Preferential Intercalation of Human Amyloid-Î² Peptide into Interbilayer

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Preferential Intercalation of Human Amyloid-# Peptide into Inter-Bilayer Region of Lipid-Raft Membrane in Macromolecular Crowding Environment Mitsuhiro Hirai, Satoshi Ajito, Shouki Sato, Noboru Ohta, Noriyuki Igarashi, and Nobutaka Shimizu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b08006 • Publication Date (Web): 26 Sep 2018 Downloaded from http://pubs.acs.org on September 28, 2018

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The Journal of Physical Chemistry

Preferential Intercalation of Human Amyloid-β Peptide into Inter-Bilayer Region of Lipid-Raft Membrane in Macromolecular Crowding Environment Mitsuhiro Hirai1*, Satoshi Ajito1, Shouki Sato1, Noboru Ohta2, Noriyuki Igarashi3, and Nobutaka Shimizu3 1

Graduate School of Science and Technology, Gunma University, 4-2 Aramaki,

Maebashi, Gunma 371-8510, Japan. 2

Japan Synchrotron Radiation Research Institute, 1-1-1, Kouto, Sayo-cho, Sayo-gun,

Hyogo 679-5198, Japan. 3

Institute of Materials Structure Science, High Energy Accelerator Research

Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan

* Corresponding Author E-mail: [email protected] (TELEFAX) INT+81 272-20-7551 (PHONE) INT+81 272-20-7554

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

Abstract This study focuses on the interaction of human amyloid β-peptide (Aβ) with a lipid-raft model membrane under macromolecular crowding conditions that mimic the intracellular environment. Aβ is central to the development of Alzheimer's disease (AD) and has been studied extensively to determine the molecular mechanisms of Aβ-induced cellular dysfunctions underlying the pathogenesis of AD. According to evidence from spectroscopic studies, ganglioside clusters are key to the fibrillization process of Aβ. Gangliosides are a major component of glycosphingolipids and are acidic lipids of the central nervous system known to form so-called lipid rafts. In this study, the small unilamellar vesicle (SUV) membrane, composed of monosialogangliosides, cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, did not show any structural changes after the addition of Aβ under non-crowding conditions. However, the addition of Aβ under crowding conditions induced shape deformation and aggregation to SUV resulting in multi-lamellar stacking. The time evolution of the lamellar peak suggested the preferential cohesion or intercalation of the Aβ peptide into the inter-bilayer region. This phenomenon was only observed at the gel (L ) phase. These results suggest that an β

intracellular crowding environment promotes Aβ–membrane interaction and a selective accumulation of Aβ peptides into the inter-bilayer regions.


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1. Introduction The amyloid β-peptide (Aβ), which exists in fibrillar forms as a major component of senile plaques, is central to the development of Alzheimer's disease (AD).1,

2

Aβ

fibrillization has been extensively investigated to elucidate the molecular mechanisms of Aβ-induced cellular dysfunctions underlying the pathogenesis of AD. Particularly, the importance of ganglioside clusters in the fibrillization process of Aβ has been highlighted.3 Gangliosides, which are major components of glycosphingolipids (GSLs), are acidic lipids and are rich in the central nervous system. GSL-cholesterol forms microdomains in cell membranes, so-called lipid rafts. Lipid rafts are fluctuating nanoscale assemblies of sphingolipid, cholesterol, and proteins and involved in a platform for various membrane-associated events such as signal transduction, cell adhesion, and lipid/protein sorting.4-6 Due to the presence of a large hydrophilic head portion, gangliosides and lipid mixtures containing gangliosides showed unique structural and dynamical physicochemical features, including; temperature-dependent reversible hydration-dehydration of the sugar-head region accompanying the bending of the head portion and the change of dissociation degree of sialic acids7-12; preferential localization of gangliosides at the outer-leaflet of liposomes13,

14

; microdomain

formation rich in gangliosides and cholesterol on the liposome surface15; osmotic pressure-dependent reversible transition from unilamellar to double-layered lamellar vesicle16; control the diffusional motions (undulation/bending motions) of micelles and liposomes,17, 18 and so on. According to accumulating evidence using spectroscopic methods, it was shown that the interaction between monosialogangliosides (GM1) and Aβ promotes conformational changes to the cross-beta structure in Aβ and the seeding in the process of Aβ polymerization to amyloid fibril.19, 20 Recently, we reported that the interaction between Aβ peptide (1-40) and the raft model membrane containing GM1 ganglioside at the liquid-crystal phase significantly suppressed the bending-diffusional motion of the liposome membrane, suggesting the possibility of non-receptor-mediated disorder in signaling through a modulation of a membrane dynamic induced by the association of amyloidogenic peptides on a plasma membrane.21 However, most of the previous studies on the amyloidogenic reactions of the Aβ peptide and its interaction with the membrane have been conducted in diluted solutions. The inside of living cells are known as molecular-crowding environments, where large amounts of different molecules coexist.22 As a result, this crowding environment is thought to affect the equilibrium states of proteins and membranes, but our understanding of molecular-crowding is poor due to a lack of experimental studies treating the effect by macromolecular crowding. However, recent progress in large-scale molecular dynamic (MD) simulations has allowed us to study the effect of ACS Paragon Plus Environment


