Hydrophobic Interface on Amyloid-Î² Peptides

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Effects of a Hydrophilic/Hydrophobic Interface on Amyloid-# Peptides Studied by Molecular Dynamics Simulations and NMR Experiments Satoru G. Itoh, Maho Yagi-Utsumi, Koichi Kato, and Hisashi Okumura J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b11609 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 17, 2018

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The Journal of Physical Chemistry

Eects of a Hydrophilic/Hydrophobic Interface on Amyloid-β Peptides Studied by Molecular Dynamics Simulations and NMR Experiments Satoru G. Itoh,

†,‡,¶

†,‡,§,∥

Maho Yagi-Utsumi,

Okumura

†

Koichi Kato,

†,‡,§,∥

and Hisashi

∗,†,‡,¶

Institute for Molecular Science (IMS), National Institutes of Natural Sciences, Okazaki, Aichi 444-8585, Japan

‡

Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Okazaki Aichi 444-8787, Japan

¶

Department of Structural Molecular Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8585, Japan

§

Department of Functional Molecular Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8787, Japan

∥

Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi 465-8603, Japan

E-mail: [email protected] Phone: +81 (0)564 55 7277. Fax: +81 (0)564 55 7025

1

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Abstract Oligomer formation of amyloid-β peptides (Aβ ) is accelerated at a hydrophilic/hydrophobic interface. However, details of the acceleration mechanism have not been elucidated. In order to understand the eects of the interface on oligomerization at the atomic level, we performed molecular dynamics simulations for an Aβ 40 monomer in the presence and absence of the hydrophilic/hydrophobic interface. Nuclear magnetic resonance experiments of Aβ 40 peptides with gangliosidic micelles were also carried out. In the simulations and experiments, the hydrophobic residues of Aβ 40 bound to the interface stably. Moreover, we found that Aβ 40 formed a hairpin structure at the interface more readily than in bulk water. From these results, we discussed the acceleration mechanism of the oligomer formation at the interface.

INTRODUCTION Amyloid-β peptides (Aβ ) form soluble oligomers and insoluble amyloid brils spontaneously. These oligomers and amyloid brils are associated with Alzheimer's disease. posits of Aβ peptides are observed in the brain of Alzheimer's patients, and the brils are toxic to neuron cells.

58

3,4

1,2

In fact, de-

and the oligomers

However, the mechanisms of the oligomer and

bril formation have yet to elucidate, even though an understanding of these mechanisms is essential to remedy Alzheimer's disease. There have been many experimental and computational studies for Aβ including its fragments.

926

Various structures of the full-length Aβ peptides and the fragments were

reported by experiments.

911,21,2729

As for a full-length Aβ , Aβ 40, which consists of 40

amino-acid residues, it was found that two intermolecular

β -sheet

structures were formed

in the amyloid bril by the solid-state nuclear magnetic resonance (NMR) experiments. The two intermolecular (β2).

Most of the

β1

β -sheets and

β2

11

are composed of residues 1022 (β1) and residues 3040

regions consist of hydrophobic residues.

models of Aβ brils were also reported, in which the

2

β -sheet


Other structural

regions are dierent.

For

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example, Lu and co-workers reported the model of Aβ 40 that had three intermolecular sheet structures.

27

These sheet structures consisted of residues 1213, residues 1819, and

residues 3536, respectively.

Xiao and co-workers reported the model of Aβ 42 which is

composed of 42 amino-acid residues. three intermolecular

28

In this model, resides 1218, 2433 and 3640 formed

β -sheet structures.

formed three intermolecular 4041.

β-

β -sheet

In the other model reported by Gremer et al., Aβ 42

structures that consists of residues 322, 2835, and

29

Several experiments recently reported that the oligomer and bril formation were accelerated at a hydrophilic/hydrophobic interface such as a water/air interface and an interface between a sugar-head group of a glycolipid and a hydrocarbon chain.

3034

For example, Aβ

bound to GM1 exhibited an extremely high potential to accelerate Aβ assembly.

30,31

GM1

is a glycosphingolipid and abundant in neuronal membranes. The monomer conformation of Aβ 40 was determined by NMR experiments when it bound to carbohydrate-lipid interfaces of GM1 and lyso-GM1 micelles. of Aβ 40, the

32,35

In the binding conformation, two hydrophobic regions

β1 and β2 regions, bound to the micelles.

