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Molecular Insight into the Inhibition Effect of Trehalose on the Nucleation and Elongation of Amyloid β-Peptide Oligomers Fu-Feng Liu, Luo Ji, Xiao-Yan Dong, and Yan Sun* Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin UniVersity, Tianjin 300072, China ReceiVed: June 15, 2009; ReVised Manuscript ReceiVed: June 30, 2009
Soluble amyloid oligomers are a cytotoxic species in Alzheimer’s disease, and the recent discovery that trehalose can prohibit aggregation of amyloid β-peptide (Aβ) has received great attention. However, its inhibition mechanism remains unclear. In order to investigate the molecular mechanism of the inhibition effect, molecular dynamics simulations of Aβ16-22 and Aβ40 peptides at different trehalose concentrations (0-0.18 mol/L) are performed using an all-atom model. The simulations confirmed that Aβ16-22 aggregation is prevented by trehalose in a dose-dependent manner, and it is found that the preferential exclusion effect of trehalose is the origin of its inhibition effects. Namely, there is preferential hydration on the peptide surface (3 Å), and trehalose molecules cluster around the peptides at a distance of 4-5 Å. At high trehalose concentrations, the preferential exclusion of trehalose leads to three sequential effects that prevent the nucleation and elongation of Aβ16-22 oligomers. First, the secondary structures of Aβ16-22 monomers are stabilized in the turn, bend, or coil, so the β-sheet-rich structure that is prone to forming peptide oligomers is prevented. Second, the thin hydration layer and trehalose clusters can weaken hydrophobic interactions that lead to Aβ16-22 aggregation. Third, more direct and indirect H-bonds form between trehalose and Aβ16-22, which suppress the interpeptide hydrogen bonding. Analyses of the simulation data for a single Aβ40 peptide indicate that trehalose can inhibit the nucleation and elongation of Aβ40 by a similar mechanism with that on Aβ16-22 oligomerization. The work has thus elucidated the molecular mechanism of trehalose on the inhibition of Aβ oligomeric aggregation. 1. Introduction Alzheimer’s disease (AD), the most common form of senile dementia, is pathologically characterized by the presence of amyloid plaques in the brain. The main constituents of the plaques are small peptides 39-43 amino acids long called amyloid β-peptide (Aβ).1 Although early attention was focused on the amyloid fibrils as the cause of AD, recent studies found that Aβ oligomers formed during early aggregation are the main cytotoxic agents.2 The causal relationship between aggregation morphology and the disease remains controversial. However, considerable evidence indicates that a key event in AD pathogenesis is the conversion of Aβ from its soluble monomeric state into various aggregated morphologies in the brain. Therefore, preventing the assembly of Aβ monomer into toxic oligomer or fibril is the primary goal of a number of therapeutic strategies under development or in clinical trials. Recently, major research has involved the development of compounds capable of inhibiting or reversing the Aβ aggregation process. Thus far, a number of diverse compounds have been used to prevent or reduce the aggregation of Aβ into oligomers or fibrils, such as curcumin,3 Congo red derivatives,4 antibodies,5 peptidic β-sheet breakers,6,7 and osmolytes.8 Among the osmolytes, trehalose has received special interest because it has been found to be effective in the treatment of neurodegenerative diseases associated with peptide or protein aggregation.9-12 These diseases, including AD,13,14 Huntington’s,15 and prion diseases,16 are generally associated with the formation of amyloid deposits of protein or peptide arranged in fibrils with a characteristic cross-β structure. Despite its widespread use, * Corresponding author. Phone: +86 22 27404981. Fax: +86 22 27406590. E-mail:
[email protected].
