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Human Apolipoprotein A1 at Solid/Liquid- and Liquid/Gas-Interfaces Susanne Dogan, Michael Paulus, Yury Forov, Christopher Weis, Matthias Kampmann, Christopher Cewe, Irena Kiesel, Patrick Degen, Paul Salmen, Heinz Rehage, and Metin Tolan J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b12481 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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The Journal of Physical Chemistry

Human Apolipoprotein A1 At Solid/Liquid - And Liquid/Gas - Interfaces E-mail: *[email protected] *[email protected]

Susanne Dogan+*, Michael Paulus+*, Yury Forov+, Christopher Weis+, Matthias Kampmann+b, Christopher Cewe+, Irena Kiesel+a, Patrick Degena, Paul Salmen+, Heinz Rehagea and Metin Tolan+

+

Fakultät Physik/DELTA, TU Dortmund, 44221 Dortmund, Germany

a

Fakultät Chemie, TU Dortmund, 44221 Dortmund, Germany

b

present address: Deutsches Elektronen-Synchrotron, 22607 Hamburg, Germany

Abstract An x-ray reflectivity study on the adsorption behaviour of human apolipoprotein A1 (apoA1) at hydrophilic and hydrophobic interfaces is presented. It is shown, that the protein interacts via electrostatic and hydrophobic interactions with interfaces resulting in the absorption of the protein. pH dependent measurements at the solid/liquid-interface between silicon dioxide and aqueous protein solution show that in a small pH rage, between pH 4 and pH 6 adsorption is increased due to electrostatic attraction. Here the native shape of the protein seems to be conserved. In contrast, the adsorption at the liquid/gas-interface is mainly driven by hydrophobic effects, presumably by extending the hydrophobic regions of the amphipathic helices and results in a conformational change of the protein during adsorption. However, the addition of differently charged membrane-forming lipids at the liquid/gas-interface illustrates the ability of apoA1 to include lipids resulting in a depletion of the lipids from the interface.

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Introduction Proteins are the basic modules of human life as they are involved in many important biological processes. They are responsible for structure stabilization, mass transport and act as enzymes in human metabolism and as messenger proteins or hormones. As their function is mainly given by their three dimensional structure, structural changes induced by external stimuli or the interaction between proteins and interfaces are in the focus of current research.1-6 For example, different diseases like the Alzheimer’s disease or type-2 diabetes mellitus can be linked to the formation of protein fibrills.7-9 Here the role of interfaces is crucial, because they can act as nucleation sites for fibril formation.10, 11 The human apolipoprotein A1 (apoA1) is a transport protein and the proteinous part of the High-density lipoprotein (HDL) which is responsible for the transport of lipids, mainly cholesterol from the cell membranes to the liver, where the lipids are dismantled. As cholesterol is essential for the survival of cell membranes, an abundance of cholesterol can result in serious diseases like arteriosclerosis and coronary artery diseases.12, 13 Reduced blood plasma levels of apoA1 and HDL are key risk factors. The anti-arterogenic properties of apoA1 arise primarily through its important roles in the pathway of the reverse cholesterol transport (RCT).14, 15 Apolipoprotein A1 exists in lipidfree, lipidpoor and lipidrich states in plasma. It consists of 243 amino acids and has a weight of 28.3 kDa. There are three isoforms present.16 Lipidfree and wild-type apoA1 are the main constituent of amyloid deposits found in atherosclerotic and senile plaques, an acquired type of amyloidosis.17, 18 The isoelectric point of the most present isotype (with 70% of apoA1) is 5.7, resulting in a negative net charge at pH 7.19 But also it holds neuroprotective properties, such as inhibition of amyloid-aggregation. Low plasma apoA1 concentrations are associated with Alzheimer’s disease.21 The lipoprotein apoA1 has an exon 3 (1-43) and 4 (44-243) encoded region, that contains 10 11/22-mer tandem repeat amino acid component. These segments are predicted to form class A and Y amphipathic α-helices (AαHs).20 These α-helices are the lipid binding motif of apoA1 and the structural basis for the multiple functions like the anti-arterogenic, -inflammatory and -oxidative effects. A lot of studies suggest that the lipid-free apoA1 fold a 4-helix bundle and is split in domains.22 The N-terminal domain


