Effects of Al3+ on Phosphocholine ... - ACS Publications

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Effects of Al on phosphocholine and phosphoglycerol containing solid supported lipid bilayers Hannah K. Wayment-Steele, Yujia Jing, Marcus J. Swann, Lewis E Johnson, Björn Agnarsson, Sofia Svedhem, Malkiat S Johal, and Angelika Kunze Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b03999 • Publication Date (Web): 19 Jan 2016 Downloaded from http://pubs.acs.org on January 24, 2016

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Effects of Al3+ on phosphocholine and phosphoglycerol containing solid supported lipid bilayers

Hannah K. Wayment-Steele1, Yujia Jing2, Marcus J. Swann3, Lewis E. Johnson,1,4 Björn Agnarsson2, Sofia Svedhem2, Malkiat S. Johal1, and Angelika Kunze2,5*

1

Department of Chemistry, Pomona College, 645 North College Ave. Claremont, CA 91711, USA

2

Department of Applied Physics, Chalmers University of Technology, 412 96 Göteborg, Sweden

3

Swann Scientific Consulting Ltd. 110 Sandy Lane, Lymm, Cheshire, UK

4

Department of Chemistry, University of Washington, 109 Bagley Hall, Seattle, WA 98195, USA

5

Institute of Physical Chemistry, University of Göttingen, 37077 Göttingen, Germany

*

corresponding author: [email protected]

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Abstract Aluminum has attracted great attention recently as it has been suggested by several studies to be associated with increased risks for Alzheimer’s and Parkinson’s disease. The toxicity of the trivalent ion is assumed to derive from structural changes induced in lipid bilayers upon binding, though the mechanism of this process is still not well understood. In the present study we elucidate the effect of Al3+ on supported lipid bilayers (SLBs) using fluorescence microscopy, the quartz crystal microbalance with dissipation (QCM-D) technique, dual-polarization interferometry (DPI), and molecular dynamics (MD) simulations. Results from these techniques show that binding of Al3+ to SLBs containing negatively charged and neutral phospholipids induces irreversible changes such as domain formation. The measured variations in SLB thickness, birefringence and density indicate a phase transition from a disordered to a densely packed ordered phase.

Introduction Aluminum, the third most abundant element in the earth’s crust, is found in daily life as a contaminant in food products as well as in medical and cosmetic products.1 The aluminum ion Al3+ is of health-related interest for numerous reasons; Al3+ has been implicated in blood, liver, kidney, and bone diseases including encephalopathy, microcytic anemia, and osteomalacia (aluminum bone disease).2 Importantly, it has been shown that Al3+ can cross the blood-brain barrier, accumulating in several regions of the brain. Several studies have suggested that this accumulation of Al3+ is correlated with increased risks for neuronal disease such as Alzheimer’s and


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Parkinson’s disease.3-5 These diseases are characterized by dysfunction in neuronal communication steered by synapses.6 Membranes at the synaptic interface are enriched in lipid domains, which are likely to organize and maintain synaptic proteins.7 These lipid domains are a consequence of phase separation of different combinations of lipid molecules (such as cholesterol, ceramide, sphingomyelin) within the membrane. In general, the lipids of the cell membrane can exist in several different phase states, characterized by the diffusivity of the lipid molecules and the order parameters of the acyl chains. Below the main phase transition temperature Tm, the lipids are found in the solid gel phase so (high order parameter, low diffusivity). Above Tm they can exist either in a liquid disordered ld (low order parameter, high diffusivity) or a liquid ordered lo (high order parameter, high diffusivity) phase. As the phase separation of the lipid molecules (forming lipid domains) at the synaptic interface seems to be of crucial importance for neuronal communication,7 it is imperative to understand the effect of Al3+ on the cell membrane and its associated phases. Liposomes and solid supported lipid bilayers (SLBs) are two common model systems used to mimic the complex cell membrane. The former of these model systems, liposomes, has been used in previous studies for the investigation of Al3+ interactions with phospholipids. Deleers et al. showed that Al3+ leads to aggregation of negatively charged liposomes in solution.8 Furthermore, Verstraeten et al. suggested a change in lipid packing and domain formation upon binding of Al3+ to liposomes composed of a mixture of neutral and negatively charged lipids.8, 9 10 However, due to the liposome aggregation reported in these studies, the observed effects cannot be unambiguously attributed to interaction of the lipids within the bilayer (termed cis-interactions) but may include interactions between lipids of separate bilayers from adjacent liposomes


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(trans-interactions). In contrast, SLBs allow for the investigation of solely cisinteractions without interference with lipids of another adjacent bilayer. Thus, results obtained in studies using SLBs can more reliably be attributed to lipid-lipid interactions within the bilayer. Another advantage of using SLBs as model systems for cell membranes is that the surrounding bulk solution can easily be exchanged, which aids in probing the reversibility of binding effects. Furthermore, SLBs provide the opportunity to apply highly sensitive surface analytical techniques to characterize the physical properties of the membranes. Until now, the effects of Al3+ binding to SLBs have not been studied. In this work, we elucidate the effect of Al3+ binding to lipid bilayers using a combination of different experimental techniques such as fluorescence recovery after photobleaching (FRAP), dual-polarization interferometry (DPI), and quartz crystal microbalance with dissipation monitoring (QCM-D) supported by molecular dynamic (MD) simulations. The combination of these techniques allows for the study of changes in physical characteristics in lipid bilayers including diffusivity, lipid packing, and bilayer thickness. These effects were investigated with negatively charged SLBs containing a mixture of 50 mol% POPC and 50 mol% negatively charged POPG (referred to henceforth as POPC/POPG bilayers) and neutral SLBs containing only zwitterionic POPC.

