POPC Bilayers Supported on Nanoporous ... - ACS Publications

Jun 10, 2016 - Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, Florida 33620, United States. ‡. ...
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POPC Bilayers Supported on Nanoporous Substrates: Specific Effects of Silica-Type Surface Hydroxylation and Charge Density Nalvi Duro,† Marion Gjika,† Ahnaf Siddiqui,† H. Larry Scott,‡ and Sameer Varma*,† †

Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, Florida 33620, United States Department of Physics, Illinois Institute of Technology, Chicago, Illinois 60616, United States

‡

S Supporting Information *

ABSTRACT: Recent advances in nanotechnology bring to the forefront a new class of extrinsic constraints for remodeling lipid bilayers. In this nextgeneration technology, membranes are supported over nanoporous substrates. The nanometer-sized pores in the substrate are too small for bilayers to follow the substrate topology; consequently, the bilayers hang over the pores. Experiments demonstrate that nanoporous substrates remodel lipid bilayers differently from continuous substrates. The underlying molecular mechanisms, however, remain largely undetermined. Here we use molecular dynamics (MD) simulations to probe the effects of silica-type hydroxylation and charge densities on adsorbed palmitoyl-oleoylphosphatidylcholine (POPC) bilayers. We find that a 50% porous substrate decorated with a surface density of 4.6 hydroxyls/nm2 adsorbs a POPC bilayer at a distance of 4.5 Å, a result consistent with neutron reflectivity experiments conducted on topologically similar silica constructs under highly acidic conditions. Although such an adsorption distance suggests that the interaction between the bilayer and the substrate will be buffered by water molecules, we find that the substrate does interact directly with the bilayer. The substrate modifies several properties of the bilayerit dampens transverse lipid fluctuations, reduces lipid diffusion rates, and modifies transverse charge densities significantly. Additionally, it affects lipid properties differently in the two leaflets. Compared to substrates functionalized with sparser surface hydroxylation densities, this substrate adheres to bilayers at smaller distances and also remodels POPC more extensively, suggesting a direct correspondence between substrate hydrophilicity and membrane properties. A partial deprotonation of surface hydroxyls, as expected of a silica substrate under mildly acidic conditions, however, produces an inverse effect: it increases the substrate− bilayer distance, which we attribute to the formation of an electric double layer over the negatively charged substrate, and restores, at least partially, leaflet asymmetry and headgroup orientations. Overall, this study highlights the intrinsic complexity of lipid−substrate interactions and suggests the prospect of making two surface attributesdipole densities and charge densities work antagonistically toward remodeling lipid bilayer properties.

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from continuous solid/polymeric supports,12−20 which provide continuous contacts to membranes. Experiments demonstrate21,27−30 that nanoporous substrates remodel macroscopic lipid bilayer properties differently from continuous supports. They can alter lipid diffusion by an order of magnitude, induce domain formation under conditions that are not conducive to free-standing bilayers or in bilayers adsorbed on continuous supports, decrease phase-transition temperatures, and reduce membrane stabilities drastically under acidic conditions that unsupported vesicles can withstand. The underlying molecular mechanisms, however, remain undetermined. Molecular dynamics (MD) simulations have been used in the past to gain molecular insight into membrane remodulation by nanoporous substrates.33,34 However, results from these simulations cannot be extrapolated to interpret

INTRODUCTION The physical properties of lipid bilayers can be remodeled by many intrinsic and extrinsic constraints.1−20 Intrinsic constraints include, for example, diffusion barriers in the form of membrane proteins, pore-forming antimicrobial peptides, preferentially binding lipids, sterols, and alcohols. Extrinsic constraints originate from the ionic strength, cytoskeletal anchors, and the nonintercalating contact points provided by cytoskeletal proteins and synthetic solid/polymeric supports. Although considerable knowledge about the effects of several of these constraints exists, recent advances in nanotechnology21−32 bring to the forefront a new class of extrinsic constraints for remodeling lipid bilayers. In this next-generation technology, membranes are supported over porous materials that have pore sizes on the order of nanometers. These pore sizes are not large enough for lipid bilayers to follow the surface topology of the underlying substrate; consequently, the bilayers hang over the pores, making only intermittent contact with the underlying substrate. Such supports are physically different © 2016 American Chemical Society

