Article pubs.acs.org/Langmuir

Formation of Lysozyme Oligomers at Model Cell Membranes Monitored with Sum Frequency Generation Spectroscopy I. I. Rzeźnicka,*,†,‡ R. Pandey,§ M. Schleeger,§ M. Bonn,§ and T. Weidner§ †

Department of Chemistry, Graduate School of Science, 6-3 Aramaki Aza-Aoba, Aoba-ku, Tohoku University, Sendai, Japan Institute for International Education, Tohoku University, Sendai, Japan § Department of Molecular Spectroscopy, Max-Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany ‡

S Supporting Information *

ABSTRACT: A growing number of studies suggest that the formation of toxic oligomers, precursors of amyloid fibrils, is initiated at the cell membrane and not in the cytosolic compartments of the cell. Studies of membrane-induced protein oligomerization are challenging due to the difficulties of probing small numbers of proteins present at membrane surfaces. Here, we employ surface-sensitive vibrational sum frequency generation (VSFG) to investigate the secondary structure of lysozyme at the surface of lipid monolayers. We investigate lysozyme aggregation at negatively charged 1,2dipalmitoyl-sn-glycero-3-(phospho-rac-1-glycerol) (DPPG) lipid monolayers under different pH conditions. The changes in the molecular vibrations of lipids, proteins, and water as a function of pH and surface pressure allow us to simultaneously monitor details of the conformation state of lysozyme, the organization of lipids, and the state of lipid-bound water. At pH = 6 lysozyme induces significant disordering of the lipid layer, and it exists in two states: a monomeric state with a predominantly α-helix content and an oligomeric (za-mer) state. At pH ≤ 3, all membrane-bound lysozyme self-associates into oligomers characterized by an antiparallel β-sheet structure. This is different from the situation in bulk solution, for which circular dichroism (CD) shows that the protein maintains an α-helix conformation, under both neutral and acidic pH conditions. The transition from monomers to oligomers is also associated with a decreased hydration of the lipid monolayer resulting in an increase of the lipid acyl chains ordering. The results indicate that oligomerization requires cooperative action between lysozyme incorporated into the lipid membrane and peripherally adsorbed lysozyme and is associated with the membrane dehydration and lipid reorganization. Membrane-bound oligomers with antiparallel β-sheet structure are found to destabilize lipid membranes.

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INTRODUCTION Many neurodegenerative as well as non-neuropathic disorders are associated with deposits of proteins aggregates (amyloid plaques) in various parts of human organs and tissues. For this reason, research efforts have been focused on understanding molecular-scale events leading to formation of these toxic deposits. The formation of amyloid fibrils is a multistep process involving conformational changes and aggregation of the protein. Several aggregation states have been identified for amyloidogenic proteins, including soluble oligomers and insoluble fibrils. Insoluble well-defined fibrils are found to be organized in a parallel β-sheet conformation, whereas soluble aggregates display features attributed to an antiparallel β-sheet structure.1 For a long time it was assumed that only fibrillar plaques have pathogenic effects, but in recent years, it is becoming increasingly accepted that soluble oligomers are more toxic than fibrils and have a direct role in amyloid pathogenesis.2 Recent studies have also shown that contact of amyloidogenic proteins with surfaces, in particular cell membranes, can promote protein misfolding and cause extensive aggregation.3−5 The majority of studies focused on extracted, insoluble well-defined fibrils. Studies on soluble oligomeric intermediates have been limited due to their © 2014 American Chemical Society

transient character, small size, and quantities below detection limits. Despite recent progress little is known about the molecular details involved in the transformation of native proteins into amyloid aggregates at lipid membranes. In order to better understand the molecular-level details of membrane-induced protein aggregation, it is necessary to study the intricate interplay between lipids, proteins, ions, and water, which is challenging experimentally. While fluorescence labeling studies have been extremely useful to quantify the association of proteins with membranes, such studies are unable to provide information about protein conformation at the interface. Important techniques available to study matured fibrils include the thioflavin T (ThT) assay, transmission electron microscopy (TEM), and atomic force microscopy (AFM). Nuclear magnetic resonance (NMR) and X-ray diffraction reveal details of solid-state fibril structure, and Fourier transform infrared spectroscopy (FTIR) together with measurement of circular dichroism (CD) provide information on fibril secondary structure in bulk solutions. However, these methods cannot Received: March 21, 2014 Revised: June 17, 2014 Published: June 18, 2014 7736

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water interface. The protein structure was only altered in the presence of a lipid monolayer, illustrating the important role of lipids in amyloid formation.

probe proteins specifically at membrane surfaces. Vibrational sum-frequency generation spectroscopy (VSFG), with its inherent surface specificity and sensitivity toward molecular vibrations, offers a unique way to study protein secondary structure at interfaces. VSFG can provide details of the interplay between lipids, proteins, and water - at the molecular level and label-free. Successful examples of its use include the investigation of model peptides,6−9 lysozyme at the air−water interface,10 and other large proteins.11−14 Here, we apply VSFG spectroscopy to study changes in the secondary structure of hen egg-white lysozyme (HEWL) in the presence of the negatively charged surface of DPPG lipids at various pH values. Hen egg-white lysozyme is a well-studied globular protein with a well-defined three-dimensional structure and high thermodynamic stability.15 In terms of structure and the charge properties, HEWL is homologous to human lysozyme (HL), whose mutations are linked to non-neuropathic systemic amyloidosis.16 There is evidence that the amyloidogenic lysozyme mutants share the same sequence region which has been found to be the most aggregation-prone segment of HEWL.17 Lysozymes have been widely used as a model protein to study the mechanism of fibril formation. Lysozyme fibrils and short oligomeric species can be formed in vitro under extreme conditions, such as in highly concentrated alcoholic solutions,18 acidic pH (1.6−2.0)19 and elevated temperatures.15 Under these conditions, matured fibrils are formed over a period of several days or few weeks. CD and FTIR data show a significant decrease in α-helix content and an increase in β-sheet content during the process of fibril formation.20 The results of fluorescence studies suggest aggregation of lysozyme upon association with negatively charged phospholipids.4 Cell membranes may be involved in the oligomerization and subsequent fibrils formation by amyloidogenic proteins. Studies on model amyloidogenic proteins and lipid vesicles have shown that fibril formation is largely determined by the composition of the cell membrane and the nature of protein−lipid interactions. In particular, anionic phospholipids are known to enhance fibril formation in several amyloid-forming proteins such as αsynuclein, Aβ protein, lysozyme, and insulin.21,22 HEWL is a strongly basic protein (pI = 11.0) possessing a net positive charge over a broad pH range. Its binding to negatively charged lipids is mainly driven by electrostatic interactions.23 Some results indicate also the existence of hydrophobic interactions of lysozyme with the lipids.24 Studies on lipid vesicles and lipid monolayers show extensive destabilization of the membrane surface and membrane leakage in the presence of adsorbed lysozyme, features associated with its antimicrobial properties.25 Both pH and ionic strength have been reported to affect the physicochemical properties of both lysozyme and the regions of lipid membrane involved in the interaction.23 A growing number of studies reveal that the association of lysozyme with membranes is a multistep process, including (1) initial adsorption at the membrane surface driven by electrostatic and hydrophobic interactions, (2) partial insertion into the hydrophobic core of the lipid bilayer, (3) alteration of protein conformation, and (4) reorganization of the lipid phase, possibly due to membrane dehydration.4,24 The present study examines the conformational stability of lysozyme upon interaction with anionic lipids using VSFG and CD. A distinct conformational change of lysozyme bound to DPPG lipids was observed at low pH (pH < 3). The pH-driven refolding was not observed in bulk solution or at the bare air−