Material and Methods Chemicals 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-snglycero-3-phospho-(1’-rac-glycerol) (POPG), and 1-palmitoyl-2-(6-[(7-nitro-2-1,3-


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benzoxadiazol-4-yl)amino]hexanoyl)-sn-glycero-3-phosphocholine (NBD-PC) were purchased from Avanti Polar Lipids Inc. (AL, USA). Structures of these lipids are given in Figure 1. All other chemicals were of analytical grade, were obtained from commercial sources and were used without further purification. Water was deionized (resistivity > 18 MΩ⋅cm-1) and purified using a MilliQ unit (MilliQ plus, Millipore, France). Tris buffer (pH 8.0) contained 10 mM Tris and 100 mM NaCl. Tris-CaCl2 and Tris-AlCl3 buffers were made from Tris buffer together with 10 mM CaCl2 and 10 mM AlCl3, respectively. Buffers were degassed before use. Liposome preparation Lyophilized lipids were dissolved in chloroform (stock solutions were stored at 20°C) and placed in round bottom flasks. The solvent was first evaporated under a gentle stream of nitrogen while gently rotating the flask to form a thin lipid film on the wall of the flask, and then further dried under vacuum for 3 hours. The lipid films were hydrated in Tris buffer. After vortexing, the solutions were extruded 13 times through 30 nm filters (Nucleopore Track-Etched Membrane, Whatman, USA). Following this protocol the prepared liposomes typically measure 80-100 nm in diameter, as determined by dynamic light scattering. The liposome suspensions were stored refrigerated under N2. FRAP In FRAP, a defined spot of a biological sample with a uniform dispersion of fluorophores is bleached for a short time with a high-intensity light source. The subsequent diffusion of unbleached fluorophores to the bleached spot, and the associated recovery of the fluorescence signal, is monitored and the diffusion coefficient Ddiff is extracted.


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FRAP experiments were carried out employing a Zeiss Axioplan 2E MOT microscope (Zeiss, Jena, Germany) microscope equipped with a mercury lamp using a 40x water immersion objective. NBD-PC was selected as a fluorescent lipid probe; it was selected due to a low probability of the chromophore interfering with ionheadgroup coordination, as the chromophore is located on the tail of the lipid. To create fluorescent SLBs, 2% NBD-PC in the POPC bilayer fraction was used, a concentration that has been demonstrated to not significantly affect bilayer physical properties.11 SLBs were formed in accordance with the “adsorb-rupture-fusion” method12 as follows. Liposomes were added to a petri dish containing a sputter-coated SiO2 crystal (same surface as used for QCM-D experiments) immersed in the respective buffer and incubated for one hour. For POPC SLBs, POPC liposomes were added to Tris buffer. After then thoroughly rinsing the bilayer with Tris buffer to remove excess liposomes, Tris buffer containing 100 mM AlCl3 was added in aliquots to obtain the desired final concentration and FRAP experiment were conducted. Thereafter the bilayer was rinsed with Tris buffer, and FRAP data was collected again (data referred to as ‘after rinse’). This process was repeated at increasing concentrations of AlCl3. Similar experiments adding Tris-CaCl2 were performed for comparison. POPC/POPG SLBs were formed similarly, except as an initial step, POPC/POPG liposomes were added to 10 mM Tris-CaCl2 buffer. The Ca2+-ions were add to promote SLB formation acting as bridging ions between the negative charges on the SiO2 surface and negatively charged lipid headgroup. After thoroughly rinsing the bilayer with Tris buffer FRAP experiments for various concentrations of AlCl3 and CaCl2 were performed.


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FRAP diffusion measurements were performed for each new buffer condition using the following procedure. Prior to bleaching, 5 images of the lipid bilayers were taken to compensate for uneven illumination over the sample. Thereafter a spot (35 µm in diameter) was bleached for 10 s under illumination from a mercury lamp. After photobleaching, the lamp intensity was lowered and a series of 30 images was recorded at 5 s intervals. Diffusion was determined from three discrete spots on each SLB. Image analysis was performed using the Matlab script “frap-analysis” described in detail by Jönsson et al.13 DPI DPI experiments were performed using an AnaLight 4D instrument (Farfield Group Ltd., U.K.). One day prior to the experiment, the sensor surface (silicon oxynitride) on the AnaChip (Farfield Group Ltd., U.K.) were treated in UV/ozone for 30 min, and then stored in water overnight before being mounted in the instrument. All experiments were performed at 22°C. The running buffer (20 µL/min) in the experiments was Tris buffer, and all injections were performed using a 200 µL injection loop (setting a limit to the maximum volume used). Data evaluation was performed using the AnaLight Explorer software. The sensor waveguide thickness and refractive index (RI) was calibrated using the bulk RI change between 80% ethanol(w/w in water) and water solutions, and the phase response was “linearized” to correct for small periodic errors in the phase values using the 80% ethanol to water transition. The measured TE and TM phase change data is used to calculate the SLB thickness, dSLB, the isotropic refractive index, nSLB, and birefringence using Maxwell’s waveguide equations with a single slab layer model in which one of the three parameters is


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fixed.14-16 The mass is determined from the refractive index and thickness using the de Feijter equation17 mSLB = dSLB(nSLB - nbuffer)/(dn/dc)lipid where mSLB is the mass per unit area of the SLB in g·mm-2 and (dn/dc)lipid is the refractive index increment of the lipid (0.135 cm3·g-1).18, 19 The mass value is essentially insensitive to the value of the fixed parameter in the model. Using a fixed thickness or isotropic refractive index value enables the birefringence of the layer to be calculated.16, 19 QCM-D QCM-D experiments were performed using a Q-Sense E4 instrument (Q-Sense AB, Sweden). AT-cut quartz crystals with a fundamental frequency of 5 MHz coated with SiO2 were purchased from Q-Sense AB. Prior to the experiment the crystals were cleaned in a 10 mM sodium dodecyl sulfate aqueous solution (overnight), rinsed thoroughly with water, dried under N2, and treated with UV/ozone for 3 x 15 min with rinsing and drying in between. The measurements were carried out at 22°C using a flow rate of 100 µL/min. Frequency and dissipation data from the 7th harmonic was selected for further analysis based on its superior signal to noise ratio. Frequency shifts were normalized to the fundamental frequency by dividing the values by 7. MD Simulations All lipid bilayer simulations were performed with a total of 128 lipids in an initial box size of roughly 6 x 6 x 10 nm with periodic boundary conditions in all directions. Coordinates for both POPC and POPC/POPG bilayers were generated using MemGen20 and were equilibrated for 20 ns before adding ions. After an initial energy minimization using a steepest-descent algorithm with an RMS gradient of 1000 kJ mol-1 nm-1, systems were simulated for 100 ns. Data from the first 30 ns were excluded to allow for equilibration. The Stockholm lipids forcefield for POPC21 and


