in Figure S1 of the Supporting Information. Graphite Surface. The graphite surface was constructed by stacking six graphene sheets on top of each other along the z direction, with the sheet-to-sheet separation of 3.354 Å. The single graphene sheet was constructed by duplication of basic hexagonal unit cells of a = b = 1.415 Å and γ = 120° in both x and y directions to achieve a surface dimension of 76.572 × 88.418 Å2. The size of the graphite is sufficiently large enough to guarantee a minimal distance of 10 Å between any edge of graphite and any atom of Aβ. Given this sheet-tosheet distance of 3.354 Ǻ and a VDW cutoff distance of 12 Ǻ , six graphene sheets were a minimum value to achieve fully intermolecular interactions between Aβ peptides and all graphene sheets. The Lennard-Jones parameters for carbon atom in the graphite surface were borrowed from carbon atom of benzene (atom type is CA) in the CHARMM27 force field, with r0 = 1.9924 Ǻ and ε0 = 0.07 kcal/ mol.23,39 Aβ−Graphite Systems. The adsorption of Aβ peptides on the graphite surface depends on the initial orientation of the peptides relative to the surface, but all-atom explicit-solvent MD simulation generally prevent significant peptide rotation within nanosecond time scale. To overcome this limitation, we used the PatchDock and FireDock programs to determine the most favorable initial orientations for α-helical Aβ monomer and dimer on the graphite surface. The top two orientations ranked by the highest docking scores were selected as 6596

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representative starting configurations for simulating Aβ adsorption on the graphite surface. In the case of β-hairpin Aβ monomer and oligomers, since all of Aβ oligomers contain two parallel β-sheet (i.e., C-terminal β-sheet and N-terminal β-sheet), to avoid the biased orientation of Aβ oligomers on the graphite surface, all of Aβ oligomers were orientated in a way that only bottom peptides were contacted with the graphite surface; i.e., fibril axis of Aβ oligomers was parallel to the surface normal (i.e., z-axis). Once the orientation of Aβ on the graphite surface was determined in all Aβ−graphite systems, Aβ peptides were initially placed at ∼5 Å above the graphite surface to mimic preadsorbed state. There were ∼2 water layers between the Aβ backbones and the graphite surface, allowing peptides to freely rotate and adjust their orientations and to reduce the diffusion time approaching the graphite surface. In all cases, Aβ peptides were laid parallel to the graphite surface. Such parallel orientations not only allow Aβ peptides to achieve maximal but different initial contacts with the graphite surface but also enable to examine the effect of initial orientation on conformational dynamics and adsorption behavior of Aβ peptides on the graphite. Each Aβ-graphite system was solvated in a TIP3P water box, and any water molecule within a radius of 2.4 Å from the non-hydrogen atoms of Aβ and graphite was deleted, resulting in 10 000−12 000 water molecules. The box length of each system in the z direction was set up to ∼70 Ǻ to ensure that any atom of Aβ was at least 25 Ǻ away from the image of the graphite. Counterions (Na+ and Cl−) were added to the water box to achieve electroneutrality and ionic strength of ∼200 mM. The resulting system was energy minimized to remove any bad contacts using the conjugate gradient method for 5000 steps with the peptide backbones constrained and the graphite atoms fixed, followed by additional 5000 steps with only graphite atoms fixed. Here, we constructed eight representative Aβ-graphite models, including one α-helical Aβ monomer, one α-helical Aβ dimer, one β-hairpin Aβ monomer, three β-hairpin Aβ dimers with three different orientations, and two βhairpin Aβ trimer and tetramer with U-turn orientation on the graphite surfaces. MD Simulation Protocol. All of MD simulations were carried out using NAMD 2.7 program40 with CHARMM27 force field.41 After energy minimization, the systems were gradually heated from 50 to 300 K in 100 ps and equilibrated at 300 K for 500 ps to adjust the system size and water density in the z direction under NPT ensemble. Then, 40 ns MD simulations were performed in the canonical NVT ensemble (T = 300 K) with periodic boundary conditions. Temperature was controlled by the Langevin thermostat method with a damping coefficient of 1 ps−1. All graphite atoms were fixed and all covalent bonds involving hydrogen were constrained using the RATTLE method during simulations, which allow a 2 fs time step in the velocity Verlet integration. van der Waals (VDW) interactions were calculated by the switch function with twin-range cutoff distances of 10 and 12 Å, while long-range electrostatic interactions were calculated by the particle-mesh Ewald method with a grid size of ∼1 Ǻ and a real-space cutoff of 12 Ǻ . Each system was run twice with the same (β-hairpin oligomers) or different (monomers and α-helical dimer) initial coordinates and different initial velocities to assess reproducibility. Structures were saved every 2 ps for analysis. Analyses were performed using tools within the CHARMM, VMD,42 and inhouse TCL codes. Simulation systems are summarized in Table 1. AFM Images of Aβ1−42 Aggregation in Bulk Solution and on the Graphite Surface. Highly ordered pyrolytic graphite (HOPG) was purchased from PSI Supplies (West Chester, PA). Synthetic Aβ1−42 was obtained from American Peptide (Sunnyvale, CA) and stored at −80 °C as arrived. We used a protocol in our previous work27 to purify and obtain a homogeneous solution of Aβ monomers. Briefly, Aβ1−42 was dissolved in 99.9% 1,1,1,3,3,3-hexafluoro-2propanol (HFIP, Sigma-Aldrich, St. Louis, MO) for 2 h, sonicated for 30 min to remove any preexisting aggregates or seeds, and centrifuged at 4 °C and 14 000 rpm for 30 min. 75% of the top Aβ solution was subpackaged and frozen with liquid nitrogen and then dried with a freeze-dryer. The dry Aβ1−42 powder was lyophilized at −80 °C. Immediately prior to use, the HFIP-treated and lyophilized Aβ1−42 powder was aliquoted in DMSO (Sigma-Aldrich, St. Louis,

