Articles

Influence of Aromatic Residues on the Material Characteristics of Ab Amyloid Protofibrils at the Atomic Scale Hyun Joon Chang, Inchul Baek, Myeongsang Lee, and Sungsoo Na*[a] Amyloid fibrils, which cause a number of degenerative diseases, are insoluble under physiological conditions and are supported by native contacts. Recently, the effects of the aromatic residues on the Ab amyloid protofibril were investigated in a ThT fluorescence study. However, the relationship between the material characteristics of the Ab protofibril and its aromatic residues has not yet been investigated on the atomic scale. Here, we successfully constructed wild-type (WT) and mutated types of Ab protofibrils by using molecular dynamics simula-

tions. Through principle component analysis, we established the structural stability and vibrational characteristics of F20L Ab protofibrils and compared them with WT and other mutated models such as F19L and F19LF20L. In addition, structural stability was assessed by calculating the elastic modulus, which showed that the F20L model has higher values than the other models studied. From our results, it is shown that aromatic residues influence the structural and material characteristics of Ab protofibrils.

1. Introduction Amyloid proteins are the major cause of a degenerative disease known as amyloidosis.[1] Furthermore, amyloids are associated with neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, spongiform encephalopathies, and prion-related Creutzfeldt–Jakob disease.[2–5] Such neurodegenerative and degenerative diseases involve misfolding and membrane-mediated aggregation of amyloid proteins.[6–8] These structures disrupt the normal functioning of cells by removing the lipid layer of membranes.[9] Furthermore, fibrillar, oligomeric and plaque structures of various amyloids, for example, Ab, b2-lactoglobulin, b2-microglobulin, and prion proteins, have proven to have a hierarchical structure through studies including atomic force microscopy (AFM), scanning electron microscopy (SEM), cryo-electron microscopy (CryoEM), nuclear magnetic resonance (NMR) spectroscopy, and transmission electron microscopy (TEM).[10–14] Such experimental techniques have revealed that oligomeric species of Ab can form fibrillar structures.[15] Especially, oligomeric amyloid structures (including prefibrillar and annular assembly, soluble Ab (i.e. dimer and trimer), and Ab-derived diffusible ligands) have more toxic characteristics than the fibrillar structures.[16–18] Preand proto-fibrillar structures of amyloids are composed of highly ordered cross-beta strands, stacked along the perpendicular axis.[5] These amyloid prefibrillar structures exist in various sizes, ranging from nano- to microscale due to the repeti[a] H. J. Chang,+ I. Baek,+ M. Lee, Prof. S. Na Department of Mechanical Engineering Korea University Seoul 136-701 (Republic of Korea) E-mail: [email protected]

[+] These authors contributed equally to this work. Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/cphc.201500244.

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tive accumulation of fractured protofibrils under temperature and thermal fluctuations.[19] Repetitively formed amyloid fibrillar structures are not easily decomposed under physiological conditions owing to the hydrogen bond and steric zipper effect, which is a native contact formed from a cross-betastrand pattern of a fibrillar structure.[20–22] Developed amyloid proteins have structural stabilities and mechanical properties that have been revealed by AFM studies and by using optical tweezers.[23] According to Kim et al., a high rupture force for Ab-40 monomers exists, as revealed by AFM experiments under different pH conditions.[24] The authors analyzed the high reaction energy barriers by fitting them with Bell’s model. Recently, Hane et al. also revealed the high unbinding force of wild-type Ab-42 monomers and copper-ionized Ab-42 monomer structures by using the same experimental techniques used by Kim et al.[25] They found a higher range of force distributions on copper-ionized Ab-42 monomers compared with those of controlled Ab-42 amyloid monomer structures. In addition, Welland’s group measured the Young’s modulus of an insulin fiber, which is a fiber sharing a common structural characteristic with Ab and prion amyloid fibrils, and found it to be up to approximately 5 GPa by using AFM bending experiments.[26] The Young’s modulus of insulin reveals the increased material strength and stiffness, which is comparable to biological materials such as spider silk.[27, 28] These experimental studies highlight the high internal binding forces and interesting material properties of amyloid structures. Computational studies can be used to analyze the structural strength and stability of amyloid proteins; however, they have an additional advantage of overcoming the limitation of experimental studies by observing the phenomena at the nanoscale. Buhler’s group revealed the material properties of Ab amyloid fibrils with computational methods by using molecular dynam-

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Articles ics (MD) and the elastic network model (ENM).[29–33] They investigated the bending, torsion, and axial modes of Ab amyloid fibrils through ENM studies with material property tendencies under various length scales.[29] Through quasi-static MD and steered molecular dynamics (SMD) methods, they also revealed the various material properties of Ab amyloid fibrils. Overall, they used MD simulations to reveal the different material properties and behaviors according to different cross-sectional areas.[34] In a similar manner, Na’s group revealed the mechanical characteristics of polymorphic human islet amyloid polypeptide (hIAPP; i.e. homo- and hetero-) and their material behavior by using SMD techniques.[35–38] Through the use of a constant force SMD simulation, they also observed the ductile and brittle characteristics of hIAPP fibrils by counting the extent of hydrogen bond fracture. The number of fractured hydrogen bonds and nonbonded energies, such as VDW and electrostatic energies, supported the conclusion that there was a difference in the structural characteristics of the homo- and heterostructures of polymorphic hIAPP fibrils. Similarly, Choi et al. revealed the structural characteristic of hIAPP at different sizes by using equilibrated MD and SMD.[39] Their results showed the stability of hIAPP fibrils and also demonstrated the variation of properties of hIAPP with respect to the fibril length. To summarize, computational studies can reveal the structural properties and characteristics of amyloid fibrils in detail. Considerable effort has also been focused on determining the sources of stability and instability of amyloid fibrillar structures, by using a range of experimental techniques. DeToma investigated the instability of amyloid oligomers by adding naturally obtained flavones,[40] whereas Brender et al. studied the stabilization of prefibrillar oligomers by NMR spectroscopic and AFM analysis upon adding a metal ion.[41, 42] In a similar manner, other computational studies have shown that (¢)-epigallocatechin-3-gallate (EGCG) agents destabilize oligomeric amyloid structures, including prefibrillar structures.[43, 44] In the case of partial mutation studies, including experimental and simulation approaches, salt-bridge and aromatic residue effects have been shown to be one of the sources of stability for amyloid fibrillar structures. Reik’s group observed the partially mutated 3D Ab (17–42) structure by using electron microscopy.[45] Remarkable fibrillar structure variation was revealed through the salt-bridge area mutation. Based on the experimental results reported by Reik’s group, Buehler’s group also revealed the variation of fibrillar structure of the salt-bridge area by mutating it at the nanoscale using MD. The pitch length variation of the salt-bridge-mutated fibril was observed and compared with an unchanging wild-type fibrillar structure through the MD equilibration.[46] In addition, they revealed the different material properties of both salt-bridge-mutated and wild-type fibrillar structures by using twisted-angle and hydrogen-bond analysis. On the other hand, the effects of the aromatic residues of amyloid structures have been investigated in EM and MD studies. Marshall et al. revealed EM images and fluorescence results that showed that aromatic residues, such as phenylalanine, have an effect on the amyloid protein structure.[47] Their study showed that the aromatic residues affect the size and formation of the fibrillar structure. Similarly, several groups ChemPhysChem 2015, 16, 2403 – 2414