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POPG22 was used along with the TIP3p water model.23 Lennard-Jones parameters for Al3+ were taken from Faro et al.24 Simulations were performed using GROMACS 4.6.1. Initial velocities were randomly generated from the Maxwell distribution. A time step of 2 fs was used for all simulations with the Leap-Frog integrator. The LINCS algorithm was used to constrain all bonds in the system. Membranes were simulated in the NPT ensemble, with constant pressure at 1.013 bar and constant temperature of 310 K. Pressure was controlled via Parinello-Rahman semi-isotropic coupling, with the pressure in the xy plane (the plane of the membrane) coupled independently from the pressure along the z axis. The barostat used a coupling constant of 0.5 ps and an isothermal compressibility of 4.5 x 10-5 bar-1 for all axes. Temperature was regulated by a NoseHoover thermostat. The water and ions were coupled separately from the membrane system, with both using a coupling constant of 0.5 ps. Long-range electrostatic interactions were treated with Particle-Mesh Ewald summation. The comparison between MD simulations and experimental data must be carried out in a qualitative manner and for that there are several reasons: To simulate Al3+, we use the pair-wise Lennard-Jones-type parameters developed by Faro et al. These parameters were optimized for Al3+ in water, and although they have been used by others to probe Al3+ interacting with biomolecules,25 such pair-wise Lennard-Jonestype interactions have been demonstrated to underestimate the strength of electrostatic interactions of divalent, trivalent, and tetravalent cations.26 A forcefield that includes electronic polarizability or other higher-order effects would provide a better representation of the interactions of the highly-charged Al3+ ion with biomolecules, but such a forcefield has not yet been developed to include Al3+. Secondly, lipid forcefields are optimized to represent the fluid phase of the bilayer, and runs using


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these forcefields are typically conducted at high temperatures to maintain the fluid phase. In our experiments the bilayers are believed to enter a gel-phase-like state, that we do not believe to be very well represented by a lipid forcefield that is optimized for the fluid phase. Furthermore, MD simulations only account for interactions between the lipids and the ions, disregarding interactions between the lipids of the SLB and the underlying substrate.

Results Fluorescence Microscopy Fluorescence microscopy was employed to determine the effect of Al3+ on the distribution and on the diffusivity of the lipid molecules within the bilayer. The latter was determined by FRAP, a frequently used method for the characterization of bilayer fluidity.13, 27 FRAP measurements were performed with POPC/POPG and POPC bilayers at increasing Al3+ concentrations to quantify the effect of Al3+ concentration on the diffusivity of the lipid molecules. For comparison, additional FRAP experiments were carried out in Tris buffer supplemented with 10 mM CaCl2. FRAP results for POPC bilayers In Figure 2a, characteristic microscopy images for POPC bilayers immersed in TrisCaCl2, Tris, Tris-AlCl3 (at 10 mM AlCl3), and again Tris, but after incubation with Al3+ are shown. From left to right, images are given for prior to bleaching, immediately after bleaching, and 150 s after bleaching. The corresponding diffusion coefficients obtained under these 4 conditions are listed in Table 1. POPC bilayers in Tris and Tris-CaCl2 resulted in the nearly identical diffusion coefficients of 2.5 and 2.4 µm2·s-1, respectively. These values are in agreement with values from literature.13, 10 Environment ACS Paragon Plus

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In TRIS buffer containing 10 mM AlCl3, the diffusion coefficient was found to be

about one order of magnitude lower (Ddiff = 0.3 µm2·s-1) compared the previous cases. However, this change was observed to be reversible (Figure 2a), as the diffusivity almost fully recovered after rinsing with Tris (removing Al3+ ions). Experiments with varying Al3+ concentration showed that the decrease in the diffusion coefficient for POPC depends on Al3+ concentration in solution (see SI, Figure S1). FRAP results for POPC/POPG bilayers Figure 2b shows characteristic microscopy images of the POPC/POPG bilayer in TrisCaCl2 and Tris-AlCl3 prior to bleaching, immediately after bleaching, and 150 s after bleaching. The diffusion coefficient of the bilayer in Tris-CaCl2 was measured to be 2.1µm2·s-1, a value very close to the diffusion coefficient of POPC in Tris-CaCl2 (2.5 µm2·s-1). Upon removal of Ca2+ ions by rinsing with Tris the diffusion coefficient was observed to increase to 5.3 ± 1.4 µm2·s-1 indicating that the bilayer had become decoupled from the SiO2 substrate, a phenomenon that has been demonstrated previously.29 In the case of Tris buffer supplemented with AlCl3, the POPC/POPG bilayers were immobile for the time scale under all experimental conditions. This can be observed visually by comparing in Figure 2b the bilayer in Tris-AlCl3 upon bleaching and the bilayer 150 s after bleaching (conditions marked with *). Even after 2 hours we did not observe any visible recovery. After rinsing with Tris, fluorescence recovery was again observed with the measured diffusivity depending on the concentration of Al3+ added in the previous step (see SI, Figure S1). Interestingly we did not only observe the bilayer to become immobile immediately upon the addition of Tris-AlCl3 (at all AlCl3 concentrations probed), we also observed lipid domains varying in brightness in the POPC/POPG bilayer (Figure 2c).


