Triggers for Î²-Sheet Formation at the HydrophobicâHydrophilic

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Triggers for β-Sheet Formation at the HydrophobicHydrophilic Interface: High Concentration, In-Plane Orientational Order, and Metal Ion Complexation Maria Hoernke,† Jessica A. Falenski,‡ Christian Schwieger,§,|| Beate Koksch,‡ and Gerald Brezesinski*,† †

Department of Interfaces, Max-Planck-Institute of Colloids and Interfaces, Potsdam, Germany Institute of Chemistry and Biochemistry - Organic Chemistry, Freie Universit€at Berlin, Berlin, Germany § Department of Chemistry, Martin-Luther-Universit€at Halle-Wittenberg, Halle (Saale), Germany UR1268 Biopolymeres Interactions Assemblages, Institut National de la Recherche Agronomique, Nantes, France

)

‡

bS Supporting Information ABSTRACT: Amyloid formation plays a causative role in neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease. Soluble peptides form β-sheets that subsequently rearrange into fibrils and deposit as amyloid plaques. Many parameters trigger and influence the onset of the β-sheet formation. Early stages are recently discussed to be cell-toxic. Aiming at understanding various triggers such as interactions with hydrophobichydrophilic interfaces and metal ion complexation and their interplay, we investigated a set of model peptides at the airwater interface. We are using a general approach to a variety of diseases such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes that are connected to amyloid formation. Surface sensitive techniques combined with film balance measurements have been used to assess the conformation of the peptides and their orientation at the airwater interface (IR reflectionabsorption spectroscopy). Additionally, the structures of the peptide layers were characterized by grazing incidence X-ray diffraction and X-ray reflectivity. The peptides adsorb to the airwater interface and immediately adopt an α-helical conformation. This helical intermediate transforms into β-sheets upon further triggering. The factors that result in β-sheet formation are dependent on the peptide sequence. In general, the interface has the strongest effect on peptide conformation compared to high concentrations or metal ions. Metal ions are able to prevent aggregation in bulk but not at the interface. At the interface, metal ion complexation has only minor effects on the peptide secondary structure, influencing the in-plane structure that is formed in two dimensions. At the airwater interface, increased concentrations or a parallel arrangement of the α-helical intermediates are the most effective triggers. This study reveals the role of various triggers for β-sheet formation and their complex interplay. Our main finding is that the hydrophobichydrophilic interface largely governs the conformation of peptides. Therefore, the present study implies that special care is needed when interpreting data that may be affected by different amounts or types of interfaces during experimentation.

’ INTRODUCTION Amyloids play an important role in various common diseases such as Alzheimer’s disease (AD) or Parkinson’s disease. They are products of misfolding of normally soluble peptides that transform into β-sheets and rearrange into fibrils that end up as amyloid deposits. Recently, the early stages of amyloid formation, oligomers, and the transition processes are discussed to impose the cytotoxicity.14 However, numerous factors influencing these processes are not fully understood to date. It is agreed that an increase in (local) peptide concentration results in a higher propensity for aggregation. Besides, it is agreed that small amounts of misfolded peptides and other templates are able to act as nuclei for further aggregation. Furthermore, interactions of peptides with membranes and other surfaces and interactions of peptides with metal ions are recently discussed as factors influencing β-sheet formation. These will be the topic of this Article. Many studies focus on interactions of amyloidogenic peptides with lipid membranes and deal with various lipids.57 However, r 2011 American Chemical Society

special emphasis has recently been put on hydrophobic surfaces among which the hydrophobichydrophilic airwater interface can be regarded as the simplest model. Hydrophobic surfaces are discussed as a possible cause for bad reproducibility of folding kinetics in different studies.813 In in vitro experiments aiming at other parameters than hydrophobic surfaces, the amount and nature of hydrophobic surfaces are usually not controlled. Air water interfaces occur in flasks and experimental setups or upon shaking, filling, and mixing of solutions. Consequently, together with the special properties of two-dimensional systems compared to bulk, the alignment or orientation of adsorbed peptides is investigated as an additional trigger for aggregation.6,8,14 Another major interest is in the influence of transition metal ion complexation to peptides, since Cu2+ and Zn2+ are, for example, Received: August 2, 2011 Revised: October 10, 2011 Published: October 19, 2011 14218

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Langmuir regularly found in AD deposits. Generally, Zn2+ is discussed as inducing fast aggregation alongside neuroprotection, while for addition of Cu2+ to amyloidogenic peptides both enhancing and inhibiting effects are reported.15 Results differ due to various metal ion binding site arrangements in the peptides and experimental conditions. There is, for example, an intense debate concerning the binding mode and binding affinities in Alzheimer Aβ. Reported effects range from rapid, amorphous aggregation to inhibition of β-sheet formation.1623 Although considerable effort has been put into the understanding of these factors, especially when combinations of them complicate clear conclusions, the complex interplay of several triggers is still poorly understood. When they occur at the same time, effects can oppose each other, enhance each other, or change their influence. We focus on the factors hydrophobichydrophilic interface and metal ion binding and their interplay. Our approach includes an increase in local concentration due to the adsorption of peptides, conformational restriction of the peptides, and orientation relative to the interface as well as orientational order within the peptide layer. Concerning metal ion binding, their preferred binding geometry and the binding modes provided by the peptide are studied. Since natural amyloidogenic peptides and their aggregation process are challenging to study, we use model peptides that are more easy to handle and provide a better basis for interpretation. Therefore, the tailor-made model peptides are short and follow a periodic design. They were designed to transform into β-sheets within days.29 The most important feature of the model peptide system is that the peptides adopt two different secondary structures. The α-helical conformation is stabilized by the coiled coil design, while the transformation into β-sheets is slow enough to be followed experimentally. According to our goal to investigate the metal ion complexation at the same time as surface interactions, we chose a model system that reacts to metal ion complexation according to the corresponding design. Without taking into account a specific sequence, we are using a general approach to a variety of diseases such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes that are connected to amyloid formation. Given our focus on the influence of hydrophobichydrophilic interfaces and restricted dimensionality, a well suited system to be investigated are peptide layers at the airwater interface of a Langmuir film balance. Film balance setups allow for the adjustment of important parameters. First, like their natural examples, the tailor-made model peptides are surface active and adsorb to the airwater interface.24 The formation of a peptide surface layer implies an increase in local concentration as discussed as a possible trigger. Additionally, the hydrophobichydrophilic airwater interface acts as an hydrophobic template: inducing or stabilizing preferential local conformations. Second, changing the compression state of the adsorption layer, the peptides can be aligned in-plane. We demonstrate the importance of orientational order as a trigger for β-sheet transformation. Finally, the effects of metal ion complexation on the conformation of the peptide can easily be investigated at the interface using surface sensitive methods. The metal ion binding to the two-dimensional peptide layers was previously characterized.25 One of the best methods to investigate these surface layers concerning their conformation is IR reflectionabsorption

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spectroscopy (IRRAS). It allows a secondary structure determination in a time dependent manner. Furthermore, the orientation of the peptides can be assessed. Only very recently, chiral sum frequency generation spectroscopy was developed.26,27 It additionally renders the elimination of H2O signals from the vapor phase unnecessary. We complement the knowledge of behavior and orientations of the amyloidogenic peptides at the airwater interface by investigating the structure of the layers in-plane and normal to the interface. For this purpose, the two X-ray techniques, grazing incidence X-ray diffraction (GIXD) and specular reflectivity, were applied under different conditions including compression states and metal ions. In conclusion, the investigated factors influencing β-sheet formation are compared and ranked.

