Effects of Lithium and Other Monovalent Ions on Palmitoyl Oleoyl

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Effects of Lithium and Other Monovalent Ions on Palmitoyl Oleoyl Phosphatidylcholine Bilayer James Kruczek, See-Wing Chiu, Eric Jakobsson, and Sagar A. Pandit Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04166 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 18, 2017

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Effects of Lithium and Other Monovalent Ions on Palmitoyl Oleoyl Phosphatidylcholine Bilayer James Kruczek,∗,† See-Wing Chiu,‡ Eric Jakobsson,¶ and Sagar A. Pandit∗,† Department of Physics, University of South Florida, Tampa, Florida 33620 , Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, Illinois 61801, and Department of Molecular and Integrative Physiology, Beckman Institute for Advanced Science and Technology, Department of Biochemistry, Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois 61801 E-mail: [email protected]; [email protected]

Abstract Interations of monovalent salts with lipid membranes are explored with Molecular Dynamic (MD) simulations. The simulations included the monovalent ions Na+ and K+ , for their importance in physiology, Li+ for its small size and importance in several medical conditions including bipolar disorder, and Rb+ for its large size. All the simulations included Cl− as counter ions. One bilayer was simulated without salt as a control. POPC bilayers experienced reductions in area per lipid with the addition of salt, the smaller the ion the smaller the area, with the exception of Li+ . Li+ exhibited unique binding affinities between phosphates and Sn–2 carbonyls that lowered the order of the top part of Sn–2 chain, which increased the area per lipid, compared to other ionic simulations. Further, we observe that monovalent salts alter ∗ To

whom correspondence should be addressed of South Florida ‡ Beckman Institute for Advanced Science and Technology, University of Illinois ¶ Center for Biophysics and Computational Biology, University of Illinois † University

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bilayer properties through structural changes and not so much though the changes in surface potential.

Introduction Lipid membranes are a crucial component of the cell. These membranes separate cell interior from exterior and regulate many cell functions.

1

Physiologically, lipid membranes are solvated

with water and are often surrounded by salt ions. These ions can play an important role in the properties of the bilayer. The most common inorganic ions in human biology are Na+ , K+ , Ca2 +, Mg2 + and the anion Cl− . Additionally, Li+ is interesting because it is an alkali earth metal just like Na+ and K+ , but it has dramatic effects on our biology. Lithium has been used as a treatment for bipolar disorder since the 1950s. 2 The mechanism by which Li+ aids with bipolar and other disorders is not completely understood. Some evidence suggests its role as an inhibitor of inositol monophosphatase (IMPase). 3 IMPase is important because it regenerates inositol, an important signaling mechanism, from inositol monophosphate. 4 In addition to a treatment for bipolar disorder, Li+ has many other impacts on human physiology. 5 de Freitas et. al. suggest that a major mode of action of lithium is its ability to compete with magnesium in magnesium-bound enzymes, of which IMPase is one. 6 The functional effect of lithium is to reduce the activity of these enzymes. A particularly well studied magnesium-bound protein is Glycogen Synthase Kinase 3-beta (GSK-3β ). Glycogen Synthase Kinase derives its name from its ability to phosphorylate, and hence inhibit, glycogen synthase. Glycogen synthase activity, and glycogen synthesis,is thus increased producing an effect similar to insulin, but by a completely different molecular mechanism. There are estimated to be over three thousand human proteins with magnesium binding sites. 7 This large number, combined with lithium’s ability to compete for magnesium binding sites, may account for the fact that lithium’s effects are so diverse and complex. In the current paper we consider a less-well studied aspect of lithium, namely its interactions