Received: March 24, 2016 Revised: June 6, 2016 Published: June 10, 2016 6766

DOI: 10.1021/acs.langmuir.6b01155 Langmuir 2016, 32, 6766−6774

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is a minimum. The potential energy is determined by describing the LJ spheres using parameters corresponding to the CH4 moiety in the Gromos 43a1 force field.42 We note that such substrate geometry is unlike that of amorphous silica; however, the goal of this study is not to predict bilayer remodeling by amorphous silica but to understand the specific effects of silica-type surface hydroxylation and charge densities on bilayers. Nevertheless, this 3D geometry is identical to that employed in our previous work,34 and retaining this geometry allows us to connect findings from this study to those from our previous study. We also note that our choice of parameters for the LJ spheres causes the substrate constructed at this stage to be hydrophobic. We know that such a hydrophobic substrate does not adhere to a POPC bilayer,34 as observed in experiments,19 so in that sense this topology serves as a control for surface templating. In the next step, a cylindrical channel parallel to the z axis is carved in the 3D grid of CH4 moieties by removing spheres that lie within a distance of 13.5 Å from the central z axis of the grid. Placing this carved grid of LJ spheres in a box with XY dimensions of 127 × 127 Å2 yields a XY substrate coverage roughly equal to one-half the XY area of the box. This corresponds to a porosity of 50%. Note that the >15-Åwide gap between the substrate XY edges and box edges not only serves to control the substrate porosity but also allows the adsorbed membrane sufficient freedom to undergo changes in surface area. Finally, the surface of the grid is functionalized. Hydroxyl groups are incorporated by replacing randomly selected CH4 spheres on the surface with CH3OH molecules. Force field parameters for CH3OH are taken from Walser et al.43 Negative charge is introduced on the substrate by deprotonating CH3OH molecules. The partial charges for the deprotonated CH3O− molecule, qCH3 = 0.13 eu and qO = −1.13 eu, are taken as the Breneman charges obtained from the electron density computed using the B3LYP/aug-cc-pvtz density functional theory.44,45 Figure 1 shows the topology of the substrate that has a surface density

bilayer remodulation by nanoporous substrates employed in many synthetic/biomimetic nanodevices22−32 as there are several differences between experimental and simulated constructs, including the substrate dipole/charge density, substrate porosity, and lipid chemistry. Here we focus on understanding experimental observations concerning lipid remodulation by nanoporous silica. As it stands, experimental observations30 seem somewhat in contrast to extrapolations derived from MD simulations.34 Neutron reflectivity studies21 show that nanoporous silica adsorbs a POPC bilayer at a distance of 5.1 Å, which is smaller than the range of distances (6−30 Å) reported for PC bilayers supported on continuous substrates.17,35−37 Experiments also show that nanoporous silica increases the transition temperatures of PC bilayers and induces domain formation in binary DSPC/DOPC mixtures.30 Additionally, experiments show that the bilayers adsorbed on nanoporous silica exhibit lateral diffusion rates that are 10 times slower compared to those of free-standing bilayers.30 In contrast, whereas MD simulations34 also suggest that POPC bilayers are adsorbed on hydroxylated nanoporous substrates at short distances (6−7 Å) and exhibit a 10-foldslower lateral diffusion rate, they suggest that substrates do not affect bilayer structure, yielding essentially no molecular basis for the remodulation of phase-transition properties of bilayers adsorbed on nanoporous silica. Nevertheless, we note three main differences between the experimental silica constructs21,30 and the simulated model constructs:34 (i) although amorphous silica has a surface density of 4.6 hydroxyls/nm2, the simulated substrates have surface densities ≤1 hydroxyls/nm2; (ii) although lipid properties were measured experimentally at a pH where the silica surface is partially deprotonated (or negatively charged),38−41 the simulated constructs are neutral; (iii) the simulated substrates have higher porosities than experimental substrates (75 vs 50%). Furthermore, we note that the neutron reflectivity experiments used for measuring lipid−substrate distances were conducted under highly acidic conditions21 where the silica surface is expected to be fully protonated, leaving the lipid−substrate distance unknown under less acidic conditions in which bilayer properties were determined.30 To gain molecular insight into lipid remodulation by nanoporous silica and also narrow the existing gap between experiments and simulations, we carry out a new set of MD simulations. The goal here is not to predict from simulations the properties of POPC bilayers adsorbed on amorphous silica, an exercise that has already been undertaken and has yielded important molecular insights into correlated effects.33 Rather, the goal is to understand the isolated and combined effects of silica-type surface charge densities and hydroxylation on POPC bilayers. We therefore simulate POPC bilayers adsorbed on model substrates whose physical and chemical properties are varied systematically. This allows us to disentangle causalities from correlated effects. Additionally, it allows us to relate the findings from these studies to those from our previous studies34 and broaden the phenomenological understanding of bilayers adsorbed on nanoporous substrates.