however, a detailed molecular picture of how trehalose inhibits the protein or peptide aggregation remains unknown. Investigating the molecular mechanism of the inhibition effects of trehalose on Aβ aggregation presents a major challenge, as it may lead to novel approaches in anti-AD drug discovery. However, it seems unlikely that experimental approaches can provide the molecular details of how trehalose inhibits peptide or protein aggregation, so we herein employ all-atom molecular dynamics (MD) simulation to address this issue. MD simulation can be used to supplement experiment and fill in some of the gaps in our knowledge about a molecular picture of the dynamics of the early events in the route to amyloid oligomer or fibril, which are often inaccessible from most of the current sophisticated experimental approaches.17,18 Up to now, MD simulations have been used to study the oligomerization of Aβ16-22,19,20 to research the role of the stability of a monomeric intermediate in control of amyloid formation,21 to explore the conformational transition of Aβ40,22 to investigate the role of electrostatic interactions in full-length Aβ oligomer formation,23 and to probe the dual binding modes of Congo red to the amyloid protofibril surface.24 The aim of the present work is to understand how trehalose inhibits the seed formation and elongation at the molecular level of the Aβ16-22 fragment (Ace-Lys-Leu-Val-Phe-Phe-Ala-GluNH2). The choice of Aβ16-22 is based on its medical significance and on past experimental and simulation studies.25,26 Aβ16-22, the shortest fragment from the full-length Aβ, can form antiparallel or parallel β-sheet-rich fibrils.27 Many experimental and theoretical studies on Aβ16-22 have established that an antiparallel alignment of the peptides is more stable.28,29 It is significant to note that its small size and rapid kinetics make
10.1021/jp905580j CCC: $40.75 2009 American Chemical Society Published on Web 07/21/2009
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Figure 1. Initial structures of Aβ16-22 peptides used for MD simulations. (a) Three peptides with random coil conformations and orientations. (b) One antiparallel three-strand β-sheet extracted from the MD trajectory of the trimer formation and one monomer with random coil conformation and orientation. (c) Two antiparallel three-strand β-sheets extracted from the MD trajectory of the trimer formation. The backbone and side chains of the peptides are shown by a red NewCartoon and thin sticks, respectively. Atoms are colored red for oxygen, blue for nitrogen, white for hydrogen, and green for carbon. The snapshots are plotted with the visual molecular dynamics (VMD) software (http://www.ks.uiuc.edu/Research/ vmd/).
TABLE 1: Summary of Simulation Systems
its computational study attractive.30 In addition, biochemical mutational studies indicated that this region is critical for the full-length Aβ assembly.31 That is, the assembly property of the short peptide can represent that of the full-length Aβ. Furthermore, in order to examine the generality of the inhibition mechanism of trehalose and the consistency with experiments, we have also simulated a single Aβ40 peptide, a key pathogenic component in AD. Our simulation data reveal that the preferential exclusion effect of trehalose leads to its inhibition effects. Furthermore, analyses of the conformational transition, contributions of hydrophobic interactions, and hydrogen bonds (Hbonds) have unveiled a molecular mechanism for the inhibition effect of the disaccharide on the early aggregation of Aβ. 2. Methods Simulation Systems. Initial coordinates for the Aβ16-22 monomer used in the simulations were extracted from mode 1 of the Aβ10-35 peptide available in the Protein Data Bank (ID code 1HZ3).32 The N and C termini of Aβ16-22 are capped, respectively, by neutral acetyl and amide groups, and the simulated sequence is Ace-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2. For the Aβ40 monomer, the initial coordinates were taken from mode 2 of PDB entry 1BA4.33 The initial structure of trehalose was obtained from the GlycoSciences Database (http://www. glycosciences.de/). Force field parameters and the topology for the trehalose molecule were generated using the PRODRG2 server (http://davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg_beta).34 Four types of systems were simulated. (1) Three Aβ16-22 monomers with random coil conformations and orientations (Figure 1a). The peptides were separated from each other by at least 3.4 nm. (2) One Aβ16-22 monomer and one preformed antiparallel Aβ16-22 trimer (Figure 1b). The preformed antiparallel Aβ16-22 trimer was extracted from the MD trajectory of the trimer formation in water with the nematic parameter P2 over 0.9 (see below for the definition of P2). The structure was further visualized and validated using the Visual Molecular Dynamics (VMD) software.35 The system was built by putting the preformed ordered antiparallel trimer and a monomer with random conformation and orientation into the simulation box with a separation distance of at least 3.4 nm. (3) Two preformed antiparallel Aβ16-22 trimers (Figure 1c). It was created by adding two preformed ordered trimers with a separation distance of at least 3.8 nm. (4) One Aβ40 monomer. Table 1 summarizes the important data for the simulation systems.