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with bilayer membranes as compared to the interactions of other ions. There are three primary ways ions affect lipid bilayers, mechanical, electrostatic, and specific binding on bilayer and proteins. 8 In this paper we focus only on the interactions of ions with lipids. Experimental work on the effects of ions on pure zwitterionic lipid bilayers is sparse. Pabst et. al. used x-ray diffraction, EPR spectroscopy, dilatometry and velocimetry, and differential scanning calorimetry on POPC bilayers and found that at higher concentration of NaCl (about 500 mM) bilayers thicken and their areas shrink. 9 Böckmann et. al. used fluorescence correlation spectroscopy and Calorimetry on POPC bilayers which showed with increasing ion concentration the lateral lipid self-diffusion decreases. 10 Binder et. al. used infrared spectroscopic and showed deep penetration of cations into the polar region of POPC bilayers. They also note that Li+ dehydrates the phosphate and carbonyl groups better then Na+ and K+ . 11 Eisenberg et. al. measured the ζ potentials of phosphatidylserine vesicles in solutions containing monovalent cations. 12 McLaughlin et. al. demonstrated that the Stern equation predicted the absorption of divalent cations to phosphatidylcholine bilayer membranes. 13 Simon et. al. measured the hydration repulsive pressure between phosphatidylcholine bilayers and determined that hydration pressure depended on the dipole potential of the bilayer. 14 There are several MD simulations that have improved our understanding of lipid-ion interaction. Pandit et. al. simulated Dipalmitoylphosphatidylcholine and found a small decrease in the area per lipid of the bilayer and an increase in the order parameter of the carbon chains with the addition of NaCl. 15 Work by Gurtovenko et. al. and Cordomi et. al. simulated many cations, with different force fields, with viarious lipids, and showed two major points. First, monovalent ions with smaller radii, bind in grater numbers to the lipid bilayer, and thereby further ordering the bilayer. Second, that the larger dipole potential created by larger numbers of smaller ions is nearly counter balanced by an increase in the opposing dipoles of the lipid head group and water. 16–18 All of these simulations have shown that cations penetrate the head group while anions are weekly bound to the top of the bilayer. An important factor in an lipid bilayer-ion simulation is the ionic force field. The decision


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of which Lennard-Jones parameters to use can affect the binding properties of ions and therefore many measurable properties like area per lipid. 17 It has been shown that some of the traditional ionic force fields caused unphysical states in MD simulations, such as salt crystals forming below their solubility limit. 19 Recent work by Joung et. al. re-optimizes the ion force fields for various commonly used water models. These force fields were validated by calculating the hydration free energy, lattice energy, and lattice constants 19 for various salts. We used these force fields in our simulations. Quantitative study of lipid bilayers in ionic solvents require long simulation time. 8 Equilibration of ionic binding is limited by two major factors: i. diffusion of ions in bulk and ii. the thermodynamic balance of association and dissociation process at the bilayer surface. smaller ions, such as Li+ , have been shown to be particularly difficult since so many of the ions prefer to be bound to the bilayer. 16 In an effort to ensure all runs reached an equilibrium state with the new force fields described above, each of our simulations were run for 0.5 µ s.

Simulation Methods Molecular Dynamics (MD) simulations were performed on hydrated palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers. We simulated 5 systems (please see table 1 For details). The first simulation, henceforth referred to as POPC (No Salt), was used as a base case to compare the subsequent salt simulations. It contained 200 POPC lipids, 100 per leaflet, and 50 waters per lipid. The other four simulations used the same 200 POPC lipid bilayer, but with the addition of an initial concentration of 200 mM of Chloride (Cl) anions and 200 mM of the respective cation: Lithium (Li), Sodium (Na), Potassium (K), and Rubidium (Rb). Ionic simulations require large quantity of water to get correct bulk water behavior. A ratio of 150:1 water to lipid, was used as described in table 1. Since the lipid bilayer adsorbs a significant portion of the free ions in water, the effective bulk concentration(table 1 column 4) of each cation was determined from the remaining concentration in bulk water.