’ MATERIALS AND METHODS All solutions were prepared using Milli-Q Millipore water with a resistivity of 18.2 MΩ cm. Peptide Synthesis, Purification, and Characterization. The peptides i,i+1 and i,i+7 were synthesized using a SyroXP-I peptide synthesizer (Multi-SynTech GmbH) on a 0.05 mM scale according to standard Fmoc (9-fluorenylmethoxycarbonyl)/tBu chemistry. Preloaded Fmoc-Leu-Wang residue (0.64 mmol/g; Novabiochem-Merck) was used for all peptides. For standard couplings, a 4-fold excess of amino acid and coupling reagents TBTU (O-(1H-benzotriazol-1-yl)-N, N,N0 ,N0 -tetramethyluroniumtetrafluorborat)/HOBt (1-hydroxybenzotriazole) as well as an 8-fold excess of DIEA (N,N-diisopropylethylamine) relative to resin loading were used. All couplings were performed as double couplings (30 min). The coupling mixture contained 0.23 M NaClO4 to prevent on resin aggregation. A mixture of DBU (diazabicyclo[5,4,0]-undec-7-ene) and piperidine (2% each) in DMF (dimethylformamide) was used for Fmoc deprotection (4 5 min). Peptides were cleaved from resin by treatment with 2 mL of TFA (trifluoracetic acid)/TIPS (triisopropylsilane)/H2O (90/9/1) for 3 h. The resin was washed twice with TFA (1 mL) and DCM (dichloromethane; dry, 1 mL), and excess solvent was removed by evaporation. The peptides were precipitated with cool diethyl ether, centrifuged, and dried in vacuum. Purification of all peptides was carried out by preparative reversed phase HPLC equipped with a Luna C8 (10 u, 250 21.20 mm, Phenomenex) column. Eluation solvents were ACN (acetonitrile)/0.1% TFA and water Millipore/0.1% TFA. The flow rate was 20 mL min1. Purified peptides were characterized by analytical high-performance liquid chromatography and electospray ionization mass spectrometry and used after removal of trifluoroacetate (lyophilized three times with 0.5 mM HCl and once with Milli-Q Millipore water28). The molecular weights are i,i+4 = 3349 g/mol, i,i+1 = 3201, and i,i+7 = 3160. The peptide was dissolved in hexafluoroisopropanol (HFIP, Aldrich; 1 mg per mL) to revert possible aggregation and dried in vacuum or under N2-stream directly before being dissolved in 10 mM PBS (Fluka) containing 150 mM NaCl (Fluka, tempered at 600 °C before use), and the corresponding concentration of metal ions (0.27 μM) at pH 7.4 yielded a 0.3 μM or 0.5 μM peptide solution unless stated differently. The solution was used immediately; shaking was avoided to prevent aggregation. ZnCl2 (Sigma) and CuCl2 (Fluka) were added from aqueous stock solutions of 1 mM without further purification. i,i+4 was synthesized and characterized as in ref 29. At the N-terminus of each peptide, an o-aminobenzoic acid (Abz) group was introduced. The full sequences are as follows: i,i+1, Abz-LKVELEVLKSELEKLHHELVKLKSEL; i,i+7, Abz-LKVELEVLHSELEKLHSELVKLKSEL; and i,i+4, Abz-LKVELEKLKSELVVLHSELHKLKSEL. Film Balance Measurements. To record πt and πA isotherms, a polytetrafluoroethylene (PTFE) Langmuir trough equipped with a microbalance (R&K, Potsdam, Germany) and filter paper Wilhelmy 14219

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plate was used and thermostatted at 20 °C (E1Medingen, Leipzig, Germany). The peptide solution was filled into the trough, and the adsorption isotherm was recorded. Monolayers were compressed symmetrically from two sides with a speed of 8.2 cm2/min. IRRAS and Simulation of IRRA Spectra. Infrared reflection absorption spectra (IRRA spectra) were recorded using an IFS 66 FT-IR spectrometer (Bruker, Ettlingen, Germany) and an external reflectance unit containing the film balance trough (R&K, Potsdam, Germany) inside a container flushed with dry air to stabilize the relative humidity in the optical path. The trough system comprises a sample trough with two movable barriers and a reference trough to allow recording of sample and reference spectra within short time ranges using a shuttling technique. Before hitting the water surface, the beam is polarized by a KRS-5 wire grid polarizer to create parallel (p) or perpendicular (s) polarized light. The incidence angle was varied between 30° and 70° relative to the normal of the surface. The use of mirrors allows the infrared beam from the external port of the spectrometer to hit the water surface at an adjustable angle of incidence. After reflection from the water surface, the beam is collected at the same angle and led to a narrow band mercury cadmium telluride (MCT) detector cooled with liquid nitrogen. Reflectanceabsorbance spectra with a resolution of 8 cm1 were obtained using log(R/R0), with R being the reflectance of the sample and R0 the reflectance of the reference, the surface of the respective buffer solution. For each single beam spectrum with p-polarized light, 400 scans (200 scans for s-polarized light, scanner velocity 20 kHz) were added, apodized using Blackman-Harris 3-term function and fast Fourier transformed after one level of zero filling. Before representation, all spectra were corrected for atmospheric interference using the OPUS software and baseline corrected using a constant value. The spectra are not smoothed.30,31 Simulation and a global nonlinear least-squares fit of IRRA spectra were performed using a program written by Christian Schwieger in MATLAB. The optical model of Flach et al.30 was adapted. The molecular director of the respective peptide conformations is defined parallel to the α-helix and normal to the plane of the β-sheet. Tilt angles θ are given with respect to the layer normal. Therefore, a tilt of 90° indicates α-helices parallel to the interface and a tilt = 0° indicates helices standing upright. Contrarily, the CdO groups of a β-sheet are parallel to the interface at tilt = 0° and oriented normal to the interface with a tilt angle of 90°. The twist angle is not considered; that is, we suppose free rotation of the molecules about the molecular director. For β-sheets, the director is defined perpendicular to the plane of the β-sheet, more precisely perpendicular to the CdO groups involved in hydrogenbonding. This results in an isotropic in-plane orientation for β-sheets oriented with the CdO groups parallel to the interface. To simulate spectra with multiple bands, real and imaginary parts of the complex refractive index were calculated according to k¼

∑ ki

n ¼ nmax

∑ ni

were i denotes the band number. The reflectivity of the film was then calculated with these values for n and k. Spectra are measured and simulated with p-polarized and s-polarized light and angles of incidence between 30° and 70° in steps of 5° omitting 50° and 55°. The optical constants of the water subphase are taken from ref 32 to create a baseline. The effective film thickness d and the refractive indices no = neo are fitted using the OH stretching vibration at 38003000 cm1. The polarizer quality is set to Γ = 0.018 in all simulations. d, no, and Γ were constant in the fit of the amide I vibrations. For each band in the amide region, the exact wavenumber ν0, the vibration strength kmax, the full width at half-maximum (FWHM), and the tilt angle of the peptide θ at the interface were varied. The amide I bands were simulated with two components representing β-sheet and

α-helical or unstructured peptide strands. The global fit of all spectra (recorded with p-polarized light and s-polarized light and all angles of incidence) was performed using the trust-region-reflective algorithm. The square errors of spectra recorded with s-polarized light were weighted doubly to account for their low intensity. Spectra were fitted in the range 15701700 cm1. In order to account for the overall shape of the spectra due to the amide II vibrations, two further bands were added in the simulations at wavenumbers below 1570 cm1. All simulations were performed with an initial tilt of 45°. When a nonparallel configuration yielded a local minimum, the fit was additionally started with a configuration parallel to the interface. Dichroic ratios (DRs) are calculated from spectra taken with an incidence angle of 40° at either 1655 cm1 for α-helices or 1624 cm1 for β-sheets. They are normalized to the theoretical dichroic ratio of isotropic distributions of all orientations within the layer. DR norm ¼