trehalose no. of trehalose no. of water simulation system concn (mol/L) molecules molecules lengtha (ns) 1b
2b 3b 4b
0 0.01 0.032 0.065 0 0.032 0.065 0 0.065 0.13 0 0.06 0.18
0 3 10 20 0 10 20 0 20 40 0 18 54
16985 16945 16819 16657 16944 16783 16602 16843 16461 16154 16915 16638 16067
300(2) 300(2) 300(2) 300(2) 300(2) 300(3) 300(3) 300(2) 300(2) 300(2) 300(2) 300(2) 300(2)
a The number of repetitive simulation runs is given in parentheses. b 1, three Aβ16-22 monomers; 2, one Aβ16-22 monomer + one ordered Aβ16-22 trimer; 3, two ordered Aβ16-22 trimers; 4, one Aβ40 monomer.
The peptides were first placed in a cubic box with periodic boundary conditions. The size of the cubic box throughout the simulations was 8 nm with negligible volume fluctuations, where the distance between the peptide and the box edges was chosen to be about 1.8 nm. This allows us to rule out any unwanted effects,36 which may arise from the applied periodic boundary conditions. Then, trehalose molecules were located and oriented randomly around the peptides. Finally, water molecules nonoverlapping with either the peptides or trehalose molecules were randomly added into the simulation box. Initial configurations were minimized in three steps, first keeping the peptides and trehalose fixed, then keeping only the peptides fixed, and finally keeping all of the molecules free. After 1000 steps of energy minimization, the system was equilibrated for 100 ps at constant pressure (1 atm) and temperature (300 K) using the Berendsen coupling procedure. The system was then further equilibrated for 100 ps at the temperature and at a constant volume. The convergence of potential energy indicates equilibrium (data not shown). To make certain that the effect of trehalose on the peptide aggregation at different trehalose concentrations is the intrinsic character of trehalose rather than a stochastic output of simulations, two or three MD simulations of 300 ns were conducted for each system under different initial conditions by assigning different initial velocities on each atom of the simulation systems.
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MD Simulations. All MD simulations were carried out using the GROMACS 3.3 package37 together with the GROMOS96 force field.38 The simple point charge (SPC) model was used to describe water.39 A 2 fs time step was used to integrate the equations of motion with the Verlet leapfrog algorithm.40 The long-range electrostatic interactions were treated with the particle mesh Ewald method.41 A nonbond pair list cutoff of 0.9 nm was used, and the pair list was updated every four steps. The LINCS algorithm was used to constrain all bond lengths.42 Temperature (300 K) and pressure (1 atm) were controlled by the Berendsen thermostat and barostat with coupling constants of 0.1 and 1.0 ps, respectively.43 For all simulations, the atomic coordinates were saved every 0.5 ps for analysis. MD simulations were run on a 64-CPU Dawning A620r-F server (Dawning, Tianjin, China). Analyses. The simulation trajectories were analyzed using several auxiliary programs provided with the GROMACS 3.3 package. The programs include g_dist for calculating the distance between the centers of mass of two groups of atoms as a function of time and g_hbond for the H-bond interactions between hydrogen donors and acceptors. H-bonds are considered to be intact if the donor-to-acceptor distance is less than 0.35 nm and the donor-hydrogen-acceptor angle is within 30° of linearity. The contact maps were generated using the g_mdmat program provided with the GROMACS 3.3 package. In these calculations, two residues are considered in contact if the minimum distance between any heavy atoms of each residue is lower than 0.45 nm.44 The nematic order parameter P2 was used to characterize the ui oligomeric state of Aβ16-22.45 In terms of the unit vector b linking N and C termini for the ith peptide, P2 is given by i N |b r NC | 0 1 P2 ) P N i)1 Li 2
∑
(1)
N
P02 )
∑
3 1 1 d )2 (u b ·b N i)1 2 i 2
(2)