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EXPERIMENTAL SECTION

Materials. A powder of lysozyme from hen egg-white (molecular weight 14 300 Da) was purchased from Sigma-Aldrich (Catalog. No. L6876). It contained 10% of acetate buffer. Originally, 1 g of lysozyme powder was dissolved in 10 mL of NaCl (0.01 M, prepared with MilliQ water passed through a 0.22 μm pore-size filter). The solution was centrifuged at 3000 rpm for 5 min and then transferred onto dialysis membrane (MWCO 8000) and dialyzed against 1 L of Milli-Q water at 4 °C, while stirring. After 2 h, the water was changed, and dialysis continued for another 2 h. The resultant lysozyme concentration of the stock solution was 25 mg mL−1, determined spectrophotometrically at 280 nm using an extinction coefficient ε = 36 000 M−1 cm−1. The 1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-1-glycerol) (DPPG) lipid (Avanti No.840455, Tc = 41 °C) was purchased from Avanti Polar Lipids Inc. (Alabama, AL), and used without further purification. The lipid was dissolved in a mixture (70:30 v/v) of chloroform (Fisher Chemicals) and methanol (Acros Chemicals) to a total concentration of ∼1.4 mM. The subphase for the VSFG studies was composed of D2O (Cambridge Isotope Laboratories, Inc., 99.93% purity, used without further purification). Hydrochloric acid (Wako, 35-37%) was used to adjust the pH. All reported pH values are uncorrected meter readings. Lipid Monolayers. All experiments were performed on lipid monolayers at room temperature (22 °C) in a commercial microtrough (Delta Pi, Kibron, Finland, 80 mm in diameter). Droplets of lipid solutions were spread onto a D2O subphase (19 mL) using a Hamilton microsyringe equipped with a repeating dispenser. The surface pressure was measured with a Dynaprobe instrument (Kibron, Finland), the tip of which consisted of a thin metal wire. The surface pressure was adjusted by changing the amount of lipid solution that was spread at the air−water interface. No barrier was used in the experiment. The monolayer was allowed to relax and experiments were performed after the surface pressure reached a constant value (typically after 3 min). The initial monolayer surface pressure (before injection of lysozyme) was set in the range of 15−25 mN m−1, corresponding to a lipid condensed phase.26 According to the pressure vs surface area isotherms for DPPG, the molecular density of lipid at a surface pressure of 20 mN m−1 is about 1.7 × 1014 lipid molecules/ cm2. For the trough used in the experiments, the number of molecules corresponding to one monolayer is ∼8.4 × 1017. After equilibration, VSFG spectra were recorded on pure lipid monolayers and after injection of lysozyme into the aqueous and pH-adjusted subphases. 20−25 μL of 25 mg mL−1 lysozyme solution (in water) was injected beneath the lipid monolayer into 19 mL volume trough using a Hamilton microsyringe. The theoretical 1 ML saturation coverage is 20 lipid molecules per protein corresponding to 4.2 × 1016 protein molecules.4 Surface Activity of Lysozyme. Lysozyme is a globular protein with rather low surface activity at low concentrations (< 0.035 mM).27 For this reason the amount of the stock lysozyme solution used in experiments on a pure water subphase was higher (max. 870 μL in 19 mL volume) than the amount used in experiments on lipids monolayers. With this amount of lysozyme the monolayer surface pressure reached by lysozyme was ∼13 mN m−1. Circular Dichroism. Far-UV CD spectra were recorded with a circular dichroism spectropolarimeter J-815 (JASCO Germany GmbH, Gross-Umstadt, Germany). Samples were maintained at 20 °C in quartz cuvettes with a 1 mm path length. Spectra were obtained from 195 to 260 nm and averaged over five scans. No data corrections were performed on the presented CD spectra. Vibrational Sum-Frequency Generation (VSFG). The VSFG setup has been described previously.28 Briefly, a Ti:sapphire fs-laser oscillator (MaiTai, Spectra-Physics) and a regenerative amplifier (SpitFire PRO, Spectra-Physics) pumped by a Nd:YLF laser (EMPower, Spectra-Physics) were used to generate a 4.85 mJ pulse 7737

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at 800 nm with a 40 fs duration at a repetition rate of 1 kHz. One part of the output was used to pump an optical parametric amplifier (OPA) system (TOPAS, Light Conversion) to generate IR pulses tunable from 3.1 to 6.1 μm. The second part of the amplifier output was passed through a Fabry-Perot etalon to generate a narrow band pulse (∼15 cm−1), which was used as the visible pulse for sum-frequency generation. The visible and IR beams were spatially and temporally overlapped on the sample surface with incident angles of 36° and 41°, respectively, with respect to the surface normal. For the amide region, the IR pulse energy was 1.8 mW, and for the O−D stretch region it was 4.2 mW. The visible pulse energy was 20 mW. The VSFG signal was focused into a spectrograph (Acton Spectra Pro 2300i, Princeton Instruments) in which it was dispersed, via a grating, and focused onto an electron multiplied charge coupled device (emCCD) camera (Newton, Andor). All VSFG spectra were recorded under ssp polarization conditions (SFG and visible s, IR p polarizations). In the present study, VSFG measurements were recorded in the amide I region 1500−1800 cm−1, O−D stretching region 2400−2800 cm−1, and C−H stretching region 2800−3100 cm−1. D2O was used since the setup was found to perform better at O−D than at O−H stretch frequencies. The use of D2O also avoids the overlap between the amide I band and the H2O bending mode. The SFG sample area and the IR beam path were flushed with nitrogen to avoid artifacts due to absorption of the IR light by water vapor. All the VSFG spectra were normalized using reference spectra obtained from z-cut quartz. The measured VSFG intensity is proportional to the square of the secondorder nonlinear susceptibility χ(2) of the sample and the intensities of the visible and infrared beams.