Ip I isotropic DR ¼ sisotripic DR isotropic Is Ip

DRisotropic corresponds to an average in-plane angle of 45° and was calculated from simulated spectra of α-helices or β-sheets, lying flat at the surface but having an isotropic in-plane distribution. The normalization allows for the calculation of the experimental and theoretical dichroic ratios without the knowledge of the optical parameters of the film. While DR depends on n, ϕ, k, and the in-plane orientation, DRnorm depends only on the in-plane orientation. The corresponding in-plane angle was estimated by comparison of the normalized dichroic ratio to calculated values. GIXD and X-ray Reflectivity. Grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity were performed at the liquid surface diffractometer of the undulator beamline BW1 at HASYLAB (DESY, Hamburg, Germany) that is equipped with a Langmuir trough with a single movable barrier and a microbalance with a filter paper Wilhelmy plate. The trough was thermostatted at 20 °C. It is located in a hermetically closed container which was flushed with helium. The synchrotron beam was monochromated by a beryllium (002) crystal (wavelenght: λ ≈ 1.3 Å) before striking the liquid sample surface at an incidence angle of αi = 0.85 3 αcr = 0.85 3 0.13° (αcr: critical angle for total external reflection). For GIXD, a linear position-sensitive detector (PSD-70-M, MBraun, Garching, Germany) or a MYTHEN detector system (PSI, Villigen, Switzerland) was rotated around the sample to detect the intensity of the diffracted beam as a function of the vertical and horizontal scattering angles (αf and 2θ). A Soller collimator was located between the sample and the detector. The Bragg rods and Bragg peaks were background subtracted before evaluating peak positions and FWHM values. The vertical and horizontal scattering vector components were calculated using Qz = 2π/λ sin(αf) and Qxy = 4π/λ sin(2θ/2). For X-ray reflectivity, the vertical scattering vector component Q z = (4π/λ)sin αr in the interval 0.010.85 Å1 was recorded as a function of the vertical incidence angle αi with the help of a NaI scintillation detector, while the vertical reflection angle equals the vertical incidence angle αr = αi. The reflectivity was normalized to the Fresnel reflectivity (R/RF). To access the electron density profile normal to the interface, a model independent approach including linear combinations of b-splines was used.33,34

’ RESULTS AND DISCUSSION For the onset or acceleration of β-sheet formation in peptide monolayers, we consider (a) the enhancement of the peptide concentration by adsorption at the interface, (b) the effect of the orientation of the peptides at the interface, and (c) metal ion complexation (especially Cu2+ and Zn2+ ions) as possible triggers. 14220


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Figure 2. Adsorption isotherms of i,i+1 (black), i,i+7 (red), and i,i+4 (green). The small oscillations are caused by the shuttling process in IRRAS experiments (0.3 μM peptide, 10 mM PBS, pH 7.4, 150 mM NaCl, 20 °C).

Figure 1. (A) Helix view of i,i+1 and i,i+7 as monomers and i,i+429 as coiled coil dimer. This view emphasizes the design principle, but not the conformation that is most stable. The design is based on heptad repeats and a leucine zipper in positions a and d, and contains opposing charges in positions c and g, b and e as well as g and e0 and e and g0 . (B) Schematic structure of the peptides as elongated β-sheets. Hydrophobic stretches are highlighted by gray boxes. This is probably not the real arrangement of the peptides as β-sheets.

The Building Principles of the Model Peptides and Their Surface Activity. The peptides used in this study are designed to

exhibit an α-helical conformation. This is achieved by coiled coil formation. Therefore, their sequences follow the heptad repeat with a leucine zipper motif in positions a and d and additional matching of opposite charges in positions e/g0 and g/e0 as well as e/b and g/c (Figure 1).35 Incorporation of three Val residues into the sequence (Figure 1)29 makes the peptides prone to transformation into β-sheet conformation within hours to days. Additionally, to enable the investigation of metal ion complexation, the peptides possess metal ion binding sites (His residues) in different positions. Using the positions of the His binding sites at the residues i and i+N as names for the peptides, three peptides were studied: N = 1,7 and N = 4. The effect of metal ion addition (Cu2+ and Zn2+) was examined in bulk (ref 29 and Supporting Information). The first set of peptides has His binding sites at the positions i,i+1 and i,i+7. The peptides combine the heptad repeat with the His distances of Aβ. In Aβ, there are three His residues in positions i,i+1 and i,i+7. In order to study the effects of both Hisbinding geometries separately, the simultaneous incorporation of three His residues that are discussed as metal ion binding sites was avoided. His binding sites are introduced in an i,i+4 geometry into the other peptide.29 In i,i+4, two His binding sites for transition metal ions are close to each other in the α-helical conformation.

In the β-sheet, His residues within one peptide are not able to bind to one and the same ion, thereby forming a chelate. This is because the His residues within one peptide are not close enough to each other. However, in adjacent strands of β-sheets, the distance between His residues might be more favorable for chelate formation. The aim is to study the possible stabilization of either the α-helix or the β-sheet systematically. We use the hydrophobichydrophilic airwater interface for our investigations of the onset and early stages of β-sheet and amyloid formation. As recently discussed, hydrophobic surfaces are a strong trigger for amyloid formation and can be responsible for changed aggregation kinetics, that are not fully understood to date.11,13 The Langmuir film balance is a very simple and widely used tool to study layers at the airwater interface. The surface tension can be determined to monitor the time-dependent adsorption behavior by a Wilhelmy film balance. Additionally, movable barriers can be used to compress the film laterally. In this way, the surface density increases and an in-plane orientation of the peptides is induced. All model peptides adsorb to the airwater interface. The adsorption process starts immediately and follows a sigmoidal πt isotherm. The equilibrium pressure of about 20 mN/m is reached after 12 h (Figure 2). All peptides have similar equilibrium surface pressures. The equilibrium surface pressure is also independent of the bulk concentration in the range studied (0.30.5 μM) and independent of metal ion binding within experimental errors (Figure S 3 and Figure S 4, Supporting Information). Starting from the peptide adsorption layer, the aim of further studies is to examine transitions of the peptide conformation from α-helical or unfolded to β-sheet. Trigger (a). Increasing the Local Peptide Concentration: i,i+1 and i,i+7. High concentrations of amyloidogenic peptides promote β-sheet formation and aggregation. Adsorption and enrichment of the peptide at the airwater interface result in an increase of the local concentration compared to bulk. Starting with 0.3 μM in bulk, the two-dimensional concentration on the surface is some orders of magnitude higher.9 In bulk, this concentration provokes immediate aggregation of amyloidogenic peptides. IRRAS has often been used to assess the secondary structure of peptides at the airwater interface, as well as the change of secondary structure over time.3642 The amide I band in the IR spectrum is used to determine the secondary structure of the peptides at the interface. It is assigned to the CdO stretching vibration of the amide group that is sensitive to hydrogen bonds. 14221


Langmuir The position of the band reflects the hydrogen-bond environment of the respective groups and allows to determine the conformation.37 The amide I band is found at 1655 cm1 for α-helical structures, at 1645 cm1 for unordered structures, and at 1625 cm1 for β-sheet conformations.43 Therefore, the band intensity of the component at 1625 cm1 can be used to follow β-sheet formation. The amide II band is usually not used to determine secondary structure elements. It is found between 1570 and 1530 cm1. The peptides i,i+1 and i,i+7 adsorb to the interface and adopt mainly α-helical conformations at concentrations accessible in IRRAS experiments (Figure 3, black curves; see the Supporting

Figure 3. Time dependent and surface pressure dependent IRRA spectra of (A) i,i+1 and (B) i,i+7 at the airbuffer interface. The peptide layers were compressed to 30 mN m1 after reaching the equilibrium surface pressure and waiting for ≈1 day. Gray lines indicate the wavenumbers of bands at 1655 cm1 assigned to α-helix and 1625 cm1 assigned to β-sheet (0.3 μM peptide, 10 mM PBS, pH 7.4, 150 mM NaCl, 20 °C).