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Optical experiments using dual-polarization interferometry Optical experiments using DPI were performed to characterize changes in the mass, density, thickness and birefringence of POPC and POPC/POPG bilayers upon addition of Al3+. DPI measures the change in effective refractive index, n, in two different polarization modes (transverse electric (TE) and transverse magnetic (TM)) caused by the adsorption of a layer on the DPI sensor surface. In particular, the magnitude of the measured TE or TM phase change is proportional to the mass of the layer and the ratio TE/TM is proportional to the refractive index n of the adlayer, or inversely proportional to the birefringence.30 Together with the refractive index increment of dn/dc for the material under study (0.135 cm3·g-1 for lipids),19 the refractive index gives the density of material in the adsorbed layer18 and consequently the added (dry) mass (see also above Materials and Methods). A further benefit of DPI is that it allows for the characterization of anisotropic properties of the adlayer, such as the order parameter of the lipids, as discussed in the introduction.19, 31, 32 A higher value of birefringence indicates an aligned arrangement of lipids – such as in the well-ordered gel phase or liquid ordered lo phase – whereas a lower birefringence is characteristic for a less ordered arrangement – such as in the liquid disordered ld phase. Thus, DPI can be employed to investigate not only density, thickness and mass of adlayers, but also the phase transition behavior of lipid bilayers.19, 31 DPI experiments were performed following an analogous sequence as was described for FRAP experiments: Firstly, bilayer formation was performed by adsorption and spontaneous rupture of liposomes containing POPC or POPC/POPG respectively (indicated by * in Figure 3) on silicon oxynitride coated DPI sensors, followed by changing buffers to expose the bilayers to Tris-AlCl3 and then rinsing again with Tris. Figure 3a and 3b show the monitored TE and TM signal and the corresponding


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TE/TM ratio of the linearized signal for the POPC and POPC/POPG bilayers, respectively. The values for the thickness, refractive index, density, birefringence and mass of the two bilayers under the different ionic conditions are summarized in Table 2. DPI results for POPC bilayer Upon injection of liposomes, a rapid change in the optical signal was observed for both types of liposome solutions (Figure 3). In the case of POPC, the signals plateau at TE=11.2 and TM=14.8 rad (Figure 3a). This signal change, together with the dn/dc value for the material (0.135 cm3·g-1),19 corresponds to a mass of 4.73 ng·mm-2. These values are in agreement with values described before for the formation of a lipid bilayer on a DPI sensor.19, 31, 32 Subsequent buffer changes from Tris to TrisCaCl2 and Tris-AlCl3 buffer leave the signal essentially unaffected (Figure 3a), with only a small increase evident in the TE/TM ratio in presence of 10 mM AlCl3. The birefringence of the POPC bilayer in Tris-CaCl2 can be calculated using an isotropic refractive index of n = 1.47.19 This gives a birefringence value of 0.0180 in Tris, which is in the expected range for a fluid, birefringent lipid bilayer.19, 31, 32 When changing the ionic conditions, the calculated birefringence (assuming a constant n) (Table 2) shows a small reversible decrease to 0.0178 in the presence of Ca2+ and a slightly larger, but still small and reversible, decrease to 0.0168 in the presence of Al3+. DPI results for POPC/POPG bilayer In the case of the POPC/POPG SLB, larger phase changes than for the formation of the POPC SLB are observed, with TE = 16.9 and TM = 22.1 rad (Figure 3b). Changing to Tris buffer led to a significant decrease in the monitored TE and TM


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phase changes and the corresponding TE/TM ratio. These changes are mostly reversible (see Figure S2 in SI) and about 10 times the change of the bulk shift of a buffer change from Tris-CaCl2 to Tris buffer on a bare DPI sensor. The TE/TM ratio in Tris buffer is also significantly lower than that for POPC in Tris buffer, indicating a significantly less densely packed bilayer. In contrast to the case for Ca2+, addition of Al3+ to the POPC/POPG SLB in Tris buffer leads to a mostly irreversible increase in the DPI signal (Figure 3b), indicating irreversible binding effects of the trivalent cations to the lipid bilayer. The TE/TM ratio is also increased by 0.0165 which is significantly larger than was the case for Ca2+ (0.0083), and which indicates an increase in bilayer density, as well as the significant increase of the bilayer mass per unit area. It is also worth noting that the TE/TM ratio values did not stabilize as quickly as they did for Ca2+, which may indicate a slow rearrangement of the bilayer structure (possibly related to the domain formation observed via fluorescence microscopy) following solution exchange. Birefringence, thickness, and density values for the bilayers under different buffer conditions (listed in Table 2) were calculated through an iterative process based on the monitored TE and TM phase change. Acoustical sensing of Al3+ binding effects on SLBs Acoustical sensing via QCM-D has become a frequently used method for the characterization of lipid bilayer formation and the study of lipid bilayers with biological entities. Here, QCM-D was employed to further investigate the effects of Al3+ binding to POPC/POPG bilayers, providing complementary results to the optical methods described above. The signal of the QCM-D provides two parameters; the (resonance) frequency shift ∆f which is related to the bound mass, including hydrodynamically coupled water 14 Environment ACS Paragon Plus

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molecules, and the (energy) dissipation shift ∆D which is related to the viscoelastic properties of the adsorbed material.33 34 Together, ∆f and ∆D may provide information about the bilayer thickness 33 or the phase state of a liposome layer.35 Figure 4 shows the time curves of the QCM-D experiment for the formation of POPC (Figure 4a) and POPC/POPG (Figure 4b) SLBs and the subsequent addition of Al3+. In the first step of the experiment (indicated by *), SLBs were formed on the negatively charged SiO2 surface of QCM-D sensors by adsorption and spontaneous rupture of liposomes in Tris buffer supplemented with 10 mM CaCl2. The bilayer formation follows for both cases (POPC and POPC/POPG) a typical behavior as reported many times before resulting in characteristic values for the frequency shift ∆f ~ -27 Hz and the dissipation shift ∆D < 0.5·10-6.34, 36 After the formation of the bilayer was complete, SLBs were rinsed with Tris buffer to remove bivalent cations prior to addition of Al3+. Figure 5 shows average ∆f and ∆D values for POPC/POPG bilayer at the different steps of the experiments (different ionic conditions). QCM-D results for POPC bilayer In the case of the POPC bilayer (Figure 4a), essentially no changes in the QCM-D signal were observed when buffer was changed from Tris-CaCl2 to Tris buffer. Upon injection of Tris-AlCl3 buffer in the next step the signal remained essentially constant. However, rinsing with Tris buffer at the end of the experiment resulted in a decrease in frequency and increase in dissipation. QCM-D results for POPC/POPG bilayer In the case of the POPC/POPG bilayer (Figure 4b), the first buffer change from TrisCaCl2 to Tris buffer yielded a dissipation shift ∆D ~ 3.5·10-6 whilst the frequency remained almost unchanged similar to observations reported and discussed in detail in 15 Environment ACS Paragon Plus