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investigated the aromatic residue, for example, phenylalanine, because of its tendency to lead to the formation of fibrillar structures. Some research groups reported that supramolecular biological materials could be applied as novel biomaterials, a template for understanding amyloid-like phenylketonuria (PKU) symptoms, and in technological applications in nanomedicine.[48–51] For example, the group of Gazit investigated toxic fibrils that were assembled around the phenylalanine residue and assessed their influence in PKU.[52] For the study of neurodegenerative diseases, MD techniques have been used to investigate the effect of the hIAPP amyloid fibrillar structure at the nanoscale.[53] Yoon et al. revealed the difference in material properties and structural characteristics of polymorphic hIAPP fibrils based on the mutation studies[54] of phenylalanine to leucine. Additionally, Ndlovu et al. identified structural differences of hIAPP (20–29) fibrils, such as those from wild-type, F4L, A6P, and I7V mutated models.[55, 56] From their studies, the role of the aromatic residues in depreciating material properties and structural stability was notable. Therefore, many researchers now focus on the tendency of aromatic residues to destabilize amyloid structures. Recently, the role of the aromatic residues on Ab amyloid fibrils was studied by using experimental techniques. Vivekanandan et al. examined Ab amyloid structures by using NMR techniques and reported that Phe19, in hydrophobic region, retained the Ab species and a-helices structures by interacting with the C-terminal region.[57] In addition, Cukalevski et al. investigated the effect of the aromatic residue through ThT fluorescence and cryo-TEM studies.[58] They observed the different structural formation time of wild-type, F19L, F20L, and F19L20L models of Ab amyloid along with different monomer content. However, the structural stability and properties of Ab amyloids in relation to the aromatic residue has not been investigated. In this study, we investigated on the nanoscale by using MD simulations and principal component analysis (PCA) how the structural characteristics and material properties of Ab amyloid protofibrils (17–42) are affected by partial mutation. Four types of protofibrillar structural models, wild-type, F19L, F20L, and F19LF20L, were constructed and were observed after equilibration for 50 ns. The structural difference was supported by molecular mechanics/Poisson–Boltzmann solvent analyses (MM/ PBSA), the hydrogen bond count, and by twist-angle analysis. Furthermore, we revealed the differences in the structural properties of the Ab (17–42) wild-type and mutated models by determining the bending, torsion, and axial elastic modulus, obtained from PCA. Our study shows the relationship between the structural stability of Ab amyloid protofibrils and their aromatic residues. Moreover, our results are compared with those of our previous studies concerned with the role of aromatic residues in determining the properties and structural characteristics of hIAPP fibrils.

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Articles Materials and Methods Materials The growth mechanisms of protofibrillar structures of amyloids are related to their structural stability. Formation of amyloid protofibrils proceeds through a repetitive sequence of elongation and fracturing processes under physiological conditions, such as internal flow and thermal fluctuations. In order to measure the stability of the amyloid structures, we needed to construct a fibril that can resist a solvent from accessing the internal section. Therefore, we constructed 18 layers of Ab amyloid protofibrils. Recently, Kahler et al. revealed the stability of Ab amyloid oligomers and protofibrils of various sizes by using Lìhr’s model.[59] From their results, the folded structure of O12 that consists of 12 layers of beta strands was the most stable structure found. However, to apply the continuum models, such as the Euler–Bernoulli beam model, the O12 protofibril structure is rather too short to allow an evaluation of their stability and their properties. Thus, we decided to construct the Ab protofibril using 18 layers for the application of the continuum models, as shown in Figure 1. To synthesize the amyloid protofibril structures, the basic building-block was the Ab(17–42) protein oligomer structure, which is based on NMR spectroscopic data [protein data bank (PDB) ID: 2BEG].[45] The Ab protofibril that was used

in this study consisted of the Ab (17–42) residue because of the unstable structure of the Ab amyloid protein residues 1–16. This protein structure generally consists of a beta-turn-beta strand topology. The composition of this structure is rather different from that of the steric zipper Ab amyloid model. Subsequently, we constructed four different models: one is the original structure of the Ab(17–42) wild-type (WT) model (Figure 1 < (e)xfigr1 >) and the other three are mutation structures of the phenylalanine (F) amino acid at residue 19 mutated into the leucine (L) amino acid (F19L) (Figure 1 (f)), F amino acid at residue 20 mutated into the L amino acid (F20L) (Figure 1 (g)), and both 19 and 20 F residues mutated into the L residue (F19LF20L) (Figure 1 (h)). We then stacked each of the four models using up to 18 layers of beta-sheets to construct the amyloid fibril structure along the axis direction (Figure 1 (a–d)) with a stacking distance of 4.8 æ. The molecular structure of the amyloid fibrils were mutated by using the VMD Mutator plugin.[60]

Methods Simulation of Equilibrium Molecular Dynamics In this work, we performed an explicit solvent equilibrium MD simulation for all atoms to compare our result with those of Cukalevski et al. In this regard, protofibril formation tendency and the thermodynamic stability of the amyloid fibril model including the WT and the mutated model associated with the aromatic residue were investigated. We built a simulation box to allow solvent to surround the amyloid fibril at a distance of 1.5 nm in all directions using the TIP3P water molecule. The system solution was neutralized with randomly placed Na + and Cl¢ ions at a salt concentration of 0.1 m. The system was energy minimized by using the steepest descent algorithm until the maximum force acting on the system was less than 100 kJ mol¢1 nm¢1. The temperature of the energy minimization (EM) system was then stabilized at 300 K by using the velocity-rescaling thermostat temperature-coupling method. Additionally, the system was stabilized under a pressure of 1 bar by using Parrinello–Rahman pressure coupling. With the use of EM, temperature, and pressure stabilization processes, all heavy atoms in the protein structure were in a position-restraint condition. To stabilize the Ab protein structure, we performed equilibrium dynamics simulations using the leap-frog algorithm with NVT ensemble. The equilibration was carried out for a total period of 50 ns with a time step of 2 fs. The trajectory and energy were saved every 1 ps for structural characteristics and property analyses. The long-range electrostatic interactions were calculated by using the Particle Mesh Ewald (PME) algorithm with a cubic interpolation scheme, with a grid spacing of 0.16 nm, and a grid-based neighbor-searching algorithm. All MD simulations were carried out with the GROMACS 4.6.5 package[61] along with the CHARMM27 force-field. During the entire simulation process all bond lengths were fixed by using the linear constraint solver (LINCS) algorithm and the edge effect of the system was minimized by using periodic boundary condition (PBC) with triclinic unit cells.

Analysis of Intermolecular Interactions in Amyloid Protofibrils Figure 1. Construction of the Ab model and the basic layer of the Ab structure through the representation of phenyalanine and leucine residue of a, e) WT, b, f) F19L, c, g) F20L, and d, h) F19LF20L.