Table 1. Structural Characterization of Aβ Aggregates in Solution and on the Graphitea system

name

rmsd (Å)

bulk

A1 B1 A2 B2 B3 B4 A1-P B1-P A2-P B2-C B2-N B2-U B3-U B4-U

6.6 10.4 12.5 10.2 8.1 9.2 9.4 14.4 8.0 6.1 9.1 7.3 6.8 8.1

graphite

a

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.8 0.7 1.4 1.1 0.3 0.3 0.4 0.3 0.4 0.2 0.4 0.3 0.5 0.2

Rg (Å) 14.4 21.0 14.0 19.2 21.9 21.8 16.4 19.5 19.4 20.2 19.6 19.7 20.4 20.8

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.9 1.6 1.4 0.9 0.3 0.5 0.6 0.2 0.4 0.4 0.5 0.2 0.3 0.2

All data are averaged from the last 5 ns simulations.

MO) for 5 min. The initiation of 10 μM Aβ1−42 aggregation in solution was accomplished by adding an aliquot of the concentrated DMSOAβ1−42 solution to PBS buffer followed by immediate vortexing to mix thoroughly. For time sequential AFM imaging of Aβ morphology change in bulk solution, 10 μL of Aβ1−42 solution was deposited onto a freshly cleaved mica substrate for 1 min, rinsed three times with 50 μL of deionized water to remove salts and loosely bonded Aβ, and dried with compressed N2 for 5 min before AFM imaging. To characterize Aβ aggregation on HOPG, freshly pealed the HOPG was placed into Aβ1−42 solution at the same time it was prepared. After incubation for a given time, the chips were taken out of the solution, rinsed by three times of 50 μL of deionized water, and dried with compressed N2 for 5 min before AFM imaging. AFM images were acquired by using a multimode Nanoscope III (DI) equipped with a 15 μm E scanner. Commercial Si cantilevers (NanoScience) with an elastic modulus of ∼40 N m−1 were used. All images were acquired as 512 × 512 pixel images at a typical scan rate of 1.0−2.0 Hz with a vertical tip oscillation frequency of 250−350 kHz. Representative images of each sample were collected by scanning at least six different locations.

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RESULTS AND DISCUSSION For clarity and convenience, the Aβ-graphite systems are denoted by a character sequence: the initial conformational characteristics of Aβ (i.e., A represents α-helical Aβ and B-for βhairpin Aβ), the aggregation status of Aβ (i.e., 1 represents for Aβ monomer, 2 for dimer, 3 for trimer, and 4 for tetramer), and initial orientation of Aβ (i.e., C for C-terminal β-strands facing toward the graphite surface, N for N-terminal β-strands facing toward the graphite surface, U for the bottom peptide facing toward the graphite surface, and P for other parallel orientations of Aβ peptides relative to the graphite surface). For example, A1-P indicates that the α-helix Aβ monomer adopts the parallel orientation relative to the graphite, while B2C represents that the β-hairpin Aβ dimer is initially oriented parallel to the graphite using C-terminal β-strands. We also performed additional MD simulations of Aβ peptides in bulk solution for comparison with those in the presence of graphite surface. The bulk systems are denoted without orientation information. Simulation details are summarized in Table 1. Structural Stability of Aβ Peptides in Bulk Solution. It is very computationally demanding to study a complete adsorption process of biomolecules from aqueous solution to the surface using all-atom explicit-solvent MD simulations within nanosecond time scale. Instead, we simulated Aβ peptides both in aqueous solution and on the graphite surface 6597