i is the end-to-end where N is the number of peptides and b rNC vector that connects two CR atoms from the termini of the ith monomer. The end-to-end distance in the fully stretched state is computed by Li ) (Ni - 1)a, where Ni is the number of amino acids in the ith monomer and a (≈0.4 nm) is the distance between two adjacent CR atoms. b d, defining the preferred direction of alignment, is the eigenvector associated with the largest eigenvalue of the ordering matrix QRβ46
N
QRβ )
∑(
3 1 1 u - δRβ , b u b N i)1 2 iR iβ 2
)
R,β ) x,y,z
(3)
where δRβ is the Kronecker delta function. The time-averaged normalized ratio of water oxygen atoms, gNOW, was used to interpret the local distribution of trehalose around the backbone of the target peptide.47 gNOW is computed by
gNOW )
nOW(NOW + NOS) (nOW + nOS)NOW
(4)
Figure 2. (a) Time dependence of P2 in the absence (black) and presence of 0.01 mol/L (green), 0.032 mol/L (blue), and 0.065 mol/L (red) trehalose. The lower panel shows two snapshots of the peptides extracted from the MD trajectories corresponding to the representative structures with P2 values of 0.91 (b) and 0.05 (c). The illustrations of the snapshots are the same as those described in the caption to Figure 1.
where nOW and nOS are the local numbers of water oxygen atoms and trehalose hydroxyl group oxygen atoms, respectively, located at a minimum distance from the backbone of Aβ. NOW and NOS denote the total numbers of water oxygen atoms and trehalose hydroxyl oxygen atoms, respectively, in the simulation box. Simulation trajectories were visualized, and all of the snapshots in this article were prepared using the VMD software. The simulation data plotted in the figures are averaged over two or three simulation trajectories, except those for secondary structures. Secondary structure analyses were carried out employing the dictionary secondary structure of proteins (DSSP) method.48 The secondary structures are calculated by only one trajectory, and the repeated trajectory showed similar results. 3. Results and Discussion 3.1. Inhibition Effect on Aβ16-22 Oligomer Nucleation and Elongation. Aβ16-22 Trimer Nucleation. The formation of amyloid oligomer or fibril is a dynamic process and known to occur through three basic steps: nucleation, monomer addition, and oligomer connection.49 To characterize the inhibition effect of trehalose on the early step of Aβ16-22 nucleation, we probe the inhibition of trehalose on the Aβ16-22 trimer assembly process from initial random coil conformations (Figure 1a and Table 1). The nematic liquid crystalline order parameter P2 can discriminate between ordered and disordered conformations. Therefore, it is used to represent the assembly extent of three Aβ16-22 monomers. If P2 > 0.5, the peptides have a propensity to be in an antiparallel or parallel ordered state; if P2 g 0.9, all peptides are stretched and aggregated into an antiparallel or parallel ordered state.50 The values of P2 are displayed as a function of simulation time in Figure 2a. It can be seen that P2 in water increases rapidly at the initial stage and reaches about 0.91 in 36 ns. It indicates that the three Aβ16-22 monomers have assembled into an ordered antiparallel trimer in which the backbone of each of the monomers is in a plane (Figure 2b). In the subsequent simulations, P2 in water fluctuates greatly because of the finite size effects; that is, the ordered oligomers are only marginally stable. The phenomena are also observed by Nguyen et al.50 In the cases of trehalose solutions, the values of P2 decrease with increasing concentration of trehalose, and
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Figure 3. Time evolution of the distance between the added monomer and the ordered trimer (a) together with a histogram of distance value distribution (b). The dashed line indicates the distance of the interpeptide separation within a β-sheet layer. The two boxes in the inset of Figure 3b show the two representative structures from the most populated clusters shown in red NewCartoon.