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All the simulations were performed using the GROMACS package, version 4.5. 20–23 Force field parameters for the lipids were taken from work of Chiu et al. 24 Additionally, we used ion force field parameters developed by Joung et al. 19 The cross terms between ion and lipid parameters were explicitly calculated using Lorentz-Berthelot rules. 19 Temperature was held constant at 300 K using Nose-Hoover temperature coupling scheme, 25 while pressure was held at 1 atm using Parrinello-Rahman semiisotropic pressure coupling. 26 The LINCS algorithm was used to constrain all bonds in the system. 27 This allowed an integration time step of 4 fs. Periodic boundary conditions were used in all three dimensions. The long-range electrostatics were calculated using the SPME algorithm 28 with a real-space cutoff of 10.0 Å. Van der Waals interaction was computed with a cutoff of 16 Å. Initial configuration for simulations were generated by constructing two 100 lipid leaflets. A block of SPC/E waters, in the amount described in table 1, were placed above the bilayer. For the 4 salt simulations, 216 random water molecules were replaced with 108 Cl− and 108 appropriate cations. The systems were energy-minimized to remove bad contacts resulting from overlapping hard sphere surfaces and over stretched bonds. All the systems were annealed to ensure proper thermalization of the hydrocarbon chains. The Annealing steps involved a 200 ps 290 K run to ensure the bilayer did not break down during annealing. Further, a set of NVT runs at 500 to 420 K in steps of 20 K and 400 to 300 K in steps of 10 K, followed with NPT runs from 400 to 300 K in steps of 10 K for 50 ps each step. After annealing, 0.5 µ s of continuous MD simulations were performed for each of the systems. Box areas were monitored throughout the simulation.


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Results and Discussion Analysis Methods and Base Case Results Structural Properties We have demonstrated the validity of our lipid force fields in previous works. However we have not really established the stability of the lipid bilayer under long simulation runs and investigated the agreement of the simulated structures with the experimental data. 29 Table 2 reports various structural properties of the simulated bilayers. These properties are important because they can be measured in x-ray and neutron scattering experiments. The volumes of hydrocarbon chains, Vc (table 2 row 1), head group and glycerol backbone, VHG (table 2 row 2), and water Vw , where computed by the method proposed by Petrache et. al. 30 In this method we optimized the function

F(vi ) = ∑ ns (1 − ∑ 3ni (z j )vi ), zj

i=1

with respect to the partial molecular volumes vi of the components, where ni (z j ) is the number density of the i–th type. These partial molecular volumes were multiplied by the number of atoms in the group for a single lipid, 32 for Vc and 20 for VHG , to determine the volume per lipid. By adding Vc and VHG the volume of the lipid, Vl (table 2 row 3), was calculated. The volume of the POPC lipid thus computed to be 1216 ± 0.61 Å3 per lipid, which agrees well with experimental data. 31 The thickness of the hydrocarbon region, 2Dc (table 2 row 4) was calculated by finding the distance between the Gibbs surfaces formed by the number density of the hydrocarbon chains. 32,33 Db was calculated as the distance between the Gibbs surfaces formed by the water density. The 2Dc for POPC in absence of salt, was 26.73 ± 0.53 Å, and Db was 35.80 ± 0.72 Å, both match well with the experimental data. 31 In scattering experiments, the reported area is the ratio of the hydrocarbon volume and hydro6 ACS Paragon Plus Environment

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carbon thickness. Thus, instead of reporting the geometric area per lipid, we reported Al = Vc /Dc in table 2 row 6. The geometric area per lipid is usually smaller then the Al as discussed in literature. 34,35 Our reported value of the area per lipid is 67.6 ± 1.36 Å2 for POPC without salt, which matches well with experimental area. 31 Experimental structural parameters come from fitting a model to raw form factor data. In a simulation we have direct access to the electron density which can be used to deduce the expected form factor by taking the cosine transform of the symmetrized electron density. 36 This raw form factor data is independent of a particular model and therefore presents an ideal way to compare the simulation. The electron density was calculated using Gromacs analysis tools as seen for no salt in figure 1a. These data were used to find the peak to peak distance in electron density DHH (table 2 row 7). For pure POPC this value was determined to be 37.0 ± 0.4 Å, which matches well to x-ray scattering data. 36 The minimum of these densities were used as the center of the respective bilayer. Figure 1b shows the agreement of the computed form factor for POPC with the experimental x-ray form factor. 31