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different peptide packing also showed a similar loss of α-helical conformation at intersection region. As compared to relative stable α-helical Aβ peptides, the βhairpin Aβ monomer quickly lost its U-bend shape and converted into a disordered structure, with a small helix formed by His14-Val18 near the N-terminal region (Figure 1b). The structural instability of the β-hairpin Aβ monomer could result from the significant loss of intrapeptide hydrogen bonds, as evidenced by ∼9 intra-peptide hydrogen bonds for the βhairpin Aβ monomer as compared to ∼21 hydrogen bonds for the α-helix monomer. However, when two β-hairpin monomers were assembled into a dimer with an in-register peptide packing, β-hairpin structure was remained to some extent because extra inter-peptide hydrophobic side chain contacts and hydrogen bonds enhanced the association of two peptides. Meanwhile, both peptides had one side completely exposed to solvent, causing a slow unfolding and loss of initial intact βhairpin conformation, in congruence with a slight decrease in βstructure content from 38.6% to 36.0% within 40 ns. As Aβ oligomer size increased to trimer and tetramer, overall structural integrity of these oligomers was well-preserved with a large population of β-hairpin conformation of 64.4% for trimer and 58.7% for tetramer (Figure 1c,d), which could be attributed to the enhanced stabilization of intermolecular hydrogen bonds between adjacent Aβ peptides. Aβ Adsorption on the Graphite Surface. Visual inspection of MD trajectories has clearly shown that all of the different Aβ species with different sizes and conformations can rapidly adsorb on the graphite surface within 10 ns, but they experienced different conformational and orientation changes upon adsorption. The final adsorbed conformations of Aβ aggregates are presented in Figure 2. Specifically, the adsorption behaviors of different Aβ species on the graphite surface are described below. α-Helical Aβ Monomer and Dimer. The extent of Aβ adsorption on the graphite surface is quantified by the number of contacts per peptide, defined as the number of non-hydrogen Aβ atoms within 4.5 Å of the surface. It can be seen in Figure 3 that in both simulations of Aβ monomer and dimer on the graphite surface, as Aβ rapidly approached the graphite surface, the number of adsorbed peptide atoms quickly increased to 400−500 at the first 5 ns and remained at ∼880 and ∼980 with small fluctuations, though there were some variations in which residues or atoms adsorbed, for the rest of the MD simulations. Upon Aβ adsorption on the graphite surface, both Aβ monomer and dimer gradually but significantly lost their initial helical contents spreading from N-/C-terminal residues to the middle region (Figure 2a,c). In Figure 4b, for Aβ monomer, the helical conformation was greatly reduced from 41.7% to 19.6% while unstructured conformation was conversely increased from 58.3% to 80.4%. Similarly, Aβ dimer also experienced the overall decrease in helicity from 53.2% to 41.0%, resulting from 33% helicity in one peptide and 49% helicity in the other. The unstructured conformation of α-helical Aβ dimer increased from 46.8% to 59.0%, but with much less extent. As the simulation was extended to 80 ns, the helical population of Aβ dimer was continuously decreased to 25% at 80 ns from 41% at 40 ns (Figure S2). In the repeated MD simulation of α-helical Aβ dimer on the graphite, one peptide almost completely lost its helical conformation, while the other retained ∼57% helical structure (data not shown). Therefore, α-helical structure is not favorable conformation on the graphite surface, while interpeptide interactions will delay the secondary transition from

separately, followed by the comparison of the structure and dynamics of Aβ peptides in different environments to illustrate the surface-induced changes in Aβ conformation, oligomerization, and polymorphic energy landscape upon adsorption. Aβ monomer and dimer are the two smallest building blocks that can further self-assemble into higher-order oligomeric species at the very early aggregation stage. MD trajectories showed that the α-helical Aβ monomer quickly straightened its initial semicircle shape to an extended helical structure at the first 4 ns and then remained this conformation with ∼60% of helicity unchanged for the remainder of simulations (Figure 1a). Similarly, for the α-helical Aβ dimer, two peptides largely remained the helical content of 56% and 46%, respectively. Meanwhile, we also observed that at the intersection region between two peptides one peptide tended to lose a small fraction of α-helical conformation between Gly25 and Ile31. Repeated MD simulations of another α-helical Aβ dimer with

