Large Scale Membrane Movement Induced by a Cation Switch
A biomembrane sample system where millimolar changes of cations induce reversible large scale (≥ 200 Å) changes in the membrane-to-surface distance is described. The system composes of a freefloating bilayer (FFB), formed adjacent to a selfassembled monolayer (SAM). To examine the membrane movements, differently charged FFBs in the presence and absence of Ca 2+ and Na + , respectively, were examined using neutron reflectivity (NR) and quartz crystal microbalance (QCM) measurements, alongside molecular dynamics (MD) simulations. In NR the variation of Ca 2+ and Na + concentration enabled precision manipulation of the FFB-to-surface distance.Simulations suggest that Ca 2+ ions bridge between SAM and bilayer whereas the more diffuse binding of Na + , especially to bilayers, is unable to fully overcome the repulsion between anionic FFB and anionic SAM.Reproduced NR results with QCM demonstrate the potential of this easily producible sample system to become a standard analysis tool for e.g. investigating membrane binding effects, endocytosis and cell signalling.
Introduction<!>Modulation<!>Complementary Analysis on QCM-D Sensor Surfaces<!>MD Simulations Reveal Differences in Ion Binding between COOH-OEG-SAM and Membranes<!>14<!>Discussion<!>Conclusion
<p>Comprised predominantly of a lipid bilayer with embedded and bound proteins from a large variety of classes, biological membranes are the key structural material of life at the cellular level. Membranes define the external limit of each cell and, within eukaryotic cells, the many organelles, which carry out a multiplicity of cellular functions. Membranes control molecular transport, both into and out of the cell. Therefore, membrane associated proteins are involved in almost all biochemical pathways and account for around ~40% of drug targets [1] . Studying membranes in vivo is challenging due to their complexity and small transverse size. Model membranes have therefore been key in gaining a molecular level understanding of membrane biochemistry [2], [3] . These systems allow for precision structural and biophysical studies to occur on systems of reduced (whilst defined) complexity compared to those found in vivo.</p><p>Planar supported lipid membrane samples at interfaces offer an ideal model membrane system due to the variety of benchtop techniques suitable for analysing this sample type [4] and the relative ease of fabrication of these samples by vesicle rupture [5] , solvent exchange [6] or monolayer transfer techniques [7] . The simplest models consist of bilayers deposited directly onto a 2 solid support material such as silicon, glass or mica [8], [9], .</p><p>More advanced forms of planar membrane models use soft polymer supports [10] or surface tethers [11] to reduce surface influence and thus have water on both sides of the membrane model. Floating supported bilayers achieve this through a combination of repulsive and attractive forces between the membrane and the surface [12], [13] . This effect causes the membrane to float ~2 nm away from a bulk interface. Consequently a layer of 'bulk' solution is present on both sides of the planar membrane without the need for either tethering or interacting with surface bound material. Recently we discovered that membranes which float 1-3 nm away from a solid interface can be self-assembled via vesicle rupture adjacent to carboxylate terminated oligo (ethylene glycol) alkanthiol self-assembled monolayer surfaces (OEG-SAM) [14] . The self-assembly of the free floating bilayer (FFB) onto the support surface significantly reduces the complexity of the FFB generation protocol, thus opening this sample system up to wider utilisation. This is in contrast with previous systems which in general required Langmuir-Blodgett/Langmuir Schaefer deposition to form high quality membranes, and therefore required specialist deposition apparatus and expertise , [15], [16], [17] , . OEG-SAMs have gained significant interest due to their use in anti-fouling surface coatings [18] , with this effect related to the repulsive hydration forces resulting from water layers bound to the EG groups of the SAM [19], [20] .</p><p>The most commonly studied OEG-SAM contains a terminal hydroxyl group and has, according to the "Whiteside" rules of hydrophilicity, no net charge [21], [22], [23] . Here, and in previous studies on FFB systems, we have used carboxyl terminated (COOH-) OEG-SAMs as a support for FFBs. These do not obey these rules due to partial ionisation of the SAM terminal carboxyl groups [24] . Charged surfaces are known to bind oppositely charged counter ions at the solid liquid interface, which plays a key role in many technological processes such as water purification [25] and cation exchange chromatography [26] . However, the interactions of metal cations with partly negatively charged COOH-OEG-SAMs and its resulting surface charge modulation have not been studied.