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Figure 1. Topology of a 50% porous substrate with a surface density of 4.6 hydroxyls/nm2. The red spheres represent hydroxyl oxygens, and the green box indicates the unit cell dimensions. of 4.6 hydroxyls/nm2 and is embedded in a box in which the substrate covers half of its XY area, making the construct 50% porous. Molecular Dynamics. All MD simulations are carried out under isobaric−isothermal conditions. Substrate topologies in MD simulations are preserved by restraining harmonically (force constant, 1000 kJ/mol/nm2) and individually the substrate’s CH4 molecules as well as the CH3 moieties of the protonated/deprotonated methanol molecules. No restraints are placed on the hydroxyl group or on the oxygen atom in the deprotonated methanols. The pressures in the lateral and transverse directions are maintained separately at 1.013 bar using an extended-ensemble approach46,47 and with a coupling constant of 1 ps and a compressibility of 4.5 × 10−5 bar−1. Additionally, because a subset of particles is restrained harmonically in MD simulations, reference coordinates of all particles are scaled with the scaling matrix of pressure coupling. An extended ensemble approach48 is also used to maintain the temperature at 303 K, although a smaller coupling constant of 0.2 ps is employed. Electrostatic interactions are computed using the particle mesh Ewald scheme49 with a Fourier grid spacing of 0.15 nm, a sixth-order interpolation, and a direct space cutoff of 10 Å. van der Waals interactions are computed

METHODS

Substrate Modeling. Substrates are constructed in three stages. First, a set of uncharged Lennard-Jones (LJ) spheres are placed equidistant from each other on a 3D grid of size 87.4 × 91.5 × 29.1 Å3. The separation between two adjacent LJ spheres is set at 4.16 Å, which corresponds to a distance at which the potential energy between them 6767

DOI: 10.1021/acs.langmuir.6b01155 Langmuir 2016, 32, 6766−6774

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Langmuir explicitly for interatomic distances of up to 16 Å. The bonds in lipid molecules are constrained using the P-LINCS algorithm,50 and the geometries of the water molecules are constrained using SETTLE.51 These constraints permit the use of an integration time step of 2 fs. Water molecules are described using SPC/E parameters,52 and the lipid molecules and lipid−water interactions are described using the recently refined GROMOS 43A1-S3 parameter set,53,54 which yields lipid component volumes, cross-sectional areas, form factors, and electron densities in agreement with experiment. The unit cells of freestanding bilayers contain 64 lipids/leaflet and ∼58 waters/lipid. The unit cells of supported bilayers contain 256 lipids/leaflet and ∼80 waters/lipid, yielding systems that contain over 150 K particles. All molecular dynamics simulations are carried out using Gromacs 4.5.3.55 The simulation of the free-standing POPC bilayer embedded in salt solution is initiated from a conformation of the bilayer taken from a pre-equilibrated trajectory in pure water.34 Salt is introduced by replacing randomly selected water molecules with Na+ and Cl− ions but ensuring that no “structural” waters within 10 Å of the lipids are chosen for replacement. This free-standing bilayer is equilibrated for 500 ns, and the final 200 ns is used for analysis. The simulations of supported bilayers are initiated by placing pre-equilibrated freestanding bilayers 16 Å from the substrate. The supported bilayers are simulated for different time scales, depending on their respective equilibration times, and the final 75 ns of each trajectory is used for analysis. The equilibration of MD simulations is monitored by tracking the time evolution of potential energies, lattice constants, and pressure tensors. Additionally, in MD simulations involving bilayers supported on substrates, equilibration is also monitored by tracking the time evolution of substrate−bilayer distances as well as the number of water molecules occupying the interstitial region between substrates and bilayers, just as we did in our earlier study.34 In all simulations of supported bilayers, the distance decreases and levels out to smaller values, and so does the numbers of interstitial water molecules. Unless stated otherwise, all standard deviations are obtained via block averaging, which indicates that the trajectory lengths used are sufficient for the types of analyses presented in this work. Tessellated Lipid Areas. Typically, two different approaches are employed for computing bilayer areas. In one approach, bilayer areas are determined as the ratio A = νC/2dC, where νC is the partial volume of the hydrocarbon tail56 and 2dC is the half-width of the number density of the hydrocarbon core.57 In the other approach, bilayer areas are determined by dividing the lateral area of the unit cell by the number of lipids/leaflet and averaging over the trajectory (A2D). Both techniques, however, assume that lipid bilayers are geometrically planar. To determine the surface area of a nonplanar corrugated bilayer, we need to account for the displacement of lipids along the transverse (z) direction. To accomplish this, we first connect the phosphorus atoms of a leaflet via triangular tessellation and then estimate the 3D surface area of the tessellation. This is done separately for each snapshot in the trajectory, and then the tessellated areas are averaged. Note, however, that such a tessellation will omit the space between the phosphorus atoms lying at the periphery of the unit cell and the boundary of the unit cell and will therefore produce an underestimated value for the leaflet surface area. To correct for this artifact, we also include in the tessellation a set of points that constitute the lateral edges of the unit cell, (0, y = {0,..., | b|}, z), (|a|, y = {0,..., |b|}, z), (x = {0,..., |a|}, 0, z), and (x = {0,..., |a|}, | b|, z), where a and b are unit cell vectors. The z coordinates of the four points (x = {0, |a|}, y = {0, |b|}, z) are all assigned the same value, which is the average of the z coordinates of the phosphorus atoms closest to them. This follows from the condition imposed by unit cell periodicity. The z coordinates of the remaining edge points are taken as respective averages of the z coordinates of two phosphorus atoms one inside the unit cell and the other in the adjoining lateral image of the unit cellboth of which are the closest to the edge point. Also, to ensure periodicity, the z coordinates of (0, y = {0,..., |b|}, z) are set equal to those of (|a|, y = {0,..., |b|}, z) and the z coordinates of (x = {0, ..., |a|}, 0, z) are set equal to those of (x = {0, ..., |a|}, |b|, z). Such