Figure 1. MD snapshots of (a) α-helical Aβ monomer and dimer, (b) β-hairpin Aβ monomer and dimer, (c) β-hairpin Aβ trimer, and (d) βhairpin Aβ tetramer at 40 ns in bulk solution. N- and C-terminus are presented by red and white balls, respectively. Color codes: hydrophobic residues (white), hydrophilic residues (green), negatively charged residues (red), and positively charged residues (blue). 6598

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Figure 2. MD snapshots of (a) A1-P, (b) B1-P, (c) A2-P, (d) B2-C, (e) B2-U, (f) B2-N, (g) B3-U, and (h) B4-U on the graphite surface at 40 ns. Both top and side views are shown.

Figure 3. Number of contacts between Aβ and the graphite. An Aβ− surface contact is counted if separation distance between nonhydrogen atoms of Aβ and the top graphene sheet is less than 4.0 Ǻ .

helix to unstructured conformation. In contrast to wellpreserved α-helical conformation of Aβ monomer (60.4%) and Aβ dimer (52.3%) in solution (Figure 4a), surface-induced conformational changes of Aβ monomer and dimer from αhelical conformation to unstructured conformation allowed a larger number of residues to interact with the surface. This was confirmed by increased number of peptide−surface contacts and radius of gyration, which in turn enhance peptide−surface interactions to induce peptide adsorption on the surface. β-Hairpin Aβ Monomer to Tetramer. Independent of initial Aβ orientation and size, all of β-hairpin Aβ monomer and oligomers were able to adsorb on the graphite surface within 40 ns simulations (Figure 2). During the adsorption, the number of adsorbed peptide atoms increased to ∼860 for Aβ monomer, ∼580, ∼690, and ∼980 for Aβ dimer with C, N, and U orientations, ∼780 for Aβ trimer, and ∼1200 for Aβ tetramer (Figure 3). By taking together of common adsorbed Aβ species and different peptide−surface contacts, this reflects that a threshold value of peptide−surface contacts is required to

Figure 4. Secondary structural comparison of α-helix and β-structure for Aβ aggregates between the first 5 ns (left column) and the last 5 ns (right column) MD simulations (a) in bulk solution and (b) on the graphite surface.

realize Aβ adsorption. Upon peptide adsorption, the integrity of β-hairpin structures was significantly disrupted for Aβ monomer with 0% of β-structure, well-preserved for B2-C and B2-N dimers with 39.0% and 34.1% of β-structure, and marginally disturbed for Aβ dimer with 28.0% of β-structure, trimer with 32.3% of β-structure, and tetramer with 41.9% βstructure for the U-bend orientation (Figure 4b). In contrast with significant secondary structure transition of α-helical Aβ dimers on the graphite, β-hairpin dimer showed much higher structural integrity. Increased structural stability of Aβ oligomers as size could be attributed to the enhanced stabilization of intermolecular hydrogen bonds between 6599