</p><p>In a previous study, we observed that the presence of 200 mM NaCl in the bulk solution reversibly increased the bilayer-to-SAM distance but could not identify the cause of this effect [14] . Here, we demonstrate a reversible phenomenon where removing and re-adding a calcium chloride (CaCl2) concentration one hundred fold smaller than the NaCl concentration previously studied (2 mM vs. 200 mM) induced substantial reversible changes to the bilayer-to-SAM distance on length scales of greater than 20 nm. As electrolytes seem to have a major effect on the behaviour of the FFB system, and given that the self-assembly process of the FFB onto the COOH-OEG-SAM requires CaCl2 present in the buffer solution, the interactions of Ca 2+ and Na + , as well as the water density within the system were of particular interest in this study. Therefore, Ca 2+ or Na + in the buffer solution and FFB lipid compositions with differing levels of biomemetic accuracy were used in the sample systems investigated.</p><p>Neutron reflectometry (NR) was used to examine the interfacial membrane constructs, due to its sensitivity as a structural probe for samples buried within complex environments, such as the solid-liquid flow cells used here. This technique is able to structurally resolve complex assemblies of macromolecules through the use of differential protium/deuterium labelling of the interfacial samples and bulk solution [15] , [27] . To obtain a molecular level understanding of the membrane</p><!><p>Self-assembled free floating membranes (FFBs) of different lipid compositions adjacent to COOH-OEG-SAMs were generated (Table 1). The complexity of the lipid composition was stepwise increased in order to investigate the feasibility of the system for studies of membrane systems of near-biological complexity.</p><p>Therefore, starting from a POPC-membrane and a POPC/POPS mixture (8:2, mol:mol), a model of the inner leaflet of the mammalian plasma membrane (POPC/POPS/POPIP3 7:2:1, mol:mol) [28][29] and a model approximating the plant plasma membrane (POPC/POPS/Chol/DGDG 5:2:2:1, mol:mol) [30][31] were produced. As the self-assembling process of FFBs needed 2 mM CaCl2 present in the buffer solution and a previous study suggested a significant impact of cations on the bilayer-to-SAM distance, the role of the Ca 2+ and its interactions within the system were of particular interest. Therefore, each system was treated with 1 mM EDTA to remove Ca 2+ after the membrane deposition.</p><p>Then, to investigate the difference between divalent Ca 2+ and monovalent Na + , 200 mM NaCl was added to the system. Finally, the system was again flushed with a 2 mM CaCl2 containing solution. NR measurements at three isotopic contrasts (H2O, AuMW and D2O) were carried out for each system directly after the FFB deposition and after each single treatment. The data fitting quality was high for all the samples studied, which allowed us to resolve the relative distributions of the COOH-OEG-SAM, the membrane, and the water components under all conditions.</p><p>An example of a full NR data set, including the model data fits and the neutron scattering length density (nSLD) profiles, are shown for the mammalian plasma model membrane (POPC/POPS/DOPIP3 7:2:1, mol:mol) in Figure 1. For all FFB samples the bilayerto-SAM distance, together with the FFB roughness, are listed in Table 1. Here, the bilayer-to-SAM distance is defined as that between the COOH-OEG-SAM/water interface and the adjacent bilayer head-group/water interface. Therefore, the bilayer-to-SAM distance is equivalent to the thickness of the water interlayer between the FFB and the COOH-OEG-SAM surface.</p><p>Further key structural parameters of the NR experiments are listed in Table S2. All FFBs showed high bilayer coverage and were in good agreement with previous studies [14], [32], . ). In all cases the reversible movement of the FFB away from and back to the COOH-OEG-SAM surface did not result in measurable loss of the total membrane coverage, suggesting these to be fully reversible processes (see Table S2).</p><p>Though the trends in bilayer-to-SAM distance change against solution salt conditions were similar, the magnitude differed between FFBs. For example, whilst after Ca 2+ sequestration the POPC bilayer moved to ~200 Å from the COOH-OEG-SAM surface (see Table 1), the anionic eukaryotic membrane models (i.e.</p><p>POPC:POPS and POPC:POPS:DOPIP3 ) were found to move to significantly higher distances relative to the COOH-OEG-SAM in this condition (~600 and ~400 Å respectively, see Table 1). This suggested a correlation between membrane anionic character and the bilayerto-SAM distance change after Ca 2+ sequestration. A similar trend was seen with 200 mM NaCl. Here, the POPC sample returned to a close membrane-to-SAM surface distance (~35 Å, compared to ~10 Å in 2 mM CaCl2) whereas the anionic POPC:POPS and POPC:POPS:DOPIP3 membranes returned to a larger bilayer-to-SAM distance of ~100 Å. Interestingly, when comparing the two mammalian anionic membrane models with each other, the bilayer-to-SAM distances during all applied salt solution buffer conditions were greater for the POPC:POPS membrane than for the POPC:POPS:DOPIP3 membrane, although the latter had a higher net negative charge (see Table 1).