choices make the lateral projection of the 3D tessellated area equal to the lateral area of the unit cell, or A2D × N, where N is number of lipids in a leaflet (Figure S1 in the Supporting Information). We have implemented the Delaunay algorithm for tessellation58,59 using Gromacs APIs.55 The source code can be downloaded from https:// simtk.org/home/conf_ensembles.

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RESULTS AND DISCUSSION Bilayer Supported on a Neutral Hydroxylated Substrate. To understand the effect of the surface chemistry of nanoporous silica substrate on the POPC bilayer, we consider first a nanoporous substrate functionalized with hydroxyl groups. We chose a 50% porous substrate and functionalized its surface with 4.6 hydroxyls/nm2; these two attributes match those of the silica substrates used in experiments.21,30 This is also a natural starting point as it is an extension of our previous MD study34 where we had simulated POPC bilayers supported on nanoporous substrates functionalized with lower surface hydroxyl densities of 0.5 and 1.0 nm−2. Together, these studies will inform us of the systematic effect of surface hydroxylation on membrane properties. Additionally, this simulation will provide a direct contact point with experiments. Neutron reflectivity studies21 report that under acidic conditions (pH 2) of the D2O/D2SO4 buffer, where the surface of a 50% porous sol−gel silica substrate will be fully protonated or charge neutral,38−41 the sum of the widths of the interstitial water and the POPC headgroup is 14.6 Å. Assuming a standard POPC headgroup width of 9.5 Å60 and that the silica substrate and the acidic conditions do not alter the headgroup width, the substrate adsorbs a POPC bilayer at a distance of 5.1 Å. We discuss the implications of this assumption below. Figure 2 shows the time evolution of the average distance of POPC phosphates from the substrate surface. The distance

Figure 2. Time evolution of the average distance between the phosphates of POPC and the surface of a 50% porous substrate with 4.6 hydroxyls/nm2. Note that during averaging we consider only the phosphates of the leaflet proximal to the substrate.

decreases from an initial value of 16 Å to a final time-averaged value of 4.5 ± 0.1 Å, a result consistent with neuron reflectivity experiments.21 Although such an average distance suggests that the interaction between the POPC bilayer and the substrate will be buffered by water molecules, radial distributions of POPC headgroup atoms around the substrate hydroxyl oxygens indicate that about 20% of the substrate hydroxyls interact directly with the POPC bilayer (Figure 3). This substrate− POPC interface is somewhat different from that of POPC bilayers adsorbed on substrates functionalized with lower surface hydroxyl densities,34 where we had noted a completely water-buffered interaction between POPC bilayers and substrates. For substrates functionalized with lower surface hydroxyl densities of 1.0 and 0.5 nm−2, the POPC bilayer was adsorbed, respectively, at larger distances of 6.1 ± 0.6 and 7.0 ± 6768