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To analyze the structural stability of each of the four amyloid protofibrils, including the WT and mutated models, depending on phenylalanine residue, we obtained the root-mean-square deviation (RMSD), twist angle, order parameter (OP), number of hydro-

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Articles gen bonds (H bonds), and protein structure energy, such as the molecular mechanics Poisson–Boltzmann surface area (MM/PBSA), during 50 ns of equilibration in MD simulations. The RMSD measures the average distance between the atoms and constructs the protein structure through the entire simulation time. The RMSD equation is given in Equation (1): RMSD ¼

rﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 Xn d i¼1 i n

ð1Þ

where n is the total number of atoms that construct the protein structure, and d is the distance between the equivalent atoms of the initial reference structure. The twist angle is an average dihedral angle between all upper and lower beta-sheets that face each other. Given that the stacking component of the Ab amyloid protofibril has a beta-turn-beta strand structural form, we selected for the specific beta strand a twist angle that ranged from the 18th valine residue (V18) to the 26th serine residue (S26). To calculate the twisting angle, which is known as the dihedral angle, we extracted the Ca atom locations of V18 and V26. We then measured the dihedral angle based on Equation (2): i ¼

Xm¼17 i¼1

cos¢1

li ¡ liþ1 jli jjliþ1 j

ð2Þ

where length l is the distance between the V18 and S26 residues. In addition, li and li + 1 are the inter-beta-strand lengths between the ith and the i + 1th layer, and m is the total number of betasheet layers of amyloid protofibrils, respectively. To evaluate the structural stability over the 50 ns equilibration timescale, we measured the OP after obtaining the dihedral angles. The OP is a measure of the amount of dispersion from the average twist angle. The OP is defined by Equation (3): OP ¼ STDðcos i Þ

ð3Þ

where fi is the ith twist angle. The H bond has been known to constitute the stability component for the amyloid protofibrils. Correspondingly, the stability characteristics and properties of different amyloid protofibrils can be elucidated by counting the number of H bonds. To obtain the H-bond data, we extracted data from a number of H bonds every 10 ps using the g hbonds plugin of GROMACS program version 4.6.5. The raw data was then smoothed through the weighted-movingaverage estimation every 100 data sets. Intramolecular interaction energies in amyloid protofibrils were analyzed based on MM-PBSA free-energy calculations using the g mmpbsa plugin of GROMACS.[62] The vacuum potential energy (EMM) was calculated by using the molecular mechanics (MM) forcefield parameter, and the polar solvation energy (Gpolar) was calculated by using the Poisson–Boltzmann (PB) equation. The protein structure of the Ab amyloid protofibrils was divided in half. Each section used nine beta-sheet layers when the EMM and Gpolar values were calculated. The nonelectrostatic solvation free-energy (Gnonpolar) was calculated by using the solvent-accessible surface area (SASA). The Gnonpolar was defined by Equation (4): Gnonpolar ¼ g SASA þ b

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ð4Þ www.chemphyschem.org

where g is a solvent surface tension constant and b is the fitting parameter. By using a value of 0.14 nm for the water probe parameter, values for g = 0.54 kcal mol·nm2 and b = 0.92 kcal mol are obtained. During the calculation of the geometric characteristic parameters, such as RMSD, twist angle, OP, intrasheet distance, number of H bonds, and MM/PBSA, data were extracted every 10 ps. The average and standard deviation values of the energy data were calculated and ranged from 40 to 50 ns.

Principal Component Analysis Vibrational properties from fluctuating amyloid fibril data can be extracted from the atomic coordinates of the equilibrated Ab structures. We thus performed principal component analysis (PCA) to measure the natural frequency of the fluctuating protein structure; this approach required the construction of a fluctuation matrix Q that is defined as a covariance matrix, given by Equation (5):[63] Q ¼< ðrðtÞ¢ < rðtÞ >Þ ðrðtÞ¢ < rðtÞ >ÞT >

ð5Þ

where r(t) is the coordinate of the backbone structure of the amyloid, comprised of Ca atoms. Ca atom coordinates of Ab amyloid protofibrils were extracted every 10 ps. The symbol is the tensor product result of the covariance matrix. The angled brackets indicate an ensemble average over time. The number of structural configurations was set to be equal to 3N Õ 3N matrix forms that have full-rank nonzero eigenvalues (excluding the six rigid-body modes), where N is the total number of Ca atoms in a given structure. The fluctuation matrix Q can also be expressed in a summation form Equation (6): Q¼

X3N¢6 k¼1

ð6Þ

xk nk nk

where vk, and xk are the kth eigenvector and eigenvalue of the fluctuation matrix Q, respectively. Each eigenvalue represents the positional deviation; therefore, the eigenvalues were sorted in order of decreasing value to prioritize the large fluctuation mode (i.e. the highly collective mode). The stiffness matrix K of the amyloid fibril that can be obtained from the fluctuation matrix can be expressed by using the relationship between the eigenvectors and eigenvalues of the fluctuation matrix in the form of Equation (7): K ¼ kB T

X3N¢6 j¼1

x¢1 j nj nj

ð7Þ

where kB is the Boltzmann constant (1.38294 Õ 10¢23 kcal mol¢1) and T is the temperature (300 K).[64] To determine the natural frequency of the amyloid structures, the equation of motion of a fluctuating amyloid fibril is expressed in terms of the stiffness matrix K through the quasi-harmonic approximation given by Equation (8): Mì þ Ku ¼ 0

ð8Þ

where M and u are the mass matrix and the displacement vector of the fibril, respectively. In addition, the dot notation above ì indicates the second partial derivative with respect to time t. Note that the quasi-harmonic approximation applied in Equation (8) yields accurate results only under a low-frequency range of motion.

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Articles Based on the fluctuating motion of the amyloid protofibrils, we assumed the displacement vector u(t) to have the periodic form given by Equation (9): uðtÞ ¼ z ¡ exp½iwt¤

ð9Þ

where pﬃﬃﬃﬃﬃﬃzﬃ is the modal vector, i is an imaginary number defined as i = ¢1, and w is the natural frequency of the amyloid fibril. Substituting Equation (9) into Equation (8) and solving the resulting eigenvalue problem yields the natural frequency of the amyloid fibril for the ith normal mode [Eq. (10)]: wi ¼

rﬃﬃﬃﬃﬃﬃﬃﬃﬃ kB T xi Mc

ð10Þ

2.2.4. Continuum Beam Model To calculate the mechanical properties (e.g., the bending rigidity, torsional shear modulus, and the axial elastic modulus) of the amyloid fibril in terms of the natural frequency obtained from PCA, we introduced the continuum beam model that is written as Equations (11 a)–(11 c): [email protected] w ðx; t Þ þ EB [email protected] wðx; tÞ ¼ 0

ð11aÞ

[email protected] ðx; t Þ ¢ GT [email protected] ðx; tÞ ¼ 0

ð11bÞ

[email protected] lðx; t Þ ¢ EX [email protected] lðx; t Þ ¼ 0

ð11cÞ

where Equations (11 a)–(11 c) are applied for the bending mode, torsional mode, and the axial stretching mode, respectively. Note that Equation (11.a) is also known as the Euler–Bernoulli beam model equation.[65] Constants 1, A, EB, I, J, GT, and EX, denote the mass density, cross-sectional area, bending elastic modulus, crosssectional moment of inertia, cross-sectional polar moment of inertia, torsional shear modulus, and the axial elastic modulus of an amyloid fibril, respectively. Correspondingly, the variables w(x,t), f(x,t), and l(x,t), represent the transverse deflection, twist angle, and axial displacement, as a function of fibrillar axis and the longitudinal direction of the fibril’s x-coordinate and time t, respectively. Solving Equation (11) for the natural frequencies of each deformation mode yields Equations (12 a)–(12 c):

p wT ¼ L wX ¼

sﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ GT 1

ð12aÞ

ð12bÞ

sﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ p E L

X

ð12cÞ

1

where wB, wT, wX, l, and L are the natural frequencies for the bending, torsion, stretching, weighted natural frequency, and the length of an amyloid fibril, respectively. Identification of the deformation modes, on the other hand, requires a geometrical observation in relation to the mode shapes. After evaluating the mode shapes of the first 30 lowest frequenChemPhysChem 2015, 16, 2403 – 2414

2. Results 2.1. Geometrical Characteristics of Ab Amyloid Protofibrils Depending on Mutation

where Mc is the molecular weight of the Ca atom.