ISFG ∝ |χ (2) |2 IVISIIR

Figure 1. Far-UV CD spectra of hen egg-white lysozyme in D2O at different pH values. Spectra were taken at 20−22 °C with lysozyme concentration of 18.5 μM in D2O.

solution structure of lysozyme we employed VSFG to investigate the lysozyme structure at interfaces. Conformation of Lysozyme at the Air/Water and Lipid/Water Interfaces: VSFG in the Amide I Region. The assembly and conformation of lysozyme was first monitored at the air/water interface. 700 μL of lysozyme solution (25 mg mL−1) was injected into a trough containing 19 mL of D2O, to give a final concentration of 7 × 10−5 M. No lag time for the adsorption of lysozyme at the interface was observed at this concentration, consistent with the report by Alahverdjieve et al. and contrary to experiments performed at lower concentrations.27,34 Upon lysozyme injection, the surface pressure reached 13 mN m−1 indicating adsorption of lysozyme at the interface. Figure 2a shows a VSFG spectrum of lysozyme at the air/D2O interface in the amide I region. The spectrum has a broad peak with a maximum at ∼1660 cm−1 indicative of a high α-helical content in the lysozyme secondary structure adsorbed at the interface.3,11 According to X-ray diffraction35 and FTIR36 studies, the secondary structure of the native form of lysozyme consists of 40% α-helices, 7% β-sheets, 40% turns, and 13% unordered structures. Thus, the appearance of the peak at ∼1660 cm−1 indicates there are no significant changes in the lysozyme secondary structure when adsorbed at the air/water interface. The observation of a strong amide band in the VSFG spectrum implies that lysozyme molecules tend to orient at the air/water interface, since molecular order is a prerequisite for SFG detection; for a disordered monolayer, the signals from oppositely oriented CO dipoles would cancel out in the far field. This is in accordance with neutron reflectivity results which show that at concentrations above 10−6 M and at neutral pH lysozyme is adsorbed in the edge-on conformation, exposing its hydrophobic residues to air.37 Figure 2b shows VSFG spectra in the amide I region for 20 μL of lysozyme solution (25 mg mL−1) injected into the 19 mL D2O subphase of a liquid-condensed layer of DPPG at pH = 6.0. The surface pressure increased from 20 to 33 mN m−1 upon lysozyme injection, indicating that lysozyme penetrates the lipid monolayer. A strong amide I protein signal was observed, despite the fact that only 3% of the amount of lysozyme used at the air/water interface was injected. The signal strength indicates a much higher affinity of lysozyme toward DPPG than to the air/water interface. The higher SFG signal for lysozyme at the DPPG/water interface may in part

(1)

When the frequency of the incident infrared field is resonant with the vibrational mode n, the VSFG field can be resonantly enhanced. Thus, the susceptibility χ(2) consists of nonresonant (NR) and resonant (RES) terms: (2) (2) χ (2) = χNR + χRES = ANR eiφNR +

∑n

An ωn − ωIR − i Γn

(2)

where ANR represents the amplitude of the nonresonant susceptibility, φNR is its phase, An is the amplitude of the nth vibrational mode with resonant frequency ωn, and Γn is the line width of the vibrational transition.29 Equation 2 was used to fit the measured VSFG spectra. All fittings parameters have been summarized in the Supporting Information.

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RESULTS Conformation of Lysozyme in the Bulk Water Solutions: Circular Dichroism Study. The far-UV CD spectra, at pH = 2 and 6, are shown in Figure 1. The spectra were taken at lysozyme concentration of 18.5 μM in D2O. There are no apparent significant differences between the two CD spectra, both characterized by minimum at 207 nm which is characteristic of proteins with a high content of α-helix in their secondary structure and are consistent with the CD spectra of native lysozyme.30,31 CD spectra for lysozyme fibrils, formed from a native lysozyme exposed to ethanol solutions of high concentration, have been characterized by a single negative peak with minimum at ∼215 nm, which is correlated with the β-sheet-rich structure.18 By comparison, the CD spectrum of heat-denaturated lysozyme shows a minimum ellipticity at 202 nm, associated with the random-coil structure.32 Given these assignments, we conclude there is no major secondary structure change of the lysozyme under the reported experimental conditions. The aggregation of the lysozyme in the presence of DPPG lipid micelles were not studied due to a known difficulty in separating micelle curvature-induced interactions from direct attractions between proteins.33 After investigating the bulk 7738

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Figure 2. VSFG spectra in the CO stretch region for (a) a pure lysozyme monolayer at a solution concentration of 64 μM, (b) a DPPG monolayer in the presence of lysozyme at a solution concentration of 2 μM and pH = 6.0, and (c) a pure DPPG monolayer. The monolayer surface pressure is indicated in the upper right-corner of each graph. The solid lines represent fits to the data using a Lorentzian model.

Figure 3. VSFG spectra in the CO stretch region for (a) a pure lysozyme monolayer at a solution concentration of 80 μM at pH = 3; (b) a DPPG monolayer in the presence of lysozyme at a solution concentration of 2.3 μM and pH = 3.0; and (c) a DPPG monolayer in the presence of lysozyme at a solution concentration of 2.3 μM and pH = 1.5 (monolayer surface pressure and data fit cf. Figure 2).

that observed for lysozyme at pH = 6.0 (Figure 2a), indicating there is no change in the lysozyme secondary structure. In addition, a broad peak is observed at 1750 cm−1, which can be assigned to the CO vibrations of protonated aspartic acid.42 DPPG remains negatively charged at pH = 3. 0, since the pKa = 1.9 of the phosphate group in the DPPG molecule. The pKa values of major acidic amino acid residues of lysozyme are above 3; thus at pH = 3.0 they are predominately protonated. CD spectra, taken at the same pH conditions, are consistent with these VSFG results and show that lysozyme mostly preserves its native conformation, as also evident from Figure 1. Interestingly, the secondary structure of membrane-bound lysozyme changed dramatically when the pH was reduced to 3 or less (at bulk lysozyme concentrations of 0.03 mg mL−1). We observed a sharp transition (within minutes) from the α-helix to the β-sheet structure. The VSFG spectrum in the amide I region is shown in Figure 3b for pH = 3.0, revealing a pronounced peak at 1685 cm−1, attributed also here to small amorphous aggregates of lysozyme.38,40 No significant change of the monolayer surface pressure was observed, suggesting that these oligomers are formed between the lysozyme molecules incorporated into lipid layer and those peripherally adsorbed, whose concentration is higher at low pH. Further decrease of the pH value to 1.5 (Figure 3c) causes a drop of the signal intensity for both aggregates and lipids, suggesting departure of lipid−lysozyme aggregates domains from the interface. Lipids Dehydration upon Lysozyme Oligomerization: VSFG in the O−D Stretch Region. Water molecules play an important role in modulating the interactions of proteins with lipids. Interfacial water molecules in the hydration shells of lipid headgroups are characterized by an altered geometry and hydrogen bonding strength compared to bulk water molecules. A significant restructuring of water in the hydration layer of lipids is expected to occur when proteins approach the lipid