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molecular crowding on the protein structure.23 Dastidar et al. demonstrated a sharp decrease in the interaction energy between the water layers and protein beyond 4 Å from the protein surface.24 The study of the effect of protein crowding on the structure and the dynamics of water by Harada et al. suggested significant changes in both structure and dynamics of water under highly crowded conditions.25 An all-atom MD simulation study by Wang et al. on the influence of the size of the crowding proteins on the hydration structure indicated changes in the accessible volumes and water locations in three well-defined regions, while the water densities as a function of the distance from protein surfaces remained the same.26 The development of MD simulation indicates the importance of quantitative experimental evidence, which is enough to compare with theoretical simulation results. However, the study of the protein– membrane

interaction

under

macromolecular

crowding

environment

is

still

experimentally challenging. In the present study, we have focused on the Aβ-membrane interactions in a mimic intracellular environment by using time-resolved small- and wide-angle X-ray scattering (SWAXS). The membrane used was the small unilamellar vesicle (SUV) membrane, composed of monosialogangliosides, cholesterol, and phospholipids, and was used as a model of lipid raft. The mimic intracellular environment, so-called crowding environment, was realized by the addition of the high-molecular weight neutral polymer (polyvinyl-pyrrolidone, PVP). PVP is a water-soluble polymer made from N-vinylpyrrolidone, and is used as a non-toxic additive in various industrial products, such as pharmaceutical tablets, cosmetics, and foods. The use of PVP for studying hydration forces between lipid membranes is well established.27-29 The study resulted in the following observations: in a non-crowding environment, the interaction between SUV and Aβ peptide proceeds mostly without affecting the SUV membrane structure, while in a crowding environment, the presence of crowders induces the deformation of the SUV shape and the interaction between SUV, where the addition Aβ peptide causes further aggregation to form the lamellar stacking. The time evolution course of the lamellar distance and the lamellar-peak height suggests the preferential cohesion or intercalation of Aβ peptides in the inter-bilayer region. These findings indicate the importance and need for studies on amyloidogenic reactions under macromolecular crowding environment. 2. Experimental Methods Amyloid β-peptide (Human, 1-40) purchased from Peptide Institute Inc. (Osaka, Japan) was used without further purification. Monosialoganglioside (GM1) from bovine brains, cholesterol, and 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) ACS Paragon Plus Environment

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were

purchased

from

Avanti

Polar

Lipids

Inc.

(Alabaster,

AL,

USA).

Polyvinylpyrrolidone (PVP, Mt. 40,000) was purchased from SIGMA Chemical Co. (USA).

These

materials

were

also

used

without

further

purification.

GM1-cholesterol-DPPC mixed vesicles were prepared according to the method described by Sillerud et al.,30 with the following modifications. GM1, DPPC, and cholesterol were separately dissolved in chloroform/methanol mixture solvent (1/1 (v/v)). These solutions were mixed with the appropriate molar ratios. After removal of the organic solvent under a nitrogen stream, the lipid mixtures were dried and annealed in vacuo overnight at 45°C (above the gel-to-liquid crystal transition temperature of DPPC). The dried mixtures were suspended in water solvent (10 mM HEPES, at pH 7.4, with 50 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 2 mM CaCl2), vigorously stirred for ~20 minutes, and stored overnight to obtain giant multilamellar vesicle (GMLV) solutions. The GMLV solutions were subjected to sonication for 10 min at 45°C using a high-power probe-type ultrasonicator (Model UH-50, Shimazu Co., Japan). Finally, we obtained the substantially transparent homogeneous small unilamellar vesicle (SUV) solutions. The molar ratios [GM1]/[cholesterol]/[DPPC] of the mixtures were 0.1/0.1/1 and 0.2/0.2/1. The phospholipid concentration was 2% w/v. These SUV solutions were used as stock solutions. PVP stock solutions with different concentrations were also prepared. Both the SUV and PVP stock solutions were mixed at an appropriate volume ratio. Before carrying out scattering measurements, Aβ peptide was dissolved in the water solvent and then mixed with the SUV solutions. The final phospholipid concentration was 0.82% w/v (0.011 M). The Aβ peptide was added the SUV solution to be the molar ratios of [Aβ]/[GM1] = ~1/6 and [Aβ]/[total lipids] = ~1/42, corresponding to ~0.16% w/v (0.37 mM). SR-WAXS measurements were carried out using a BL-40B2 spectrometer at the Japan Synchrotron Radiation Research Institute (JASRI, Harima, Japan) and a BL-10C spectrometer at the High Energy Accelerator Research Organization (KEK, Tsukuba, Japan). The X-ray wavelength and the sample-to-detector distance were 0.75 Å for 38.9 cm and 1.0 Å for 412 cm at BL-40B2; 1.49 Å for 190 cm at BL-10C, respectively. The X-ray scattering intensity was recorded by the R-AXIS IV detector (30x30 cm2 in area, 100-µm in pixel-resolution, from RIGAKU Co.) at both facilities. The exposure time was 10 seconds at BL-40B2 and 30 seconds at BL-10C. The temperature of the solutions contained in the sample cells was controlled using temperature controller mK2000 (INSTEC Co.). The background correction of SWAXS data was carried out according to the method described in detail in another study.31, 32