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Information for details). Approaching the equilibrium surface pressure, they spontaneously transform into β-sheets. Assuming constant orientation, the intensity of the band at 1625 cm1 is a direct measure of the β-sheet content in the interface layer (Figure 4). The peptide i,i+1 transforms partially into β-sheet conformation already during the adsorption process (black open symbols in Figure 4 A and black line in Figure 3 A). However, the spectra taken ≈24 h after starting the adsorption process (Figure 4 B and Figure 3 A) clearly show bands both, at 1655 cm1 and 1625 cm1, proving the presence of a mixed layer containing α-helices and β-sheets. The adsorption of the peptide i,i+7 to the airbuffer interface as α-helix is followed by immediate β-sheet formation when the surface coverage increases (Figure 3B). i,i+7 transforms almost completely into β-sheets, in contrast to i,i+1 forming mixed layers of α-helices and β-sheets with a fixed ratio. This different behavior suggests the importance of local conformation or charge repulsion for the intermolecular interactions of these peptides especially at the interface, where the peptides are confined to two dimensions. A difference of the two peptides is one additional negative charge in i,i+7. However, Lepere et al.44 showed that a difference in peptide charge does not influence the aggregation process. Therefore, we assume an additional salt bridge and not a difference in total charge to be the decisive difference between i,i+1 and i,i+7. Our hypothesis is that i,i+1 has a higher propensity to adopt an α-helical conformation compared to i,i+7, because of one additional intramolecular salt bridge between the residues K9 and E13 in the α-helical conformation (indicated by a gray broken line in Figure 1A). These favorable electrostatic interactions are abolished in i,i+7 by the replacement of K9 for H9. The stabilization of a coiled coil by one salt bridge is in the order of 4 kJ/mol45 and therefore in the order of differences of conformational energies. Additional to the experiments with adsorption surface layers, the surface films used for this study can be laterally compressed by movable barriers. Compression leads to two effects, namely, a further increase in the concentration and increased in-plane orientational order of the peptides relative to each other. First, the absolute intensities increase on compression, due to the larger amount of compressed material in the footprint area. Second, an increase in the ratio of band intensities at 1625 cm1 and at 1655 cm1 is observed after compression from ≈20 to 30 mN m1 (Figure 3). This is indicative for the transition from

Figure 4. (A) Reflectionabsorption intensity at 1625 cm1 versus time. Absolute intensities are taken from IRRA spectra of i,i+1 (open symbols) and i,i+7 (filled symbols) at the airbuffer interface. The gray box indicates the duration of the adsorption process. (B,C) IRRA spectra taken 1 day after adsorption of i,i+1 (B) or i,i+7 (C) to the airwater interface. The layers are not compressed. Uncomplexed peptide (black), interacting with Cu2+ (red) or Zn2+ (green) (p-polarized light at 40°, 0.3 μM peptide, 10 mM PBS, pH 7.4, 150 mM NaCl, 20 °C, with 0.27 μM Cu2+ions or Zn2+ions, respectively). 14222


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Figure 5. Time dependent and surface pressure dependent IRRA spectra of i,i+4 at the airbuffer interface. The peptide layer was compressed to 30 mN m1 after reaching the equilibrium surface pressure and waiting for ≈1 day. Gray lines indicate the wavenumbers of bands at 1655 cm1 assigned to α-helix and 1625 cm1 assigned to β-sheet (p-polarized light at 40°, 0.3 μM peptide, 10 mM PBS, pH 7.4, 150 mM NaCl, 20 °C).

α-helices to β-sheets in the i,i+1 and i,i+7 layers. While i,i+7 transforms almost completely, the further increase of concentration and orientational order does not lead to a complete transition of the i,i+1 layer into β-sheets. To sum up, the transition of i,i+7 to β-sheet structure occurs spontaneously at the airsolution interface even without compression, and is almost complete after compression (Figure 3B). This is a significant difference from the case of i,i+1, which only transforms partially and slowly, even after compression (Figure 3A). The population of an α-helical conformation in the beginning of all experiments (see the Supporting Information) supports the hypothesis that amphiphilic amyloidogenic peptides pass through an α-helical state upon adsorption at hydrophilic hydrophobic interfaces.4650 Sometimes, this state cannot be monitored by IRRAS, as in the case of Aβ(140),51 because the conformational change occurs already at low concentrations of the peptide at the surface, which are below the detection limit of this method. Trigger (b). Increasing the In-Plane Orientational Order of Peptides: Compression of i,i+4 Layers. Interestingly, i,i+4 retains α-helical conformation after adsorption to the interface for up to 24 h as indicated by the band position and intensity of the amide I band (Figure 5). Compared to the spontaneous transition of i,i+1 and i,i+7 at the interface, this phenomenon is a strong indication for a stabilization of the α-helical conformation by the hydrophobichydrophilic interface. In its initial surface conformation, the peptide can arrange in a manner that the hydrophobic side chains of the helical positions a and d (Figure 1) point into the air. The rearrangement of the peptide into its β-sheet conformation needs a further trigger. In contrast to i,i+1 and i,i+7, the β-sheet formation can be induced only by compression of i,i+4 layers (Figure 5). It is interesting to note that even surface concentrations up to the order of mM are not high enough to overcome the ability of the interface to stabilize the α-helical conformation. Concentrations in the order of mM immediately induce visible aggregation of the peptide in bulk. By compression from ≈20 mN m1 (equilibrium) to 30 mN 1 m , the increase in concentration at the interface is e4-fold (as estimated from the decrease in available area). Because tightly packed β-sheet structures need ≈1/3 less space than α-helices (see the Supporting Information for more details), a β-sheet

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formation leading to an energy gain is expected. However, compared to the increase in concentration due to adsorption, this is only a small additional factor. Therefore, we suggest that the increase in orientational order of the α-helical peptides with respect to each other is the more important effect. When peptides are aligned parallel to each other, the probability for hydrogenbond formation is increased, favoring intermolecular β-sheet formation. The orientational order results from an energy gain due to a first-order two-dimensional isotropic to two-dimensional nematic transition.52 The increasing orientational order of the α-helices can be surveyed using the dichroic ratio DRnorm at 1655 cm1 normalized to the dichroic ratio of isotropic films. For an isotropic layer, the normalized dichroic ratio is 1. DRnorm < 1 would indicate that the α-helices are oriented more perpendicular to the barriers, while DRnorm > 1 indicates orientation of the α-helices more parallel to the barriers. The normalized dichroic ratio increases from 1.5 at 22 mN m1 to 2.7 after compression of the i,i+4 layer above 27 mN m1. Since the normalized dichroic ratio at 22 mN m1 is >1, the two-dimensional isotropic to two-dimensional nematic transition may have occurred already during adsorption. Furthermore, the β-sheet formation is not complete even after compression and absorption bands for α-helical or unfolded conformation persist. This might indicate slow kinetics that cannot be followed reasonably by the experiment anymore. After 30 h, perturbations of the peptide structure by dust particles cannot be excluded. Trigger (c). Metal Ion Complexation. The model peptides examined in this study are designed to have different metal ion binding modes. His residues as metal ion binding sites are introduced in various geometries. The His residue arrangements in the peptides i,i+1 and i,i+7 are derived from Alzheimer Aβ. i,i+4 is designed to have two His residues in close proximity in its α-helical conformation, but not in its β-sheet conformation. The two transition metal ions Cu2+ and Zn2+ are regularly found in the plaques of Alzheimer’s patients. Cu2+ and Zn2+ are therefore used in the present study. In bulk, the formation of β-sheet structures is prevented when either Cu2+ ions or Zn2+ ions are added to i,i+1 or i,i+4, respectively (see the Supporting Information and ref 29 for CD data). This is achieved through a stabilization of the α-helical conformation. When added to i,i+7, the metal ions have different effects. While Zn2+ also prevents β-sheet formation, Cu2+ ions do not change the slow transition of the coiled coil into β-sheets. Metal ion complexation does not influence the kinetics of adsorption and the equilibrium surface pressure reached when peptides adsorb to the interface as shown in Figures S 3 A and S 4 A (Supporting Information). The adsorption of the peptides at the airwater interface can counteract or enhance the effects of metal ion binding observed in bulk. In order to examine the conformation of the peptide layers at the interface complexed with metal ions, a slightly substoichiometric amount of either Cu2+ or Zn2+ is added to the peptide solution. Avoiding high local concentrations of metal ions and precipitation in the phosphate buffer, the ions are added to the buffer directly before dissolving the peptide. The IRRA intensity at 1623 cm1 is taken as a measure for the β-sheet content of the peptide layer. In Figure 4A, the band intensities are depicted together with those of peptide layers without addition of metal ions. Comparing the intensity versus time plots of i,i+1 complexed with Cu2+ (red open symbols) or Zn2+ (green open symbols), respectively, to that corresponding 14223