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can cause a given external stimulus to interact differently with the bilayer depending on whether it originates closer to the distal leaflet or the proximal leaflet. A prominent feature of this remodulation is an enhancement in peak heights, with compensating reductions in half-widths. The sharpened distribution of water charges suggests an increased alignment of water dipoles along the bilayer normal. We confirm this by estimating the effect of the substrate on the transverse component of the water dipoles (Figure S2 in the Supporting Information). The modulation in the charge densities of lipids can be understood partly by noting that the substrate alters the phosphate−choline (PN) dipole orientations (Figure S3 in the Supporting Information). Specifically, the substrate induces a shift in the orientation of the PN dipole toward the bilayer normal. We had noted a similar but less pronounced reorientation of PN dipoles in POPC bilayers adsorbed on substrates functionalized with low surface hydroxyl densities.34 A similar observation was also made in an MD simulation of a PC bilayer supported on nanoporous amorphous silica.33 The enhanced sharpness in the lipid charge distribution can also be attributed to a substrate-induced reduction in the transverse fluctuations of lipid headgroups (Figure S4 in the Supporting Information). Because the widths of the number distributions are related to statistical (or Shannon) entropy,61 it can be argued that the sharpening of the number densities suggests a loss of entropy. Such a loss of entropy along the transverse direction was also noted in coarse-grained MD simulations of bilayers supported on continuous supports.61 Note, however, that whereas there is a loss of statistical entropy, it is clearly not large enough to prevent the bilayer from staying juxtaposed to the nanoporous substrate. Overall, the nanoporous substrate modifies headgroup dipole orientations and lipid transverse fluctuations, which together result in altered charge distribution profiles. The substrate also dampens significantly the lateral diffusion of the lipids, as also observed in experiments of bilayers on various different nanoporous constructs.28,30 Furthermore, the drop in diffusion is much more pronounced in the leaflet proximal to the substrateWhen the diffusion coefficient drops by a factor of 6 in the leaflet proximal to the substrate, the diffusion of lipids in the distal leaflet drops by a factor of 2 (Figure S5 in the Supporting Information). The lateral diffusion coefficients are estimated from the limiting case, D = limΔt→∞⟨Δr2⟩/4Δt, where Δr2 is the lateral mean-square displacement. The effect of the substrate on the diffusion of lipids in the distal leaflet can be attributed to the long-range nature of the electrostatics34 but not to increased lipid interdigitation (Figure S6 in the Supporting Information). The differential diffusion of lipids in the two leaflets has, to our knowledge, not been reported in the case of bilayers adsorbed on continuous supports, and may therefore be a unique feature of bilayers adsorbed on nanoporous substrates. Bilayer Supported on a Charged Hydroxylated Substrate. In the simulation above, the POPC bilayer adheres to the hydroxylated substrate at a distance similar to that observed in experiments21 involving a nanoporous silica substrate. We note that the experimental distance was measured under very acidic conditions (pH 2) where the silanonl groups on the silica surface are fully protonated,38−40 just as in our simulated construct. An increase in pH will deprotonate a fraction of the silica surface hydroxyls, introducing explicit negative charge onto the surface. This poses the general

Figure 3. Microenvironment of the hydroxyl oxygens on the surface of the neutral substrate that interacts with the POPC bilayer. (a) Integrated radial distributions n(r) of water oxygens and POPC headgroup atoms around the surface hydroxyl oxygens. (b) Representative (150 ns) snapshot of an MD trajectory highlighting a portion of the substrate−POPC interface. The substrate is drawn as a vdW surface, and the surface of the hydroxyl oxygens is colored pink.