Moreover, these hydrophobic regions

formed the helical structures although these regions have intermolecular in the amyloid bril. Furthermore, it has been demonstrated that

β -like

β -sheet

structures

conformation was

induced at the C-terminus of Aβ 40 upon binding to the GM1 micelles. By contrast, the lysoGM1 micelle could not induce such

β -like

structure formation although topological modes

of interaction of Aβ 40 with micelles were almost identical between the GM1 and lyso-GM1 micelles under their excess conditions.

36

The diameters of the GM1 and lyso-GM1 micelles

have been estimated as 12 nm and 8 nm, respectively, by dynamic light scattering. Thus, the sizes and curvatures of the micelles are supposed to be determining factors for the number of Aβ molecules that can be accommodated on their hydrophilic/hydrophobic interface and the occurrence of Aβ Aβ interactions coupled with

β -structure

formation.

As for the computational studies for Aβ , most of the studies employed systems in bulk water.

12,1416,18,19,22,23,3745

Several studies reported the monomer conformation of Aβ 40 at the

3



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hydrophilic/hydrophobic interface by performing molecular dynamics (MD) simulations.

46,47

Miyashita et al. employed an implicit model for both of the hydrophilic and hydrophobic regions.

46

They showed the

β1

and

hydrophilic/hydrophobic interface.

β2

regions of Aβ 40 were located in the vicinity of the

Vahed et al.

performed MD simulations of Aβ with

explicit water molecules and a GM1-containing membrane.

47

They showed that H13 and

H14 played an important role in binding to the sugar-head group of GM1. To understand eects of the hydrophilic/hydrophobic interface, it is essential to see the dierence between Aβ at the interface and that in the bulk water.

In this paper,

therefore, we performed MD simulations for Aβ 40 in the presence and absence of the hydrophilic/hydrophobic interface. We employed a water/vapor interface as the hydrophilic/hydrophobic interface to mimic the water/air interface. Furthermore, NMR experiments with gangliosidic micelles were carried out to investigate Aβ 40 conformations at the interface and to compare the results with the simulation results. Since the GM1 micelles are larger than the lyso-GM1 micelles and have a atter interface, they are a better model as the water/air interface or a cell surface.

MATERIALS AND METHODS

Molecular dynamics simulations To investigate monomer structures of Aβ 40 at a hydrophilic/hydrophobic interface, we performed MD simulations for an Aβ 40 molecule. The amino-acid sequence of Aβ 40 is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV. The N-terminus and C-terminus did not have caps such as Ace- and Nme-groups. The Aβ 40 molecule was put in a cubic unit cell with explicit water molecules. The hydrophilic/hydrophobic (water/vacuum) interface was prepared by removing water molecules located in the lower half of the cubic unit cell (see Fig. 1). We employed three dierent initial positions of Aβ 40 to remove their dependencies. The three positions were at the interface, in water solvent, and in vacuum, as shown in

4


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Fig. 1. The initial structure of Aβ 40 was an extended structure for all positions. The side length of the cubic unit cell was 108.0 Å and periodic boundary conditions were utilized. The AMBER parm99SB force eld

48

and the TIP3P rigid-body model

49

were employed for

the Aβ 40 molecule and for the water molecules, respectively. The SHAKE algorithm was utilized to constrain bond lengths with the hydrogen atoms of Aβ 40 and to x the OH and HH distances of the water molecules during the simulations. The cuto distance for the Lennard-Jones potential energy was 12.0 Å. The electrostatic potential energy was calculated by the particle mesh Ewald method. Nosé-Hoover thermostat.

5154

50

Temperature was controlled at 350 K by the

The multiple-time-step method

55

was employed, and the time

steps were taken to be 4.0 fs for interactions between the water molecules and 1.0 fs for other interactions. Three dierent initial velocities were employed for each Aβ 40 position. That is, we employed nine dierent initial conditions (three positions

× three velocities).

For each

initial condition, an MD simulation was performed for 230.0 ns after an equilibration run for 10.0 ns. The production run was conducted for 2.07

µs (= 230

ns

×

9) in total. We remark

that the water/vapor interface were obtained after the equilibration run because the water molecules evaporated to the vacuum region during the equilibration run. For the purposes of comparison, we also performed MD simulations of Aβ 40 without the hydrophilic/hydrophobic interface (in the bulk water). The side length of the cubic unit cell was 91.1 Å, and the initial structure of Aβ 40 was the extended structure.

Nine dierent

initial velocities were employed. For each initial condition, an MD simulation was performed for 230.0 ns after an equilibration run for 10.0 ns, again.

Experiments The expression and purication of isotopically labeled recombinant Aβ 40 and its mutant with an extra-cysteine residue at its C-terminus (Aβ 40-Cys) were performed as described previously.