2 sﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ﬃ l EB I wB ¼ L 1A

cies, we selected four modes that exhibited the four main deformation modes: soft bending deformation, stiff bending deformation, torsional deformation, and axial stretching deformation. Herein, the bending mode terms ‘soft’ and ‘stiff’ are used to differentiate one bending direction of the fibril structure from another. Once those modes were selected, we considered the corresponding natural frequencies, which were calculated from the eigenvalues, to be equal to the natural frequencies defined in Equation (12). Based on these associations, we calculated the mechanical properties.

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To gain an insight into the structural stabilities of the amyloid protofibrils, we first observed the final formation of the structures. Figure 2 shows the conformation of WT and the partially

Figure 2. Fibrillar structure of Ab after 50 ns equilibrated simulation: a) WT, b) F19L, c) F20L, and d) F19LF20L models.

mutated Ab amyloid protofibrils after 50 ns of equilibrated MD simulations using VMD.[60] The simulation time lasted for 50 ns, which was sufficient for the fibrils to become energetically favorable, and thus adopt a structurally stable form. It should be noted, however, that F19L adopted a bent form, indicating the instability of the structure with respect to F19L mutation. Except for F19L, the structure of which was unstable because of its bent conformation, other models yielded stable conformations after 50 ns of equilibrated MD simulations. Among them, F20L clearly showed a stable conformation compared with WT and F19LF20L. In the case of F19LF20L and WT models, fluctuations existed at the ends of the beta strands, as shown in Figure 2. These fluctuations can be used to determine the relative instability compared with F20L. To verify the stability of the amyloid protofibrils in a more quantitative manner, we monitored the RMSD values of the amyloids (Figure 3). The RMSD value, which relates to the average structure deviation rate, of F19L stabilized at approximately 30 ns with an average value of approximately 0.8 nm, whereas that of F20L was consistent at approximately 4 ns at a rate of approximately 0.6 nm. The lowest RMSD values of WT were approximately 0.6 nm, whereas those of F19LF20L were around 0.4 nm. However, as the simulation continued, the two values converged at a value of approximately 0.7 nm, manifesting a similar structural stability.

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Articles

Figure 5. Mode shape of WT Ab protofibrils a) soft bending mode, b) stiff bending mode, c) torsion mode, and d) axial mode.

Figure 3. RMSD result of WT and mutated Ab protofibrils after 50 ns equilibrated simulation: WT, F19L, F20L, and F19LF20L models.

2.2. Vibrational Characteristics of Ab Amyloid Protofibril Based on the PCA Method

To determine the vibrational characteristics of Ab amyloid protofibrils elicited by the partial aromatic residue, we employed PCA, which is a method that is used to measure the natural frequencies of the amyloid protofibrils. As a result, we were able to obtain the 30 lowest eigenvalues and the eigenvectors, i.e. the 30 modes that exhibit the most collective motions, based on the amyloid fibril’s stiffness matrix K. Each vibrational mode possesses the corresponding mode shapes that represent the continuum structure deformations, such as soft bending, stiff bending, twisting, and stretching. The corresponding results are shown in Figures 5 and S1 (see the Supporting Information) for the F20L and F19L, WT, and F19LF20L, respectively. Furthermore, we assessed the geometrical conformations Figure 4. a) Twist angle of WT and mutated Ab protofibrils during 50 ns equilibrated simulation. b) Order parameso as to investigate the modal ter of WT and mutated Ab protofibrils, which were obtained in the last 5 ns. indices concerned with the principal deformations. As a consequence, the equivalent modes spectively) exhibited smaller angle fluctuations during the simwere obtained. It was also verified that the vibrational characteristics of the Ab amyloid fibril follow the tendencies of a conulation. Based on the RMSD and twist angle results, the F20L tinuum elastic beam model. model was found to be the most stable for the Ab amyloid fibril. This result was supported by the OP values, as shown in To understand the behavior of the selected vibrational modes, we compared the mode index numbers and their natuFigure 4 (b). The OP values of the amyloids, that is, statistical values representing the twist angle fluctuations with respect ral frequencies with respect to the deformation mode presented in Figure 6. It is important to note that all the deformation to time, were calculated as the standard deviation value of the twist angle during the last 5 ns of the simulation. The OP modes are greater in magnitude than the seventh mode bevalues of WT, F19LF20L, and F20L, were less than or equal to cause the first six modes signify the rigid-body motions of the amyloid structures. Additionally, the mode indices of each of 0.01, whereas the OP value of F19L was larger (ca. 0.08; Figure 5 (b)). It should be noted that OP values greater than 0.07 the four structures are soft bending, stiff bending, twisting, and stretching, in increasing order. In the case of soft and stiff are considered to be relatively unstable, disordered fibril structures. In addition to the OP value, the error value for F19L was bending and twisting, the corresponding mode indices of all four amyloid fibril structures are in the low range of the 7– 0.02, implying a large twist angle variation. 12th modes. In contrast, the axial mode indices are in the high-frequency range of the 17–24th modes, indicating a disThe structural stability can also be explained through the dihedral angle and OP (Figure 4). The twist angle of F19L (plotted by a red line in Figure 4 (a)) increased within the 0–30 ns range and up to approximately 108. On the other hand, WT, F19L20L, and F20L (depicted as green, yellow, and blue, re-

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Articles in regard to the F20L model, a higher value of natural frequencies was observed compared with those of the WT, F19LF20L, and F19L. For our studies, the axial deformation modes are in the high-frequency range, which is consistent with the results of the previous study.[35] The torsional mode indices, however, are in the low-frequency range, as are the bending modes. This may be due to the different layer structure with respect to the beta-turn-beta topology of the Ab amyloid.