</p><p>The anionic plant plasma membrane model  1).</p><p>In all cases, the bilayer roughness, which describes the height-height correlation function across a layer boundary [14] , showed a proportional increase with bilayer-to-SAM distance (see Table 1). In a continuous planar material such as a supported lipid bilayer the largest and therefore overriding source of roughness is likely to be displacement of the bilayer from its mean averaged position due to the fluctuations in the plane of the membrane [33] . This increase in the roughness value is therefore likely to be related to an increased in the fluctuation amplitude of the floating membrane as it moves away from the bulk interface [8], [13], . Changes in the bilayer-to-SAM distance can be seen through changes in the Gaussian distribution of the FFB (here equal to unlabelled distribution in SLDs). The largest changes caused the EDTA mediated Ca 2+ sequestration, resulting in very rough FFBs, far away from the COOH-OEG-SAM surface. 200 mM NaCl returned the POPC bilayer almost completely, the anionic membranes only partly back towards their initial positions. In presence of negatively charged phospholipids the changes in bilayer-to-SAM distance were larger after Ca 2+ sequestration (purple) and Na + addition (green), but similar after Ca 2+ addition (SLD profiles overlaid in orange).</p><p>Experimental data and model data fits used to produce these SLD profiles are given in supporting information Figures S3 to S6.</p><p>Finally, as the NR data suggested that Ca 2+ brings the FFB much closer to the COOH-OEG-SAM surface than Na + , the effect of Ca 2+ on the bilayer-to-SAM distance in the presence of physiological concentrations of Na + was investigated. To this end, a POPC FFB was sequentially examined in buffer solutions containing 100 mM NaCl with and without 1 mM CaCl2. The solution without CaCl2 contained 0.5 mM EDTA to remove any contaminating Ca 2+ cations from the previous buffer solution. The NR analysis (see Figure 3) revealed that the bilayer-to-SAM distance was significantly smaller with the buffer solution containing both Ca 2+ and Na + than in the presence of Na + only (~23 Å vs. ~41 Å). These data demonstrate the strong influence of divalent cations on the bilayer-to-SAM distance, even in the presence of a one hundred times higher concentration of monovalent cations.</p><p>Furthermore, these results suggested the possibility of fine-tuning the bilayer-to-SAM distance by using combinations of mono-and divalent cations. (Figure S7 shows an independent repeat of this experiment, confirming the reproducibility of these effects).  S4.</p><!><p>QCM-D is a surface sensitive technique utilising the piezoelectric effect for real-time measurements of surface interactions. The change in the resonant frequency (∆f) is inversely proportional to the amount of mass at the sensor surface, as given by the Sauerbrey 9 equation [34], [35] . To complement NR measurements and to demonstrate utilisation of this sample system in studies using benchtop analytical techniques, FFBs of POPC:POPS (8:2, mol:mol) were self-assembled onto an COOH-OEG-SAM coated QCM sensor chip surface.</p><p>Measurements of changes in the frequency (∆f) and dissipation (∆d) were used to monitor the sample response after changing the solution salt condition. This is based on the observation that the solution between the FFB and the COOH-OEG-SAM surface is trapped and therefore part of the total layer mass [5] . Therefore, the changes in bilayer-to-SAM distance will be associated with a decrease in ∆f and an increase in ∆d. This assumption is supported by the observation that the water inside a vesicle adsorbing onto a QCM sensor adds to the adsorbed mass measured and its loss can be measured during vesicle rupture [36], .</p><p>The QCM-D results were complementary to those observed with NR, showing a large decrease in ∆f and increase in ∆d when the buffer condition was changed from 2 mM CaCl2 to 0 mM CaCl2 by EDTA mediated Ca 2+ sequestration. This implies that a thicker water layer was trapped between the COOH-OEG-SAM and the FFB in case of the Ca 2+ free state compared to the initial state given in the presence of Ca 2+ (see Figure 4). Interestingly, ∆f often after an initial decrease showed a slow increase, whereas ∆d increased always to a plateau after Ca 2+ sequestration. It is possible that this effect was due to the movement of the FFB to a large distance, which was outside the frequency depth sensitivity range of QCM-D (which is inversely proportional to square of the resonant frequency) and/or at which the water was given for the 3 rd (black), 5 th (red) and 7 th (green) overtones. For the control data set with no membrane adjacent to the COOH-OEG-SAM only the 3 rd overtone is given (grey line). Repeated data sets are given in Figure S8-9.