36

The reaction of Aβ 40-Cys with nitroxide spin label MTSL (1-oxy-2,2,5,5-

tetramethyl-D-pyrroline-3-methyl) methanethiosul-fonate (Toronto Research Chemicals) was

5



carried out as described previously.

35

Page 6 of 33

Aβ 40-Cys spin-labeled with MTSL (Aβ 40-Cys-MTSL)

was puried by reverse-phase chromatography an octadecylsilane column (TSKgel ODS80TM, TOSOH). Lyophilized Aβ 40 and Aβ 40-Cys-MTSL was dissolved at an approximate concentration of 2 mM in 0.1 % (v/v) ammonia solution then collected and stored in aliquots

◦ at -80 C until use. Powdered lyso-GM1 and GM1 was purchased from Takara Bio Inc. and Carbosynth Ltd., respectively. These glycolipids were suspended at a concentration of 12 mM in 10 mM potassium phosphate buer (pH 7.2), and then mixed by vortexing. To observe paramagnetic relaxation enhancement (PRE) eects brought out by the Cterminal spin label,

2

H- and

15

N-labeld Aβ 40-Cys-MTSL was dissolved at a concentration

of 0.2 mM in 10 mM potassium phosphate buer (pH 7.2) containing 10 % (v/v) 2H2O in the presence of 6 mM lyso-GM1 micelles and subjected to using a JEOL EC-920 spectrometer at a GORIN application.

56

1

1

15 H- N TROSY measurement

H observation frequency of 920 MHz employing

Signal assignments for Aβ 40 in the presence of lyso-GM1 micelles

were obtained by triple-resonance NMR experiments, as reported previously.

32

The unpaired

electron of MTSL was subsequently reduced using 4 mM ascorbic acid. PRE eects were measured from the peak intensity ratio between two TROSY spectra of Aβ 40-Cys-MTSL acquired in the presence and absence of its nitroxide radical. One-dimensional carbonyl

13

C

spectra were recorded on a Bruker AVANCE III-400 spectrometer using Aβ 40 labeled with

13

C selectively at the carbonyl group of Lys, Val, Ile, Tyr, and Met. The probe temperature

◦ was set to 37 C for all measurements.

NMR spectra were processed using the software

Topspin (Bruker BioSpin Co.) and NMRPipe

57

and analyzed with the software Sparky.

58

RESULTS AND DISCUSSION The previous experiments reported that Val12Gly25, Ile31Val36, and Val39Val40 of Aβ 40 bound to lyso-GM1 micelles. and the

β2

32

The

β1

region almost consists of the residues Val12Gly25,

region includes Ile31Val36 and Val39Val40. Therefore, the

6


β1

and

β2

regions

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bound to the lyso-GM1 micelles. It was also concluded that the Aβ monomer has an upand-down shape at the hydrophilic/hydrophobic interface as shown in Fig. 2(a). To see the shape of Aβ 40 at the hydrophilic/hydrophobic interface in our simulations, we calculated the averaged z-axis values of the Cα atoms of the amino-acid residues. Here, the z axis was set to be perpendicular to the interface. The z-axis values of the interface is 0. When the value is positive (negative), the Cα atom is in the hydrophilic (hydrophobic) region. The denition of the interface is described in the supporting information. Figure 2(b) shows the averaged z-axis values.

Here, the errors were estimated by the jackknife method.

59

The number of

bins for the jackknife method was two for each MD simulation, and the total number of bins was 18 because nine MD simulations were conducted. As seen in the experiments, Aβ 40 had an up-and-down shape. The residues that bound to the GM1 micelles in the experiments existed in the vicinity of the interface also in the simulations. A typical conformation at the interface is shown in Figure 2(c). In this conformation, the

β1

and

interface. The N-terminal region and the linker region between

β1

β2

regions bound to the

and

β2

were exposed in

the water solvent. The number of hydration water molecules was counted for each residue.

Here, when

the distance between the oxygen atom of a water molecule and any atoms of the residues (except for the hydrogen atoms) was less than 5.0 Å, the water molecule was regarded as a hydration water molecule. In Fig. 3 the averaged numbers of hydration water molecules for amino-acid residues are shown. The averaged numbers of hydration water molecules were increased and decreased almost the same as the averaged z-axis values of the Cα atoms in Fig. 2(b). Although the curve in Fig. 2(b) was smooth, that in Fig. 3 was not smooth. For example residue 16 and residue 22 had the larger numbers than reside 17 and residue 21, respectively.