Langmuir to uncomplexed i,i+1 (black open squares), no difference between the complexed and uncomplexed layers is observable. In other words, the addition of Cu2+ or Zn2+ does not influence the conformation of i,i+1 at the interface. As also shown in Figure 4B, the spectra with and without addition of metal ions are indistinguishable for at least 1 day. This proves that the transformation of α-helices to β-sheets over time is independent of metal ion complexation. This does make a significant difference to the behavior observed in bulk where metal ions prevent βsheet formation. On the one hand, metal ion binding could be disfavored when the energy gain by metal ion binding is too small to change the conformation that is imposed on the peptide by the interface. On the other hand, it is possible that, although the metal ions do bind, they do not have an influence on the conformation of the peptide. The local conformation required for metal ion binding could be the same as the conformation imposed by interaction with the hydrophobichydrophilic interface. As a quantitative measure for the conformation, the ratio of the strength of vibration kmax of the bands at 1656 cm1 (α-helical) and 1625 cm1 (β-sheet) obtained in the simulations of IRRA spectra is ≈1.4 independent of metal ion addition. The strength of the respective vibrations kmax as obtained by spectra simulations (see below) is independent of the orientation. Therefore, the amount of α-helices and β-sheets in the molecules can be assessed taking into account the extinction coefficients for normalization.53 A layer of i,i+1 is composed of 60% residues in an α-helical conformation and 40% residues incorporated into β-sheets. With this data, it is not possible to decide whether these are distributed in coexisting α-helical or β-sheet domains or if all peptides transformed to a certain degree from α-helical into βsheet. For coexisting domains of either α-helices or β-sheets, usually cooperative transition is expected47 but is not observed. Therefore, we favor the hypothesis of a peptide layer, where each peptide transforms to a certain degree. From the present data, it cannot be reasonably speculated which residues may adopt an αhelical conformation and which residues may adopt β-sheet conformation. Similar to the case of i,i+1, the intensity versus time plots of i,i+7 complexed with Cu2+ (red filled symbols) or Zn2+ (green filled symbols) shows no remarkable difference compared to the curve corresponding to uncomplexed i,i+7 (black filled squares in Figure 4). A slightly higher intensity of the band at 1625 cm1 can be recognized when Cu2+ is present in the subphase. Whether this is in the range of experimental error cannot be securely decided. Similar to the case of i,i+1, the conformation of i,i+7 at the interface is not influenced by the presence of Cu2+ or Zn2+. Cu2+ is not expected to interfere with the peptide conformation at the interface, since in bulk Cu2+ ions do not influence the slow transition of i,i+7 into β-sheets. This is in agreement with Cu2+ being able to bind to a single His residue without the need to form a chelate that would restrict the local conformation of the molecule. In contrast, Zn2+ ions stabilize the α-helical conformation of i,i+7 in bulk but do not slow down or restrict the β-sheet formation at the interface. Zn2+ binding to the His residues of the model peptides depends on the possibility to form chelates.25 This possibility is always given in β-sheets where parallel peptide strands are close to each other. In the α-helix, Zn2+ binding depends on the distances between the His residues. In α-helices of i,i+7, the distance between two His residues is only enabling chelate formation, when the His residues bend strongly. Since the

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adsorbed peptides are presumably monomers with the hydrophobic side facing air and restricted in their motion, bending of the His residues might be disfavored at the interface. Therefore, Zn2+ ions might not bind in the same way as in bulk and may lose their ability to stabilize α-helices at the interface. Obviously, the acceleration of the β-sheet formation by the confinement to the interface is strong enough to compensate for the restrictions imposed by the metal ion complexation. After transformation to β-sheets, Zn2+ ions can bind, forming chelates with His residues belonging to adjacent peptide strands. In summary, metal ion binding has no influence on the conformation of i,i+1 or i,i+7 at the airwater interface. Our explanation is that either the interface imposes the same local conformation as metal ion complexation or the conformational restriction by the interface is stronger than the energy gain achieved by possible metal ion binding. Here, the interface clearly dictates the conformation of the peptides at the interface. Furthermore, upon lateral compression, all peptide layers transform into β-sheets, supporting our hypothesis of parallel alignment being a strong trigger. Different from i,i+1 and i,i+7, the model peptide i,i+4 was not derived from Aβ, but designed to respond to metal ion complexation by retaining an α-helical conformation. The intensity versus time plots of i,i+4 complexed with Cu2+ (red symbols) or Zn2+ (green symbols), respectively, are shown in Figure 6 together with the curve corresponding to uncomplexed i,i+4 (black symbols). The IRRA intensity of the band at 1623 cm1 is the same for uncomplexed i,i+4 and i,i+4 complexed with Cu2+ (Figure 6). This means that the α-helical conformation is stabilized at the interface even at high local peptide conformations, also in the presence of Cu2+. Contrarily, when i,i+4 is complexed with Zn2+, the IRRA intensity of the band at 1623 cm1 increases rapidly even before the adsorption process has reached equilibrium (green symbols in Figure 6). Since the depicted band intensity is a measure for the β-sheet content at the interface, Zn2+ ions are found to accelerate β-sheet formation at the interface. This is the opposite effect of the behavior observed in bulk where Zn2+ stabilizes an αhelical conformation of i,i+4.29 This effect is not expected, because Zn2+ is able to bind exclusively to the His residues forming a chelate. As revealed by total reflection X-ray fluorescence,25 Zn2+ ions are able to bind to the α-helix as well as to the β-sheet of i,i+4. Still, small differences in binding affinities could lead to a preference of the β-sheet structure. Our explanation for the preference of the β-sheet structure when Zn2+ is present is again the slight difference in HisHis distances in the α-helical structure (56 Å) of i,i+4 compared to the β-sheets (e4.7 Å). We previously found a similar effect of Zn2+ on a model peptide that is very similar to i,i+4 except that the peptide i,i+2 is unable to form a chelate with Zn2+ ions in its α-helical conformation.24 The fact that i,i+2 transforms into β-sheets upon addition of Zn2+ shows that the Zn2+ binding brings an energy gain large enough to trigger a conformational change of this kind of model peptide. Interestingly, Talmard et al.48 discuss an enhanced transition from an α-helical intermediate of Aβ to β-sheets due to Zn2+ binding. The difference in binding affinity as well as the different binding geometries of Zn2+ and Cu2+ play an important role in peptide complexation at the hydrophobichydrophilic interface. Additionally, there is a delicate interplay with the arrangement of the binding sites in the peptides. As a consequence, in contrast to 14224