0.4 Å.34 While a general trend seems to emerge in that the bilayer−substrate distance is related inversely to the substrate hydroxyl density, we note that the previously simulated substrates also had a higher porosity of 75%. Just as we noted in the case of bilayers adsorbed on substrates with low surface hydroxyl densities,34 we find that the substrate with a high hydroxyl density selectively perturbs only specific structural and dynamical properties of the POPC bilayer. The bilayer properties that remain unaffected are the partial volumes of lipid components, bilayer widths, and lipid areas (Table S1 in the Supporting Information). This finding also supports the assumption that the silica substrate in the neutron reflectivity studies21 does not affect the structure of the adsorbed POPC bilayer. The bilayer properties that are remodeled extensively by the substrate are the charge distribution, headgroup dipole orientation, lipid transverse fluctuations, and lateral diffusion coefficients. Additionally, we find that the substrate remodels the properties of the proximal leaflet more extensively compared to the leaflet distal to the substrate. Figure 4 shows the distribution of charges in the supported POPC bilayer. As we observed in the case of bilayers adsorbed on substrates with low surface hydroxyl densities,34 the substrate with a high hydroxyl density induces more extensive modulations in the leaflet proximal to the substrate as compared to the distal leaflet. We expect that such asymmetry

Figure 4. Charge densities in bilayer adsorbed on a 50% porous substrate with 4.6 hydroxyls/nm2. The charge densities of the water molecules and the POPC lipids are plotted separately as red and blue lines, respectively, and the total charge densities, which are the sums of the charge densities of water molecules, lipids, and the substrate hydroxyl groups, are drawn as solid black lines. 6769

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Figure 5. Transverse number densities of lipid carbonyl groups, Na+ ions, and Cl− ions in (a) a free-standing POPC bilayer, (b) a bilayer supported on a neutral substrate, and (c) a bilayer supported on the charged substrate. (d) 400 ns snapshot of the MD simulation of a bilayer supported on the charged substrate showing the distribution of ions. The substrate is drawn as a vdW surface.

ionic strength of 100 mM corresponds to the concentration of Na+ ions, and there are relatively few Cl− ions to compensate for the negative charge on the substrate. As controls, we also carry out two additional MD simulations−one of a freestanding POPC bilayer in 100 mM NaCl and the other that employs the same hydroxylated substrate we used above but with a construct that is embedded in a 100 mM NaCl solution. Note that in the analysis that follows we refrain from comparing systems containing salt with systems containing no salt to avoid the overinterpretation of ion-induced effects.62−64 We initiate the two supported bilayer MD simulations by placing the bilayer at a distance of 16 Å, just as we did in the case of the supported bilayer construct we simulated above. We expected that the introduction of explicit charge on the substrate, which increases the substrate hydrophilicity, would cause the POPC bilayer to adhere to the substrate even more closly than for the neutral hydroxylated substrate. Surprisingly, however, we find that the POPC bilayer adheres to the charged substrate at a distance (8.6 ± 0.1 Å) that is almost twice as large compared to that of the neutral substrate (4.5 ± 0.1 Å) simulated under the same salt conditions (Figure S7 in the Supporting Information). Although the bilayer supported on the charged substrate does not interact directly with the substrate, the bilayer supported on the neutral substrate interacts directly with the substrate (Figure S8 in the Supporting Information). We attribute the larger substrate−bilayer distance in the case of the charged substrate to the formation of an electric double layer on the substrate. Figure 5 shows the transverse distributions of Na+ and Cl− ions superimposed over the

question of the effect of surface charge on the substrate−bilayer distance and bilayer remodulation. As such, we note that the type of bilayer remodulation induced by the charge-neutral substrate does not entirely match the type of remodulation observed in experiments of silica-supported membranes that were carried out under mildly acidic conditions of pH 5−7 (personal communication with Atul N. Parikh).30 Although FRAP experiments30 show that nanoporous silica dampens the lateral diffusion of lipids significantly, as also noted in studies of charge-neutral nanoporous constructs,28 nanoporous silica increases the phase-transition temperature of a DOPC bilayer and induces domain formation in binary DSPC/DOPC mixtures, implying that nanoporous silica must be remodeling the molecular organization of the bilayer. Although our hydroxylated construct does not induce any large changes in lipid volumes or areas (Table S1 in the Supporting Information), we do note changes in headgroup orientations (Figure S3 in the Supporting Information). Could these differences emanate from the fact that the nanoporous silica substrate is partially negatively charged under the experimental conditions? To examine the effect of explicit surface charge, we deprotonate a fraction of the hydroxyls in the substrate we employed above. At a pH of 7, experiments38−40 indicate that 19% of the surface hydroxyl groups are deprotonated; however, recent studies also show that the extent of deprotonation is saltdependent.41 Given the uncertainty in the pH in experiments that measure bilayer remodulation by nanoporous silica and the fact that the pH is mildly acidic, we chose to deprotonate 10% of the hydroxyls. We conduct simulations in 100 mM NaCl salt solution, which matches the experimental conditions.30 The 6770