This is because residues 16 and 22 have electric charges and these residues

prefer to have more hydration water molecules. We calculated the probability distribution of the z-axis values of the representative Cα atom in each region to see the tendency of its position with respect to the interface. Here,

7



Page 8 of 33

Tyr10, Val18, Ser26, and Leu34 were employed as the representative residues in the Nterminal region, the

β1

region, the linker region between

β1

and

β2,

and the

β2

region,

respectively. The probability distributions of the z-axis values are presented in Fig. 4. The distributions for Tyr10 and Ser26 took a wide range of positive values.

This means the

N-terminal and linker regions uctuated in the water solvent. As for Val18 and Leu34, the distributions were narrow and took values around zero. This indicates that the

β1

and

β2

regions not only bound to the interface but also kept the binding states stably (see Movie 1). The stable binding states were observed in all MD simulations despite the initial positions. Let us consider that there are multiple Aβ 40 molecules. Most of Aβ 40 molecules are expected to gather in the vicinity of the interface because of the stable binding to the interface. Therefore, the local concentration at the interface increases. Since Aβ 40 aggregates rapidly with a high concentration, the aggregation of Aβ 40 accelerates at the hydrophilic/hydrophobic interface. This is one of possible reasons why the hydrophilic/hydrophobic interface accelerates Aβ aggregation. In order to investigate the eects of the interface on Aβ 40 structures, we calculated contact probabilities of Cα atoms from our simulations.

Figure 5(a) and 5(b) show the

contact probabilities in the presence of the interface and in the absence of the interface, respectively.

Here, when the distance between a pair of Cα atoms was less than 6.5 Å,

it was regarded as a contact.

60

Figure 5(a) can be considered as the contact probabilities

when Aβ 40 bound to the interface because Aβ 40 almost bound to the interface during the simulations as seen in Fig. 4. At the interface, the

β1 and β2 regions formed helix structures.

This is consistent with the experimental data with the lyso-GM1 micelle.

32

Not only the helix

structures but also a hairpin structure was formed by forming the contacts between the and

β2

β1

regions. As for in the bulk water (in the absence of the interface), both regions had

helix structures, as at the interface (see Fig. 5(b)). However, the probability of the hairpin structure in the bulk water was lower than that at the interface.

This dierence in the

forming ability of the hairpin structure between at the interface and in the bulk water would

8


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cause dierence in the forming ability of the oligomer. In fact, two of the authors reported that a

β -hairpin 18

fragments.

structure readily formed intermolecular

Namely, the

intermolecular

β -sheet

β -hairpin

structure.

computational works showed the formation.

β -sheet

structures with other Aβ

structure accelerated formation of an oligomer with the Not only our works but also several experimental and

β -hairpin

structure played an important role in oligomer

6163

A possible reason why the

β -hairpin structure is formed at the interface more readily than

in the bulk water is as in Fig. 6. As shown in Fig. 4, the

β1

and

β2

regions get trapped at

the interface. These regions can move only at the interface. Therefore, relative motion of the

β1 the

region to the

β1

of the

β2

region is suppressed two-dimensionally. In the bulk water, conversely,

region can move three-dimensionally.

β1

region relative to the

β2

By having various conformations (positions

region), the entropy increases in the bulk water.

At

the interface, however, increase of the entropy is suppressed because of the two-dimensional motion. Therefore, lower enthalpy is preferred in order to decrease the free-energy. Formation of hydrogen bonds between the

β1

and

β2

regions decrease the enthalpy. As a result, the

β-

hairpin structures are formed. To explain this mechanism in more detail, the time sequenced snapshots are presened in Fig. 7. Initial conformation of Aβ 40 was fully extended structure (Fig. 7(a)). In Fig. 7(b), the

β1 and β2 regions formed helix structures.

These regions bound

to the interface stably and moved only at the interface. The helix structure in the was then broken as seen in Fig. 7(c). The extended and the

β2

β -bridge

β1

and

region got close to the

β2

region,

was formed between these regions (Fig. 7(d)). The helix structure in the

region was broken although the

β -hairpin

β1

β1 region

β -bridge

kept being formed, as shown in Fig. 7(e). The

structure was nally formed as in Fig. 7(f ). Thus, hydrogen bonds between the

β2

regions were fromed step by step, changing the helix structures to the extended

structures. We carried out NMR experiments with GM1 micelles to investigate the structure of Aβ 40 at the interface. A series of 1D-NMR spectra for Aβ 40 are shown in the supporting informa-