I(q) =

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% (1− cva ) " 1 % 1 " 1 1 1 I (q) − I (q) − I (q) − I (q) # & # & sol cell solv cell sol solvl I peak BcelllTcelll BcellTcell I peak $ BsolTsol ' $ BsolvTsolv '

(1) where I

sol peak

and I

solvl peak

are the water-correlation peak intensities of the solution and the

solvent; c and va are the concentrations of the lipids and those partial specific volumes; I(q)sol, I(q)solv, and I(q)cell are the observed scattering intensities of the solution, the solvent, and the blank cell; Tsol, Tsolv, and Tcell are those transmissions, respectively. Here the va values used were obtained from a handbook.33 To determine the SUV structures, we carried out a fitting procedure using the following model scattering function, I(q,R). I(q,R) describes a multi-shelled ellipsoid particle with a size-distribution, as shown previously.13, 14 ∞

∫

I(q) =

(2)

I s (q, R)D(R)dR

Rmin

where Rmin is a lower limit of particle radius determined by the lipid-bilayer thickness of the SUV; D(R) is the number distribution function of the particle radius R; and Is(q, R) is the spherical averaged scattering function of an ellipsoidal particle with the radius R composed of n shells (ith shell with average excess scattering density ρ (so-called contrast), radius Ri and Vi). Is(q, R) and D(R) are given as follows. 2

n ) # &, € I (q, R) = ∫ +3 $ ρ1V1 j1 (qR1 ) / (qR1 ) + ∑ ( ρ i − ρ i−1 )Vi j1 (qRi ) / (qRi )'. dx s (€ 0* % i=2 1

(3)

where j1 is the spherical Bessel function of the first rank. Ri is defined by

(

Ri = ri 1+ x 2 (ν i2 −1)

1/2

)

(4)

€ where ri and νi are the semi-axis and its ratio of ith ellipsoidal shell, respectively. For a spherical-shelled particle (νi = 1, Ri = ri), equation 3 is simplified as n # & I (q, R) = 9 $ ρ1V1 j1 (qR1 ) / (qR1 ) + ∑ ( ρ i − ρ i−1 )Vi j1 (qRi ) / (qRi )' s % ( i=2

2

(5)

As a function of D(R), we adopted the Gaussian distribution function given by " (R − R)2 % 1 D(R) = exp #− & 2σ 2 ' 2πσ $

(6)

where R and σ are the average radius and its standard deviation, respectively. The Gaussian function is used as a size-distribution function in many cases of small unilamellar vesicle systems.34 For shell-model fitting, we introduced the cut-off of the minimum radius in D(R) because the size of the liposome should be larger than the lipid bilayer width.


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3. Results and Discussions 3.1. Determination of SUV structure Figure 1 depicts the SWAXS curve of small unilamellar vesicle (SUV) membrane composed of gangliosides, cholesterol, and DPPC at 30°C, where the thin and thick lines are [ganglioside GM1]/[cholesterol]/[DPPC] = 0.1/0.1/1 and 0.2/0.2/1, respectively. The q range observed ranged from ~0.0027 Å-1 to ~2.5 Å-1, with the structural information ranging from ~2300 Å to ~2.5 Å in the real-space distance. The measured q-range covered all hierarchical structures of an SUV at its different hierarchical levels. Namely, in Figure 1, the scattering curves below ~0.02 Å-1, in the range from ~0.04 Å-1 to ~0.6 Å-1, and at ~1.4-1.5 Å-1 reflect the globular shape and size distribution of the SUV, the lipid-bilayer structure (thickness, internal scattering density distribution), and the ordering of the alkyl-chain packing (gel or liquid crystalline (fluid) phase), respectively.14 The scattering curve ranging from ~0.025 to ~0.035 Å-1 corresponds to the crossover region between the liposome shape and its lipid-bilayer structure. When a transition from a unilamellar to a multilamellar structure (approach of lipid-bilayers) occurs, an inter-bilayer correlation peak (so-called lamellar diffraction peak) appears in the middle q-range, depending on the lamellar spacing. Comparing the two scattering curves, we identified changes depending on the molar ratio of lipids. With an increasing ganglioside content, the slope below ~0.005 Å-1 became high and the peak position of the broad hump at ~0.1 Å-1 shifted to a slightly lower q-value. These changes suggest enlargements in the liposome size and in the width of the bilayer due to the increase of ganglioside and cholesterol contents. The change of the radius of gyration Rg from 235.1±0.1 Å to 260±1Å supports such an enlargement. The peak position and its width (FWHM) at ~1.5 Å changed from 1.511±0.001 Å-1 to 1.486±0.001 Å-1 and from 0.125±0.001 Å-1 and 0.164±0.001 Å-1, respectively. This means that the increase of ganglioside and cholesterol contents caused a slight disordering of the alkyl-chain packing. Using the shell-modeling method (equations 3, 6, and 7), we fit the theoretical scattering curve in the experimental curve and determined both the size-distribution and the lipid-bilayer structure. In Figure 2A, the experimental and theoretical scattering curves are overimposed, where the insets are the size distribution functions obtained by the fitting. Figure 2B depicts the structural parameters of SUV bilayers (width of each shell and its relative value of the contrast) obtained by the shell-model fitting analysis. The liposome containing gangliosides is an asymmetric bilayer structure due to the preferential distribution of ganglioside molecules on the outer-leaflet of the lipid bilayer. Namely, the hydrophilic head (sugar) portions of ganglioside molecules tend to protrude from the membrane surface.13, 14 Figure 2B clearly shows this tendency. ACS Paragon Plus Environment