they are smaller than that in water, except the one in 0.01 mol/L trehalose solution, which has the same tendency as that in water. At high trehalose concentrations (g0.032 mol/L), the values of P2 are always lower than 0.5 throughout the whole simulations. It means that the peptides cannot aggregate into ordered oligomeric forms in high trehalose solutions. In addition, the values of P2 in 0.065 mol/L trehalose are even lower than that in 0.032 mol/L trehalose. For example, P2 is only about 0.05 at the end of the simulation. The three monomers are shown in disordered states (Figure 2c), and they have no propensity to aggregate into an ordered trimer. The results indicate that ordered trimer nucleus formation is suppressed by trehalose molecules at high concentrations (g0.032 mol/L). Aggregation of a Free Aβ16-22 Monomer to a Preformed Ordered Trimer. Previous studies have shown that preformed amyloid oligomer or nucleus with β-sheet-rich structures possesses seeding capacity and is elongated by adding individual monomers to the end of the nucleus.51 In order to study the inhibition effect of trehalose on the elongation of the Aβ16-22 nucleus, an assembled antiparallel ordered trimer is supplied as an amyloid nucleus (Figure 1b and Table 1). As known previously, a free monomeric peptide deposits itself to one of the lateral peptides of the nucleus during the elongation of Aβ.52 To investigate whether trehalose can prevent a nascent monomer from aggregating to the preformed oligomer, we calculated the distance between the main chains of the monomer and the lateral peptide, which is on the side of the nucleus and interacts directly with the added monomer. Figure 3 shows the time evolution of the distance between the added monomer and the peptide of the trimer together with a histogram of the corresponding distance values. Within 10 ns after interaction with the trimer, the distance between the added monomer and the peptide of the trimer in water decreases dramatically from 3.4 to 1.2 nm. The distance continues to decrease slowly over a period of tens of ns and fluctuates around 0.5 nm in the subsequent simulation. It clearly shows that the distance distribution in water is sharply peaked around 0.5 nm, as shown in Figure 3b (indicated by a dashed line). It corresponds to the structures of an ordered antiparallel tetramer (see the snapshot indicated by the arrow in the inset of Figure 3b) with interpeptide separation within a β-sheet layer of 0.5 nm, which is in good agreement with the distance 0.47 nm determined by X-ray powder diffraction.26 Similar phenomena are observed in 0.032 mol/L trehalose solution except that the distance between the added monomer and the peptide of the trimer increases dramatically and then decreases quickly in the initial simulation. It is considered due to the interactions between trehalose and peptides because a similar phenomenon is also observed in 0.065
mol/L trehalose solution. However, the distance between the added monomer and the peptide of the trimer in 0.065 mol/L trehalose solution is always much larger than that in water and in 0.032 mol/L trehalose solution (Figure 3a). It means that the nascent peptide cannot assemble into an ordered trimer in 0.065 mol/L trehalose solution. There is a significant difference in the distance distributions in 0.065 mol/L trehalose, being smaller on average and broader in fluctuation, than the distributions for the structure in lower trehalose concentrations (e0.032 mol/ L). The snapshot of the representative disordered structure is shown in the inset of Figure 3b. In addition, the inhibition effect of trehalose on the elongation is also reflected in the dynamics of P2 (Figure S1 in the Supporting Information). Aggregation of Two Preformed Aβ16-22 Trimers. The nuclei or seeds may link together and form a larger oligomer or protofibril when many ordered nuclei occur. Therefore, MD simulations are performed to explore the inhibition effect of trehalose on the aggregation of two preformed Aβ16-22 trimers at different trehalose concentrations (Figure 1c and Table 1). This is also illustrated by the reduction of the distance between the two trimers (Figure S2 in the Supporting Information). In water and 0.065 mol/L trehalose, there is a sharp peak