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our previous study.29 We concluded this observation to result from a combination of different effects like changes in the interfacial water layer, structural and geometric changes, as well as a slip mechanism. Most importantly, it was shown that the dissipation shift is completely reversible, indicating that the bilayer remains intact after buffer exchange. Here, subsequent addition of Al3+ by rinsing the SLB with TrisAlCl3 buffer resulted in a small drop in the frequency (5.0 Hz, see Figures 4b and 5) and a significant decrease in the dissipation, returning to 0.7·10-6 (see Figures 4b and 5). Changing back to Tris buffer (final step of the experiment) led to a small decrease in frequency (4.1 Hz) and no significant change in dissipation. This observation, that the QCM-D signal at the end of the experiment (final rinse with Tris buffer, with removal of unbound and reversibly bound Al3+) does not return to values observed before in Tris buffer prior to exposure to Tris-AlCl3, agrees with FRAP and DPI results suggesting the Al3+ ions bind partially irreversibly to the lipid bilayer. Molecular Dynamics Results In order to observe Al3+-induced changes to bilayer structure at the molecular level, MD simulations of POPC and POPC/POPG membranes in Na+ and Al3+ ionic conditions were conducted. The simulations and their respective ionic conditions are listed in Table 3. The systems were observed to equilibrate after roughly 30 ns, as measured via the equilibration of the average area per lipid (see SI, Figure S3). After energy minimization and equilibration, the membranes simulated adopted a structure in agreement with previous simulations of fluid-phase POPC membranes (Table 3). The first 30 ns of data from each trajectory were excluded from analysis to allow for equilibration. For pure POPC bilayers, the presence of Al3+ ions caused no significant changes in the average area of lipid , bilayer thickness dP-P, or diffusion coefficient Ddiff 16 Environment ACS Paragon Plus

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(Table 3). However, for mixed POPC/POPG bilayers, the presence of Al3+ ions caused a decrease in , an increase in thickness, and a significant decrease in the diffusion of both the POPC and POPG lipids. The diffusion coefficients are plotted in Figure 6. The average areas per lipid, bilayer thickness, and diffusion coefficients for the control (Na+) conditions are in agreement with previous MD simulations of POPC and POPC/POPG bilayers using different force fields.37-39 Two methods were used to compare the affinities for ions binding to the headgroups. At the end of the 100 ns runs (to allow for full equilibration), the number of bound cations was counted (quantified as < 0.7 nm from the P atoms of the bilayer). For these bound cations, the number of POPC lipids and POPG lipids for each bound cation were also counted. Additionally, the net average interaction energies between ions and lipid species were calculated. These results are tabulated in Table 4. To better characterize the geometry of Al3+ to POPC and POPG lipids, radial distribution functions were calculated for the Al3+ ion and oxygens in the POPC/POPG lipids. The RDFs for the non-bridging phosphoryl oxygens (referred to as P=O oxygens), which were most involved in coordinating to Al3+, are plotted in Figure 7a. Two different binding modes were observed for Al3+-lipid coordination. Figure 7b demonstrates oxygen atoms from two POPG lipids coordinating within the inner solvation shell of Al3+. Figure 7c demonstrates P=O oxygen atoms from both POPC and POPG lipids coordinating within the second solvation shell of Al3+.

Discussion In this study, we observed both reversible and irreversible changes in the POPC and POPC/POPG bilayers upon exposure to Al3+. From fluorescence microscopy, we


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observed domain formation for the POPC/POPG bilayer and reduced lipid diffusion in both bilayers, which is supported by MD simulations. From DPI optical characterization, we observed increases in lipid packing density and bilayer thickness in the POPC/POPG bilayer that were further supported via QCM-D and MD simulations. We now wish to discuss the various effects that were observed and the underlying interactions causing them. Diffusivity In both the POPC and POPC/POPG bilayer, FRAP experiments demonstrated reduction in diffusion with increased Al3+ concentration. However, the lipid diffusion of the two bilayers showed differences in reversibility. For POPC, the diffusion reduction was completely reversible, whilst for POPC/POPG diffusion was observed to be irreversibly reduced. Furthermore, the POPC/POPG bilayer was completely immobile in Tris-AlCl3 whereas the POPC bilayer was still mobile. Diffusion coefficients calculated from MD simulations indicated that for POPC bilayers, Al3+ binding had little on POPC lipid diffusion. In contrast, in the POPG bilayer, lipid diffusion was significantly reduced: POPC diffusion was reduced by 26% and POPG diffusion was reduced by 38% (see Figure 6). In both SLBs, the Al3+ ions can interact with the bilayer in two ways that would hinder diffusion. Firstly, it may coordinate with both the SiO2 substrate and lipids, bridging the bilayer and the substrate, which we term a lipid-Al-substrate interaction. Secondly, Al3+ may coordinate only with lipid molecules within the bilayer, which we term a lipid-Al-lipid interaction. Lipid-Al-substrate interactions are an artifact of using a solid support, and it has been reported before that because of this the diffusivity of lipids within an SLB is lower than within a free bilayer. This reduced mobility is