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3.2. Effect of macromolecular crowding on SUV structure Figure 3 presents the effect of macromolecular crowding on the SWAXS curve of the SUV ([GM1]/[cholesterol]/[DPPC] = 0.2/0.2/1) at 30 °C, where the PVP concentration was varied from 0 to 20 % w/w. By increasing the PVP concentration, the change of the scattering curve below q = 0.02 Å-1 started at first, then the scattering profile in the range from ~0.04 Å-1 to ~0.3 Å-1 changed from a simple broad peak-shape to a modulated one. The presence of PVP creates both an excluded volume effect and osmotic pressure. The appearance of the shoulder at ~0.01 Å-1 indicates and inter-SUV correlation peak with an average distance of ~600 Å due to a so-called excluded volume effect by PVP. The double-hump profile at ~0.1 Å-1 is attributable to the appearance of a nearest lipid-bilayer correlation by an approach of SUVs. This would be occurred due to the change of osmotic pressure and the depletion effect caused by PVP.15 These changes are qualitatively explained by the deformation of the spherical SUV shape to an oblate one accompanying the approach of the inter-SUV distance as shown in the following section. 3.3. Interaction of SUV with amyloid-β peptides under macromolecular crowding environment Figure 4 presents the time evolution of the SWAXS curve of SUVs after the addition of amyloid-β peptide: (A) SUV ([GM1]/[cholesterol]/[DPPC] = 0.1/0.1/1) at 30°C, non-crowding; (B) SUV ([GM1]/[cholesterol]/[DPPC] = 0.1/0.1/1) at 30°C in 15 % PVP (w/w); (C) SUV ([GM1]/[cholesterol]/[DPPC] = 0.2/0.2/1) at 30°C in 15% PVP (w/w); (D) SUV ([GM1]/[cholesterol]/[DPPC] = 0.2/0.2/1) at 45°C in 15% PVP. The molar ratios of [Aβ]/[GM1] and [Aβ]/[total lipids] were ~1/6 and ~1/42, respectively. These ratios were much lower than those of the previous spectroscopic studies.3, 19, 20 The setting temperatures of 30°C and 45°C corresponded to the gel phase (L phase) and the β

liquid-crystalline phase (L phase) of the base lipid, DPPC. As described above, the α

former and later ranges reflect the shape of the SUV with a size distribution, the lipid-bilayer structure (thickness and internal contrast), respectively. In a non-crowding environment at L phase (Figure 4A), the addition of amyloid-β peptide caused little β

changes in the scattering curve of SUV in the whole q-range. This result agrees well with the previous results of a large unilamellar vesicle (LUV) containing GM1.20 Comparing with Figure 4B and 4C, at the L phase, the SUV containing more GM1 β

(Figure 4C) showed significant changes in the small-q (< 0.02 Å-1) and medium-q (0.04-0.3 Å-1) ranges. In particular, the broad hump at q = ~0.08 Å-1 changed to a ACS Paragon Plus Environment

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diffraction peak at q = 0.0714 Å-1, with the second diffraction peak at q = 0.142 Å-1. This suggests that in a crowding environment at L phase, the interaction between the β

SUV and amyloid-β peptides greatly depends on ganglioside content and proceeds to form a lamellar (stacking of lipid bilayer) structure. On the other hand, in a crowding environment at L phase (Figure 4D), such a trend of lamellar formation and its growth α

was not observed. It means that not only the content of ganglioside but also the fluidity of the lipid bilayer membrane is an essential factor for the interaction between the lipid raft and amyloid-β peptide in a macromolecular crowding environment. Figure 5 depicts the time evolution of the q-value of the first hump or peak position and of its peak intensity. The q-value of the broad hump and/or the diffraction peak appearing in a crowding environment reflects the inter bilayer distance between the closely

spaced

liposome

membrane.