3.3. Physical Properties of Ab Amyloid Protofibrils Based on the Euler–Bernoulli Beam Model

Figure 6. a) Mode index of Ab protofibrils and b) natural frequency of Ab protofibrils.

tinct inclination in tensile motion (Figure 6 (a)). According to Yoon et al., the bending modes tend to occur in the low-frequency range because the bending motions are mainly caused by the thermal fluctuation behavior of amyloids.[54] Unlike torsional or axial deformation, the elastic modulus of which may be dependent on the layer–layer chemical interaction, the bending deformation is the dominant motion occurring in similar amyloid protein models; that is, for Ab and hIAPP amyloid fibrils. Results with similar tendencies were also obtained from geometrical information, such as RMSD, twist angle, and OP. Based on these results, we found that mutation of the aromatic residue affects the vibrational characteristics of the fibril; that is, the mode index and the natural frequency values. Specifically,

Given that the protofibrillar motion abides by the motion predicted by the continuum elastic beam model, we have determined the mechanical characteristics, bending rigidity, axial elastic modulus, and torsional shear modulus. In effect, we applied a continuum mechanical theory for the Ab amyloid protofibrils, which associates the natural frequency with the mechanical properties, as shown in Figure 7. In the case of the soft bending mode, the soft bending rigidities of WT and F19LF20L are approximately 0.03 Õ 10¢27 N·m2 and approximately 0.10 Õ 10¢27 N·m2, respectively, whereas the rigidity for F19L is approximately 0.01 Õ 10¢27 N·m2. Conversely, the soft bending rigidity of F20L is as high as approximately 0.22 Õ 10¢27 N·m2, which is 22 times larger than that of F19L. The stiff bending rigidity also exhibited a similar tendency to that of the soft bending rigidity, but with a higher overall value. For example, the stiff bending elastic modulus of F20L is approximately 0.25 Õ 10¢27 N·m2, which is approximately 20 % higher than the soft bending rigidity of F20L. The torsional shear modulus, on the other hand, exhibited a different behavior, whereby its value for F19L was measured to be approximately 65 MPa. This value was greater than that of WT and F19LF20L (i.e. ca. 30 and 40 MPa, respectively). This inconsistency may be due to the structural characteristics attributed to the composition of beta-turn-beta structures. For axial stretching, the elastic modulus of F20L was approximately 400 MPa, whereas that for F19L was approximately 120 MPa. The modulus for WT and F19LF20L yielded a similar value of approximately 280 MPa, thus resulting in a similar pattern of soft bending and stiff bending. Comparison of this work with the results of our previous studies using the equivalent method applied on the hIAPP mutated fibrils, has shown that the bending rigidities, torsional shear modulus, and the axial elastic moduli values have a similar order of magnitude (Table 1).

Figure 7. Material properties of Ab protofibrils: a) soft bending, b) stiff bending, c) torsion, and d) axial modulus.

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Articles a similar tendency in the elicited RMSD results. In addition, the twist angle value of F20L exhibTorisional elastic modulus Axial elastic modulus Model (Method) Bending rigidity its the lowest value, indicating [10¢27 Nm2] [GPa] [GPa] the stability of F20L amyloid prohIAPP, WT[54] (MD) 0.05–0.47 0.1–0.3 0.29–0.70 tofibril structures. OP further hIAPP, F20L[54] (MD) 0.02–0.25 0.07–0.27 0.14–0.54 strengthens the stability status Ab, WT (our method) 0.03–0.07 0.03 0.27 of WT and mutated amyloid proAb, F20L (our method) 0.02–0.25 0.07–0.08 0.13–0.41 tofibrils; except in the case of F19L, for which the OP value was found to be critically unsta3. Discussion ble. Thus, through analysis of RMSD, configuration, and twist angle of WT and mutated Ab amyloid protofibrils, we con3.1. Aromatic Residue Effect on Ab Protofibril Formation firmed that F19 residues independently have an effect on the Supported by Geometrical Characteristics formation of amyloid protofibrils on an atomic level. The structural stability can also be delineated based on the result of The aim of this study is to reveal the effect of the aromatic resMM/PBSA analysis, as shown in Table 2. The total energy of idue on the formation of the Ab amyloid protofibrils and their stability at the atomic scale, reflecting the experimental results reported by Cukalevski et al.[58] Understanding the mechanism Table 2. MM/PBSA results of WT and mutated Ab models. MM/PBSA data of amyloid fibril development is fundamentally important for obtained in the last 5 ns equilibrium sections. advancing the treatment of neurodegenerative diseases. This mechanistic study is concerned with examining the structural Enonpolar EMM Etotal Model Epolar stability on an atomic scale as reflected by the partial mutation [kcal mol¢1] [kcal mol¢1] [kcal mol¢1] [kcal mol¢1] of the aromatic residue. Furthermore, when fragmented amyWT ¢3616.5 35.8 127.5 1.4 492.0 23.5 ¢2996.9 41.3 loid protofibrils, which are classified as oligomeric structures, F19L ¢3979.4 46.1 131.8 1.3 610.1 22.1 ¢3237.6 48.8 F20L ¢4104.3 37.0 117.7 1.0 528.6 24.8 ¢3458.1 46.7 exist under physiological conditions that include temperature, F19LF20L ¢3544.5 47.2 124.8 1.4 379.1 24.5 ¢3040.6 48.8 pH, and thermal fluctuation, amyloid growth accelerates from [19, 22] fibril to plaque structures. To reveal how the structural stability is related to the growth mechanism of the partially mutated Ab amyloid protofibril, we measured the geometric paF20L is more favorable than the energy of the other models. rameters, such as the RMSD and the twist angle, after 50 ns of To be more specific, polar solvation energy, which is the main equilibrated MD simulations. According to Cukalevski et al., stability factor for the Ab amyloid protofibrils, was dominant in the aggregation kinetics of F20L, F19L, F19LF20L, and WT Ab all models compared with the nonpolar solvation and molecuamyloid models was revealed through ThT fluorescence by lar mechanics energies. Additionally, the total solvation energy varying the amounts of Ab amyloid monomers.[58] From their of F20L Ab amyloid fibril was found to be the most stable among all the equilibrated models. These results can be comresults, F20L showed the most rapid amyloid fibril formation rate at the various amounts of amyloid monomer concentrapared to the other similar aromatic residue mutation studies of tions (2, 4, and 6 mm). Our results also show the rapid RMSD WT and F2L mutated hIAPP amyloid fibrils, based on the equilibrated MD study by Yoon et al.[54] These authors noted an aroconvergence phenomenon of F20L model, which, as shown in Figure 3, converged after 1 ns. The remaining WT, F19LF20L, matic residue effect through the observation of the differences and F19L models, gradually converged after 4, 30, and 40 ns, of RMSD, twist angle, MM/PBSA analysis, and bend angle values. This tendency can also be compared with the work of respectively. These values are also comparable to the results obtained by Cukalevski et al. The lowest RMSD value of the Ndlovu et al., in which the effect of the mutated aromatic resiF20L indicates its structural stability, whereas the remaining due under various loading-mode types such as peel, stretch, and slide was investigated.[55, 56] Through the relationship bemodels show instability. These elicited RMSD values can be verified by the last configuration of the WT and mutated Ab tween the geometric characteristics and MM/PBSA, the influamyloid protofibrils (Figure 3). Except for F19L, which exhibits ence of changes of the aromatic residue on the structural fora bending configuration, the remaining models retain the mation of Ab amyloid protofibrils and their internal structural overall stable protofibrillar structures after 50 ns of equilibrated stability was assessed. MD simulations. Among them, F20L clearly sustains the protofibrillar structure, whereas WT and F19LF20L show relatively un3.2. Influence of the Aromatic Residue on the Material Charstable structures because of the fluctuation of their betaacteristics of Ab Protofibrils strand sides. We also measured the twist angle of the Ab amyloid protofiThe mechanical properties of amyloid protofibrils are imporbril to support the formation of WT and partially mutated amytant for the growth mechanism of the amyloid because the loid protofibrils. As shown in Figure 4 (a), F20L also showed protofibril undergoes a repetitive process of fragmentation rapid convergence of the twist angle after 4 ns, indicating and elongation under various physiological conditions.[19] NuTable 1. Mechanical properties of Ab and hIAPP protofibrils analyzed by PCA methods such as bending rigidity, torsion elastic modulus, and axial elastic modulus.