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mainly caused by frictional forces40 and bridging effects of divalent ions between the substrate and the bilayer.41-44 Thus, it is obvious to assume that Al3+, a trivalent cation with a much higher affinity to the lipid molecules (and the substrate), would also produce a strong bridging effect and as a result hinder lipid diffusion. Lipid-Al-lipid interactions would implicate Al3+ ions coordinating to multiple lipids. Under this condition molecules are no longer free to move individually but rather diffuse as a unit, thus hindering their free diffusion. In POPC, the decrease in diffusion is likely due primarily to lipid-Al-substrate interactions. We claim this because the diffusion coefficient calculated from POPC MD simulations, a system in which there is no substrate interaction, revealed no significant difference for POPC diffusion with and without the presence of Al3+. In contrast, the decrease in diffusion present for POPC/POPG membranes does arise to a certain extend from lipid-Al-lipid interactions, since MD simulations demonstrated that there is significant diffusion reduction for the POPC/POPG membrane with the presence of Al3+ (Figure 6). In the POPC/POPG bilayer, lipid diffusion was significantly reduced for both types of lipids: POPC diffusion was reduced by 26% and POPG diffusion was reduced by 38% (see Figure 6). Since Al3+ had little effect on the diffusion of POPC lipids in a purely POPC bilayer, the reduction in the diffusion of POPC in the mixed POPC/POPG SLB is likely due to the POPC lipid being constrained by neighboring constrained POPG lipids. Additionally, domain formation observed via fluorescence microscopy upon the addition of Al3+ in POPC/POPG bilayers (discussed next) was irreversible upon rinsing with Tris buffer. Since domain formation is caused by lipid-Al-lipid interactions this observation also supports that the irreversible diffusion reduction is largely due to lipid-Al-lipid interactions.


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We posit that the immobility of bilayers in Tris-AlCl3 before rinsing with Tris is due to lipid-Al-substrate interactions, since both FRAP results after rinsing and MD simulations showed that Al3+ decreased diffusion significantly, but did not result in an entirely immobile bilayer. Al3+ coordination to POPC and POPG The difference in the effect of Al3+ ions on POPC and POPC/POPG diffusion reflects a difference in the underlying affinity for Al3+ to POPC and POPG. MD simulations provided two methods to analyze the observed difference in Al3+ affinity for POPC and POPG: via the number of POPC and POPG lipids coordinated per ion bound to the bilayer, and via the interaction energies. Both of these methods indicate that in the POPC/POPG bilayer, Al3+ has a higher affinity for POPG than for POPC. Each bound Al3+ ion coordinated on average to 1.9 POPG lipids and 1.2 POPC lipids (Table 4). The interaction energy for Al3+-POPG (-7.7 kJ/mol) is also significantly higher than the Al3+-POPC interaction energy (-5.7 kJ/mol) (Table 4). A radial distribution analysis was performed to identify which moiety of the headgroup the Al3+ ion coordinates (Figure 7a). This analysis revealed that the phosphodiester oxygens in both POPC and POPG lipids are involved in coordinating to Al3+. Phosphate oxygens from both lipid types were observed to coordinate at radii corresponding to both the first and second solvation shell, indicating two possible modes of Al3+ binding. These two modes are displayed in snapshots (Figure 7b and 7c, respectively). Domain formation Domain formation was observed via fluorescence microscopy in the POPC/POPG bilayer at all Al3+ concentrations. This confirms a previous assumption made by Verstraeten et al. that domain formation may occur in mixed lipid bilayers upon


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addition of Al3+.9 This domain formation is likely due to a difference in Al3+ affinity for POPC and POPG, given the different charges in the two lipids. This difference in affinity is confirmed by the differences observed with respect to the reversibility of the effects measured in all experimental techniques. In case of POPC the observed effects of Al3+ were essentially all reversible, whilst in case of POPC/POPG the effects were mostly irreversible. This can be explained by the higher affinity observed for Al3+ coordinating to POPG than to POPC. The observed domain formation may also be reflected in our DPI results via the small evolution of the TE/TM ratio to higher values over time in Al3+, possibly indicating a density increase or birefringence decrease that may occur during slow rearrangement of the domains. Given that the POPC/POPG bilayer is not homogeneous after Al3+ exposure, DPI and QCM-D measurements must be viewed as averages for the entire bilayer. Lipid packing and bilayer thickness Al3+ coordinating to multiple lipids would be presumed to increase the lipid packing of the membrane to best minimize the distance between multiple lipid headgroups and the Al3+ ion. If the lipid packing is increased, the footprint available for lipid tails is decreased, and the overall membrane thickness must subsequently increase to account for the volume of the lipid tails. An increase in lipid packing density and membrane thickness would cause an increase in birefringence as measured in the DPI. The observed increase in birefringence from (0.0152 in Tris to 0.0208 in Tris-AlCl3) for POPC/POPG bilayers in the presence of Al3+ ions, compared to the preceding bilayer in Tris buffer, is even in the same range as changes of the birefringence reported for the transition of lipid layers from the liquid disordered ld phase to the ordered gelphase so,19, 31 indicating a phase transition of POPC/POPG SLB upon addition of Al3+.


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Furthermore, an increase in lipid packing density and bilayer thickness would lead to an increased mass per area, which would cause a frequency decrease in the QCM-D. This is observed in our POPC/POPG experiments: upon switching from Tris to TrisAl3+, there was a frequency decrease of approximately 5 Hz (Figure 4b and 5). Since changes observed for POPC bilayers via DPI and QCM-D upon addition of Al3+ were mostly reversible, thickness and lipid packing density may be assumed to be essentially constant. There was a small increase in the TE/TM ratio upon changing to Tris-AlCl3. This change may be attributed to some bound Al3+ that would also be responsible for the reduced diffusivity discussed above. These results are also reflected in MD simulations. There was no significant difference in area per lipid and membrane thickness in POPC with Na+ and Al3+ ions, but for POPC/POPG, there was a reduction in area per lipid and an increase in membrane thickness (Table 3). Additionally, POPC/POPG bilayers in Al3+ had higher acyl chain order parameters than POPC/POPG bilayers in Na+, indicating that Al3+ binding induces a denser, more ordered state for the bilayer (See Figure S4 in SI). Taken together, QCM-D and DPI show that binding of Al3+ to a POPC/POPG SLB (mediated by the interaction with the negatively charged lipid) irreversibly change the thickness, refractive index, density and birefringence of the SLB. The changes in thickness and density are also reflected in MD simulations. Interfacial water layer Due to an interplay of van der Waals, electrostatic, steric and hydrophobic interactions a multimolecular thin water layer is trapped between the lipid bilayer and the underlying substrate. The thickness of this layer has been reported to be 1-2 nm.4547

However it is reasonable to assume that this value depends on lipid-ion-substrate

interactions discussed above. Accordingly Ca2+ and Al3+ may affect the thickness of 22 Environment ACS Paragon Plus