also result from the protein molecules being more ordered. The increased alignment is most likely explained by attractive electrostatic interactions between the negatively charged headgroup of DPPG and the positively charged part of the lysozyme (lysozyme carries a net positive charge at pH values below the isoelectronic point pI = 11.3). In addition, hydrophobic interactions are likely to further promote protein alignment. The spectrum in Figure 2b has been fitted with two peaks in the 1650−1690 cm−1 frequency region: one with a maximum at 1660 cm−1 and another at 1685 cm−1. The second peak, observed as a shoulder at 1685 cm−1, has been assigned to the antiparallel β-sheets content.11 The latter feature may be attributed to native β-sheet content exposed to the air interface upon binding to the lipids, or it may originate from protein aggregates.38 Experiments performed at lower pH (presented below) suggest the second explanation is more likely here. A peak at 1685 cm−1 has been observed in oligomeric species of Aβ-(1-42) peptide,1 human islet amyloid peptide,39 and human serum albumins.40 The observation of this peak thus indicates that in the presence of negatively charged lipids a fraction of lysozyme undergoes aggregation. A third peak observed in Figure 2b, found at 1732 cm−1, is assigned to the CO stretch vibration of the lipid ester group, as referenced by the spectra of a pure DPPG monolayer, shown in Figure 2c. The frequency shift of the lipid carbonyl group in different environments does not exceed 10 cm−1 and thus is not expected to overlap with the amide I group frequencies of the protein.41 The aggregation of lysozyme was then investigated at acidic pH, at which lysozyme is known to form elongated amyloid fibrils.19 Spectrum (a) in Figure 3 shows the VSFG signal of the amide I region for 870 μL of lysozyme solution (25 mg mL−1) injected into 19 mL of D2O subphase at pH = 3.0 (adjusted with HCl of 120 mM ionic strength). The spectrum resembles 7739

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experiments at pH values as low as 1.5 (the data for this experiment are included in the Supporting Information). Figure 4b shows the O−D region spectra at the DPPG/water interface. Recent experimental and theoretical efforts have shown that the water molecules experience a quite heterogeneous hydrogen-bonding environment at the lipid water interface,44,45 which explains the large width of the SFG resonances. In comparison to the pure water surface the intensity of the signal in the hydrogen-bonded O−D stretch of interfacial water around DPPG is strongly enhanced. This enhancement has been explained as being due to the ordering of water dipoles by lipid headgroup charges.46 In addition to various types of hydrogen-bonded water molecules, the dangling OD has also been found to be present at the lipid/ water interfaces.43 In Figure 4b this band is observed as a small feature near 2730 cm−1. Figure 4c shows a vibrational response in the O−D stretch region recorded at the DPPG/water interface in the presence of lysozyme. The intensity of the signal has decreased over all frequency regions with the highest decrease observed on the strongly hydrogen-bonded side of the spectrum, related to water molecules associated with the lipid phosphate group.44 This decrease of the signal intensity may be the result of screening of the negative phosphate charges by positively charged lysozyme sites which decreases the net order in the adjacent water layer. A similar decrease of the interfacial water signal was also observed upon addition of HCl into the subphase below the DPPG monolayer, as shown in Figure 4d. In analogy to lysozyme, this decrease may be caused by an accumulation of protons near the negatively charged lipid layer and/or by dehydration of lipid headgroups. Figure 4e shows the vibrational spectrum in the OD region for the DPPG/water interface in the presence of lysozyme at pH = 3.0. The signal in the hydrogen-bonded OD region has almost completely disappeared, with only some dangling OD intensity remaining. Although a decrease of the signal in this region is also observed for the DPPG/water interface at pH 3.0 (Figure 4d), the nearcomplete disappearance of the signal is only observed when lysozyme is present at the lipid surface. There are two effects that can cause a decrease in the interfacial water signal in the SFG spectrum. One is the loss of preferential orientation of water molecules due to accumulation of protons or positively charged lysozyme molecules at the surface, screening the negative charges in the DPPG layer. The other is a significant loss of water molecules associated with the lipid headgroups through displacement of water by lysozyme at the membrane surface. These effects may both be associated with the lysozyme aggregation apparent from the VSFG signal in the amide region (Figure 3b). Studies with fluorescently labeled lipids show that the association of lysozyme with phospholipid vesicles is accompanied by membrane surface dehydration.23,47 Gramicidin A, for example, has been reported to induce dehydration of the lipid headgroups upon insertion into bilayers.48 Dehydration of lipids can also affect the melting transition and thus result in a change of molecular organization of the lipids within a membrane. Using VSFG, the dehydration of lipids can be further investigated by studying lipid organization in the monolayer. Lysozyme-Induced Lipids Reorganization: SFG in the C−H Stretch Region. Depending on the temperature, lipid monolayers can exist in a liquid expanded or a liquid condensed (gel) phase. These phases are characterized by a different molecular packing density and a different lipid order. Lipid

membrane surface. This restructuring may involve changes in (1) the binding geometry of water molecules, (2) the distribution of hydrogen bond strengths, and (3) the number of hydrating water molecules. Vibrational spectroscopies allow probing of the hydrogenbonding network of water molecules via the O−H stretching modes, which are strongly affected by the hydrogen bond strength. Lower frequency indicates stronger hydrogen bonding. Before discussing the vibrational characteristics of water around DPPG and lysozyme, the features of the air/heavy water (D2O) interface, shown in Figure 4a, are first

Figure 4. VSFG spectra in the O−D stretch region for (a) pure D2O, (b) a pure DPPG monolayer, (c) a DPPG monolayer in the presence of lysozyme at a solution concentration of 2.3 μM and pH = 6.0, (d) a DPPG monolayer at pH = 3.0, and (e) a DPPG monolayer in the presence of lysozyme at a solution concentration of 2.3 μM and pH = 3.0 (monolayer surface pressure and data fit cf. Figure 2).