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is structural inflexible and the less organized structure of the C-terminal possess the highest lipid binding affinity.23 The 4-helix bundle shows a dimension of 88 x 25 x 27 Å3.22 Apolipoprotein A1 is a monomer in solution under a concentration of 0.1 mg/ml. Several studies showed that the lipidfree apoA1 self-associates at membranes in human plasma.24 Via hydrophobic interaction of the non-polar sites of the protein, apoA1 is able to bind cholesterol and triglycerides. The outer part is hydrophilic accomplishing a sufficient solubility in aqueous solutions, and forms stable micellar complexes with phospholipids, cholesterol, triglycerides, and cholesteryl esters.25, 26 Furthermore apoA1 is characterized as a molten globule and a loosly fold protein.27, 28 Due to the anisotropic surface (polar and apolar α-helices), apoA1 is able to interact with surfaces and interfaces via different interaction mechanisms including hydrophobic and electrostatic interactions. Investigations that provide information about the adsorption behaviour of apoA1 at different surface pressures and microenvironments can improve the understanding of the structure/function relationship as well as the membrane/protein interaction. Surface sensitive X-ray scattering methods are suitable for these experiments. The advantage is that it is possible to investigate proteins and polypeptides label free and in-situ. In the presented study the adsorption of apoA1 at hydrophobic liquid/gas- and hydrophilic solid/liquidinterfaces is investigated by means of x-ray reflectivity (XRR). Structural information of interfaces is gained with Ångström resolution.3, 29 By analyzing the data we are able to provide a deeper knowledge on changes of the three dimensional structure induced by protein/interface interactions. In an XRR experiment, the intensity of a specular reflected x-ray beam is monitored as a function of the incident angle α. Due to this scattering geometry, the wave vector transfer in such an experiment 𝐪𝐪 has only one component, which is 𝑞𝑞𝑧𝑧 =

4𝜋𝜋 𝜆𝜆

𝑠𝑠𝑠𝑠𝑠𝑠(𝛼𝛼 ), perpendicular to the sample surface or interface. In the first

Born approximation the reflected intensity is given by30

𝐼𝐼( 𝑞𝑞𝑧𝑧 ) ∝

1

𝑞𝑞𝑧𝑧4

�∫

𝑑𝑑𝜌𝜌𝑒𝑒 (𝑧𝑧) 𝑑𝑑𝑑𝑑

2

exp(𝑖𝑖𝑞𝑞𝑧𝑧 𝑧𝑧)𝑑𝑑𝑑𝑑� .



Thus, this technique is sensitive to changes of the electron density perpendicular to the samples 𝑑𝑑𝜌𝜌𝑒𝑒 (𝑧𝑧) ). 𝑑𝑑𝑑𝑑

surface (

The XRR technique is a widely used method for interface characterization. It is utilized

for many applications. In the Ångström regime it provides an exactness in terms of layer thickness, roughness, and electron density. We show that the adsorption of apoA1 at hydrophobic interfaces goes in hand with an unfolding and denaturation of the protein, resulting in thin layers of approximately 12 Å thickness, while at hydrophilic interfaces, the protein structure is affected less strongly. Moreover, the interaction of apoA1 with a cationic lipid monolayer structure led to a complete reduction of the electron densities without changing the layer thickness.

Experiment Liquid/gas-interface The measurement of surface tension was used to monitor the adsorption kinetics at the liquid/gas-interface. A pendant drop set-up (OCA 20, Dataphysics, Fielderstadt, Germany) was utilized to monitor the surface tension over a time span of 3500 s at different protein concentrations between 10-3 mg/ml and 5 ∙ 10-2 mg/ml. A surface pressure/area isotherm was recorded for the apoA1 concentration of c = 0.01 mg/ml including a subsequent relaxation monitoring of the compressed film.

The phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA, anionic, Sigma Aldrich, Germany) and a 1:1 mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, zwitterionic, Avanti, USA) and 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA, cationic, Avanti, USA) were used in order to study the electrostatic interactions between charged interfaces and apoA1. The combination of DOPC and DOTMA was used because pure DOTMA films were not stable under compression. The surface pressure start value of the DOPC/DOTMA film was π = 15 mN/m. A NIMA Langmuir trough was used for this purpose. The adsorption of apoA1 at the liquid/gas-interface was studied with different surface modifications. First, the interface between a bare aqueous solution and air was used as a model system of an extreme hydrophobic interface. Apolipoprotein A1 concentrations between c = 0.0005 g/l and 0.05 g/l in a 10 mmol/l Na2HPO4-NaH2PO4 buffer with a pH 7 were used.