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the interfacial water layer. Although, the experimental methods of our study does not allow for a direct measure of this value, QCM-D may indicate thickness changes of the interfacial water layer as it measures not only the dry lipid mass, but also hydrodynamically coupled water, such as interfacial water. In the QCM-D results for POPC at the last rinse with Tris buffer (after Al3+ exposure) a small decrease in frequency accompanied by an increase in dissipation is observed. This increase in acoustical mass is not reflected at this stage in the DPI data, indicating that the observed signal has to be related to hydrodynamically coupled water. Thus, we assume that this change in the QCM-D signal may be due to an increase in the thickness of the interfacial water layer between the bilayer and the underlying substrate. Let us assume that during the last rinse with Tris the bound Al3+ ions are reversibly rinsed away (which is presumed from the FRAP and DPI results), an influx of Na+ cations and coupled water would be required to replace the small, highly charged Al3+ ions. More buffer volume would therefore be required to replace the volume previously occupied by Al3+ and its coupled water molecules. This effect is similar to the increased dissipation observed for POPC/POPG bilayers upon switching from Tris-CaCl2 to Tris, but in the present case we exclude the possibility of a decoupling, since the POPC SLB is neutrally charged and there would be no electrostatic repulsion as there is for the POPC/POPG SLB. Accordingly, FRAP indicates that the diffusivity of this POPC bilayer after rinsing is equivalent to preAl3+ values, and the dissipation is approximately 50% of the dissipation of the uncoupled POPC/POPG bilayer. In POPC/POPG, we do not observe comparable significant changes in frequency and dissipation upon rinsing with Tris as we see in POPC (∆ f = 4.1 ∆D ~ 0, Figure 5). We attribute this to the higher affinity of Al3+ to POPG than to POPC. As we see in


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FRAP experiments, the lipid diffusivity is not fully reversible after rinsing Al3+ away, and from DPI, we see that the induced changes in lipid packing and bilayer thickness are also not reversible. Due to this observed irreversibility, we can conclude that a sufficient number of Al3+ ions remain to retain the thickness of the interfacial water layer. Possibility of phase transition We posit that the observed domain formation, reduced diffusion, increased lipid packing density, and increased thickness are all suggestive of a phase transition in the bilayer from the fluid phase to the gel phase. Interestingly, the density increase of the lipids in this study (22% higher density for the POPC/POPG SLB in Tris-AlCl3 compared to the bilayer in Tris) is of the same order of magnitude as changes in the density observed for waxes in transition from liquid to solid phases.48 Thus it is feasible that we are observing a phase transition between ld and so states upon addition of Al3+ ions to the POPC/POPG SLB. However, confirming this transition is beyond the scope of SLB experimental methods and would require further study via calorimetry and other techniques.

Conclusion Understanding Al3+ bilayer interactions is crucial for a fundamental understanding of the mechanism of aluminum toxicity in biological systems. Using a combination of fluorescence microscopy, acoustic, and optical sensing techniques supported by MD simulations we have demonstrated that Al3+ binding to SLBs containing 50 mol% negatively charged lipids and 50 mol% neutral lipids induces domain formation accompanied by a reduced diffusivity, a change in bilayer thickness and lipid packing density. These different observed effects are indicatives of a phase transition of the


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charged bilayer upon binding of Al3+ ions. If our findings are relevant for interactions between Al3+ and lipids in the natural cell membrane, as a consequence the function of many proteins in biological systems would be affected, possibly including the synaptic interface involved in neuronal communication.

Acknowledgement This work was funded by the Dorothea Schlözer Programme of Georg-August University Göttingen and the Beckman Scholars Program. Simulations were conducted via allotments from the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI⋅1053575, and the Gesellschaft für wissenschaftliche Datenverarbeitung mbH Göttingen (GWDG). The authors would like to gratefully acknowledge Dr. Jochen Hub for helpful and informative discussions and Prof. Burkhard Geil for his technical help with running the MD simulations.

Supporting Information Available

Effect of varying Al3+ concentration on the diffusion coefficient, reversible binding effect of Ca2+ on POPC/POPG SLBs observed with DPI, details on equilibration of MD simulations and determination of the acyl chain order parameters. This information is available free of charge via the Internet at http://pubs.acs.org/.


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Figures Figure 1:

Figure 1: Chemical structure of phospholipids used in this study.


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Figure 2:

Fgure 2: Representative FRAP data from varying buffer conditions for (a) POPC and (b) POPC/POPG membranes on SiO2. Pre-bleach (t = -5 s), bleach (t = 0 s), and post bleach (t = 150 s) are shown. Top row represents FRAP experiment in Tris-CaCl2 buffer, second row experiments in Tris, third row experiments after addition of 10 mM AlCl3, and the bottom row shows typical FRAP after final rinsing with Tris buffer. (c) Close-up image of representative observed lipid domains in a POPC/POPG membrane in TRIS-AlCl3 buffer.


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Figure 3

Figure 3: DPI time curves and corresponding TE/TM ratio for the SLB formation and the subsequent binding of Al3+ ions. First (indicated by a *) an SLB was formed by liposomes containing (a) POPC and (b) 50 mol% POPC and 50 mol% POPG in Tris and Tris-CaCl2, respectively, followed by buffer changes: (A) Tris buffer TrisCaCl2 buffer Tris buffer 10 mM Tris-AlCl3 buffer Tris buffer and (B) TrisCaCl2 buffer Tris buffer Tris-AlCl3 buffer Tris buffer.


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Figure 4

Figure 4: QCM-D frequency (top) and dissipation (bottom) shifts as function of time during SLB formation and the subsequent binding of Al3+ ions plus schematic illustration. First (indicated by a *) an SLB was formed by liposomes containing (a) POPC and (b) 50 mol% POPC and 50 mol% POPG in Tris-CaCl2 buffer followed by buffer changes: Tris-CaCl2 buffer Tris buffer 10 mM Tris-AlCl3 buffer Tris buffer.


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Figure 5

Figure 5: Mean values and corresponding standard deviations of QCM-D frequency (left hand side) and dissipation (right hand side) shifts for the POPC/POPG SLB for varying buffer conditions: (blue) Tris-CaCl2, (light-grey) Tris, (red) Tris-AlCl3, and (dark-grey) Tris after the SLB had been exposed to Tris-AlCl3.