Chain Order Parameters An important structural property of hydrocarbon chains is the NMR order parameter profile. The order parameter tensor, S, is defined as 1 Sαβ = h3 cos (θα ) cos θβ − δαβ i, 2

(1)

where the α and β are the molecular axis (taking values either x, y, or z), and θα is the angle made by the α –th molecular axis with respect to the bilayer normal. The NMR experiments routinely report the deuterium order parameters for a deuterated hydrocarbon chain. In absence of explicit hydrogens (or deuteriums), as in united atom model, these order parameters can be obtained using the molecular order parameter tensors of the neighboring carbon atoms in a chain using the relation derived by Douliez et al. 37 Figures 2a and 2b show the order parameter profiles for Sn–1 and Sn–2


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chains respectively. These profiles agree well with no salt POPC simulations previously reported.

Pressure Profile The mechanical stresses in lipid bilayer manifest in terms of surface tension, which can be calculated from the pressure profile, or local pressure tensor. As argued by Jähnig, in absence of osmotic pressure, if force fields are accurate, and simulations are equilibrated then the net surface tension across the lipid bilayer must be zero. 38 The computed surface tension for POPC bilayer without salt is 3.14 ± 15 dyn/cm, which is essentially zero, as it must be because the pressure coupling algorithm used (same pressure set points normal to and parallel to the membrane plane) require zero surface tension. The accuracy of the simulations is attested to by the fact that these boundary conditions produce structural features in agreement with experiments. The computation of local pressure tensor is a very complex issue and deserves some elaboration. The local pressure tensor, is comprised of a kinetic and configurational components. αβ

αβ

Pαβ = PK + PC . The configurational contribution to the local pressure tensor is αβ

PC (~r) = ∑ fiα i

fiα is the force acting on the i–th particle.

Z

~ β. δ (~r −~l ) ds

C0i

~ ) dsβ is the line integral of a delta function

R

C0i δ (~r − l

along an arbitrary path C0i , from a common reference point r0 to the particle i at ri . ~l is the position vector of the line element and ~s is the line segment of the contour. To simplify the expression, we can assume that the forces acting on particle i can be broken into a set of pairwise interactions ~fi j . Additionally, if we force the contour though the location of particle j the sum of the product of the contour C0 j with the pairwise interaction ~fi j , must be zero, for the configurational energy to be invariant under translation. 39,40 For multibody interactions, such as angles and dihedral terms, force decomposition is necessary to determine the pairwise components. The method of this de8 ACS Paragon Plus Environment

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composition is not unique; some recent papers have show differences in the pressure tensor from different decompositions. 41 Applying these steps, we are left with αβ

PC (~r) = −

1 fiαj 2 i6∑ =j

Z

δ (~r −~l ) dsβ .

Ci j

The contour Ci j is not uniquely defined. The most common choice for Ci j is the Irving-Kirkwood contour (IK), which is the straight line vector~ri j . The limitation to this contour is that the method of calculating long range electrostatic interactions by Gromacs ( i.e. PME and Edwald sums) do not have an explicit source, ~r j , for the interaction and therefore can not be used in calculating the pressure tensor. To compensate for this, the electrostatic cutoff for our local pressure tensor calculations was set to 22 Å. The kinetic contribution to the local pressure tensor is simply the sum of, for each particle in the region, the outer product of velocity ~vi with itself, times the mass of the particle. β

PK (r)αβ = ∑ mi vαi vi δ (r − ri ), i

For a heterogeneous, isotropic bilayer, the local pressure tensor can be computed, for each slice z parallel to the bilayer with a volume of V , using β