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adjacent Aβ peptides. Meanwhile, the interactions between Aβ peptides appear to compete against surface-induced interaction to prevent Aβ peptides from large conformational changes. Structural comparison of Aβ peptides in solution and on the graphite surface reveals that within 40 ns simulations Aβ monomer prefers to adopt α-helical or unstructured conformation in solution, but α-helical conformation is continuously converted to unstructured conformation upon the adsorption of Aβ monomer on the graphite. Although the exact atomic structure of Aβ monomer is not determined yet, a number of structural studies from CD, FTIR, NMR, and MD simulations have shown that monomeric Aβs mainly adopt random coil and α-helix conformations in solution.43−46 Meanwhile, once the higher-ordered β-sheet-rich Aβ oligomers are formed in solution, the adsorption of these Aβ oligomers from solution to the graphite surface has a little influence on the primary structural integrity and secondary β-sheet content of these Aβ oligomers. These results suggest that the presence of the graphite surface indeed changes the polymorphic structure and energy landscape of Aβ aggregates. Aβ Orientation on the Graphite Surface. It is interesting to determine whether Aβ peptides, particularly β-structure-rich oligomers, have the preferential orientation to be adsorbed on the graphite surface, which is very important for understanding Aβ adsorption mechanism at the very early stage. For β-hairpin Aβ dimer that is the smallest Aβ oligomer, three typical orientations relative to the graphite surface were considered: the hydrophobic C-terminal β-strand region facing toward the graphite (referred as C-orientation), the hydrophilic N-terminal β-strand region facing toward the graphite (i.e., N-orientation), and both β-strands of the hairpin in contact with the graphite (i.e., U-orientation). For Aβ trimer and tetramer, only Uorientation was considered. The C-, N-, and U-orientation were three main possible orientations between Aβ oligomers and graphite. Any other initial orientations (most likely being random or tilted orientations) should be in between any two of the C-, N-, and U-orientations. These three orientations were selected because (i) they can potentially achieve the maximal contacts between Aβ and graphite, (ii) they represent different peptide elongation pathways to grow into fibrils on the graphite, and (iii) usage of random or other titled orientations will be very computationally demanding for Aβ oligomers to search for a preferred orientation for adsorption. To quantify the orientation change of Aβ oligomers on the graphite surface, averaged angle between the surface normal to two β-strand vectors (one vector is from the Glu22 in the first peptide to the Glu22 in the last peptide; the other vector is from the Lys28 in the first peptide to the Lys28 in the last peptide) was used to characterize the Aβ orientation on the graphite (Figure 5), where 0° indicates the ideal U-orientation and 90° indicates the ideal C- or N-orientation. As shown in Figure 5 and MD trajectories, Aβ dimer with initial C-orientation tightly bound to the graphite surface and barely changed its initial orientation of 90°. Such stable adsorption via C-orientation can be attributed to strong hydrophobic interactions between C-terminal residues of Ile31, Met35, Val 39, Ile41, and Ala42 of Aβ dimer and the graphite. For Aβ dimer with N-orientation, hydrophilic Nterminal β-strands quickly lost initial contacts with the graphite, leading to that Aβ dimer changed its initial N-orientation of 78° toward final U-orientation of 36°. In the case of an unbiased Uorientation for Aβ dimer, trimer, and tetramer, initial contacts of Aβ oligomers with surface were established by the only

Figure 5. Orientation of β-hairpin Aβ oligomers on the graphite surface. 0° means that only bottom peptide contacts with the surface, with fibril axis parallel to surface normal (i.e., U-orientation), while 90° means that either C- or N-terminal β-strands contact with the surface, with fibril axis perpendicular to surface normal (i.e., C- or Norientation).

bottom Aβ peptide. During the simulations, all of β-hairpin Aβ oligomers eventually rotated over by ∼80°, leading to hydrophobic C-terminal residues facing toward the graphite. During the reorientation, C-terminal residues formed intensive contacts with the surface to maximize the hydrophobic interactions with graphite, so that the new orientation remained steadily until the end of the 40 ns simulation. Similar reorientation process of Aβ oligomers on a hydrophobic CH3−SAM was also previously reported.27 Because of the amphiphilic sequence of Aβ peptide and the hydrophobic nature of graphite and CH3−SAM, the reorientation of Aβ on the hydrophobic surface further confirms that Aβ oligomers prefer to adopt an energetically favorable orientation to be adsorbed on the surface via hydrophobic C-terminal residues. Moreover, during the reorientation process, all of β-hairpin Aβ oligomers with initial U-orientation were able to remain their overall structural integrity without peptide disassociation and secondary β-structures of 28.0% for Aβ dimer, 32.3% for trimer, and 41.9% for tetramer, indicating that increased peptide numbers generally preserve more β-structure in the oligomeric structures. Moreover, as compared to Aβ dimer with U-orientation, Aβ dimer with C-orientation and N-orientation displayed higher β-structure content of 38.9% and 34.2%, respectively. It appears that stable adsorption lead to the less conformation change of Aβ. As the size of Aβ increases, particularly high-ordered oligomers, protofibrils, and fibrils are very likely to be lied down and adsorbed on the surface via the hydrophobic C-terminus, rather than to be stood up on the surface via by the bottom peptide. Interactions of Aβ Peptides with the Graphite Surface. Since Aβ is an amphipathic peptide with distinct hydrophobic C-terminal residues and hydrophilic/charged N-terminal residues, a quantitative comparison of the peptide−surface interactions involving hydrophobic and hydrophilic residues enable to derive what driving forces (hydrophobic or hydrophilic forces) control Aβ adsorption on the graphite surface. It should be noted that due to the absence of partial charges in the graphite as defined by the force filed, the nonbonded interaction energy between Aβ peptides and the graphite surface only contains van der Waals (VDW) interaction. Figure 6 shows nonbonded VDW interaction energies between Aβ peptides and the graphite surface and their partition into surface interactions with hydrophobic and 6600

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Figure 6. Nonbonded VDW interactions between Aβ peptides and the graphite surface and their partition into surface interactions with hydrophobic and hydrophilic residues.