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The lipids were dissolved in chloroform or 2-propanol resulting in a concentration of 1 mmol/l. After the solution was placed dropwise on the buffer solution and after an evaporation time of several minutes, the lipids were compressed into the solid phase (DPPA, π = 35 mN/m). Surface pressure dependent measurements were performed for the cationic DOPC/DOTMA monolayer at 10, 15 and 20 mN/m π-values. All measurements at the liquid/gas-interface were performed at room temperature. The x-ray reflectivities were recorded at beamline BW131 of the synchrotron light source DORIS III at Hamburg, Germany, using the liquid scattering set-up. The photon energy was 9.5 keV. Further measurements were performed at a photon energy of 8 keV using a Bruker D8 diffractometer in θ - 2θ geometry. Here a typical x-ray reflectivity scan reaching a maximum wave vector transfer of 0.5 Å-1 took around 3 h including the measurements of the diffusely scattered radiation which was subtracted from the reflectivity data. Due to the large beam size (10 ∙ 0.1 mm2 (h x v)) and low photon flux

(5 ∙ 105 photons/s) no radiation damage is expected, which was proven by control measurements. A reflectivity curve from the bare systems without protein was recorded as a reference. Subsequent, the protein was injected into the subphase. After a waiting time of 60 minutes a reflectivity curve of the interface was measured.

Solid/liquid-interface Furthermore the adsorption of apoA1 was investigated pH dependent at a hydrophilic interface between a native SiO2 layer on top of a silicon wafer and aqueous protein solution. Here the variation of pH causes both changes to the protein charge and changes to the SiO2 zeta potential.32 As 70% of apoA1 is one isoform which has an isoelectric point of pI = 5.6 whereas the zeta potential of SiO2 tends to zero below pH 3, adsorption is mainly expected in a pH region between pH 3 and pH 6. The silicon wafers were cleaned using piranha solution ([safety warning: may explode when interacts with organic solvents] 75% sulfuric acid (96%) and 25% hydrogen peroxide (30%)) and stored under water (Milli-Q water) until use. The protein was dissolved in a 10 mmol/l Na2HPO4 - NaH2PO4 buffer solution. The concentration was c = 0.01 mg/ml. The pH of the solutions were adjusted by the addition of NaOH and HCl respectively. The measurements at the solid/liquid-interface were performed at the synchrotron light source DELTA, Dortmund, Germany, using the 27 keV set-up of beamline BL9.33 5 ACS Paragon Plus Environment


First, an x-ray reflectivity curve of the interface between the wafer and pure buffer solution was recorded at a given pH as a reference. Afterwards the protein solution was filled into the cell. The beam size was 1 ∙ 0.2 mm2 (h x v). The low beam focusing and the photon energy of 27 keV minimize beam damage

in biological material.34, 35 A reflectivity scan including the measurement of the diffusely scattered

radiation took 30 min. The photon flux was 7 ∙ 108 photons/(s∙mm2). Data analysis To analyze the data, the raw data were first normalized and then corrected by subtraction of the diffuse scattered data. The x-ray reflectivities were analyzed using the Parratt algorithm36 in combination with the effective density model.37 In our study, one layer is adequate to describe the adsorbed protein layer at the hydrophobic air/waterinterface and the hydrophilic silicon dioxide surface sufficiently. To minimize the number of fitting parameters at the hydrophilic substrates a characterization under pure buffer without protein was implemented. As no changes in the silicon wafer properties are expected during the protein adsorption afterwards, the EDP received for the substrate is kept constant when analyzing the data after apoA1 adsorption. The electron density of the silicon substrate, the silicon dioxide layer, and the water subphase are assigned to the theoretical values of 0.702, 0.663 and 0.334 e-/Å3 respectively. The electron density of the silicon dioxide layer was varied over a narrow range. The silicon dioxide layer thickness is varied between 10 and 15 Å. The significance of the resulting electron density profiles was evaluated by a comparison of the results of independent refinements. To obtain the behavior of apoA1 at the mixed DOPC/DOTMA monolayer the two-layer system which was used to fit the lipid layer without protein was adjusted.