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Figure 6. (A) Intensity of the IRRA band at 1623 cm1 of i,i+4 at the airbuffer interface versus time. The gray box indicates the duration of the adsorption process. (B) Amide I region of IRRA spectra taken after 2024 h. Uncomplexed i,i+4 (black), interacting with Cu2+ (red) or Zn2+ (green) (p-polarized light at 40°, 0.3 μM i,i+4, 10 mM PBS, pH 7.4, 150 mM NaCl, 20 °C, with 0.27 μM Cu2+ions or Zn2+ions).

i,i+1 and i,i+7, the conformation of i,i+4 at the interface is influenced by metal ions. Our results show that the onset of β-sheet formation does not primarily depend on the concentration of the peptides at the interface. We found that the adsorption to the hydrophobichydrophilic interface can lead to both stabilization of the α-helical intermediate and the expected induction of β-sheet formation. This is due to stabilization of α-helical intermediates by hydrophobic interactions as well as due to increased concentrations. The triggers leading to an onset of β-sheet formation depend crucially on the peptide primary structure. Triggers range from simply increasing the local peptide concentration during adsorption, alignment of the α-helical intermediates to metal ion complexation. The strongest trigger we observed in our study is a high orientational order of the α-helices as induced here by compression. Metal ion complexation, especially of Zn2+ ions, may lead to fast aggregation. On the other hand, at the interface, metal ions are not inhibiting β-sheet formation, even though they are able to do so in bulk. The ability of metal ions to interfere with the secondary structure of peptides critically depends on the arrangement of metal ion binding sites in the peptide as well as on the binding modes of the ion. Metal ion complexation was found to be able to influence the β-sheet formation, but not to oppose the effect induced by the interface. We found that the increase in local peptide concentration is leading to aggregation, but it may be counteracted by other factors favoring α-helical conformations. At the hydrophobic hydrophilic interface, strong hydrophobic interactions and the presence of one additional salt bridge may stabilize α-helical intermediates at concentrations that immediately lead to aggregation in bulk. The Orientation of the Peptides Is Parallel to the Interface, and In-Plane Orientational Order Increases upon Compression. To determine the orientation of the vibrating dipole with respect to the layer normal and hence the orientations of the molecules at the airwater interface, IRRA spectra recorded at various angles of incidence below and above the Brewster angle can be used.54 To obtain information about the orientation of the vibrating groups, the intensity, the position, and the composition of the amide I bands of simulated spectra are fitted to experimental ones (one example is shown in Figure 7). Fitting paramters are kmax, ν0, FWHM, and the tilt angle θ. IRRA spectra of i,i+1 and i,i+7 layers give qualitatively the same results, except for the amount of β-sheet content in uncompressed films. All orientations determined for i,i+1 and

i,i+7 in various conditions suggest that both the α-helices and the CdO groups in β-sheets are oriented parallel to the interface. The simulation of IRRA spectra of i,i+4 layers is less straightforward. For all examined layers, a local minimum was found for both α-helical and β-sheet conformations being parallel to the interface. In some cases, tilt angles cannot be excluded, because of further local minima of the fit (see the Supporting Information for more details). From simulations, a tilt angle of the α-helices of 90°65° is obtained. The CdO groups of β-sheets are most probably oriented parallel to the interface with a maximum deviation of 20°. After compression, the β-sheet content of all examined peptide films increased and simulation data indicates β-sheets with the CdO groups being oriented parallel to the interface. This is a strong indication that β-sheets are lying flat at the interface. However, we will show that there is some possibility for an overall tilt of the peptide strands in β-sheet conformation. This is not in contradiction to the CdO groups being oriented parallel to the interface, as IRRAS simulations of the amide I bands do not probe the overall tilt of the β-sheets. In a layer of i,i+4 in the presence of Cu2+ ions, there is still a remarkable amount of peptides in an α-helical conformation detectable. These helices are parallel to the interface. A discrepancy between experimental and simulated intensities of s-polarized light is most probably caused by an increased inplane orientational order of the β-sheets. The model used for the simulation and fit does only account for out-of-plane orientation θ but not for in-plane-orientation. The in-plane orientational order can be analyzed using the normalized dichroic ratio of the amide I band at 1624 cm1: DRnorm.55 For β-sheets that do not have an in-plane orientational order, the normalized dichroic ratio is 1. This was found for all layers that have β-sheet conformation before compression in this study. After compression, the normalized dichroic ratio varies from 1.95 (i,i+1, i,i+7) to 3.25 (i,i+7 and Cu2+ or Zn2+ at 40 or 45 mN m1, i,i+4 and Zn2+ at 30 mN m1). These values correspond to an average inplane angle of the CdO groups of 62°72° with respect to the direction of compression. Since the out-of-plane tilt of the CdO groups of the peptides does not change upon compression, the high values of the dichroic ratio after compression can be attributed to a high in-plane orientational order of the peptides. The CdO groups align more perpendicular to the direction of compression; that is, the peptides strands are rather parallel to the direction of compression. As indicated by the correlation length in GIXD (see below), the formed structures are longer in 14225


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Figure 7. IRRA spectra and respective best fit simulated spectra (dotted) of i,i+7 complexed with Cu2+ at equilibrium surface pressure taken after 25 h. Simulation details are given in the Supporting Information. Spectra were fitted in the range 17001570 cm1 as indicated by gray boxes (p-polarized and s-polarized light, angles of incidence from 30° to 70°, 0.3 μM i,i+7, 10 mM PBS, pH 7.4, 150 mM NaCl, 20 °C, 0.27 μM Cu2+ions).

the direction of the hydrogen bonds (CdO groups) than in the direction of the peptide strands. Therefore, the β-sheets align preferentially with the peptide strands perpendicular to the barriers used for compression, but with the long axis of the whole β-sheet structure parallel to the barriers. Electron Density Profiles Can Be Separated into Contributions of α-Helices and β-Sheets. The X-ray reflectivity normalized to Fresnel reflectivity from an ideal planar interface can be inverted to yield the laterally averaged electron density F(z) of the monolayer as a function of the vertical z coordinate. The inversion can be performed either by a model independent method, as in the present case, or by using a layer (box) model of the interface. The latter method is only useful for monolayers whose number of electrons can be easily calculated and the molecular area is known from the pressurearea isotherms. In the present case, the molecular area of the adsorbed peptides is unknown. Additionally, the monolayer structure might not be homogeneous, since the secondary structure of the adsorbed peptides changes over time or during compression. The IRRAS information does not directly correspond to the situation in an XR experiment, since the kinetics of the secondary structure transition cannot be controlled and depends on known and unknown experimental factors. One problem is, for example, the changed surface area/volume ratio of the troughs used for the different experiments. Surely, the short waiting time before compression has an influence on the amount of the respective conformations. Compression started after 2 h for X-ray reflectivity and after ≈24 h for IRRAS. Therefore, the model independent method is the best choice. The electron density F(z), written in terms of cubic-spline functions, can be extracted from least-squares fitting for agreement between the corresponding model reflectivity and the measured reflectivity. The only restriction is an upper limit for the layer thickness (60 Å). Figure 8 parts A and C show the reflectivity curves with the corresponding fits for i,i+4 at equilibrium adsorption pressure and when compressed to 30 mN m1. The experiments have