summarized. The spectrum shows peaks in two frequency regions: a narrow peak observed at higher frequencies with maximum at ∼2745 cm−1 and a broad feature at lower frequencies. The peak centered at 2745 cm−1 has been assigned to the free OD stretch, pointing into the gas phase (so-called “dangling OD” or free OD).43 The double-peak feature at lower energies originates from hydrogen-bonded OD groups. No change in the shape of water spectrum was observed in control 7740

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monolayers serve as good, relatively simple models to study the molecular properties of lipids in various phases. VSFG can report on lipid tail order by monitoring the intensity ratio of the methyl and the methylene groups in the lipid alkyl chains. When the lipids tails are ordered, such as in the condensed phase, mainly CH3 modes are observed and the intensity of CH2 stretch modes is weak.49 The CH2 intensity is low for ordered, all-trans chains because the local symmetry renders the CH2 modes SFG inactive. However, in the liquid expanded phase, gauche defects are present which cause the symmetry within the alkyl chain to be broken, and the relative intensity of the CH2 symmetric stretch mode increases. In parallel, the ensuing disorder reduces the methyl stretch intensity. Figures 5a and 5b show the VSFG spectra for a DPPG monolayer spread at the air/water interface at pH= 6.0 and 3.0,

The injection of lysozyme into the subphase below the DPPG monolayer (Figure 5c) causes the signal intensity to decrease over the entire frequency range, which is typical of an overall decrease of the lipid order at the interface. Also, distinct spectral changes occur, most notably an increase in the CH2 symmetric stretch at 2855 cm−1 and an accompanying decrease in the ∼2881 cm−1 peak intensity due to the symmetric CH3 stretch mode. These observations show that penetration of lysozyme into the lipid monolayer causes disruption of the lipid order, despite the observed increase in the surface pressure. Interestingly, the addition of HCl into the subphase below the DPPG/lysozyme layer partly restores the lipid order that has been disrupted by initial binding of lysozyme (Figure 5d): the intensity of the CH3 stretch is now again higher than the intensity of the CH2 stretching mode and the spectrum resembles that of the initial DPPG layer shown in Figure 5a.

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DISCUSSION A growing number of studies suggest that the formation of amyloid fibers and their cytotoxic effects are membrane-related processes.4,22 Membranes, in particular those containing anionic lipids, have been demonstrated to catalyze fibril formation. Gorbenko et al. have summarized four major factors that are likely relevant to understand membrane-mediated protein association.50 These are (1) alteration of the protein conformation upon contact with the lipids, (2) accumulation of protein at the lipid/water interface, (3) specific orientation of proteins upon association with the charged lipids, and (4) templating effects of the membrane. Here, we find spectroscopic evidence for a membrane-induced change of the lysozyme secondary structure. Membrane-induced conformational changes of proteins have been usually explained by a reduction of the pH at interfaces. At low pH, the increased charge at increasingly protonated basic protein sites may increase side chains charge repulsions and lead to exposure of the hydrophobic, aggregation-prone sites to the solvent. Recent electronic sum frequency generation study indeed show that value of pH at the DPPG/water interface is around 1.5 units lower than the pH of the bulk solution.51 This explanation however is not sufficient to explain aggregation of lysozyme whose bulk secondary structure has been found to be largely unperturbed even at pH = 0.6.52 While one may expect the conformational stability of lysozyme at the interface to be different, the results presented here show that lysozyme adsorbed at the air/water interface maintains a mostly native conformation down to pH = 1.5 - the lowest pH studied in this work. At pH < 3 hydrophilic lysozyme sites are likely protonated, and indeed, the peak in Figure 3a near 1750 cm−1 indicates protonated aspartic acid.42 Though the side chain repulsions are present, lysozyme molecules do not undergo association in the absence of lipids. Together, this suggests membrane effects beyond interfacial pH changes have to be considered to fully understand membrane-induced aggregation. In the present study, oligomerization of lysozyme was observed at low pH and was associated with extensive lipids dehydration and an increase in lipid order. At low pH, more lysozyme molecules may accumulate near negatively charged lipid head groups due to an increased positive charge of lysozyme. These extra lysozyme molecules do not incorporate into the lipid monolayer as no change in the surface pressure was observed (Figure 3b,c). They likely adsorb at the peripheral sites of lipids and cause dehydration of lipid head groups, a

Figure 5. VSFG spectra in the C−H stretch region for (a) a pure DPPG monolayer; (b) the same DPPG monolayer in the presence of lysozyme at a solution concentration of 2.3 μM, and pH = 3.0; (c) a DPPG monolayer at pH = 6.0; and (d) a DPPG monolayer in the presence of lysozyme at a solution concentration of 2.3 μM and pH = 3.0 (monolayer surface pressure and data fit cf. Figure 2).

respectively. Three peaks can be seen in both spectra. The most intense peak, at 2881 cm−1, is assigned to the symmetric CH3 stretch mode of the terminal CH3 group and a broad peak at around 2945 cm −1 to the CH3 Fermi resonances in combination with contributions from the asymmetric CH3 stretch mode.49 A third, weaker resonance at ∼2855 cm−1 is due to the symmetric CH2 stretch mode. The observation of a strong CH3 stretch vibration indicates a large degree of order of the lipid alkyl chains within the monolayer. This is expected as at the monolayer surface pressure of π = 20 mN m−1, DPPG is in a liquid condensed phase at room temperature.26 7741

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two parameters controlling protein association.56 Our integrated spectroscopic observation is in accordance with the models, where a change of lipid order is a decisive factor leading to protein aggregation. The toxicity of oliogomers on membranes has been considered to be similar to the action of antimicrobial peptides.57 Lysozyme has a known antimicrobial activity against Gram-negative and Gram-positive bacteria.58 It causes membrane permeabilization, but the exact mechanism of membrane disruption is still under debate.57,58 The results of this study suggest that lysozyme disrupts lipid membrane by formation of protein oligomers/pores within the lipid matrix as shown in Figure 7. During lipid reorganization, followed by protein

process typically associated with an increase of the lipid order. As the lipid order increases, the area occupied by one lipid molecule decreases, thus decreasing also the distance between neighboring lysozyme molecules anchored inside the lipid matrix. The peripherally adsorbed proteins may thus act as coupling units, connecting proteins inclusions in the membrane. Recent dissipative particle dynamic (DPD) simulations have demonstrated that attractive protein−protein interactions can be a consequence of lipid reorientation and a decrease of a distance between proteins.53 In Figure 6 we draw a schematic

Figure 6. Cartoon schematics of lysozyme and lipid monolayers under different experimental conditions: (a) organization of DPPG lipids and lysozyme at the air/water interface; (b) disordered lipid monolayer due to lysozyme binding and penetration into lipids; (c) oligomers formation and reordering of the lipid monolayer at pH = 3.0. Threedimensional structure of lysozyme was prepared by using the program KING (http://kinemage.biochem.duke.edu) with structure coordinates (PDB ID 6LYZ) file downloaded from the Protein Data Bank.