These

values

after

the

addition

of

-1

amyloid-β peptide were 0.0851±0.002 Å for the SUV of [GM1]/[cholesterol]/[DPPC] = 0.1/0.1/1 at 30°C, 0.0826±0.002 Å-1 for the SUV of [GM1]/[cholesterol]/[DPPC] = 0.2/0.2/1 at 30°C, and 0.0791±0.002 Å-1 for the SUV of [GM1]/[cholesterol]/[DPPC] = 0.2/0.2/1 at 45°C, which correspond to the real-space distances of 73.8 Å, 76.1 Å, and 79.4 Å, respectively. The above difference in the distances is attributable to the difference in the surface charge depending on ganglioside concentration and in the lipid phase. Of these, only in the case of the SUV of [GM1]/[cholesterol]/[DPPC] = 0.2/0.2/1 at L phase was there an evident change in q-value, starting from ~200 min after which β

the q-value gradually decreased to 0.0714±0.001 Å-1. For this SUV, the peak intensity also started to show a significant increase from ~200 min onwards. The above results indicate the growth of bilayer stacking that accompanied the extension of the inter-bilayer distance from 76.1 Å to 88.0 Å. On the other hand, for the other SUVs with a lower ganglioside concentration and/or at L phase, these changes were not α

observed. Because the interaction of the Aβ peptide with the lipid membrane occurs through ganglioside molecules,19,

20

this would be reasonably understood by the

characteristic physical properties of ganglioside molecules as shown previously. Namely, even in the relatively low-temperature range from 20°C to 50°C, the conformation and hydration of the sugar-head portion and the dissociation degree of the sialic-acid easily vary,7-12 and in the case of liposomes of lipid mixtures containing gangliosides, the size of the microdomain being rich in ganglioside and cholesterol (raft region) depends on temperature.15 It should be noted that the extension of the inter-bilayer distance showed the trend of the saturation after 290 min. This suggests that the preferential cohesion or intercalation of Aβ peptides in the inter-bilayer regions stopped due to a lower Aβ concentration compared with the previous studies.3, 19, 20



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Therefore, under the present experimental condition, the amyloidogenic reaction seems to stay at the oligomerization state, not at the fibrillization state. Figure 6 depicts the theoretical simulation of the scattering curve considering the disordering of the bilayer stacking, with (A) experimental scattering curves of the SUV in Figure 5C, and (B) theoretical scattering curves. In the simulation, we assumed a multi-layered flat membrane with an inter-bilayer-distance disordering. The structural parameters of the lipid bilayer used are given in Figure 2B. The disordering parameter was based on the paracrystalline theory by Hosemann.35 For an ideal paracrystalline lattice with a disorder of the second kind, when the presence probability function of the nearest-neighbor bilayer is simplified to be of the form of a Gaussian distribution, the paracrystalline lattice factor W(q) for one-dimensional alignment is given by 1− H (q)2 W (q) = 1− 2H (q)cos(qa) + H (q)2

(7)

H (q) = exp(−q 2 Δ 2 / 2)

(8)

where a is the mean inter-bilayer distance. The factor g is an index of the extent of the paracrystalline disorder defined by

g = Δ / a , where fluctuations Δ of the

paracrystalline disorder are relative to the mean adjacent inter-bilayer distance. In Figure 6B, the disorder factor g was varied from 0.26 (at the initial state) to 0.1 (at the final state). A slight difference between theoretical and experimental scattering curves is attributable to the use of the form factor of the lipid bilayer determined from the SUV scattering curve under non-crowding conditions. The time evolution of the experimental scattering curve of the SUV of 0.2/0.2/1 at L phase after the addition of Aβ can be β

qualitatively reproduced by the change of the paracrystalline disorder parameter, namely the growth of the bilayer ordering accompanying the extension of the inter-bilayer distance as shown below. Figure 7 represents the time evolution of the position and half-width at half maximum (HWHM) of the peak at ~1.4-1.5 Å-1 in Figure 4. The q-value of the peak position relates to the average correlation distance (given by d = 2π / q peak−position ) between the alkyl-chains of the lipid molecules, and the HWHM reflects the thermal fluctuation and disordering. The significant difference of the peak position and HWHM of Figure 7D with those of A, B, and C is attributable to the difference in lipid phases, namely, L and L . Except for in the case of A (in the non-crowding environment), α

β

within ~60 min after the addition of amyloid-β peptide, the increase of the HWHM values were recognized, which was most evident for C. This suggests that the shape deformation accompanying the growth of lamellar stacking is associated with a rearrangement of lipid molecules.