hydrophilic residues, averaged from the last 5 ns simulations. In all cases, Aβ−graphite interactions were energetically favorable ranging from −180 to −350 kcal/mol, consistent with MD observations that all Aβ peptides were adsorbed on the graphite surface. The size of Aβ oligomers appears to have a little effect on total Aβ−SAM interactions, strongly depending on local and specific interactions associated with the conformation and orientation of adsorbed Aβ peptides. Decomposition of total Aβ−surface interaction energies into hydrophobic and hydrophilic contributions showed that hydrophobic interactions contributed to 53.8%−68.7% of interaction forces for Aβ adsorption, while hydrophilic interactions also contributed to 31.3%−46.2% of total Aβ−surface interaction. As compared to hydrophilic interactions, hydrophobic interactions appear to be energetically more favorable and more stable in time. Such interaction distribution indicates that Aβ adsorption on the graphite surface is in a cooperative mode. To further identify which residues are critical for Aβ adsorption, Figure 7 shows interaction energies between each residue of Aβ peptides and the graphite surface. Although the residue−surface interactions were highly heterogeneous and strongly depended on the size and orientation of Aβ, some residues showed apparent preferential interactions with the graphite surface. It can be seen that in case of unstructured Aβ monomers derived from initial α-helical or β-hairpin conformation, since almost all of residues remained unstructured, residue−surface interactions from N-terminal and C-terminal residues were comparable. For the small β-hairpin Aβ dimers with three different initial orientations, the hydrophobic Cterminal residues of 30−42 generally involved stronger interactions with the graphite surface than the hydrophilic Nterminal residues of 10−22 and turn residues of 23−29. These preferential interactions with C-terminal residues became more pronounced for Aβ trimer and tetramer. The sequence of 15− 25 near the N-terminal region had no interactions with the graphite. This fact clearly supports the observation from MD trajectories that Aβ oligomers tend to reorient themselves so as to make the C-terminal residues in more contacts with the surface. Because of this reorientation, a number of hydrophobic residues near C-terminal region (Ile31, Ile32, Leu34, Met35, V40, and I41) had relative strong interactions with the surface, while some N-terminal residues lost or weaken interactions with the surface. Such hydrophobic interactions also facilitate Aβ to be stabilized on the graphite surface. Effect of Surface Hydration on Aβ Adsorption on the Graphite Surface. It has been well reported that surface

Figure 7. Nonbonded VDW interactions between each individual residue of Aβ aggregates and the graphite surface.

hydration play a critical role in controlling the conformation, adsorption, and orientation of proteins/peptides on the surface.30 The adsorption of peptides on the surface generally requires the dehydration of both peptides and the surface to maximize peptide−surface interactions. To characterize the structure and distribution of interfacial water molecules near the graphite surface, Figure 8a shows the radial distribution functions of (RDFs) of oxygen atoms of water molecules around the carbon atoms of the topmost graphite sheet. It can be seen clearly that regardless of different Aβ species adsorbed on the graphite, all RDFs were almost identical. The first hydration layer with a reduced low density (i.e., low height) of ∼0.45 g/cm3 appeared at 4.5 Ǻ from the graphite surface, as compared to a typical RDF of water−water pairs in bulk solution that usually has the first strong peak at ∼2.6 Ǻ with a much higher water density of >1 g/cm3. This fact suggests that the loss of interfacial water molecules near the graphite surface. Consistently, a visual inspection of close-up snapshots also revealed that very few water molecules were observed in the vicinity of the graphite surface (Figure 8b). Similar water RDF profiles on the hydrophobic CH3−SAM surface were found in our previous simulations, with the first hydration layer at ∼3.9 Ǻ , indicating that the graphite surface is more hydrophobic than CH3−SAM surface. The dehydration of the surface eliminates the surface interaction with water and thus facilitates Aβ peptides, especially their hydrophobic residues to directly contact with the graphite surface. Competition between Aβ− surface interactions and water−surface interactions is crucial for controlling Aβ adsorption and the orientation adjustment. 6601