been performed using the buffer with and without additional Zn2+ or Cu2+ ions (curves are overlapping). The shapes of the reflectivity curves are significantly different between the experiments with and without addition of metal ions. The derived electron density profiles are depicted in Figure 8 parts B and D. Only in the case of the peptide adsorption layer on the buffer solution without additional Zn2+ or Cu2+ ions at equilibrium pressure can the profile be described by one symmetrical electron density distribution assuming a root-mean-square roughness of 4 Å. The FWHM of the Gaussian-shaped curve amounts to 17 Å, which translates directly into the thickness of the corresponding film. This film thickness is in good agreement with the thickness of a layer of α-helical peptides lying flat at the interface. The maximum of the electron density reaches 0.4 electrons Å3 for the pure peptide layer. This is in complete agreement with the electron density of an entirely different α-helical peptide at the airwater interface.56 Comparison to the dimensions of the α-helix allows one to estimate that ≈300 H2O molecules are associated to each peptide in the peptide film. Compression of this layer leads to a changed reflectivity profile. The electron density profile cannot be described by one single Gaussian-shaped curve anymore. The deconvolution of the electron density profile into two contributions with the same roughness of 4 Å shows that two layers with different thicknesses contribute to the observed electron density. The first layer has a thickness of 17 Å with a maximum of the electron density of 0.36 electrons Å3. The second layer has a thickness of 11 Å with an electron density of maximum 0.21 electrons Å3. This corresponds well to the thickness of a β-sheet layer with the β-sheets parallel to the surface. The smaller maximum value of the electron density indicates a smaller contribution of the α-helical part due to the transformation of a certain part of the layer to β-sheets. This interpretation fits the IRRAS observation of a transition of α-helices into β-sheets on compression. The peptide i,i+4 complexed with either Zn2+ or Cu2+ ions forms a mixed layer already at equilibrium adsorption pressure. The transition 14226


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Figure 8. (A,C) Specular X-ray reflectivity, R(qz), of an i,i+4 adsorption layer on buffer solution (A) together with the best fits to the data (solid lines: equilibrium adsorption pressure (red), compressed to 30 mN m1 (blue); compression started 2 h after adsorption) and complexed with Zn2+ (C). The curve of i,i+4 complexed with Cu2+ is the same as that in the presence of Zn2+ within experimental errors. (B,D) Electron density profiles along the surface normal z of an i,i+4 adsorption layer on buffer solution (B) and complexed with Zn2+ or Cu2+ (D), derived from the corresponding reflectivity curves in (A) and (C). The different contributions have been calculated with the assumption of a symmetrical electron density distribution and using a film roughness of 4 Å (solid lines: equilibrium adsorption pressure (red), 30 mN m1 (blue); dotted lines: calculated contributions) (0.5 μM i,i+4, 0.5 μM Zn2+ or Cu2+, respectively, PBS pH 7.4, 150 mM NaCl, 20 °C).

from α-helices to β-sheets occurs much faster and not only after compression. The reflectivity curves of i,i+4 complexed with Zn2+ or Cu2+ (Figure 8C and D) match perfectly. This proves that the crystallinity of the β-sheets does not influence the reflectivity properties of the adsorption layer. We will later show that only the peptide complexed with Cu2+ exhibits crystalline β-sheets causing Bragg peaks of an additional lateral order. At equilibrium adsorption pressure, one layer has a thickness of 17 Å with a maximum electron density of 0.37 electrons Å3. The second layer is 11 Å thick with a maximum electron density of 0.15 electrons Å3. We assign the first layer to α-helices and the second to β-sheets. Compression changes the ratio between the two parts of the peptide film in agreement with the IRRAS results. Electron densities are 0.34 electrons Å3 in the 17 Å thick layer and 0.22 electrons Å3 in the 11 Å layer . X-ray reflectivity data fully enforce our finding of α-helical peptide layers that transform into β-sheets upon compression or addition of ions. The electron density profiles are also in line with the α-helical intermediates as well as the formed β-sheets oriented rather flat at the interface. Complexation with Cu2+ Ions and Compression to 30 mN 1 m Leads to Highly Ordered β-Sheet Layers. GIXD is a combination of X-ray diffraction with a total reflection setup. It was used to assess two-dimensional repeat distances in the surface layer. Only ordered structures result in Bragg peaks,

whose positions are defined by the dimensions of the twodimensional repeat pattern of the probed layer. The two Bragg peaks found in the diffraction pattern of a layer of i,i+7 in the presence of Cu2+ ions are shown in Figure 9, together with the corresponding Bragg rods. All layers that consist mostly of β-sheets (proved earlier by IRRAS experiments) gave rise to a Bragg peak corresponding to the interstrand distance of adjacent peptide strands in a β-sheet (data not shown, Figure 9A as example). It is found at Qxy ≈ 1.32 Å1, corresponding to ≈4.7 Å defined by the hydrogen bonds in β-sheets.57,58 Besides the repeat pattern, the regularity of the order is accessible by GIXD. The FWHM of the Bragg peaks is connected to the correlation length by Lcorr ¼

0:88 2 π FWHMcorr

where FWHMcorr = (FWHM2 0.0082)1/2 is the FWHM corrected for the resolution given by the Soller collimator. The correlation lengths in the direction of the β-sheets are in the order of 80 Å to >600 Å, depending on the addition of metal ions, the compression state, and random preparation effects (data not shown). The average number of peptide strands in the hydrogen bonding network of one β-sheet is 17140. Therefore, β-sheets 14227


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Figure 9. Bragg peaks (A, C) and Bragg rods (B, D) of i,i+7 complexed with Cu2+ at the airbuffer interface compressed to 30 mN m1. (A) and (B) are assigned to the distance defined by the β-sheet hydrogen-bond distance. (C) and (D) are assigned to the long repeat distance. The Bragg rods in (B) and (D) are mirrored for clarity (0.3 μM i,i+7, PBS pH 7.4, 150 mM NaCl, 20 °C).

Figure 10. Bragg peaks (A) and Bragg rod (B) of i,i+4 complexed with Cu2+ at the airbuffer interface compressed to 30 mN m1. The Bragg peaks shown in (A) are assumed to be the second and third order of a peak at Qxy = 0.17 Å1, corresponding to a repeat distance of 37.4 Å (0.3 μM i,i+4, PBS pH 7.4, 150 mM NaCl, 20 °C).

can be larger in the direction of the hydrogen bonds than in the direction of the peptide strands. Additionally, a long repeat distance can be found in twodimensional smectic phases. The uncomplexed peptide layers that transform into β-sheets only after compression do not exhibit these features. Zn2+ ions are known to lead to fast and unspecific aggregation of Aβ,59 leading to not well-defined structures. With Zn2+ ions present in the subphase of i,i+4 layers, aggregation starts almost immediately, but clearly before adsorption reaches equilibrium. However, the formed β-sheet layer does not exhibit two-dimensional smectic order. In contrast to adsorption layers of uncomplexed peptides or peptides complexed with Zn2+, layers of adsorbed peptides complexed with Cu2+ are highly ordered. They are characterized by additional Bragg peaks indicating lateral order (Figure 9 parts C and D, and Figure 10). The high order in direction of the long

repeat distance could be caused by a fixed connection of adjacent peptides via Cu2+ ions that are also able to bind to additional binding sites. We propose interpeptide binding that defines the longitudinal order assisting in a two-dimensional smectic order, being aware of the fact that cross-linking is not discussed for complexes of Cu2+ with Aβ.60 As shown in Table 1, the long repeat distances are shorter than expected for an elongated β-sheet (≈85 Å for an extended βsheet of 26 amino acid peptide). The correlation lengths in the direction of the long repeat distance are in the order of 150 to >400 Å (Table 1). Taking into account the repeat distances, a number of 4 10 of the ordered β-sheet stretches can be estimated to be correlated (Table 1). There are several possible arrangements of the peptides that lead to a repeat distance shorter than the full length of the elongated molecule. For example, elongated β-sheets may exhibit 14228