Figure 7. Cartoon schematics showing lipid-mediated lysozyme oligomerization and pores formation: (a) inserted and peripheral monomers; (b) oligomeric pores.

oligomerization, some of the lipid molecules may be trapped inside the pore. Pores formed in the lipid matrix may cause membrane permeablization and leakage. A fraction of the micellized protein−lipid ensemble may also detach from the membrane surface. Such detachment would account for our observation of a decrease of the monolayer surface pressure and the intensity of SFG signals, upon protein oligomerization (Figure 3c). The above mechanism is consistent with the observation of isolated amyloid fibrils containing up to 10% of lipids.59 Three major models have been proposed to explain permeability-increasing effects of AMPs on lipid membranes. The barrel-stave and the toroidal models explain membrane leakage by formation of transmembrane pores and the carpet model by membrane micellization relevant at high concentration of a surface-bound peptide.60 All three models assume that protein oligomerization is initiated by proteins present at the membrane periphery. Interestingly, the results presented here suggest a more cooperative action between lysozyme incorporated into the membrane and lysozyme bound to the membrane periphery.

cartoon summarizing a tentative sequence of molecular events leading to lysozyme oligomerization at the DPPG monolayer at low pH. Figure 6a shows the initial ordering of DPPG and lysozyme at the water interface. Then lysozyme incorporated at high pH leads to lipid disordering (b) and at low pH coupling of protein inclusions due to dehydration-induced lipid reorganization (c). Several theoretical studies have modeled lipid-mediated protein aggregation at different levels of physical complexity. The effective lipid-mediated protein interactions were first calculated by Marĉelja.54 In his model, the protein was treated as a rigid annulus which disturbs the order of phospholipids in direct contact with protein. Marĉelja’s model showed that the change in the lipid order gives rise to an indirect lipid mediated attractive interaction between membrane proteins. Subsequent simulations by Lagüe et al. calculated the potential of mean force between two protein inclusions in the lipid matrix.55 The results showed that at a longer distance two inclusions first experience a repulsive interaction followed by attraction at closer distances. The change in the average hydrocarbon density around proteins was postulated to give rise to the lipidmediated protein interactions. Recent calculations by Yoo et al. revealed the importance of both the distance between the two inclusions and the degree of the hydrophobic mismatch as the

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SUMMARY The present study investigates the secondary structure of lysozyme and molecular events leading to its aggregation at the interface of negatively charged DPPG lipid monolayers at various pHs using VSFG. The results show that lysozyme molecules insert into the DPPG monolayer and form an 7742

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(5) Schleeger, M.; vandenAkker, C. C.; Deckert-Gaudig, T.; Deckert, V.; Velikov, K. P.; Koenderink, G.; Bonn, M. Amyloids: From molecular structure to mechanical properties. Polymer 2013, 54 (10), 2473−2488. (6) Weidner, T.; Breen, N.; Li, K.; Drobny, G.; Castner, D. Sum frequency generation and solid-state NMR study of the structure, orientation, and dynamics of polystyrene-adsorbed peptides. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (30), 13288−13293. (7) Weidner, T.; Castner, D. G. SFG analysis of surface bound proteins: a route towards structure determination. Phys. Chem. Chem. Phys. 2013, 15 (30), 12516−12524. (8) Fu, L.; Liu, J.; Yan, E. C. Y. Chiral sum frequency generation spectroscopy for characterizing protein secondary structures at interfaces. J. Am. Chem. Soc. 2011, 133 (21), 8094−8097. (9) Rzeźnicka, I. I.; Sovago, M.; Backus, E. H. G.; Bonn, M.; Yamada, T.; Kobayashi, T.; Kawai, M. Duramycin-induced destabilization of a phosphatidylethanolamine monolayer at the air−water interface observed by vibrational sum-frequency generation spectroscopy. Langmuir 2010, 26 (20), 16055−16062. (10) Kim, G.; Gurau, M.; Kim, J.; Cremer, P. S. Investigations of lysozyme adsorption at the air/water and quartz/water interfaces by vibrational sum frequency spectroscopy. Langmuir 2002, 18 (7), 2807−2811. (11) Chen, X.; Wang, J.; Sniadecki, J. J.; Even, M. A.; Chen, Z. Probing α-helical and β-sheet structures of peptides at solid/liquid interfaces with SFG. Langmuir 2005, 21 (7), 2662−2664. (12) Chen, X.; Boughton, A. P.; Tesmer, J. J. G.; Chen, Z. In situ investigation of heterotrimeric G protein βγ subunit binding and orientation on membrane bilayers. J. Am. Chem. Soc. 2007, 129 (42), 12658−12659. (13) Fu, L.; Ma, G.; Yan, E. C. Y. In situ misfolding of human islet amyloid polypeptide at interfaces probed by vibrational sum frequency generation. J. Am. Chem. Soc. 2010, 132 (15), 5405−5412. (14) Yang, P.; Wu, F.-G.; Chen, Z. Dependence of alamethicin membrane orientation on the solution concentration. J. Phys. Chem. C 2013, 117 (7), 3358−3365. (15) Sasahara, K.; Yagi, H.; Naiki, H.; Goto, Y. Heat-induced conversion of β2-microglobulin and hen egg-white lysozyme into amyloid fibrils. J. Mol. Biol. 2007, 372 (4), 981−991. (16) Booth, D. R.; Sunde, M.; Bellotti, V.; Robinson, C. V.; Hutchinson, W. L.; Fraser, P. E.; Hawkins, P. N.; Dobson, C. M.; Radford, S. E.; Blake, C. C. F.; Pepys, M. B. Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 1997, 385 (6619), 787−793. (17) Frare, E.; Polverino de Laureto, P.; Zurdo, J.; Dobson, C. M.; Fontana, A. A highly amyloidogenic region of hen lysozyme. J. Mol. Biol. 2004, 340 (5), 1153−1165. (18) Goda, S.; Takano, K.; Yutani, K.; Yamagata, Y.; Nagata, R.; Akutsu, H.; Maki, S.; Namba, K. Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution. Protein Sci. 2000, 9 (2), 369−375. (19) Sasahara, K.; Yagi, H.; Sakai, M.; Naiki, H.; Goto, Y. Amyloid nucleation triggered by agitation of β2-microglobulin under acidic and neutral pH conditions. Biochemistry 2008, 47 (8), 2650−2660. (20) Holley, M.; Eginton, C.; Schaefer, D.; Brown, L. R. Characterization of amyloidogenesis of hen egg lysozyme in concentrated ethanol solution. Biochem. Biophys. Res. Commun. 2008, 373 (1), 164−168. (21) Kayal, A. T.; Nappini, S.; Russo, E.; Berti, D.; Bucciantini, M.; Stefani, M.; Baglioni, P. Lysozyme interaction with negatively charged lipid bilayers: protein aggregation and membrane fusion. Soft Matter 2012, 8, 4524−4534. (22) Zhao, H.; Tuominen, E. K. J.; Kinnunen, P. K. J. Formation of amyloid fibers triggered by phosphatidylserine-containing membranes. Biochemistry 2004, 43 (32), 10302−10307. (23) Zschörnig, O.; Paasche, G.; Thieme, C.; Korb, N.; Arnold, K. Modulation of lysozyme charge influences interaction with phospholipid vesicles. Colloids Surf., B 2005, 42 (1), 69−78.