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Figure 6

Figure 6: Diffusion coefficient of phospholipids derived from MD simulations of a POPC SLB (left hand side) and a POPC/POPG SLB (right hand side) for varying ionic conditions: (grey) 32 Na+, 32 Cl- for the purely POPC SLB, and 64 Na+ for the mixed POPC/POPG SLB. (red) 32 Al3+, 96 Cl- for the purely POPC SLB, and 32 Al3+, 32 Cl- for the mixed POPC/POPG SLB.


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Figure 7

Figure 7: (a) Radial distribution g(r) (solid lines) and coordination number Intg(r)(dashed lines) for Al3+ with phosphate oxygens from POPC (light grey) and POPG (dark grey) and water oxygens (red). (b) Snapshot representing phosphorus oxygens coordinating within first solvation shell of Al3+. (c) Snapshot representing phosphorus oxygens of both POPC and POPG coordinating within second solvation shell of Al3+. For (b) and (c), coloring schematic is as follows: aluminum, rose; aluminum first hydration shell water molecules, orange; oxygen, red; phosphorus, gold; hydrogen, white; carbon, cyan; nitrogen, blue.


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Tables Table 1. Mean values and the corresponding standard deviations of the diffusion coefficients Ddiff (µm2 s-1) of POPC and POPC/POPG bilayers in various buffer ionic conditions, measured via FRAP (n = 10). Tris-CaCl2

Tris

(10 mM CaCl2)

Tris-AlCl3

Tris

(10 mM AlCl3)

(after 10 mM AlCl3)

POPC

2.5 ± 0.2

2.4 ± 0.3

0.3 ± 0.2

2.2 ± 0.3

POPC/POPG

2.1 ± 0.04

5.3 ± 1.4

Immobile

0.7 ± 0.3

Table 2: Thickness, birefringence, refractive index n, mass and density of the POPC and POPC/POPG bilayer in Tris, Tris-CaCl2 and Tris-AlCl3 derived by analysis of the DPI data shown in Figure 3b and Figure S2 (SI) respectively.

a

thickness (nm)

n

mass (ng·mm-2)

density (g·cm-3)

POPC Tris

0.0180 ± 0.0002

4.7 ± 0.1

1.47a

4.73 ± 0.01

1.006a

POPC Tris-CaCl2

0.0178 ± 0.0002

4.6 ± 0.1

1.47a

4.65 ± 0.01

1.004a

POPC Tris-AlCl3

0.0168 ± 0.0003

4.7 ± 0.1

1.47a

4.70 ± 0.01

1.004a

POPC/POPG Tris

0.0152 ± 0.0004

4.0 ± 0.1

1.452b

3.52 ± 0.12

0.871c

POPC/POPG Tris-CaCl2

0.0209 ± 0.0004

4.7 ± 0.1

1.474b

4.89 ± 0.12

1.034c

POPC/POPG Tris-AlCl3

0.0208 ± 0.0007

4.7 ± 0.2

1.480b

5.00 ± 0.24

1.075c

POPC/POPG Tris post AlCl3

0.0196 ± 0.0001

4.8 ± 0.3

1.480b

5.312± 0.28

1.077c

Correspond to values that are fixed in the DPI analysis.

b c

birefringence

Adjusted by iteration from 1.470.

Adjusted by iteration from 1.000 g/cm-3.


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Table 3. Average area per lipid , bilayer thickness dP-P, and lipid diffusion coefficients Ddiff,MD from MD simulations.

Lipid conditions

Ion conditions

(nm2)a

dP-P (nm)b

POPC Ddiff, MD POPG Ddiff, MD (um2 s-1)c (um2 s-1)c

128 POPC

32 Na+, 32 Cl-

0.650 ± 0.005

3.7 ± 0.3

8.5 ± 0.7

N/A

128 POPC

32 Al3+, 96 Cl-

0.640 ± 0.006

3.6 ± 0.3

7.8 ± 0.9

N/A

0.656 ± 0.003

3.7 ± 0.3

7.8 ± 1

8.6 ± 1.2

0.619 ± 0.004

3.9 ± 0.2

5.8 ± 0.6

5.3 ± 0.6

+

64 POPC, 64 POPG

64 Na

3+

64 POPC, 64 POPG

-

32 Al , 32 Cl

a

Average area per lipid calculated via the xy-area of the bilayer over time. Thickness dP-P calculated as the average distance along the z-axis between P atoms in the two bilayer leaflets. cLateral diffusion Ddiff,MD calculated as the diffusion of the P atoms via block-averaging with overlapping 20 ns intervals.

b

Table 4. Lipid-ion coordination numbers and interaction energies from MD simulations. Lipid conditions

Ion conditions

Bound cations/ POPC lipids per POPG lipids per POPC-Na+ total cations bound cation bound cation IE (kJ/mol)a

128 POPC

32 Na+, 32 Cl-

9/32

2.0 ± 0.9

--

-1.0

--

--

--

128 POPC

32 Al3+, 96 Cl-

10/32

2.9 ± 1.4

--

--

-4.1

--

--

36/64

0.7 ± 0.6

0.9 ± 0.7

-1.4

--

-1.9

--

30/32

1.2 ± 1.0

1.9 ± 0.9

--

-5.7

--

-7.7

64 POPC, 64 POPG 64 Na+ 3+

-

64 POPC, 64 POPG 32 Al , 32 Cl

a

POPC-Al3+ IE (kJ/mol)a

POPG-Na+ IE (kJ/mol)a

POPG-Al3+ IE (kJ/mol)a

Interaction energy calculated as the sum of short-range Coulomb and Lennard-Jones energies between lipid types and ions, normalized by the total number of lipids of that type in the system (128 or 64), and the total number of bound cations (quantified as Pcation distance less than 0.7 nm) at equilibrium.


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Table of Contents graphic POPC/POPG SLB

Al3+

SiO2 solid support

Tris

Tris-AlCl3


Effects of Al3+ on Phosphocholine ... - ACS Publications

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