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Articles merous experiments and simulations have been reported for the determination of the material characteristics of Ab amyloid protofibrils with respect to their salt-bridge interaction, crosssectional area, and environmental factors.[13, 34, 46, 66–69] However, the material stability of Ab amyloid protofibrils in relation to the aromatic residue mutation has not yet been investigated. Herein, to estimate the stability of amyloid protofibrils, we constructed the thermodynamically favorable WT and partially mutated Ab amyloid fibril structures after 50 ns of equilibrium simulations. Through PCA analysis, vibrational characteristics such as natural frequencies (calculated from eigenvalues and modal analysis indices) of bending, torsion, and axial modulus were obtained. Previously, Yoon et al. revealed the bending modes of hIAPP amyloid fibrils at the lowest frequencies.[54, 70] Torsion and axial modes of hIAPP amyloid fibrils were also observed at higher frequencies. Our results show similar tendencies for bending and axial modes. In these modes, natural frequencies reached values in the ranges of 0.04–0.2 THz and 0.25–0.5 THz, respectively. However, analysis of the torsional mode gave different results to those obtained by Yoon et al.; in this case, the torsional frequencies show lower values than those of the hIAPP amyloid fibrils.[35] This noted difference with respect to the torsional mode arises from the different compositions of the Ab amyloid protofibrils, based on their beta-turn-beta strand composition. In the case of the model by Yoon et al., their cross-sectional area consisted of two face-to-face beta strands with steric zipper interfaces.[35] From this analysis, the structural properties of beta-turn-beta structures are weaker than those of the hIAPP model. This tendency arises from the lack of dry interfaces in the beta-turn-beta strand structures. In addition to the natural frequencies, we also obtained the material properties of WT and mutated Ab amyloid protofibrils through modal analysis. We compared these values with those of WT and F2L mutated hIAPP models, the material properties of which were also analyzed with PCA after explicit, equilibrated MD simulations. The bending rigidity of our Ab amyloid protofibrils were approximately 0.25 Õ 10¢27 N·m2, whereas the torsion and axial elastic modulus were approximately 90 and 400 MPa, respectively. These values are smaller than those obtained from the hIAPP protofibril models, the bending rigidities of which were approximately 0.3–0.5 Õ 10¢27 N·m2. The different cross-sectional areas of Ab and hIAPP amyloid protofibrils confirmed the different material properties. Nonetheless, considering the fact that both materials were analyzed with the same approach, the observation that these two values share a similar degree of order confirms that our models have stable material properties. In addition, these material properties of Ab amyloid protofibrils can be compared with those determined by Yoon et al. and by Ndlovu et al.; their studies revealed the structural stability and properties of hIAPP under an equilibrated MD simulation with an explicit solvent condition.[54, 55] Furthermore, for Ab amyloid fibril models, Buehler’s group found that the stability and material properties of Ab amyloid fibrils depend on various cross-sectional areas, such as two-fold and threefold symmetric amyloid fibrils by using ENM studies.[29] From their studies, the lowest frequency bending mode behavior ChemPhysChem 2015, 16, 2403 – 2414

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was found to be similar to the bending mode behavior of our model. In addition, the general trend of our results is consistent with other hIAPP results obtained by using the ENM method by Yoon et al.[70] Based on the vibrational analysis on ENM-converted hIAPP fibrils, the lowest bending modes were observed. The elastic modulus was then obtained by relating the continuum mechanics with the corresponding deformation-mode eigenvalues. Through analysis of material properties of Ab amyloid protofibrils, we identified the effect of the aromatic residue of Ab amyloid protofibrils on the bending, torsion, and axial modulus, as shown in Figure 7. F20L yielded the highest elastic modulus between WT and other mutated models. The trend of the results can be explained by considering the vibrational characteristics, such as the natural frequencies, but also by examining the H-bond count. The number of H bonds has been used to understand the material properties of amyloid protein structures and their stabilities through several MD studies.[20, 54, 71] As shown in Figure 8, we found various H-bond interactions

Figure 8. The number of hydrogen bonds during 50 ns equilibration simulation.

during the 50 ns of equilibrated MD simulations. From the results, F20L undertook the largest number of H bonds in comparison to other models. We can observe a large number of Hbond interactions in F20L Ab amyloid protofibrils (ca. 370), after 45 ns. WT and F19LF20L exhibit H-bond counts of approximately 350 and 330, respectively, and F19L possessed the lowest the number of H bonds of (ca. 310) after 45 ns. Thus, we can conclude that the role of the mutation effects of the aromatic residue changes both their structural characteristics and their material properties. 3.3. The Location of the Phenylalanine Residue Affects the Structural Characteristics Based on the physical properties obtained from equilibrated Ab amyloid protofibrils with PCA analysis, and on the geometrical characteristics, the role of the phenylalanine residue was elucidated by observing the material properties and structural characteristics of the Ab amyloid protofibrils. In addition,

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Articles based on the kinetic amyloid fibril formation using the RMSD twist angle and MM/PBSA analysis, we observed that the F20L Ab amyloid protofibrils are the most stable structures compared with F19L, WT, and F19LF20L. This structural characteristic, in addition to the mutation effect, can be supported by the material properties of each model and by the number of H bonds. These tendencies can be supported by the interaction between the 19th phenylalanine residue (F19) and the 34th leucine residue (L34). This interaction sustains the stability of the beta-turn-beta strands of Ab amyloid protofibrils. The saltbridge region, which was composed of the 23th lysine and the 28th aspartate residues, is an important agent for the stability of the amyloid oligomer and the fibril structures.[45, 46, 72] However, Ahmed et al. revealed the inner interaction between F19 and L34 of the Ab amyloid oligomer and the protofibrils, which consisted of beta-turn-beta strand structures, sustaining their stability as investigated by NMR spectroscopic analysis.[73] Based on their study, we measured the SASA values as shown in Figure 9. Here, F20L has the lowest SASA values,

by the favorable F19 and L34 interactions inside beta strands. Furthermore, in the case of the F20L model, inner interaction of F19 and L34 and outer interaction of F19 and L20 were observed to be more favorable. These interactions are manifested in the structural stability and improved material properties of the Ab amyloid protofibrils. Other models, such as F19L, WT, and F19LF20L, have less favorable interactions between leucine and phenylalanine, leading to lower structural stability and to the observed properties of the Ab amyloid protofibrils of F20L model. In addition, these tendencies can be compared with the results of similar Ab oligomer studies, confirming the SASA area of the central hydrophobic core (CHC) from residues 17, 19, and 21 (i.e. alanine, phenylalanine, and leucine).[74] These previous results revealed a decrease of SASA when the size of the oligomers increased, including the number of beta strands. Thus, the aromatic residues, which reflect the hydrophobic characteristics of the Ab amyloid protofibrils, change their structural characteristics and properties.