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comparison with those of fibrils formed in solution (Figure 10). The graphite surface was incubated with freshly prepared solution of 10 μM Aβ1−42 at 25 °C, and samples with different incubation times were collected from 1 h to 6 days. Within 1 h of incubation, a large amount of spherical Aβ particles quickly and completely covered the graphite surface, with a homogeneous diameter of ∼1 nm (Figure 9a). The diameter of aggregates slowly increased to ∼3 nm after incubation of 7 h (Figure 9b,c). Some intermediate species with 50−80 nm in length and ∼1 nm in height were observed at 7 and 9 h (Figure 9c,d). At day 1, AFM revealed the mixtures of large spherical particles and a few single-strand long fibrils (1.5−3 nm in height and 0.5−2 μm in length) in the same sample (Figure 9e). After 3 days of incubation, spherical particles were largely disappeared, and Aβ aggregates including globulars and protofibrils on the HOPG were gradually elongated into both straight and branched fibrils through addition of monomers at the edge of existing aggregates and lateral association and intertwine of two or more protofibrils together to form thicker (2−20 nm) and longer (up to 10 μm) fibrils (Figure 9f−h). For comparison, Aβ aggregation in solution was imaged after 0 h, 2 h, 7 h, 9 h, 1 day, 3 days, 5 days, and 6 days of incubation as shown in Figure 10. Dispersed globular particles with diameter of ∼1 nm were observed by AFM when Aβ solution was freshly prepared (Figure 10a), indicating a monomeric state Aβ in bulk solution at the beginning. The diameter of globular particles increased from 1 to 2−8 nm at 9 h (Figure 10b−d) and to 2−10 nm at 24 h (Figure 10e) in solution, consistent with our previous observations.27 A few short single-strand fibrils with ∼0.4 μm in length were first observed at 24 h (Figure 10e). The fibrils grew slowly from ∼0.4 to ∼2 μm within the first 5 days (Figure 10e−g). Mature fibrils were formed at day 6, with averaged heights of ∼10 nm and increased lengths of ∼5 μm (Figure 10h). A close AFM inspection of Aβ fibrillation in bulk solution and on HOPG surface reveals that the morphologies of small globular

Figure 8. (a) Radial distribution function (RDF) between oxygen atoms of water molecules and the carbon atoms of the top graphene sheet for all different Aβ−graphite systems. (b) Representative MD snapshot for the dehydrated graphite surface interacting with Aβ dimer.

AFM Studies of the Surface-Induced Aβ Aggregation. Complementary to simulation results, AFM was employed to monitor the Aβ aggregation process from an initial monomeric state to a final fibrillar state on the HOPG surface (Figure 9), in

Figure 9. Time sequential AFM images of Aβ1−42 aggregation and adsorption on the graphite surface at (a) 1 h, (b) 2 h, (c) 7 h, (d) 9 h, (e) 1 day, (f) 3 days, (g) 5 days, and (h) 6 days. 6602

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Figure 10. Time sequential AFM images of Aβ1−42 aggregation in bulk solution at (a) 0 h, (b) 2 h, (c) 7 h, (d) 9 h, (e) 1 day, (f) 3 days, (g) 5 days, and (h) 6 days.

aggregates, short protofibrils, and mature fibrils were similar to those aggregated formed in the graphite surface, but formation of large aggregates (protolfibrils and mature fibrils) in solution usually requires much longer time than on the graphite surface. The fast kinetics of fibril formation on the graphite surface is attributed to that the graphite surface increases local Aβ concentrations at the water−hydrophobic interface, facilitates structural transition to β-structure, and intensifies peptide− peptide interactions. All these effects can promote Aβ adsorption and subsequent fibril formation. Adsorption Mechanism of Aβ Peptides on Graphite Surface. Aβ adsorption and aggregation on the graphite is a complex and dynamic assembly process, involving peptide conformation change/association/reorganization, peptide adsorption/desorption/migration on the graphite, and multiple peptide interactions in solution, on the graphite, and at solution−graphite interface. All these events will affect Aβ adsorption on the surface. It is thus not feasible to simulate such complex process using conventional MD simulations with limited time scale and length scale. However, our simulations provide the first nanoscopic approximation to describe the very early adsorption of small Aβ aggregates on the graphite surface, which are not accessible by experiments. Simulation results showed that hydrophobic graphite induces the quick adsorption of different Aβ species from monomer to tetramer, regardless of their initial conformations and sizes. Upon Aβ adsorption, Aβ monomer prefer to adopt random conformation, α-helical Aβ dimers tend to reduce their helicity, and β-hairpin Aβ oligomers well retain their β-strand and U-shape topology. More importantly, Aβ oligomers have a similar preferential orientation to be adsorbed on the graphite via hydrophobic C-terminal β-strands. On the basis of our experimental and simulation observations, we proposed a nucleation−polymerization model to explain a general mechanism of Aβ adsorption and aggregation on the graphite surface (Figure 11). In solution, Aβ monomers can form different species including monomers, oligomers,