Discussion The liquid/gas-interface The time dependent surface tension measured for different protein concentrations is shown in figure 1, top. At t = 0 s the protein was injected into the buffer solution. Due to the adsorption of apoA1 at the liquid/gas-interface, the surface tension drops from the water value at γ = 72 mN/m down to e.g. γ = 49 mN/m for the highest apoA1 concentration. The adsorption kinetics depend strongly on the 6 ACS Paragon Plus Environment

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protein concentration.39 While we observe only a small linear decrease of surface tension for the two lowest concentrations, concentrations above 0.01 g/l show a convergence within 3500 s. In figure 1 bottom, a surface pressure/area isotherm of apoA1 at the liquid/gas-interface is shown. The compression was started after surface tension was stabilized. The surface pressure rises monotonically with decreasing surface area until a film collapse was observed at π = 30 mN/m. The measurement of the surface pressure behaviour of a DOPC/DOTMA film after adding apoA1 with a resulting concentration of 0.005 mg/ml, is shown in figure 2. At t = 300 s apoA1 was added and a fast increase of Δπ = 8 mN/m could be observed pointing to an adsorption of apoA1 at the interface. After t = 2000 s a slow decrease of the surface pressure can be noticed.

Figure 1: Top: Time dependent surface tension measured for different protein concentrations. Blue: 0.001 g/l, red: 0.002 g/l, black: 0.005 g/l, green: 0.01 g/l, magenta: 0.02 g/l, cyan: 0.05 g/ml. Bottom: Surface pressure/area isotherm of an aqueous apoA1 solution. The measurement was started after the surface pressure was in equilibrium.

X-ray reflectivities of the liquid/gas-interface measured for different protein concentrations are shown in figure 3 together with electron density profiles obtained by a refinement of the reflectivity data.



Figure 2: Observation of the surface pressure with time. The feedback regulation was turned off after reaching the equilibrium at π =15 mN/m and apoA1 was injected (indicated by the arrow).

It becomes visible, that at protein concentrations above 0.01 g/l a dense layer forms at the interface, while at c = 0.001 g/l no effect on the structure of the water/gas-interface is observed. However, if the surface pressure is increased by a reduction of surface area, a similar shaped film is formed also at a very low concentration of c = 0.0005 g/l. It is striking that the film thickness of around 12 Å seems to be independent of the protein concentration. Lipidfree apoA1 is an elongated protein41 having about 45% α-helical content at a pH-value around 7 here the amphipathic helical structures are pronounced.42 Predominantly, the monomeric form of apoA1 is present under a concentration of 0.1 mg/ml in solution.24 The monomeric form has an extent of 25 Å (RCSB PDB - 2A01).43 As only layer thicknesses of 12 Å were observed, we conclude that the strong hydrophobic interaction cause a denaturation or unfolding of the protein at the liquid/gas-interface. We assume, that the 4-helix bundle of the monomer unfolds and the hydrophobic sites are oriented towards the air.


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Figure 3: Top: X-ray reflectivities of the liquid/gas-interface recorded for different apoA1 concentrations. The reflectivities are normalized by the Fresnel reflectivity of an ideal flat water surface and shifted vertically for a better visualization. The solid lines are the refined reflectivities calculated from the modelled electron density profiles (bottom). Black/stars: 0.05 g/l, red/circles: 0.025 g/l, blue/diamonds: 0.01 g/l, magenta/squares: 0.001 g/l, green/triangles: buffer, cyan/crosses: 0.0005 g/l (compressed to 20 mN/m).

The attachment takes place with the hydrophobic sites of the α-helices of the long axis. The surface/areaisotherms lead us to assume that the layer thicknesses which are investigated with the XRR technique are attributable of protein molecules which are lying with the long axis at the surface.55 Apolipoprotein A1 can adopt a variety of conformations to fulfill its broad spectrum of in-vivo physiological functions. Furthermore apoA1 is characterized as a molten globule and a loosely fold protein.27 Due to this polymorphism and the unknown state of hydration, it is not possible to draw conclusion from electron densities about the conformation of the protein. Thus, we used the observed layer thicknesses to interpret the processes at the interfaces. The x-ray reflectivity curves of the DPPA layer on pure buffer phase and of DPPA on aqueous apoA1 solution show no differences in the two density profiles. Both curves could be fitted with the same model consisting of the DPPA head and tail group. Thus the electrostatic repulsion between the negative charged DPPA film and the negative charged apoA1 prevents completely the adsorption of the protein. 9 ACS Paragon Plus Environment


The x-ray reflectivities of DOPC/DOTMA films on pure buffer with different surface pressures and after adding apoA1 solution are shown in figure 4 together with electron density profiles obtained from the refinement of the data. The electron densities and thicknesses of the lipid layer changes upon compression the monolayer, as expected.