4. Conclusions By using equilibrated MD simulations, we explored how the geometrical characteristics and structural properties of an Ab amyloid protofibril are affected by changes to the crucial aroFigure 9. SASA representation of Ab monomer structures: a) WT, b) F19L, c) F20L, and d) F19LF20L models. matic residue. We also found that the aromatic residue has an which explains the low polar and nonpolar solvent energy influence on the Ab amyloid fibril formation kinetics, as revalue and thus explains why it is most stable. In addition, we vealed by the conformation, RMSD, and twist angle results. calculated the intra-beta-strand distance based on the center These insoluble Ab amyloid protofibrils, including WT and the of mass of each beta strand, as shown in Figure 10 (a). Except partial mutation model for Alzheimer’s disease, have been recfor the unstable structure of the F19L model, the F20L model ognized as pathological agents because of their high structural shows the shortest intra-beta-strand distances, followed by the stabilities. However, pathological amyloid fibrils can also be utidistances of F19LF20L and WT models. This tendency can be lized as functional templates for biomaterials.[75] For example, explained by the van der Waals (VDW) forces between the b2-lactoglobulin amyloid fibrils are used as the basic template intra-beta-strands. In the case of F20L model, VDW energies for films with biocompatibility characteristics, due to their were approximately ¢300 kcal mol¢1; however, WT and stable properties.[76] Based on these characteristics, we plan to F19LF20L models exhibited higher VDW energies compared investigate a de novo design of Ab amyloid protofibrils by with the F20L models. This phenomenon might be explained using a partial aromatic residue mutation; such studies are ex-

Figure 10. a) Intrastrand distance and b) VDW energies of Ab protofibril structure during 50 ns equilibration simulation.

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Articles pected to lead to the development of new types of biological material templates.

Acknowledgements S.N. gratefully acknowledges the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP) (No. 2007-0056094 & 2014R1A2A1A11052389). H.J.C. is grateful for the financial support from the Global Ph.D. Fellowship Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (No. 2014H1A2A1021042). Keywords: amyloid beta-peptides · mechanical properties · molecular dynamics · molecular modeling · stacking interactions [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]

M. B. Pepys, Annu. Rev. Med. 2006, 57, 223 – 241. G. Merlini, V. Bellotti, N. Engl. J. Med. 2003, 349, 583 – 596. D. Eisenberg, M. Jucker, Cell 2012, 148, 1188 – 1203. F. Chiti, C. M. Dobson, Annu. Rev. Biochem. 2006, 75, 333 – 366. C. Soto, Nat. Rev. Neurosci. 2003, 4, 49 – 60. J. R. Brender, S. Salamekh, A. Ramamoorthy, Acc. Chem. Res. 2012, 45, 454 – 462. H. R. Patel, A. S. Pithadia, J. R. Brender, C. A. Fierke, A. Ramamoorthy, J. Phys. Chem. Lett. 2014, 5, 1864 – 1870. S. A. Kotler, P. Walsh, J. R. Brender, A. Ramamoorthy, unpublished results. J. R. Brender, D. L. Heyl, S. Samisetti, S. A. Kotler, J. M. Osborne, R. R. Pesaru, A. Ramamoorthy, Phys. Chem. Chem. Phys. 2013, 15, 8908 – 8915. N. Mizuno, U. Baxa, A. C. Steven, Proc. Natl. Acad. Sci. USA 2011, 108, 3252 – 3257. J. Adamcik, J.-M. Jung, J. Flakowski, P. De Los Rios, G. Dietler, R. Mezzenga, Nat. Nanotechnol. 2010, 5, 423 – 428. N. M. Kad, S. L. Myers, D. P. Smith, D. A. Smith, S. E. Radford, N. H. Thomson, J. Mol. Biol. 2003, 330, 785 – 797. Y. Miller, B. Ma, C.-J. Tsai, R. Nussinov, Proc. Natl. Acad. Sci. USA 2010, 107, 14128 – 14133. C. Sachse, N. Grigorieff, M. Fndrich, Angew. Chem. Int. Ed. 2010, 49, 1321 – 1323; Angew. Chem. 2010, 122, 1343 – 1345. Y. Suzuki, J. R. Brender, M. T. Soper, J. Krishnamoorthy, Y. Zhou, B. T. Ruotolo, N. A. Kotov, A. Ramamoorthy, E. N. G. Marsh, Biochemistry 2013, 52, 1903 – 1912. C. Haass, D. J. Selkoe, Nat. Rev. Mol. Cell Biol. 2007, 8, 101 – 112. J. Bieschke, M. Herbst, T. Wiglenda, R. P. Friedrich, A. Boeddrich, F. Schiele, D. Kleckers, J. M. L. del Amo, B. A. Grìning, Q. Wang, M. R. Schmidt, R. Lurz, R. Anwyl, S. Schnoegl, M. Fndrich, R. F. Frank, B. Reif, S. Gìnther, D. M. Walsh, E. E. Wanker, Nat. Chem. Biol. 2012, 8, 93 – 101. C. Cabaleiro-Lago, O. Szczepankiewicz, S. Linse, Langmuir 2012, 28, 1852 – 1857. S. R. Collins, A. Douglass, R. D. Vale, J. S. Weissman, PLoS Biol. 2004, 2, e321. T. P. Knowles, A. W. Fitzpatrick, S. Meehan, H. R. Mott, M. Vendruscolo, C. M. Dobson, M. E. Welland, Science 2007, 318, 1900 – 1903. M. R. Sawaya, S. Sambashivan, R. Nelson, M. I. Ivanova, S. A. Sievers, M. I. Apostol, M. J. Thompson, M. Balbirnie, J. J. W. Wiltzius, H. T. McFarlane, A. O. Madsen, C. Riekel, D. Eisenberg, Nature 2007, 447, 453 – 457. T. P. J. Knowles, M. J. Buehler, Nat. Nanotechnol. 2011, 6, 469 – 479. K. C. Neuman, A. Nagy, Nat. Methods 2008, 5, 491 – 505. B.-H. Kim, N. Y. Palermo, S. n. Lovas, T. Zaikova, J. F. W. Keana, Y. L. Lyubchenko, Biochemistry 2011, 50, 5154 – 5162. F. Hane, G. Tran, S. J. Attwood, Z. Leonenko, PLoS One 2013, 8, e59005. J. F. Smith, T. P. J. Knowles, C. M. Dobson, C. E. MacPhee, M. E. Welland, Proc. Natl. Acad. Sci. USA 2006, 103, 15806 – 15811. A. Nova, S. Keten, N. M. Pugno, A. Redaelli, M. J. Buehler, Nano Lett. 2010, 10, 2626 – 2634.