Figure 11. A proposed schematic for Aβ misfolding, adsorption, and aggregation on the graphite surface from the aqueous solution, including (1) Aβ monomers or oligomers adsorption on the graphite surface, (2) Aβ reorientation on the graphite surface, and (3) Aβ seed formation and fibril elongation.

globulomers, protofibrils, and fibrils depending on aging of samples. Once small oligomers are formed, subsequent growth proceeds rapidly via the incorporation of the monomers into the ends of nucleated fibrils in solution. Meanwhile, all these solution-state species can adsorb on the graphite surface. The deposition of these species on the surface will lead to a series of events to promote fibril formation. First, surface-bound species experience quick conformational changes particularly for Aβ monomers, and peptide−graphite interactions are believed to be a major driving force to reduce the energy barrier for such structure transition. Small conformational changes are accompanied by a dramatic change of the inter-peptide interactions for spontaneous assembly of peptides into ordered fibrils. Additionally, different conformations derived from different intermediate species provide heterogeneous seeds to grow into 6603

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polymorphic fibrils. Second, the graphite surface also induces the orientation change of peptides as approaching to the surface, and Aβ peptides are likely to interact with the surface using hydrophobic C-terminal residues, resulting in hydrophilic and charged N-terminal residues been exposed to bulk solution. Such orientation allows peptides to readily grow along the fibril axis parallel to the surface by adding peptides to the edges of existing surface-bound aggregates. Conversely, it is not feasible for high-order Aβ aggregates to stand on the graphite using a U-orientation, so that fibril elongation would occur along the fibril axis parallel to surface normal. The twist of β-strands and reduced peptide−surface contacts will make such U-orientation for fibril growth energetically unfavorable. AFM images also support that Aβ protofibrils and fibrils lie down the surface with long length up to 10 μm, but with small heights ranging from 2 to 20 nm. Third, the graphite surface enhances Aβ peptide− peptide association. The deposition of the first layer of peptides on the surface will affect the adsorption of subsequent layers of peptide association and structural adjustment of multiple adsorption layers due to inter-peptide interactions. Unlike peptide association in bulk solution which requires tremendous conformational search and peptide collisions, the peptideadsorbed surface with confined surface dimension and low surface energy has strong tendency to recruit peptides from solution. Owing to multiple peptide adsorption and peptide aggregation pathways on the graphite, the presence of hydrophobic surface not only supports for Aβ polymorphism but also shifts energy landscape of Aβ polymorphism. In parallel, the simulations and experiments of Aβ peptides performed by our group systematically studied the Aβ aggregation behavior on OH−, CH3−, and COOH−SAM surfaces.27,47−49 These works demonstrates that regardless of surface chemistry, all of SAM surfaces accelerated the formation of both β-sheets and amyloid fibrils, but Aβ peptides are much easier to be adsorbed on hydrophobic CH3−SAM and charged COOH−SAM than on hydrophilic OH−SAM. It suggests that on hydrophobic surface the seeds formation is more ordered than hydrophilic surfaces. The experiment performed by McMasters and co-workers with highly hydrophobic SAM also indicate that hydrophobic surface can dramatically promote Aβ aggregation and seed formation as compared to the hydrophilic one.28 All of experimental results confirm that the surface induce aggregation and seeding effect of Aβ in solution.

landscape of Aβ aggregation. This study provides atomic details of Aβ adsorption and aggregation on the graphite surfaces, which may be useful for understanding the interactions between Aβ and cell membrane.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by a National Science Foundation CAREER Award (CBET-0952624) and a 3M Non-Tenured Faculty Award. This study (in part) utilized the highperformance of the Anton cluster at the National Resource for Biomedical Supercomputing.

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REFERENCES

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CONCLUSIONS In this work, a series of all-atom explicit-water MD simulations were carried out to study the adsorption, structure, orientation, and surface interaction of Aβ monomers and small oligomers on the hydrophobic graphite. Simulation results clearly show that hydrophobic surface interactions not only drive all different Aβ species adsorbed on the graphite surface but also induce preferential conformation and orientation of Aβ for adsorption. Regardless of their initial conformations of α-helical or βhairpin in solution, Aβ monomers likely adopt random structure on the graphite surface. Once β-sheet-rich Aβ oligomers are formed in solution and subsequently adsorbed on the surface, they experience minor structural changes on the graphite, but Aβ oligomers adopt more energetically favorable orientation on the graphite surface via hydrophobic C-terminal β-strands. The combination of our MD and AFM results collectively suggest that the presence of hydrophobic graphite surface indeed changes in polymorphic structures and energy 6604

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