</p><!><p>Taken together, the NR and QCM-D results indicated that Ca 2+ reversibly moves the FFB closest to the COOH-OEG-SAM, followed by Na + . These results raised the question what the difference is between Na +and Ca 2+ -interactions within the system. In particular, we wished to understand which layer preferentially interacted with cations, and how strongly the different cationic species interacted. This information would enable us to understand the observed substantive, reversible movements of the FFB away from the surface in response to changes in cation concentrations. Coarse grained (CG) simulations were used to generate initial model structures, using the well established Martini force field [37], [38] . For the CG model of the COOH-OEG-SAM a range of initial lattice constants (a0) of the hexagonal COOH-OEG-SAM-grid (0.1 Å intervals between 4.9 and 5.6 Å) were defined for a set of COOH-OEG-SAMs differing in the percentage of charged SAM-COOmolecules present (0, 5, 10, 20, 39 and 50%).  Zero on the x-axis is equal to the centre of mass of the SAM carboxyl groups.</p><p>12 Charged SAM-COOand uncharged SAM-COOH molecules showed negligible differences in order parameters <STHI-C1B> within all SAM compositions (Figure S11, A/B). This explains why <a> and <d> seemed to be independent of the percentage of charged COOH-OEG-SAM molecules (Figure S11, C/D). Thus, for further simulations the 39% charged SAM was chosen, corresponding to the COOH-OEG-SAM used in NR at 7.2 pH employing the pKa value of carboxyl terminated SAMs predicted by previous studies. [24] However, <d> and <STHI-C1B> showed a high dependency on a0. Previous studies showed that with the Martini bead definition it is not possible to reproduce the experimental alkane thiol chain tilt angle of 20-35° [39], [40] while matching the experimental lattice constant of 4.97 Å [41] on gold (111). [42] This was resolved when converting the CG presentation into an all-atom (AA) representation by rescaling the xy coordinates by 0.93x. A tilt angle of 20-35° corresponds to 0.51-0.82 <STHI-C1B> which needed a0 to be bigger than 5.1 Å (Figure S11, A/B). An initial lattice constant of 5.2-5.3 Å agreed well with the COOH-OEG-SAM thickness observed in NR (20.3-21.7 Å, Table S5 and Figure S11, D). The averaged <a> showed the overall structural stability of the packing (Figure S11, C) and revealed that both a0 of 5.2 and 5.3 Å converged due to minor rearrangements to 5.3 Å. Thus, an a0 of 5.3 Å was chosen for further simulations. The hydrophilicity of the SAM seemed to be almost independent of a0 (Figure S11, E).</p><p>The CG COOH-OEG-SAM was converted into an AA representation and simulated for up to 100 ns in the presence of different Na + and Ca 2+ concentrations. To explore the robustness of our results to the parametrization of these ions we explored a number of variations, namely: Charmm36 [43] , the electronic continuum correction (ECC) within Charmm36 [44] , and a modified ECC version developed using Charmm36, here called ECC2 [45] (see section S1.7 for details). As anticipated, we observed oscillations in the water density close to the COOH-OEG-SAM surface, extending approximately four molecular water diameters away from the surface (Figure 5, C/D), in agreement with previous studies on OEG-SAMs [46], [47] .</p><p>13 (A) Free energy profiles for cation interactions with the OEG-COOH-SAM (solid lines). These were evaluated in the presence of 400 mM Ca 2+ (red) and 400 mM Na + (black) using the ECC2 force field. Zero on the x-axis is equal to the centre of mass of the COOH-OEG-SAM carboxyl groups. Dashed lines represent the corresponding number density profiles of the terminal COO -/H groups of the SAM-molecules (B, C) Free energy profiles for Na + (B) and Ca 2+ (C) interactions with phospholipid bilayers (solid lines). The different colours represent the different bilayers: Blue: POPC, orange: POPC:POPS (8:2, mol:mol), cyan: POPC:POPS:POPIP3 (7:2:1, mol:mol). Zero on the x-axis is equal to the centre of mass of the respective bilayer. Dashed lines represent the corresponding number density profiles of the lipid headgroups, including the inner phosphates.</p><!><p>The minima of the free energy landscapes for Na + and for Ca 2+ ions above the COOH-OEG-SAM were comparable with values of ~6.7 and ~6.8 kJ/mol (Figure 6, A), but with closer approach for Ca 2+ (dMIN = ~2.3 Å for Ca 2+ and ~3.9 Å for Na + ). In each case there was a shoulder at ~ -1 Å suggesting a degree of penetration of cations between the OEG-carboxyl groups (as can be seen from the corresponding density profiles in Figure S14). However, for Ca 2+ the potential well is steeper such that cation density extends for ~7 Å from the carboxylates at the surface of the SAM, whereas for Na + the potential well is broader and bulk cation concentration is not reached until > 15 Å from the SAM surface. Comparable results for the standard Charmm36 and the ECC force field parameterization are represented in the SI.