9



Page 10 of 33

tion (Fig. S2). The chemical shift changes were observed for several residues upon addition of GM1 micelles. It is considered that the conformations of these residues were altered upon binding of Aβ 40 to the GM1 micelle. Figure 8(a) summarizes these experimental results. Downeld sifts were observed for the peaks originating from K16, V24, I31, I32, M35, and V36, while the chemical shifts of F4, Y10, V12, V18, F19, F20, and K28 were unchanged upon addition of GM1 micelles. This means that I31-V36 (the stable helix structure than the the simulations, both of the

β1

β1

β2

region) formed a more

region. As for the contact map in Fig 5(a) obtained by

and

β2

regions had the peaks corresponding to the helix

structures. To see the stability of these helix structures, we calculated the helix formation probability for each residue. Here, the DSSP criteria were employed to dene the helix structure. We regarded

α-helix, 310 -helix, and π -helix structures as the helix structures.

formation probability is shown in Fig 8(b). The formation although the probability of the residues. Therefore, the

β2

β2 region had a high probability of the helix

region was not much dierent from the other

region formed the stable helix structure at the interface. On the

other hand, the helix structure of the stability of the

β1

β1

region was not stable. To compare the structural

β1 and β2 regions more quantitatively, root mean square uctuation (RMSF)

was calcualted for each residue, as shown in Fig. 9. RMSF of the that of the

The helix

β2 regions.

Because the

β1

region was larger than

β2 region formed the stable helix structure, uctuation of

the residues in this region was suppressed. We remark that RMSF in the vicinity of residue 5 was also small. This is because residues in the vicinity of residue 5 formed both of the helix and hairpin structures as shown in Figs. 5(a) and 8(b). These results are coincident with the NMR experimental results in Fig 8(a). Indeed, NMR data previously indicated that the

β1

region of Aβ 40 bound to smaller lyso-GM1 micelle formed a stable

In contrast, the

β1

β1

structure.

32

region (H13F20) did not exhibit observable peak in the spectra due to

severe broadening in the presence of larger GM1 micelle, structure in the

α-helix

32

implying instability of the helical

region, which presumably interchanges between helical and disordered

structures on the atter surface of larger micelle as reected in our simulation results at the

10


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water/vapor interface. In our simulations, Aβ 40 had various conformations at the interface. To classify these conformations, the principal component analysis (PCA) were utilized. The details of PCA are given in the supporting information.

Figure 10(a) shows the free-energy landscape in

the presence of the interface with respect to the rst and second principal components. Five local-minimum states (state AE) were observed.

The representative structures of these

states are presented in Fig. 11(a). Here, the N-terminal region, D1E11, was omitted because this region was exible and had various conformations. Each representative structure is as follows: (State A) The

β1 and β2 regions were close to each other.

The

β1 region and the β2

region had the extended structure and the helix structure, respectively. The

β1

formed between these regions. (State B) The

and

β2

β -bridge

was

regions were closer to each other

than the structure in state A. Both regions had the extended structures, and the stable

β -hairpin

region formed the

β2

β1

structure was formed. (State C) The

β -bridge in this region.

The

region had the helix structure. The

β2

β -bridge was also formed between the β1 and

regions only in the vicinity of the linker region. Most of the residues in the two regions

had the open form. The

β2

(State D) The

β1

region had the

region formed the helix structure.

β -hairpin

There was no

(State E) Aβ 40 had the random-coil structure.

structure in this region.

β -bridge

between two regions.

In Table 1, the free energy of each state

is presented. The fractional population and the range of each state are also shown in the table. State B was the global minimum state, and the second lowest-energy local minimum state was state A. As seen in Fig. 5(a), the

β1

and

β2

regions tended to not only have the

helix structures but also form the hairpin structures as the structures in states A and B. In Fig. 10(b), the free-energy landscape in the bulk water is shown. The axes are the rst and second principal component axes determined from the simulations in the absence of the

′ ′ interface. Five local-minimum states (state A E ) were observed, again. In Figure 11(b), ′ the representative structures are as follows: (State A ) The ′ secondary structures. (State B ) Both

β1

and

11

β2

β1

and

β2

regions did not form

regions had the helix structures. (State



′ C ) The helix structure was formed in the ′ structure. (State D ) The helix structure.

β -bridge

′ (State E ) The

region. The

was formed in the

β -sheet

linker region had the helix structure. states are shown in Table 1.