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Langmuir transverse distributions of lipid carbonyl groups. In the freestanding bilayer and the bilayer supported on the neutral substrate, we note only one high-density peak for Na+ ions that coincides with the lipid carbonyl density peak, indicating that the lipids tend to interact with Na+ ions preferentially with the lipid carbonyl groups. In the bilayer supported on the charged substrate, we observe not one but two distinct high-density peaks for Na+ ions, one next to the substrate and one that coincides with lipid carbonyl groups. Additionally, we note a broadening of the Cl− ion distribution, which spans the interstitial region between the substrate and the bilayer. The Na+ ions, which are attracted to the negative charges on the substrate, bring along their waters of hydration and increase the amount of interstitial water between the substrate and the bilayer (Figure S9 in the Supporting Information). The broadened density distribution of lipid carbonyl groups in Figure 5 does not reflect systematic undulations in the membrane. Rather, it reflects a corrugated, nonplanar bilayer surface with the bilayer interacting at different distances with different portions of the substrate. In fact, when the transverse components of the phosphate−substrate distances are divided into three groupsthose between protonated surface hydroxyls and phosphates, those between deprotonated surface hydroxyls and phosphates, and those between surface CH4 moieties and phosphateswe find that phosphates are systematically closer to the protonated surface hydroxyls than to the deprotonated surface hydroxyls (Figure 6). This is consistent with what we note above and have noted previously:34 the bilayer−substrate distance depends inversely on the extent of substrate hydroxylation.

Table 1. Surface Areas of POPC Bilayers Simulated in NaCl Salt Solutiona system

A2D (Å2)

Aprox (Å2)

Adist (Å2)

free standing supported on neutral substrate supported on charged substrate

59.7 ± 0.6 62.8 ± 0.5 60.4 ± 0.1

64.7 ± 0.7 67.6 ± 0.8 67.9 ± 0.3

64.9 ± 0.5 68.1 ± 0.6 66.1 ± 0.1

a

Apro and Adist refer to the average tessellated 3D areas of lipids in leaflets proximal and distal to the substrate, respectively. A2D is the average area of a lipid obtained from the lateral (2D) projection of the tessellated 3D areas, which is essentially equal to lateral area of the unit cell divided by the number of lipids in a leaflet.

The corrugated bilayer adsorbed onto the charged substrate also has a statistically larger transverse width compared to a free-standing bilayer. The phosphate peak-to-peak distance is 37.2 ± 0.1 Å in the free-standing bilayer, 38.6 ± 0.2 Å in the bilayer supported on the neutral substrate, and 39.7 ± 0.1 Å in the bilayer supported on the charged substrate. Note first that because of the corrugation in the bilayer surface we did not estimate these distances using the standard protocol, that is, from transverse density distributions. Instead, we estimate these distances prior to a lateral averaging of phosphate number densities. The coordinates of phosphates in the trajectory are discretized on a 3D grid (spacing = 1 Å), and the peak-to-peak distance is measured separately for each lateral coordinate pair of the grid, which is then averaged. Second, although both the neutral and charged substrates increase the bilayer widths, the charged substrate increases the bilayer widths by a larger magnitude (>5%). Although the presence of charge on the substrate increases the lipid areas and bilayer widths, it restores to a great extent the PN-dipole orientation induced by hydroxylation (Figure 7).

Figure 6. Cumulative probability distributions of the transverse component of the distances (z) between the surface of the charged substrate and the phosphates of the bilayer leaflet proximal to the substrate. The phosphate−substrate distances are categorized into three groups: those between protonated surface hydroxyls and phosphates, those between deprotonated surface hydroxyls and phosphates, and those between surface CH4 moieties and phosphates. Figure 7. Angle distribution (p(θ)) of phosphate−choline (PN) dipoles in POPC bilayers simulated in 100 mM salt solution. θ is the angle between a PN dipole and the leaflet normal projecting outward from the membrane.