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4. Conclusion The present study found that in a non-crowding environment, the interaction between the liposome and Aβ peptide proceeded mostly without affecting the liposome membrane structure, while in a crowding environment, the presence of macromolecular crowders induced the deformation of the liposome shape and the approach between the membranes due to the excluded volume effect (depletion effect). The addition of Aβ peptides caused further ordering of the lamellar stacking with an accompanying extension of the inter-bilayer distance in spite of the presence of the osmotic pressure by the crowders. The time evolution of the lamellar distance and the lamellar-peak height strongly suggest the occurrence of the preferential cohesion or intercalation of Aβ peptides in the inter-bilayer regions. The above phenomena would be attributable to unique structural and dynamical physicochemical features of ganglioside molecules. In particular, even under non-crowding conditions, the preferential interaction between gangliosides and the Aβ peptide (1-40) significantly changed the diffusional bending motion of liposomes at L phase, namely stiffening the membrane.21 By considering the α

present results together with the previous ones, a schematic image on the Aβ–membrane interaction under macromolecular crowding is presented in Figure 8. The interaction of the Aβ peptide is assumed to be intrinsically more preferable for a rigid region such as raft region (liquid-order phase) in the membrane, and the restriction on the bending diffusional motion of the membrane will be enhanced by both the presence of crowders and the interaction of the Aβ peptide. These factors would promote further intercalation and/or assembly of Aβ peptides in the inter-bilayer region. Because the proposal of the amyloid cascade hypothesis,2 many pieces of evidence have supported a causative role for Aβ in AD, namely that the ubiquity of aggregated forms of the Aβ peptides and the apparent cytotoxicity of aggregated forms of these proteins leads to a neurodegenerative disorder in the ultimate upstream cause of AD.36 However, owing to the failure of clinical trials experimenting with anti-amyloid therapies, the problems of the amyloid cascade hypothesis were also indicated.37 To understand the molecular mechanism not only of AD but also of general amyloidosis, clarifying the fundamental physicochemical mechanism of protein misfolding and its oligomerization is necessary.38,

39

However, in particular, there seem to be less

experimental trials and pieces of knowledge from the viewpoint of protein misfolding and interaction with membranes under molecular crowding environment. The present results would suggest the possibility that a neurodegenerative disorder results from the change of dynamics of lipid rafts caused by the preferential cohesion or intercalation of Aβ peptides in the inter-bilayer region.



AUTHOR INFORMATION Corresponding Author (M.H.) *E-mail: [email protected] (TELEFAX) INT+81 272-20-7551 (PHONE) INT+81 272-20-7554 Notes

The authors declare no competing financial interest. ACKNOWLEDGMENTS The X-ray scattering experiments were performed with the approval of the Program Advisory Committee of the Japan Synchrotron Radiation Research Institute (Proposal No. 2014A1062 & 2015A1557) and the approval of the PF Program Advisory Committee (Proposal No. 2013G038 and 2014G657). The neutron scattering experiment was carried out with the approval of the Neutron Scattering Program Advisory Committee (Proposal No. 2016B0003). This research project was supported by the Grants-in-Aid for Scientific Research of JSPS (the Japan Society of the Promotion of Science) (Proposal No. 16K13722).


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(15) Hirai, M.; Hirai, H.; Koizumi, M.; Kasahara, K.; Yuyama, K.; Suzuki, N. Structure of raft-model membrane by using inverse contrast variation neutron scattering method. Physica B 2006, 385-386, 868-870. (16) Onai, T.; Hirai, M. Morphology transition of raft-model membrane induced by osmotic pressure: Formation of double-layered vesicle similar to an endo- and/or exocytosis.

J.

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doi:10.1088/1742-6596/247/1/012018. (17) Hirai, M.; Iwase, H.; Hayakawa, T. Structure and dynamics of glycosphingolipid micelles. J. Phys. Soc. Jpn. Suppl. 2001, 70, 417-419. (18) Hirai, M.; Koizumi, M.; Hirai, H.; Hayakawa, T.; Yuyama, K.; Suzuki, N.; Kasahara, K. Structures and dynamics of glycosphingolipid-containing lipid mixtures as raft models of plasma membrane. J. Phys.: Condens. Matter 2005, 17, s2965-s2977. (19) Kakio, A.; Nishimoto, S.; Kozutsumi, Y.; Matsuzaki, K. Formation of a membrane-active form of amyloid β-protein in raft-like model membranes. Biochem. Biophys. Res. Comm. 2003. 303, 514–518 (20) Matsuzaki, K.; Kato, K.; Yanagisawa, K. Aβ polymerization through interaction with membrane gangliosides. Biochim. Biophys. Acta 2010, 1801, 868–877. (21) Hirai, M.; Kimura, R.; Takeuchi, K.; Sugiyama, M.; Kasahara, K.; Ohta, N.; Farago, B.; Stadler, A.; Zaccai, G. Change of dynamics of raft-model membrane induced by amyloid-β protein binding. Eur. Phys. J. E. 2013. 36, 74, DOI 10.1140/epje/i2013-13074-3. (22) Rivas, G.; Minto, A. P. Macromolecular Crowding In Vitro, In Vivo, and In Between. Trends Biochem. Sci. 2016, 41, 970-981. (23) Musiani, F.; Giorgetti, A. Protein Aggregation and Molecular Crowding: Perspectives From Multiscale Simulations. Int. Rev. Cell Mol. Biol. 2017, 2329, 49-77. (24) Dastidar, S. G.; Mukhopadhyay, C. Structure, dynamics, and energetics of water at the surface of a small globular protein: A molecular dynamics simulation. Phys. Rev. E 2003, 68, 21921-21930. (25) Harada, R., Sugita, Y., and M. Feig. Protein Crowding Affects Hydration Structure and Dynamics. J. Am. Chem. Soc. 2012, 134, 4842−4849. (26) Wang, P.; Yu, I.; Feig, M.; Sugita Y. Influence of protein crowder size on hydration structure and dynamics in macromolecular crowding. Chemical Physics Letters 2017, 671, 63–70. (27) McIntosh, T. J.; Simon, S. A. Area per molecule and distribution of water in fully hydrated dilauroylphosphatidylethanolamine bilayers. Biochemistry 1986, 25, 4058– 4066.