β2

β2

Page 12 of 33

β1

region had the random-coil

region. The

was formed between the

β1

β1

region formed the

and

β2

regions.

The

The free energy and fractional population of these

′ The global minimum state was state D .

State E in which

Aβ 40 formed the hairpin structure was the forth lowest-energy local minimum state. The population of hairpin structure at the interface such as states A and B was three times larger than that in the bulk water. These results imply that Aβ 40 in the bulk water does not prefer to have the hairpin structure in which the

β1

and

β2

regions are close to each other.

In order to investigate the conformation of Aβ 40 bound to the GM1 cluster, we used a site-specic spin label of Aβ 40 as a source of distance information.

Intensity ratio of

Aβ 40-C-Cys-MTSL on the lyso-GM1 micelle before and after radical quenching is shown in Fig. 12(a). Upon addition of Aβ 40-C-Cys-MTSL, the peak intensities located in the vicinity of the linker region or the N-terminal region exhibited signicant peak broadening due to PRE eects. These data indicated that the C-terminus of Aβ 40 tended to be close to the linker region or the N-terminal region. For comparison, we calculated the probability of the distance between the C-terminal O atom and N atom of each residue longer than 10 Å. The results is shown in Fig. 12(b).

As the spin labeling experiment, C-terminus tended to be

close to the linker region or the N-terminal region. The structures in state A and state E in Fig. 11 had small distances between the C-terminus and the N-terminal region and between the C-terminus and the linker region, respectively.

Therefore, it is considered that Aβ 40

bound to the GM1 cluster forms the hairpin structure and the globular structure. In our simulations, the C-terminus was also present near residue K16 as the structure in state B in Fig. 11. However, intensity ratio of K16 did not decrease as those of the linker region or the N-terminal region. This might be because MTSL has a distance from the C-terminal O atom. Note that though the result with the cuto distance of 10 Å is shown, the results with other cuto distances are presented in the supporting information. The results with other

12


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cuto distances had the same tendency.

CONCLUSIONS We performed the MD simulations for Aβ 40 monomer in the presence and the absence of the hydrophilic/hydrophobic interface, to study the eects of the interface on aggregation and oligomer formation.

Moreover, the NMR experiments were conducted for Aβ 40 with

the gangliosidic micelles. In our simulations, the

β1 and β2 regions bound to the interface as

reported by the previous experiments. This binding conformation was kept stably because the

β1

and

β2

regions located only in the vicinity of the interface. If there were multiple

Aβ 40 molecules, therefore, the local concentration at the interface would increase.

It is

considered that the increase of local concentration accelerates the aggregation of Aβ 40 at the interface. As seen in the previous experimental data with the lyso-GM1 micelle,

β2

regions formed helix structures at the interface in our simulations.

the experiment with the GM1 micelles, we showed the structure, although the

β2

β1

32

the

β1

and

By carrying out

region did not have the stable

region formed the stable helix structure.

These results were

coincident with our simulation results. It was found that not only the helix structures but also the hairpin structure was formed by getting the

β1

and

β2

regions closer each other.

Such a hairpin structure was hardly formed in the bulk water. A possible reason why the

β -hairpin structure is formed at the interface is as follows:

Because the

β1 and β2 regions get

trapped at the interface, the entropy at the interface becomes smaller than that in the bulk water. To decrease the free energy, it is required to decrease the enthalpy. Lower enthalpy is realized by forming hydrogen bonds between the

β1

and

β2

regions. As a result, the

β-

hairpin structures are formed. Several experimental and computational works reported the

β -hairpin

structure played an important role in oligomer formation.

18,6163

structure can accelerate formation of an oligomer with the intermolecular

13


The

β -hairpin

β -sheet

structure.


Page 14 of 33

Since the hairpin structure is readily formed at the interface, the oligomer formed easier at the interface than in the bulk water. Our simulations results were coincident with our NMR experimental results although the interface in the simulations was dier from the GM1 micelle.

Therefore, we believe that

our results at the atomic level give useful information on the oligomer formation of Aβ at the interface. Here, we employed only Aβ 40 monomer in our simulations. To discuss the oligomerization of Aβ 40 more accurately, it is required to employ multiple Aβ 40 molecules. In the future, we will perform MD simulations for multiple Aβ 40 molecules.

Acknowledgement The computations were performed on computers at the Research Center for Computational Science, Okazaki Research Facilities, National Institutes of Natural Sciences. NMR experiments were supported by the Nanotechnology Platform Program (Molecule and Material Synthesis) of MEXT. This work was supported by MEXT/JSPS KAKENHI (JP24740296, JP17K15441, JP2510200, JP25102008, and JP26102550) and the Okazaki Orion Project of National Institutes of Natural Sciences.