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Table 1. Long Repeat Distances and Correlation Lengths of Cu2+ Complexed β-Sheet Layers Compressed to 30 mN m1 on 10 mM PBS, 150 mM NaCl at 20 °C a peptide

d/Å

Lcorr/Å

i,i+7

35.9

167

>4

i,i+4

37.4

>400

>10

no. of repetitions

The peptide concentration in the bulk phase was 0.3 μM before adsorption. a

Figure 11. Some proposed structures of i,i+7 and i,i+4 complexed with Cu2+ at the airwater interface, compressed to 30 mN m1. Layers are compressed in the x-direction. Hydrogen bonds are indicated by black lines.

a defined lateral shift between adjacent peptide strands, thereby forming ribbons of diagonal peptide strands. In order to yield a ribbon of the width of the repeat distances found in our experiments, the individual strands need to have an angle of ≈65° with respect to the ribbon direction. The fixed geometry could be imposed by Cu2+ ions bridging adjacent strands in a defined way. Such arrangements cannot be precluded from the available experimental data, but we prefer the following explanation. i,i+7 and i,i+4 complexed with Cu2+ have similar repeat distances, with a length that matches a 1011 amino acid βsheet stretch. Investigating an analogous peptide without His residues, Gerling et al.61 propose two flexible regions in the peptide, leading to arches, that have an S-shaped structure of the peptide in the aggregates formed in bulk. Although the structures of aggregates formed in bulk might be different at the interface, these residues can be expected to have a high tendency to kink at the interface. In two dimensions, the need to form hydrogen bonds within the layer plane means that arches make the peptide bend into the water face. However, this is not in-line with the low film thickness detected for i,i+4 but possibly for i,i+7. Therefore, at the interface, the flexible regions of i,i+4 may form turns, resulting in an S-shape or staple-shape lying parallel to the interface (schematic drawings in Figure 11). 6 0) of the respecFinally, the out-of-plane components (Q z ¼ tive Bragg rods (Figure 9 parts B and D, and Figure 10B) suggest

that the β-sheets are not lying entirely flat at the surface. They might have a tilt of the long repeat distance with respect to the layer normal, or, as suggested by Vaiser and Rapaport,62 the βsheets bend upon compression. In our case, a strong bending of elongated peptides or out-of-plane tilt of elongated peptides does not correspond to the low layer thicknesses found. However, the nonelongated peptide structures may be additionally tilted or bent. A tilt or bending in the direction of the peptide strands does not contradict the CdO groups being oriented parallel to the interface, as established by IRRAS. Even though metal ion complexation does not interfere with β-sheet formation, it influences the structure of β-sheet peptide layers. Presumably by intermolecular complexation, Cu2+ ion binding may lead to a well-defined interpeptide orientation. This helps the peptides to align in a two-dimensional smectic phase, enhancing crystallinity. Zn2+ ions seem to induce a less ordered layer of β-sheets. Taking together all available data, we suggest the structural molecular models depicted in Figure 11.

’ CONCLUSIONS The main interest of our study was to learn about the interplay of various triggers that lead to amyloid formation. Thereby, our goal was to follow the process of initial β-sheet formation of peptides especially at the hydrophobichydrophilic interface and their interaction with metal ions. Early stages in the aggregation process are recently considered to be the cell toxic steps in amyloid diseases. The model peptides adsorb at the airwater interface and immediately transform from unfolded structures to α-helices. Depending on the peptide, these intermediates can start to transform into β-sheets or can be extraordinarily stable compared to the fast aggregation of the peptides at similar concentrations in bulk. These films require an additional trigger in order to change peptide conformation. It is interesting to note that, even at a very high local concentration of peptides, the interface is able to stabilize a nonaggregated form, underlining the high impact of the interface on the peptide structure. The onset of β-sheet formation at the interface depends on the peptide primary sequence and may simply occur over time during adsorption due to the increase in the peptide concentration. Other peptides transform when they are complexed by transition metal ions such as Zn2+. α-Helical intermediates confined to two dimensions may help to prealign peptides parallel to each other, thus enhancing the propensity for β-sheet formation. All peptides transformed into β-sheets upon the additional parallel alignment of the α-helical intermediates by compression of the film. According to the present study, the parallel alignment of α-helices is the strongest factor that leads to β-sheet formation. The effectiveness of metal ion complexation to enhance the conformational transformation into β-sheets or to stabilize the αhelix depends strongly on the peptide primary sequence. Consequently, in some cases, metal ion complexation can enhance the transition from the intermediate to β-sheet at the interface. For example, the binding of Zn2+ ions is strongly dependent on the possibility of chelate formation. Therefore, it additionally accelerates aggregation at the interface, when chelate formation is not possible in the α-helical state.25 Contrarily, metal ion complexation was not found to prevent β-sheet formation. Our main finding is that the hydrophobichydrophilic interface largely governs the conformation of peptides according to 14229


Langmuir their sequence. The additional effect of metal ion complexation is limited at the interface, while parallel alignment of highly concentrated α-helices at the interface always leads to aggregation. An important conclusion from our study is that systematic studies of the triggers are necessary to first understand them separately before examining their interplay. Special caution is needed when interpreting experiments implicating different amounts and types of hydrophobichydrophilic interfaces. These interfaces may induce α-helical intermediates or β-sheet conformation of the peptides that may act as seeds for the peptides in bulk,10 interfering with the results of the experiment conducted. In research aiming at a molecular understanding of the onset of amyloid formation, our findings underline the importance and delicacy of the interplay of various triggers. A closer look at particular interferences of triggers may assist in understanding and treatment of common diseases related to amyloid formation (such as Alzheimer’s disease, Parkinson’s disease, or type II diabetes) in the future.

’ ASSOCIATED CONTENT

bS

Supporting Information. Further information on the conformational stability of the peptides and additional figures. This material is available free of charge via the Internet at http:// pubs.acs.org/.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT J.A.F. and B.K. thank the DFG (SFB 765, Multivalency as Chemical Organization and Action Principle: New Architectures, Functions, and Applications) for financial support. M.H. and G. B. thank HASYLAB at DESY, Hamburg, for beamtime and support. K. Pagel and U. Gerling are acknowledged for providing peptides. We thank H. Moehwald and J. J. Giner Casares for discussion of the manuscript, and U. Sommerfeld is acknowledged for language service. ’ REFERENCES (1) Conway, K. A.; Lee, S.-J.; Rochet, J.-C.; Ding, T. T.; Williamson, R. E.; Lansbury, J.; Peter, T. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 571–576. (2) Walsh, D. M.; Klyubin, I.; Fadeeva, J. V.; Cullen, W. K.; Anwyl, R.; Wolfe, M. S.; Rowan, M. J.; Selkoe, D. J. Nature 2002, 416, 535–539. (3) Kayed, R.; Head, E.; Thompson, J. L.; McIntire, T. M.; Milton, S. C.; Cotman, C. W.; Glabe, C. G. Science 2003, 300, 486–489. (4) Murphy, R. M. Biochim. Biophy. Acta, Biomembr. 2007, 1768, 1923–1934. (5) Matsuzaki, K. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 1935–1942. (6) Aisenbrey, C.; Borowik, T.; Bystroem, R.; Bokvist, M.; Lindstroem, F.; Misiak, H.; Sani, M.-A.; Groebner, G. Eur. Biophys. J. 2008, 37, 247–255. (7) Miura, T.; Yoda, M.; Tsutsumi, C.; Murayama, K.; Takeuchi, H. Yakugaku Zasshi 2010, 130, 495–501. (8) Brovchenko, I.; Singh, G.; Winter, R. Langmuir 2009, 25, 8111–8116. (9) Jean, L.; Lee, C. F.; Lee, C.; Shaw, M.; Vaux, D. J. FASEB J. 2009, 24, 309–317.

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