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(28) Nagle, S. T.; Petrache, H. I.; Nagle, J. F. X-ray structure determination of fully hydrated L-alpha phase dipalmitoylphosphatidylcholine bilayers. Biophys. J. 1988, 75, 917–925. (29) Leneveu, D. M.; Rand, R. P.; Parsegian, V. A. Measurement and modification of forces between lecithin bilayers. Biophys. J. 1977, 18, 209–230. (30) Sillerud, L.; Schafer, D. E.; Yu, R. K.; Konigsberg, W. H. Calorimetric properties of mixtures of ganglioside GM1 and dipalmitoylphosphatidylcholine. J. Bio. Chem. 1979, 254, 10876-10880. (31) Hirai, M.; Iwase, H.; Hayakawa, T.; Miura, K.; Inoue, K. Structural hierarchy of several proteins observed by wide-angle solution scattering. J. Synchrotron Rad. 2002, 9, 202-205. (32) Hirai, M.; Koizumi, M.; Hayakawa, T.; Takahashi, H.; Abe, S.; Hirai, H.; Miura, K.; Inoue, K. Hierarchical map of protein unfolding and refolding at thermal equilibrium revealed by wide-angle X-ray scattering. Biochemistry 2004, 43, 9036-9049. (33) Marsh, D. In CRC Handbook of Lipid Bilayers 1990, CRC Press, 185–197. (34) Balgavy, P.; Dubnickova, M.; Kucerka, N.; Kiselev, M. A.; Yaradaikin, S. P.; Uhrikova, D. Bilayer thickness and lipid interface area in unilamellar extruded 1,2-diacylphosphatidylcholine liposomes: A small-angle neutron scattering study. Biochim. Biophys. Acta. 2001, 1512, 40–52. (35) Hosemann, R.; Lemm, K.; Wilke, W. The Paracrystal as a model for liquid crystals. Molecular Crystals 1967. 1, 333-362. (36) Walsh, D. M.; Selkoe. D. J. Aβ Oligomers – a decade of discovery. J. Neurochem. 2007, 101, 1172–1184. (37) Ricciarelli, R.; Fedele, E. The Amyloid cascade hypothesis in Alzheimer’s disease: It’s time to change our mind. Current Neuropharmacology 2017, 15, 926-935. (38) Dobson, C. M. Protein folding and misfolding, Nature 2003, 426, 884-890. (39) Chiti, F.; Dobson, C. M. Protein misfolding, amyloid formation, and human disease: A Summary of progress over the last decade, Annu. Rev. Biochem. 2017, 86, 27-68.



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[Figure legends] Figure 1. Small-and-wide angle X-ray scattering (SWAXS) curve of the small unilamellar vesicle (SUV) membrane composed of ganglioside, cholesterol, and DPPC in aqueous solution (10 mM HEPES, at pH 7.4, with 50 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 2 mM CaCl2)

at

30°C.