Supporting Information Available The denition of the interface, The 1D-NMR spectra for Aβ 40, The details of PCA, and the probabilities of the distances between the C-terminal O atom and N atom of each residue longer than the cuto distances. at

This material is available free of charge via the Internet

http://pubs.acs.org/.

Web-Enhanced Features A Movie of typical MD simulation is available.

14


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


Table 1: The free energy, the fractional population, and the ranges of states AE at the interface in Fig. 10(a) and states A′ E′ in the bulk water in Fig. 10(b). State

Free energy (kcal/mol)

A

0.3

B

0.0

C

0.4

D

0.6

E ′ A ′ B ′ C ′ D ′ E

1.1 1.0 0.3 0.1 0.0 0.4

± ± ± ± ± ± ± ± ± ±

Population (%)

± 5.4 ± 7.9 8.3 ± 3.7 6.4 ± 4.8 2.7 ± 1.1 3.1 ± 2.2 9.8 ± 4.8 13.5 ± 5.3 16.8 ± 5.0 9.1 ± 5.6

Range

0.4

10.3

(-13.5, 4.5) < (PCA1, PCA2) < (-5.5, 12.5)

0.3

17.5

(-16.0, -4.0) < (PCA1, PCA2) < (-8.0, 4.0)

0.3 0.8 0.3 0.6 0.4 0.3 0.2 0.5

23

(-4.0, -5.0) < (PCA1, PCA2) < (4.0, 3.0) (15.0, -11.0) < (PCA1, PCA2) < (23.0, -3.0) (11.0, -3.0) < (PCA1, PCA2) < (19.0, 5.0) (-7.0, 15.0) < (PCA1, PCA2) < (1.0, 23.0) (-8.0, 5.0) < (PCA1, PCA2) < (0.0, 13.0) (0.5, 1.5) < (PCA1, PCA2) < (8.5, 9.5) (-1.0, -10.0) < (PCA1, PCA2) < (7.0, -2.0) (13.0, -7.0) < (PCA1, PCA2) < (21.0, 1.0)



(a) Interface Hydrophilic Ab40 Hydrophobic

(b) Water

(c) Vacuum

Figure 1: Three dierent initial positions. Aβ 40 molecules were located (a) at the interface, (b) in water solvent, and (c) in vacuum. The interface between water solvent and vacuum corresponds to the hydrophobic/hydrophilic interface.

24


Page 24 of 33

Page 25 of 33

(a) Experiment 1

D A E F R H

Hydrophilic D S G Y

10

E

N

K

G A I

S V L H F D G F H K E V A Q V 20

30

G G

V G I LM

V V 40

Hydrophobic

Averaged z−axis value (Å)

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


(b) Simulatoin 16 14 12 10 8 6 4 2 0

1

10

20

30

40

Residue number

(C) Typical conformation

b2 region b1 region Hydrophilic Hydrophobic

Figure 2: (a) The up-and-down topological model of the Aβ 40 monomer obtained from the NMR saturation transfer experiments. Val12Gly25, Ile31Val36, and Val39Val40 of Aβ 40 are buried in the hydrophobic interior of the lyso-GM1 micelle. Reproduced with permission from Ref. 32. Copyright (2009) Springer. (b) The averaged z-axis values of the Cα atoms of the amino-acid residues from the MD simulations. Red lines shows the residues that bound to the lyso-GM1 micelle in the experiments. (c) A typical up-and-down shape obtained from our simulations.

25


Page 26 of 33

12 10 8 6 4 2 0 1

10

20

30

40

Residue number Figure 3: Averaged number of hydration water molecules per heavy atom for each residue.

0.20

Tyr10 (N−terminal) Val18 (b1) Ser26 (Linker) Leu34 (b2)

0.15 Probability

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

Averaged number of hydration water molecules


0.10 0.05 0.00 −10

0 10 Z−axis value (Å)

20

Figure 4: Probability distributions of the z-axis values of the representative Cα atoms in the N-terminal region, the

β1

region, the

β2

region, and the linker region between

β1

The black, red, blue, and green lines show the results for Tyr10 (N-terminal), Val18 Ser26 (linker), and Leu34 (β2), respectively.