Given that the surface of the bilayer supported on the charged surface is corrugated, one could expect that it has a lateral 2D surface area smaller than that of a free-standing bilayer. We note, however, that this is not the case. The lateral 2D surface areas of the bilayer supported on the charged substrate are comparable to those of the free-standing bilayer (Table 1). We note also from Table 1 that the 3D surface area of a free-standing bilayer, obtained by tessellation, is larger than its 2D lateral surface, which informs us that the surface of a free-standing bilayer is also not ideally flat. However, when the bilayer is supported on a charged substrate, the surface corrugation increases by >5%, and we also note that the leaflet proximal to the substrate is more corrugated than the distal leaflet. The extent of this change in surface area is substantial in the sense that it is comparable to that seen in phase transitions.65,66

The difference between the lateral diffusion of lipids in the two leaflets is also somewhat reduced (Figure 8). The lateral diffusion of the lipids in the bilayer is, however, still damped and an order in magnitude smaller than the diffusion in the free-standing bilayer. This latter result matches experimental estimates. Experiments show that the diffusion coefficients of lipids in bilayers supported on nanoporous silica30 are on the order of 1 μm2/s, and the diffusion coefficients of lipids in freestanding bilayers are on the order of 10 μm2/s.67 6771

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suggests the prospect of making two surface attributesdipole densities and charge densitieswork antagonistically toward remodeling lipid bilayer properties.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01155. Tessellation of phosphorous atoms, lipid areas and partial volumes of lipid components, water orientation with respect to the bilayer normal, angle distribution of phosphate choline dipoles in a POPC bilayer, transverse distribution of lipid functional groups in the bilayer, lateral mean-square displacements of lipids in the bilayer, number distribution of terminal hydrocarbons along the transverse axis, time evolution of the average distances between the surfaces of substrates and the phosphates of the POPC lipids in the proximal leaflet, integrated radial distributions of water oxygens and POPC headgroup atoms around the surface hydroxyl oxygens of the substrate, and normalized water densities in supportedbilayer constructs simulated in salt solutions (PDF)

Figure 8. Lateral mean-square displacements of lipids Δr2 = Δx2 + Δy2 in supported POPC bilayers simulated in 100 mM salt solution. The values of the diffusion coefficients provided next to the curves are estimated by assuming a linear dependence of ⟨Δr2⟩ on Δt in the 10− 15 ns range.

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CONCLUSIONS Experiments demonstrate that nanoporous substrates remodel macroscopic lipid bilayer properties differently from continuous supports; however, the underlying molecular mechanisms remain undetermined. Here we use MD simulations to understand the isolated and combined effects of two surface properties of nanoporous silica, hydroxylation, and charge on POPC bilayers. Several general trends seem to emerge in the case of uncharged, hydroxylated nanoporous substrates. First, the higher the surface hydroxyl density of a substrate, the closer the POPC bilayer is adsorbed onto the substrate. Second, such nanoporous substrates selectively perturb only certain structural and dynamical properties of the POPC bilayer. Bilayer properties that are remodeled significantly by the substrate are the charge distribution, headgroup dipole orientation, lipid transverse fluctuations, and lateral diffusion coefficients. Bilayer properties that are affected minimally are the partial volumes of lipid components and lipid areas. Finally, the substrate remodels the properties of the proximal leaflet more extensively that it remodels the properties of the leaflet distal to the substrate, and the extent of this asymmetric remodulation increases with the level of surface hydroxylation. Together, our simulations suggest a direct correspondence between substrate hydrophilicity and membrane properties. A partial deprotonation of surface hydroxyls, as expected of a silica substrate under mildly acidic conditions, however, produces an inverse effect: it increases the substrate−bilayer distance, which we attribute to the formation of electric double layer over the negatively charged substrate. While the introduction of explicit charge on the substrate restores, at least partially, certain properties, including leaflet asymmetry and transverse charge densities, we find that lipid areas and bilayer widths, which were affected to a lesser extent by adsorption on neutral substrates, increase by >5%. We expect that such a molecular-level reorganization could explain the experimentally observed remodulations in phase-transition temperatures and domain formation induced by nanoporous silica.30 Clearly, further studies are needed to establish a detailed causal link between substrate properties and phase transitions in mixed bilayers and to also establish a general theoretical framework to enable forward predictions on bilayer remodulation by nanoporous substrates. Nevertheless, this study presents a discrete step forward in understanding bilayer remodulation by nanoporous substrates. Additionally, it

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

N.D. and M.G. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Jeff Brinker, Sagar Pandit, and David Rogers for useful discussions. We also acknowledge services provided by Research Computing at USF, funding from the USF Foundation, and NSF grant MRI CHE-1531590.

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