The

lipid

composition

of

the

SUV

is

[ganglioside

GM1]/[cholesterol]/[DPPC] = 0.1/0.1/1 and 0.2/0.2/1. The observed SWAXS curve reflects all hierarchical structures of the SUV; namely, below q = ~0.02 Å-1, size distribution and shape of SUV; q = ~0.04-0.6 Å-1, lipid-bilayer structure (thickness, internal scattering density distribution); peak at q = ~1.4-1.5 Å-1, alkyl-chain packing-order (gel or liquid crystalline (fluid) phase), respectively. The measured q-range corresponds to the real-space distance from ~2300 Å to ~3 Å. Figure 2. Determination of the lipid-bilayer structure and size distribution of the SUV by using the shell-model fitting, where (A), experimental data and theoretical scattering curve, determined size distribution function; (B), determined bilayer structure. In (B), the values of the contrast are normalized by that of the alkyl-chain region. The asymmetric profile in the normal direction of the lipid-bilayer surface shows the preferential localization of ganglioside molecules at the outer-leaflet of the SUV. Figure 3. Effect of macromolecular crowding on SUV structure ([GM1]/[cholesterol]/[DPPC] = 0.2/0.2/1) at 30°C. The crowder (PVP) concentration varied from 0 to 20 % w/w. Figure 4. Time evolution of the SWAXS curve of the SUV after the addition of amyloid-β protein. (A)

[GM1]/[cholesterol]/[DPPC]

=

0.1/0.1/1

at

30°C,

non-crowding.

(B)

[GM1]/[cholesterol]/[DPPC] = 0.1/0.1/1 at 30°C. (C) [GM1]/[cholesterol]/[DPPC] = 0.2/0.2/1 at 30°C. (D) [GM1]/[cholesterol]/[DPPC] = 0.2/0.2/1 at 45°C. (B)-(D) were under crowding conditions (15 % PVP w/w). Figure 5. Time evolution of the q-value of the first hump or peak position and of its peak intensity in the middle-q region in Figure 4, where the left and right axes correspond to the peak intensity and the peak q-value, respectively. (B)-(D) are as in Figure 4. ACS Paragon Plus Environment

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Figure 6. Theoretical scattering curve simulation of multi-layered bilayer membrane with considering with a paracrystalline disordering. (A) Experimental scattering curves of the SUV in Figure 5C. (B) Theoretical scattering curves. The paracrystalline-disorder factor g varied from 0.26 to 0.1 with shifting the inter-bilayer distance. Figure 7. Time evolution of the position and half width at half maximum (HWHM) of the peak at ~1.4-1.5 Å-1 in Figure 4. A, B, C, and D are as in Figure 4. Figure 8. A schematic image on the interaction between Aβ peptide and lipid-raft membrane under the macromolecular crowding environment. At higher crowder and ganglioside concentration, preferential cohesion or intercalation of Aβ peptides in the inter-bilayer region is promoted.



[Figure 1] SUV-0.2/0.2/1

logI(q) (arb. units)

SUV-0.1/0.1/1

bilayer structure

shape & size

chain packing

1

0.01

0.1

1

-1

q (Å )


number (-)

[Figure 2]

A

100

200 300 400 radius (Å)

500

Exp. 0.1/0.1/1 Exp. 0.2/0.2/1 Model 0.1/0.1/1 Model 0.2/0.2/1

1

0.01

0.1

-1

q (Å )

relative contrast (-)

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

0.1/0.1/1 0.2/0.2/1

B

head portion (GM1 & PC heads)

tail portion

outer-leaflet

-60

-40

head portion (PC head & hydrated water)

-20

0

inner-leaflet

20

40

60

distance (Å) ACS Paragon Plus Environment

Page 18 of 23

Page 19 of 23

[Figure 3]


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


pvp conc. 0 % w/w 7.5 % w/w 15 % w/w

1

20 % w/w

0.01

0.1

1

-1

q (Å )



[Figure 4]

tiime (min.) 1.5 23 41 60 169 229 290 348


A

B

1

1

tiime (min.) 1.5 21 39 58 168 227 289 347

C


tiime (min.) 1.5 22 40 58 168 227 289 347

tiime (min.) 1.5 38 58 77 135 195 255 315

D

1

1

0.01

0.1

1

0.01

-1

0.1

1

-1

q (Å )

q (Å )

[Figure 5]

normarized peak intensity (-)

q-value of peak (B) q-value of peak (C) q-value of peak (D)

0.090

0.080

0.070 peak-intensity (B) peak-intensity (C) peak-intensity (D)

0.060

-1

0.050 0

50

100

150

q-value of peak (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

Page 20 of 23

200

250

300

350

evolution time (min)


Page 21 of 23

[Figure 6] tiime (min.) 1.5 21 39 58 168 227 289 347

I(q) (arb. units)

A

B I(q) (arb. units)

g = 0.26 g = 0.21 g = 0.15 g = 0.1

0.06

0.08

0.1

0.12

0.14

0.16

0.18

-1

q (Å )

[Figure 7]

q-value of peak (A) q-value of peak (B) q-value of peak (C) q-value of peak (D) 0.50

-1

q-value of peak (Å )

1.50

0.40 FWHM (A) FWHM (B) FWHM (C) FWHM (D)

1.45

0.30

1.40

1.35

0

50

100

150

200

250

300

0.10 350

evolution time (min)


-1

0.20

HWHM of peak (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



[Figure 8]


Page 22 of 23

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Table of Contents (TOC) Image The following graphic will be used for the TOC:


Preferential Intercalation of Human Amyloid-Î² Peptide into Interbilayer

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