centered at about 1.0 nm corresponding to the ordered hexamer (indicated by a dashed line). It indicates that the two Aβ16-22 trimers formed into double layers, as shown by the snapshot in the inset of Figure S2b in the Supporting Information. It is also consistent with the interlayer spacing between two β-sheet layers of 0.99 nm determined by X-ray powder diffraction.26 By increasing the trehalose concentration to 0.13 mol/L, however, the peak shifts to a larger distance (1.9 nm, Figure S2b in the Supporting Information), indicating that the two trimers cannot form an ordered oligomer at the high trehalose concentration. The snapshot of the representative disordered structure is shown in the inset of Figure S2b in the Supporting Information. The results depicted above suggest that trehalose inhibits aggregation of Aβ16-22 in a dose-dependent manner. More trehalose molecules are needed to inhibit Aβ16-22 aggregation as the amount of Aβ16-22 increases. Such a behavior agrees qualitatively with the experimental finding that the aggregation of full-length Aβ was inhibited by trehalose in a dose-dependent manner.14 3.2. Preferential Exclusion of Trehalose. Preferential exclusion is commonly considered as the reason for the proteinstabilizing effect of protecting osmolytes such as trehalose. The mechanism indicates that water molecules in the hydration shell around proteins increase because osmolyte molecules are excluded from the protein/solvent surface, thus inducing thermodynamic stabilization. The preferential hydration of Aβ16-22
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Figure 4. Time-averaged normalized ratio of water oxygen atoms, gNOW, as a function of distance from the closest backbone atoms of Aβ16-22 peptides.
in trehalose solutions can be investigated by analyzing water and trehalose distributions. To this purpose, we characterized the relative local distribution of water and trehalose molecules from MD trajectories, according to the minimum distance of the water oxygen or of the sugar hydroxyl group oxygen from the closest peptide backbone atoms. We have computed the time-averaged normalized ratio of water oxygen atoms gNOW (eq 4) in a similar way to Lerbret et al.47 Class intervals are chosen at 1 Å thickness. From eq 4, we know that the ratio (gNOW) is greater than 1 in the proximity of the backbone of Aβ16-22 if trehalose is preferentially excluded from the backbone of the peptide (in other words, the peptide is preferentially hydrated). Conversely, the ratio is lower than 1 if trehalose preferentially interacts with the backbone. Data are plotted only up to 10 Å from the closest backbone atoms of the peptides; for distances larger than 10 Å, the distributions trail off due to irregular edge effects in the cubic box, which has no physical meaning. Figure 4 shows gNOW at different trehalose concentrations. It is obvious that, within a distance of about 3 Å from the closest backbone atoms of Aβ16-22, the peptides become more and more preferentially hydrated when increasing trehalose concentration (see the figure inset). For example, the ratio is 1.003 in 0.01 mol/L trehalose, only a little larger than unity, but it increases to 1.01 in 0.065 mol/L trehalose. By contrast to the preferential hydration on the peptides, at a distance of 4-5 Å, there is a significant water depletion that results from the presence of many trehalose molecules (Figure 4). It can thus be concluded that there is a thin hydration shell on the surface of the peptides in trehalose solutions. That is, trehalose molecules do not expel water molecules on the surface of the peptides; instead, water molecules are enriched as the trehalose concentration increases. Most of the trehalose molecules cluster with each other, form a continuous space-filling network, and surround the peptides at a distance of 4-5 Å. The phenomena are in good agreement with the simulation results of Lins et al.53 These observations are also consistent with the experimental observation that trehalose in aqueous solutions is totally excluded from the first hydration shell of protein.54 The preferential exclusion effect of trehalose is considered to be the origin of its inhibition effect on the nucleation and elongation of Aβ16-22 oligomers.
Liu et al.