Figure 4: Top: X-ray reflectivities of the DOPC-DOTMA/gas-interface recorded for different surface pressures and with apoA1 in a resulting concentration of c = 0.005 mg/ml. The reflectivities are normalized by the Fresnel reflectivity of an ideal flat water surface and shifted vertically for a better visualization. Red/circles: 10 mN/m, blue/squares: 15 mN/m, green/diamonds: 20 mN/m. Bottom: Electron density profiles obtained by a refinement of the reflectivity data with a scheme of the probe system.


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However, there are differences between the electron density profiles of the monolayer before and after adding apoA1. Note that the reflectivity curves were recorded after the strong increase of the surface pressure induced by the addition of apoA1 and the system reached equilibrium. The interaction of apoA1 with the monolayer structure led to a complete reduction of the electron density without changing the layer thickness. These behavior of apoA1 are surface pressure dependent and occur predominantly at low pressures. Thus, it seems that apoA1 with a negative net charge at pH 7 adsorbs at the cationic monolayer structure via electrostatic interactions and reach a physical proximity. Apolipoprotein A1 then penetrates it and finally gets the lipids into the subphase as an "in-vitro nascent discoidal particle". The typical structure of the monolayer after adding apoA1 is not affected, but the surface pressure decreased and the overall electron density decreases due to the removal of lipids by the apoA1. In the presence of lipid monolayers or lipids in general apoA1 forms dimers which are able to bind lipids. Here, the affinity and the lipid packing efficiency decline with decreasing surface pressure.44, 55 The changes of the surface pressures are documented in the inset of figure 4. We suspect that the lipid uptake especially takes place at low surface pressures. Here the lipid head groups are less tight packed and the protein is able to penetrate between the head groups. The surface pressure seems to be an important factor for adsorption, insertion and conformation of apoA1.45, 55, 56 The molecular mechanism of lipid binding of lipidfree apoA1 is not fully understood. A lot of studies have been carried out on the assembly of apoA1 with lipids and there are some lipid packing mechanisms which are present in the literature; for example a two-step mechanism57 or other models.28, 58, 59 Apolipoprotein A1 does not denaturized at these interfaces and stays bioactive.

The solid/liquid-interface X-ray reflectivities of the solid/liquid-interface between silicon wafers and aqueous apoA1 solution with a concentration of 0.01 mg/ml and buffer recorded at different pH-values are shown in figure 5 together with the electron density profiles obtained by a refinement of the reflectivity data.



Figure 5: Top: X-ray reflectivities of the solid/liquid-interface between a hydrophilic silicon wafer and aqueous apoA1 solution with a concentration of 0.01 mg/ml, measured at different pH-values. All reflectivities are normalized by the Fresnel reflectivity of an ideal flat silicon wafer interface and shifted vertically for better visualization. The solid lines are fits to the data. Bottom: Electron density profiles obtained by the fit of reflectivity data.

The measurements at hydrophilic silica surfaces show that apoA1 adsorbs in different amounts depending on the pH-value. Between pH 4 and pH 6, an adsorption window of apoA1 with different electron densities and layer thicknesses was determined, showing broad possibilities of conformations and extreme changes of the structure. As we obtain density profiles of both, the pure silica wafer/buffer system and the system with protein, we are able to extract the protein layer structure by a subtraction of the pure buffer profile. From the resulting volume fraction profile (not shown) we can calculate the relative amount of adsorbed protein (Γ) at different pH-values by integrating the area. The adsorbed amount versus the pH is shown in figure 6 where the line is a guide to the eye. 12 ACS Paragon Plus Environment

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Figure 6: Protein adsorbates (Γ) at given pH-values in order to emphasize the changes induced by the different pH-value. The results show an "adsorption window" between the pH 4 and pH 6.