26


β2. (β1),

and

Page 27 of 33

40

(b) Bulk

(a) Interface

0.8 0.7

Residue number

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


0.6

30

Hairpin

0.5 0.4

20

0.3

Helix

10

0.2 0.1

1

0.0 1

10

20

30

1

40

10

20

30

40

Residue number

Residue number

Figure 5: Contact probabilities of Cα atoms obtained from the simulations (a) in the presence of the interface and (b) in the absence of the interface. Red solid lines correspond to the residues that had helix structures in the experiments with the lyso-GM1.

Figure 6: At the interface, relative motion of the in two-dimensional space.

β1

region to the

In the bulk water, conversely, the

dimensionally.

27


β1

β2

region is suppressed

region can move three-


(a) t = 0.00 ns

Page 28 of 33

(d) t = 73.84 ns

V12

V40

V12

V40 (b) t = 8.32 ns

(e) t = 93.84 ns V40

V12

V12 V40 (c) t = 34.80 ns

(f) t = 187.24 ns

V40

V40

V12

V12

Figure 7: The time sequenced snapshots of Aβ 40 at the interface. omitted for the sake of clarity.

28


Residues D1E11 was

Page 29 of 33

(a) Experiment 1 D A E F R

H

D S

Hydrophilic

G

10

Y

E

N

K

S 20 V G L H F D F H K E V A Q V

Hydrophobic

G

A 30 G G V V I G V 40 I L M

(b) Simulatoin 0.4 Probability

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


0.3 0.2 0.1 0.0

1

10

20 Residue number

30

40

Figure 8: (a) Schematic drawing of Aβ 40 positioned on the hydrophilic/hydrophobic interface 13 of the GM1 micelle. The amino acid residues whose carbonyl C signal exhibited upeld shift, downeld shift, and unchanged upon addition of the GM1 micelle are colored by magenta, yellow, and cyan, respectively. (b) The helix formation probability of Aβ 40 at the hydrophilic/hydrophobic interface obtained by the simulations.

29



RMSF (Å)

15 10 5 0

1

10

20

30

40

Residue number Figure 9: RMSF of each residues at the hydrophilic/hydrophobic interface obtained by the simulations.

(a) Interface

30

(b) Bulk

3.0

20

A’

20 10

A C

B

0

E D

−10

2.5

B’

10

PCA2

PCA2

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

Page 30 of 33

2.0

C’

0

1.5

E’

−10

1.0

D’

−20

−20

0.5

−30 −20

−10

0

10

20

0.0 −30

−20

−10

PCA1 Figure 10:

0

10

20

30

PCA1

(a) Free-energy landscape for Aβ 40 at the interface.

The free-energy local-

minimum states are labeled as state A to state E. (b) Free-energy landscape in the bulk ′ ′ water. The free-energy local-minimum states are labeled as state A to state E . The abscissa and the ordinate are the rst principal component axis and the second principal component axis, respectively. The axes are the rst and second principal component axes determined from (a) the simulations at the interface and (b) the simulations in the bulk water. The unit of the free-energy landscape is kcal/mol.

30


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


(a) Interface A

C

B

V40 V12 V40 V12 V40

V12

E

D V40

V12

V12

V40

(b) Bulk A’

B’

C’

V40

V12 V12 V40

V12 V40

D’

E’ V12

V40

V40

V12

′ ′ Figure 11: The representative structures of states AE in Fig. 10(a) and states A E in Fig. 10(b).

31



(a) Experiment 20

10

1

30

40

1.0 㻝

0.5 㻜㻚㻡

*

*

* ** *

20

10

30

40

㻵㻟㻝

**

1

㻭㻞㻝

㻜

㻱㻝㻝

0

㻰㻝

Intensity ratio

DAE F RHD SGY E V HHQK L V F F AE DV GSNKGA I I GL MVGGVV

Residue number

(b) Simulation Probability

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

Page 32 of 33

1.0 0.9 0.8 0.7 0.6

0

5

10

15 20 25 Residue number

30

35

Figure 12: (a) Intensity ratio of the backbone amide peaks of Aβ 40-C-Cys-MTSL on the lyso-GM1 micelle before and after radical quenching. The lower intensity ratios are indicative of closer distance with the unpaired electron of the spin label. Asterisk indicates the amino acid residue that did not exhibit observable peak in the spectrum due to severe broadening even after radical quenching.

Intensity ratio are the mean

±

standard deviation of three

independent experiments. (b) Probability of the distance between the C-terminal O atom and N atom of the corresponding residue longer than 10 Å.

32


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Graphical TOC Entry V12

V40

V12

V40

V40

V40 V12 V12

33


Hydrophobic Interface on Amyloid-Î² Peptides

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