Figure 5. Secondary structures as a function of simulation time for the three monomers calculated by DSSP in the absence (a) and presence of 0.01 mol/L (b), 0.032 mol/L (c), and 0.065 mol/L (d) trehalose. The vertical coordinate represents the residue number of the three Aβ16-22 peptides by considering that the three Aβ16-22 peptides are connected. The acetyl group in the N terminal of one peptide and the amide group in the C terminal of the next peptide are regarded as one residue by the program, so there are 23 residues in total for the three Aβ16-22 peptides. The secondary structure is color-coded. The structures are analyzed every 0.6 ns.
3.3. Preferential Exclusion Inhibits the Conformational Transition of Aβ16-22 Monomer. Previous experimental and theoretical studies indicate that β-sheet formation is a crucial step in Aβ amyloidogenesis.55 To probe the conformational transition of Aβ16-22 monomers in different trehalose solutions during the nucleation, the secondary structure is determined by DSSP (Figure 5). In water and 0.01 mol/L trehalose, the conformations of the monomers begin to transform from their initial random coil to β-sheet through turn, bend, or helix conformations before the nucleation of Aβ16-22 (Figure 5a and b). The finding indicates that turn, bend, or helix conformations are possible intermediates for the Aβ16-22 aggregation process. However, the conformations of the monomers mainly adopt a turn, coil, bend, or helix in high-concentration trehalose solutions (g0.032 mol/L) (Figure 5c and d), which are unfavorable for the interpeptide interactions that could lead to aggregation. It is noted that a β-sheet is not observed during the whole 300 ns MD simulations in the high-concentration trehalose solutions. Transformations of the secondary structures of the added monomer and the ordered trimer are displayed in Figure S3 in the Supporting Information. It is observed that, in water and in 0.032 mol/L trehalose solution, the initial random coil of the added monomer transforms into β-sheet-rich structure from bend and turn. In 0.065 mol/L trehalose solution, however, the added peptide is always kept in coil or bend structure during the whole 300 ns simulations. In addition, it is noted that the conformation of the peptides in the preformed antiparallel trimer stays unchanged in the β-sheet. In other words, trehalose cannot reverse the structure of the antiparallel ordered trimer. Similar behavior is found in the simulations of two antiparallel ordered trimers; that is, the trimers keep their β-sheet conformations during the whole 300 ns simulations (data not shown). Therefore, the MD simulations suggest that, at low trehalose concentrations, the Aβ16-22 monomers convert from their initial random coil to β-sheet-rich structures and aggregate into an ordered oligomer. At high trehalose concentrations, however,
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Figure 6. Probability distributions of the number of interpeptide contacts between the side chains of central hydrophobic cluster (CHC) residues of Aβ16-22 monomers at different trehalose concentrations.
this conformational transition is inhibited and the Aβ16-22 monomers keep at the intermediate states, such as turn, bend, or helix. Thus, the preferential exclusion of trehalose prevents the Aβ16-22 monomers from forming a β-sheet-rich structure that is prone to forming peptide oligomers. 3.4. Preferential Exclusion Weakens Hydrophobic Interactions. It has been demonstrated that the central hydrophobic cluster (CHC, Leu17-Ala21) is essential for the nucleation and initial deposition of an added monomer on an oligomeric surface.56 The interpeptide hydrophobic interactions may be dominated by favorable contacts between the side chains of CHC residues of Aβ16-22, so the number of interpeptide contacts between the side chains of CHC residues can represent the intensity of hydrophobic interactions between Aβ16-22 monomers. In order to probe the hydrophobic interactions that drive and stabilize the ordered oligomer, we calculated the number of interpeptide contacts. Two atoms are considered to be in contact if any atom of one residue is e6 Å from that of the other residue.50 The distributions of the number of interpeptide contacts are shown in Figure 6. It is observed that the values in water and in 0.01 mol/L trehalose have a broad basin centered at about 50. At higher trehalose concentrations (0.032 and 0.065 mol/L), however, there is a smaller amount of contacts (