</p><p>We carried out similar calculations for interactions of the cations with the various lipid bilayer surfaces (Figure 6, B/C). Two trends are seen: as was observed with the SAM, Ca 2+ ions are more strongly localized at the surface than are Na + ions, corresponding to clear energy minima near the bilayer headgroups and a steeper potential well for Ca 2+ . Furthermore, a clear trend was observed in terms of the depth of the energy well for Ca 2+ when comparing a zwitterionic POPC bilayer with a modestly anionic POPC/POPS bilayer and with a more markedly anionic POPC/POPS/POPIP3 bilayer. With increasing average surface charge density from 0 e/nm 2 for the POPC, 0.3 e/nm 2 for the POPC/POPS to 1.2 e/nm 2 for the POPC/POPS/POPIP3 bilayer, the depth of the energy well intensified for Ca 2+ . In agreement with this trend, the SAM showed, with a surface charge density of 1.9 e/nm -2 , the deepest energy well for Ca 2+ compared to the various bilayers. Significant Na + binding was only observed in the presence of the highly negatively charged POPIP3. POPIP3 seemed to cause for Na + , as well as for Ca 2+ an intensified breadth of the energy well, suggesting strongly fluctuating binding (see also Figure S15). So there was, in the case of the POPIP3 containing bilayer, a clear Ca 2+ ion density extending more than 10 Å and a clear Na + ion density extending more than 20 Å from the bilayer surface were observed.</p><!><p>The NR results reveal a clear trend in bilayer-to-SAM distance depending on the salt solution present between the SAM and the lipid bilayer. These trends were similar for a simple POPC and more complex and biologically These results suggest attraction between bilayer and COOH-OEG-SAM is greatest in the presence of Ca 2+ , with a weaker attraction in the presence of an elevated concentration of Na + .</p><p>The interplay of four main intermolecular forces is thought to control the distance between the FFBs and the COOH-OEG-SAM surface: Van der Waals (vdW) forces give rise to weak yet long range attraction between the bilayer and the surface [48] , whilst entropic Helfrich fluctuational forces [49] , as well as hydration forces can produce a repulsion between the COOH-OEG-SAM and the FFB [13], [50], . Electrostatic forces can produce short range attractive or repulsive interactions depending on the surface charge of the SAM and the 15 FFB [48], [51] . As both the membrane head-groups and the COOH-OEG-SAM surface contained ionisable groups and given the salt solution effects it was inferred that the predominant cause of these effects were changes in electrostatics interactions of the system under differing conditions. In order to visualize the density profiles of the ions, their density was multiplied by 10. The Ca 2+ showed a significantly stronger binding to both SAM and bilayer in comparison to Na + . The density profile of Ca 2+ (B, black) suggests that Ca 2+ ions bridge between the anionic SAM and the phospholipid surface. However, the week binding of Na + , especially to the bilayer (A, black), might cause a too small bridging effect in order to overcome the repulsive force between the anionic SAM and an anionic bilayer. Again, the hydration layers above the SAM are observable in both cases (A/B, blue).</p><p>The MD results allow us to integrate these experimental results with an underlying molecular model of the COOH-OEG-SAM/FFB system (Figure 7). Simulations showed a layer of increased Ca 2+ concentration above the SAM surface with a thickness of around 7 Å and above various bilayers with a thickness of 5 to 10 Å (Figure 6). Therefore, the full width of the aqueous layer between the SAM and the bilayer surfaces observed by NR (10 to 15 Å) will contain an elevated concentration of Ca 2+ . Note that this would accommodate the three to four water layers seen at the SAM surface (Figure 5). A coarse-grained model of this resultant system is shown in Figure 7, B. From this integrative model it is evident that the Ca 2+ ions are potentially able to bridge between the anionic SAM (carboxylate) and phospholipid surfaces. It will be of interest to examine the dynamic behaviour of water and Ca 2+ ions within this nanoconfined salt solution layer in more detail by all atom MD simulations. In the case of Na + the much weaker binding, especially to the bilayer surfaces suggests a less intensified bridging effect of the interlayer cations, too small to overcome the repulsion force between the SAM and bilayer resulting in the intermediate position of the bilayer (Figure 7, A).</p><p>Additionally all atom MD simulations comparing the behaviour of Na + with that of Ca 2+ within the area between SAM and bilayer will be necessary to confirm the predicted molecular model.</p><!><p>Here we demonstrate an easily assembled floating</p>