ordered layer. Electrostatic interactions between lipid anionic polar head groups and positively charged amino acid residues as well as hydrophobic interactions are responsible for protein insertion into the lipid layer. At pH = 6 the majority of the inserted lysozyme is in a monomer form and has a native αhelix conformation. In addition to monomers, lysozyme oligomers coexist at the lipid periphery, consistent with the two-state adsorption model (monomers and z-mers). At pH = 3, lysozyme molecules bound to the DPPG layer change their secondary structure to the antiparallel β-sheet structure, associated with the formation of oligomers at the lipid surface. This refolding is mediated by the membrane and does not occur for unbound lysozyme in solution. The results show that oligomerization is a cooperative action between proteins incorporated into the lipid membrane and peripherally adsorbed proteins and is mediated by a change of lipid order due to dehydration occurring at high protein concentration.

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ASSOCIATED CONTENT

S Supporting Information *

Tables S1−S4 and Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Ph +81-22-217-5199; Fax +81-22-217-5371 (I.I.R.). Present Address

I.I.R.: Institute of Multidisciplinary Research for Advanced Materials (IMRAM) Tohoku University, 2-1-1, Katahira, Aobaku, Sendai 980-0877, Japan. Author Contributions

I.I.R., R.P., and M.S. contributed equally to this work. I.I.R. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS I.I.R. thanks to the Suntory Foundation for Life Science (2011) for financial support via a Sunbor Grant. T.W. thanks the EU Marie-Curie PEOPLE Programme for a Career Integration Grant (322124) and the Deutsche Forschungsgemeinschaft (DFG) for financial support (WE4478/2-1). This work is also a part of the research program of the Max-Planck Society. I.I.R. thanks Dr. Shusuke Nambu, Tohoku University, for help in lysozyme dialysis.

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REFERENCES

(1) Cerf, E.; Sarroukh, R.; Tamamizu-Kato, S.; Breydo, L.; Derclaye, S.; Dufrêne, Y. F.; Narayanaswami, V.; Goormaghtigh, E.; Ruysschaert, J. M.; Raussens, V. Antiparallel β-sheet: a signature structure of the oligomeric amyloid β-peptide. Biochem. J. 2009, 421, 415−423. (2) Kirkitadze, M. D.; Bitan, G.; Teplow, D. B. Paradigm shifts in Alzheimer’s disease and other neurodegenerative disorders: The emerging role of oligomeric assemblies. J. Neurosci. Res. 2002, 69 (5), 567−577. (3) Sethuraman, A.; Vedantham, G.; Imoto, T.; Przybycien, T.; Belfort, G. Protein unfolding at interfaces: Slow dynamics of α-helix to β-sheet transition. Proteins: Struct., Funct., Bioinf. 2004, 56 (4), 669− 678. (4) Gorbenko, G. P.; Ioffe, V. M.; Kinnunen, P. K. J. Binding of lysozyme to phospholipid bilayers: Evidence for protein aggregation upon membrane association. Biophys. J. 2007, 93 (1), 140−153. 7743