ChemPhysChem 2015, 16, 2403 – 2414

www.chemphyschem.org

[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] [61] [62] [63] [64] [65] [66] [67]

[68] [69]

2413

S. Keten, Z. Xu, B. Ihle, M. J. Buehler, Nat. Mater. 2010, 9, 359 – 367. Z. Xu, R. Paparcone, M. J. Buehler, Biophys. J. 2010, 98, 2053 – 2062. R. Paparcone, S. Keten, M. J. Buehler, J. Biomech. 2010, 43, 1196 – 1201. R. Paparcone, M. J. Buehler, Biomaterials 2011, 32, 3367 – 3374. R. Paparcone, M. J. Buehler, Appl. Phys. Lett. 2009, 94, 243904. M. J. Buehler, Y. C. Yung, Nat. Mater. 2009, 8, 175 – 188. M. Solar, M. J. Buehler, Nanotechnology 2014, 10, 105703. M. Lee, I. Baek, H. J. Chang, G. Yoon, S. Na, Chem. Phys. Lett. 2014, 600, 68 – 72. J. I. Kim, M. Lee, I. Baek, G. Yoon, S. Na, Phys. Chem. Chem. Phys. 2014, 16, 18493 – 18500. M. Lee, H. J. Chang, D. Kim, Y. Lee, H. Suh, N. Ahn, G. Yoon, S. Na, Biophys. Chem. 2015, 199, 1 – 8. Handbook of Proteins: Structure, Function and Methods, Vol. 2, (Eds.: M. M. Cox, G. N. Phillips Jr.), Wiley, New York, 2008. B. Choi, G. Yoon, S. W. Lee, K. Eom, Phys. Chem. Chem. Phys. 2015, 17, 1379 – 1389. A. S. DeToma, J. Krishnamoorthy, Y. Nam, H. J. Lee, J. R. Brender, A. Kochi, D. Lee, V. Onnis, C. Congiu, S. Manfredini, S. Vertuani, G. Balboni, A. Ramamoorthy, M. H. Lim, Chem. Sci. 2014, 12, 4851 – 4862. J. R. Brender, K. Hartman, R. P. R. Nanga, N. Popovych, R. d. L. S. Bea, S. Vivekanandan, E. N. G. Marsh, A. Ramamoorthy, J. Am. Chem. Soc. 2010, 132, 8973 – 8983. J. R. Brender, J. Krishnamoorthy, G. M. L. Messina, A. Deb, S. Vivekanandan, C. La Rosa, J. E. Penner-Hahn, A. Ramamoorthy, Chem. Commun. 2013, 49, 3339 – 3341. Q. Wang, J. Guo, P. Jiao, H. Liu, X. Yao, PLoS One 2014, 9, e94796. Q. Wang, L. Ning, Y. Niu, H. Liu, X. Yao, J. Phys. Chem. B 2015, 119, 15 – 24. T. Lìhrs, C. Ritter, M. Adrian, D. Riek-Loher, B. Bohrmann, H. Dçbeli, D. Schubert, R. Riek, Proc. Natl. Acad. Sci. USA 2005, 102, 17342 – 17347. R. Paparcone, M. A. Pires, M. J. Buehler, Biochemistry 2010, 49, 8967 – 8977. K. E. Marshall, K. L. Morris, D. Charlton, N. O’Reilly, L. Lewis, H. Walden, L. C. Serpell, Biochemistry 2011, 50, 2061 – 2071. E. Gazit, Nanomedicine 2014, 9, 2433 – 2436. G. Fichman, L. Adler-Abramovich, S. Manohar, I. Mironi-Harpaz, T. Guterman, D. Seliktar, P. B. Messersmith, E. Gazit, ACS Nano 2014, 8, 7220 – 7228. G. Fichman, E. Gazit, Acta Biomater. 2014, 10, 1671 – 1682. L. Adler-Abramovich, E. Gazit, Chem. Soc. Rev. 2014, 43, 6881 – 6893. L. Adler-Abramovich, L. Vaks, O. Carny, D. Trudler, A. Magno, A. Caflisch, D. Frenkel, E. Gazit, Nat. Chem. Biol. 2012, 8, 701 – 706. L.-H. Tu, D. P. Raleigh, Biochemistry 2013, 52, 333 – 342. G. Yoon, M. Lee, J. I. Kim, S. Na, K. Eom, PLoS One 2014, 9, e88502. H. Ndlovu, A. E. Ashcroft, S. E. Radford, S. A. Harris, Biophys. J. 2012, 102, 587 – 596. H. Ndlovu, A. E. Ashcroft, S. E. Radford, S. A. Harris, Beilstein J. Nanotechnol. 2013, 4, 429 – 440. S. Vivekanandan, J. R. Brender, S. Y. Lee, A. Ramamoorthy, Biochem. Biophys. Res. Commun. 2011, 411, 312 – 316. R. Cukalevski, B. Boland, B. Frohm, E. Thulin, D. Walsh, S. Linse, ACS Chem. Neurosci. 2012, 3, 1008 – 1016. A. Kahler, H. Sticht, A. H. C. Horn, PLoS One 2013, 8, e70521. W. Humphrey, A. Dalke, K. Schulten, J. Mol. Graphics 1996, 14, 33 – 38. H. J. C. Berendsen, D. van der Spoel, R. van Drunen, Comput. Phys. Commun. 1995, 91, 43 – 56. R. Kumari, R. Kumar, A. Lynn, J. Chem. Inf. Model. 2014, 54, 1951 – 1962. A. Amadei, A. B. M. Linssen, H. J. C. Berendsen, Proteins Struct. Funct. Bioinf. 1993, 17, 412 – 425. A. Kitao, F. Hirata, N. Go, Chem. Phys. 1991, 158, 447 – 472. L. Meirovitch, Fundamentals of Vibrations, Waveland Pr Inc, New York, 2001. M. Solar, M. J. Buehler, Nanoscale 2012, 4, 1177 – 1183. D. P. Smith, G. D. Ciccotosto, D. J. Tew, M. T. Fodero-Tavoletti, T. Johanssen, C. L. Masters, K. J. Barnham, R. Cappai, Biochemistry 2007, 46, 2881 – 2891. Y. Miller, B. Ma, R. Nussinov, Proc. Natl. Acad. Sci. USA 2010, 107, 9490 – 9495. S. Parthasarathy, F. Long, Y. Miller, Y. Xiao, D. McElheny, K. Thurber, B. Ma, R. Nussinov, Y. Ishii, J. Am. Chem. Soc. 2011, 133, 3390 – 3400.

Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Articles [70] G. Yoon, J. Kwak, J. I. Kim, S. Na, K. Eom, Adv. Funct. Mater. 2011, 21, 3454 – 3463. [71] S. Keten, M. J. Buehler, Nano Lett. 2008, 8, 743 – 748. [72] J. M. Borreguero, B. Urbanc, N. D. Lazo, S. V. Buldyrev, D. B. Teplow, H. E. Stanley, Proc. Natl. Acad. Sci. USA 2005, 102, 6015 – 6020. [73] M. Ahmed, J. Davis, D. Aucoin, T. Sato, S. Ahuja, S. Aimoto, J. I. Elliott, W. E. Van Nostrand, S. O. Smith, Nat. Struct. Mol. Biol. 2010, 17, 561 – 567. [74] A. H. C. Horn, H. Sticht, J. Phys. Chem. B 2010, 114, 2219 – 2226.

ChemPhysChem 2015, 16, 2403 – 2414

www.chemphyschem.org

[75] I. Cherny, E. Gazit, Angew. Chem. Int. Ed. 2008, 47, 4062 – 4069; Angew. Chem. 2008, 120, 4128 – 4136. [76] T. P. J. Knowles, T. W. Oppenheim, A. K. Buell, D. Y. Chirgadze, M. E. Welland, Nat. Nanotechnol. 2010, 5, 204 – 207. Received: March 20, 2015 Revised: April 27, 2015 Published online on June 3, 2015

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