The adsorption between pH 4 and pH 6 was mainly driven by electrostatic interactions, since the protein and surface have opposite charges in this pH region. The hydrophobic properties and electrostatic pattern on the surface of apoA1 allows the protein to adsorb at the charged interfaces22, 28. As shown in figure 7 an average film thickness of 25 Å was observed. As already mentioned, under a concentration of 0.1 mg/ml apoA1 exists predominantly in a monomeric form in solution24 and its 4helix bundle was described by Ajees et al.22 These structure of the full-length protein measures 88 x 50 x 27 Å3 at its widest point, the 4-helix bundle shows dimension of 88 x 25 x 27 Å3.

Figure 7: Layer thickness of the apoA1 adsorbates at given pH-values in order to emphasize the changes induced by the different pH-value. The error can be estimated with approx. 1 – 2 Å by variation of the refinement parameter.

Thus, we infer that the protein is not denatured completely at the solid/liquid-interface and adsorbs with the long axis parallel to the surface. Increasing the contact area with the surface by unhinging the helices 13 ACS Paragon Plus Environment


of the N- and C-terminal helices to reach the surface with the inner positive charged domains seems to be possible. Apolipoprotein A1 is conformationally flexible, elastic, and can adopt various states. Several of the conformational transitions in apoA1 are prone to be dependent on their microenvironment, like the pH-value.50

Conclusion In conclusion, the extremely surface-active human lipoprotein apoA1 denatured and unfolded at a liquid/gas-interface to cover a high surface area with the apolar sites of the amphipathic α-helices.54 Also the surface/area-isotherms lead us to assume that the layer thicknesses which are investigated with the XRR technique are attributable of protein molecules which are lying with the long axis at the surface.55 Combining the surface/area-isotherms of pure apoA1 at the gas/water-interface which indicate a monomolecular film with the results of x-ray reflectivity measurements which give the layer thickness we can conclude that apoA1 adsorbs with the helical segments of the long axes in the plane of the interface. The interaction of apoA1 with the DOTMA/DOPC monolayer structure led to a complete reduction of the electron density without changing the layer thickness. It seems that apoA1 adsorbs, penetrates and finally solubilizes the lipids into the subphase as an "in-vitro nascent discoidal particle". These actions of apoA1 are surface-pressure dependent and predominantly occur at low pressures. Here the protein is able to penetrate between the lipid head groups and the lipid uptake takes place. Other studies also describes that the surface pressure is an important factor for the conformational flexibility of apoA1 and the resulting structure.56 The interaction of lipidfree apoA1 with the DOTMA/DOPC monolayer seems to result in lipidrich apoA1 particles which are being packed and detached from the monolayer. Apolipoprotein A1 seems to be bioactive in the presence of DOPC/DOTMA layers and fulfil its in-vivo function. There are a lot of mechanisms which describes the lipid uptake.28,

57, 59

Studies of the molecular mechanisms of the lipid association and the

conformational flexibility of apoA1 are essential for understanding the structure/function relationships and pathophysiological reactions at surfaces like natural membranes or implants. At the solid/liquid-interface apoA1 adsorbs depending on the pH-value in a monomeric state with an 14 ACS Paragon Plus Environment

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adsorption window between pH 4 and pH 6. These observations at the solid/liquid-interface gives a link to the adsorption behaviour of apoA1 at implant surfaces. At a physiological pH of 7 the relative amount of absorbed apoA1 is low. Current interest in biocompatible materials has focused on surface modifications to increase healing and integration of foreign matter.60, 62 Furthermore lipidfree apoA1 plays a critical role in pathophysiological processes like in the development of amyloidosis. As a main constituent of amyloid deposits beside cholesterol and other lipids, apoA1 was found in atherosclerotic plaques, an acquired type of amyloidosis, and senile plaques.17,

18

Hydrophobic

surfaces like the air/water-interface are good models to study the behaviour of proteins which are aggregation-prone to get information about the development of amyloid fibrils and the tendency to aggregate. Apolipoprotein A1 unfolded and denatured at these interfaces. An aggregation was not observed at these pH, temperature and concentration of apoA1.

Acknowledgement We acknowledge the DELTA machine group and HASYLAB (DESY) for providing synchrotron radiation. We also acknowledge Bernd Struth (BW1) for technical support during the beamtimes. Irena Kiesel thanks NMI3-II Grant number 283883 for financial support. PS acknowledges the DFG Research Unit FOR1979 for financial support. This work is supported by the Cluster of Excellence RESOLV (EXC 1069) founded by the Deutsche Forschungsgemeinschaft.



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Gas Interfaces

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