dx.doi.org/10.1021/la5010227 | Langmuir 2014, 30, 7736−7744

Langmuir

Article

(24) Kimelberg, H. K.; Papahadjopoulos, D. Phospholipid-protein interactions: Membrane permeability correlated with monolayer “penetration”. Biochim. Biophys. Acta 1971, 223 (3), 805−809. (25) Gorbenko, G.; Trusova, V. Effects of oligomeric lysozyme on structural state of model membranes. Biophys. Chem. 2011, 154 (2−3), 73−81. (26) Majerowicz, M.; Waring, A. J.; Wen, S.; Bringezu, F. Interaction of the antimicrobial peptide dicynthaurin with membrane phospholipids at the air−liquid interface. J. Phys. Chem. B 2007, 111 (14), 3813−3821. (27) Lad, M. D.; Birembaut, F.; Matthew, J. M.; Frazier, R. A.; Green, R. J. The adsorbed conformation of globular proteins at the air/water interface. Phys. Chem. Chem. Phys. 2006, 8 (18), 2179−2186. (28) Smits, M.; Sovago, M.; Wurpel, G. W. H.; Kim, D.; Müller, M.; Bonn, M. Polarization-resolved broad-bandwidth sum-frequency generation spectroscopy of monolayer relaxation. J. Phys. Chem. C 2007, 111 (25), 8878−8883. (29) Hunt, J. H.; Guyot-Sionnest, P.; Shen, Y. R. Observation of C-H stretch vibrations of monolayers of molecules optical sum-frequency generation. Chem. Phys. Lett. 1987, 133 (3), 189−192. (30) Compton, L. A.; Johnson, W. C., Jr. Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication. Anal. Biochem. 1986, 155 (1), 155−167. (31) Sreerama, N.; Woody, R. W. Estimation of protein secondary structure from circular dichroism spectra: Comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal. Biochem. 2000, 287 (2), 252−260. (32) Arnaudov, L. N.; de Vries, R. Thermally induced fibrillar aggregation of hen egg white lysozyme. Biophys. J. 2005, 88 (1), 515− 526. (33) Reynwar, B. J.; Illya, G.; Harmandaris, V. A.; Müller, M. M.; Kremer, K.; Deserno, M. Aggregation and vesiculation of membrane proteins by curvature-mediated interactions. Nature 2007, 447, 461− 464. (34) Alahverdjieva, V. S.; Grigoriev, D. O.; Ferri, J. K.; Fainerman, V. B.; Aksenenko, E. V.; Leser, M. E.; Michel, M.; Miller, R. Adsorption behaviour of hen egg-white lysozyme at the air/water interface. Colloids Surf., A 2008, 323 (1−3), 167−174. (35) Frishman, D.; Argos, P. Knowledge-based protein secondary structure assignment. Proteins: Struct., Funct., Bioinf. 1995, 23 (4), 566−579. (36) Vedantham, G.; Sparks, H. G.; Sane, S. U.; Tzannis, S.; Przybycien, T. M. A holistic approach for protein secondary structure estimation from infrared spectra in H2O solutions. Anal. Biochem. 2000, 285 (1), 33−49. (37) Lu, J. R.; Su, T. J.; Thomas, R. K.; Penfold, J.; Webster, J. Structural conformation of lysozyme layers at the air/water interface studied by neutron reflection. J. Chem. Soc., Faraday Trans. 1998, 94 (21), 3279−3287. (38) Zurdo, J.; Guijarro, J. I.; Dobson, C. M. Preparation and characterization of purified amyloid fibrils. J. Am. Chem. Soc. 2001, 123 (33), 8141−8142. (39) Engel, M. F. M.; vandenAkker, C. C.; Schleeger, M.; Velikov, K. P.; Koenderink, G. H.; Bonn, M. The polyphenol EGCG inhibits amyloid formation less efficiently at phospholipid interfaces than in bulk solution. J. Am. Chem. Soc. 2012, 134 (36), 14781−14788. (40) de la Arada, I.; Seiler, C.; Mäntele, W. Amyloid fibril formation from human and bovine serum albumin followed by quasisimultaneous Fourier-transform infrared (FT-IR) spectroscopy and static light scattering (SLS). Eur. Biophys. J. 2012, 41 (11), 931−938. (41) Garidel, P.; Blume, A.; Hübner, W. A Fourier transform infrared spectroscopic study of the interaction of alkaline earth cations with the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol. Biochim. Biophys. Acta 2000, 1466 (1−2), 245−259. (42) Barth, A.; Zscherp, C. What vibrations tell about proteins. Q. Rev. Biophys. 2002, 35 (4), 369−430. (43) Ma, G.; Chen, X.; Allen, H. C. Dangling OD confined in a langmuir monolayer. J. Am. Chem. Soc. 2007, 129 (45), 14053−14057.

(44) Mondal, J. A.; Nihonyanagi, S.; Yamaguchi, S.; Tahara, T. Three distinct water structures at a zwitterionic lipid/water interface revealed by heterodyne-detected vibrational sum frequency generation. J. Am. Chem. Soc. 2012, 134 (18), 7842−7850. (45) Nagata, Y.; Mukamel, S. Vibrational sum-frequency generation spectroscopy at the water/lipid interface: Molecular dynamics simulation study. J. Am. Chem. Soc. 2010, 132 (18), 6434−6442. (46) Chen, X.; Hua, W.; Huang, Z.; Allen, H. C. Interfacial water structure associated with phospholipid membranes studied by phasesensitive vibrational sum frequency generation spectroscopy. J. Am. Chem. Soc. 2010, 132 (32), 11336−11342. (47) Zschörnig, O.; Paasche, G.; Thieme, C.; Korb, N.; Fahrwald, A.; Arnold, K. Association of lysozyme with phospholipid vesicles is accompanied by membrane surface dehydration. Gen. Physiol. Biophys. 2000, 19 (1), 85−101. (48) Killian, J. A.; De Kruijff, B. Thermodynamic, motional, and structural aspects of the gramicidin-induced hexagonal HII phase formation in phosphatidylethanolamine. Biochemistry 1985, 24 (27), 7881−7890. (49) Watry, M. R.; Tarbuck, T. L.; Richmond, G. L. Vibrational sumfrequency studies of a series of phospholipid monolayers and the associated water structure at the vapor/water interface. J. Phys. Chem. B 2002, 107 (2), 512−518. (50) Gorbenko, G. P.; Kinnunen, P. K. J. The role of lipid−protein interactions in amyloid-type protein fibril formation. Chem. Phys. Lipids 2006, 141 (1−2), 72−82. (51) Kundu, A.; Yamaguchi, S.; Tahara, T. Evaluation of pH at charged lipid/water interfaces by heterodyne-detected electronic sum frequency generation. J. Phys. Chem. Lett. 2014, 5 (4), 762−766. (52) Polverino de Laureto, P.; Frare, E.; Gottardo, R.; van Dael, H.; Fontana, A. Partly folded states of members of the lysozyme/ lactalbumin superfamily: A comparative study by circular dichroism spectroscopy and limited proteolysis. Protein Sci. 2002, 11 (12), 2932− 2946. (53) Morozova, D.; Weiss, M.; Guigas, G. Shape as a determinant of membrane protein cluster formation. Soft Matter 2012, 8 (47), 11905−11910. (54) Marčelja, S. Lipid-mediated protein interaction in membranes. Biochim. Biophys. Acta 1976, 455 (1), 1−7. (55) Lagüe, P.; Zuckermann, M. J.; Roux, B. Lipid-mediated interactions between intrinsic membrane proteins: Dependence on protein size and lipid composition. Biophys. J. 2001, 81 (1), 276−284. (56) Yoo, J.; Cui, Q. Membrane-mediated protein-protein interactions and connection to elastic models: A coarse-grained simulation analysis of gramicidin A association. Biophys. J. 2013, 104 (1), 128−138. (57) Trusova, V. Modulation of physiological and pathological activities of lysozyme by biological membranes. Cell. Mol. Biol. Lett. 2012, 17 (3), 349−375. (58) Ibrahim, H. R.; Thomas, U.; Pellegrini, A. A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action. J. Biol. Chem. 2001, 276 (47), 43767−43774. (59) Gellermann, G. P.; Appel, T. R.; Tannert, A.; Radestock, A.; Hortschansky, P.; Schroeckh, V.; Leisner, C.; Lütkepohl, T.; Shtrasburg, S.; Röcken, C.; Pras, M.; Linke, R. P.; Diekmann, S.; Fändrich, M. Raft lipids as common components of human extracellular amyloid fibrils. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (18), 6297−6302. (60) Huang, Y.; Huang, J.; Chen, Y. Alpha-helical cationic antimicrobial peptides: relationships of structure and function. Protein Cell 2010, 1 (2), 143−152.

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