Pαβ (z) = ∑ mi vαi vi − i∈z

1 V

β

∑ fiαj ri j w(~ri,~r j , z, dz),

(2)

i< j

where w(ri , r j , z, dz) is a function that computes the fraction of ri j that lies in slice z. We developed a stand alone analysis utility using GROMACS libraries (available to download at “https://csmlabfs1.cas.usf.edu/Sites/” under GPL) to compute pressure profile of our simulations. The software included modifications to the GROMACS libraries so that the bonded interactions could be recalculated and captured with GROMACS source code. SETTLE and LINCS constraint forces were calculated by determining the virtual forces acting on them, as described in other works. 41 SETTLE constraints were particularly important, contributing ∼30,000 bars of 9 ACS Paragon Plus Environment

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pressure in SPC/E water. As described above, multi-body interaction were decomposed using the Goetz and Lipowsky method (GLM) 39,41 and long range electrostatic interaction were calculated with a cutoff of 22 Å. It was verified, with the exception of the modification to long range electrostatics and addition of virtual restraints, our forces were exactly the same as GROMACS MD calculations. Simulation boxes were divided into 150 slices. Local pressure tensors in each slice were computed every 0.5 ps and averaged over 250 ps runs. The set of tensors for each interaction was then summed to find the total pressure tensor of the run. This process was repeated for 40, 250 ps runs, each 5 ns apart for the last 200 ns of each of the 5 simulations. For each pressure profile, the surface tension was computed by integrating over the difference between the normal pressure Pn (z) = Pzz (z) and the lateral pressure Pl (z) = (Pxx (z) + Pyy (z))/2. 42 Finally, the average and standard deviation of these 40 runs were calculated for both the pressure tensors and surface tension. Figure 3a shows the pressure profiles Pn (z) − Pl (z) for POPC bilayer without salt. As expected the normal pressure Pn (z) is almost constant over the entire length of the simulation box (data not shown). So the figure essentially shows the variation of lateral component of the pressure around the normal component. We note that the pressures (lateral and normal) are equal in hydrocarbon region and the center of the water. We can identify in general “bulk” as region that does not contribute in the surface tension. The bilayer has bulk regions in the hydrocarbon core and water outside the bilayer. Thus we can identify the thickness of the lipid bilayer from the “non–bulk” regions of the pressure profiles. We note, from the figure 3a, that the pressure profile becomes gradually zero in the bulk water. To demarcate the exact boundary point where Pn − Pl become zero, which hence forth we will call hydrostatic boundary, we used the following prescription. First, the profile was truncated at the point where the curve demonstrated monotonic decay to zero. Then the exponential function, f (x) = −ae−b(x−c) ,


(3)

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was fitted to the remaining points using Marquardt-Levenberg algorithm, with starting values of a = 400, b = 0.2, and c = 25. For the salt simulations, the peak was positive and the starting positions of a = −200, b = 0.2, and c = 25 were used. When the final values a, b, and c were determined, the length scale of the exponent,

1 b

+ c, was used as the hydrostatic boundary. For

POPC (No Salt) this value was 28.14 Å. Regions with positive Pn (z) − Pl (z) increase the surface tension while regions with negative values reduce it. For POPC (No Salt) the region where the hydrophilic head group is attracted laterally to the surrounding water has a positive contribution to surface tension. This is countered by two negative regions, the carbon tails and ordered water out side the lipid. Carbon tails reduce surface tension since they are squeezed together. This makes the Lennard-Jones increase repulsion laterally more then other balancing forces. Ordered water outside the lipid head is oriented from the typical bulk water. This slight reorientation decreases the lateral attraction while increasing the Lennard-Jones repulsion, thus decreasing the surface tension to zero.

Structure of water near the lipid bilayer Water plays crucial role in determining the structure of a bilayer. NMR experiments probe the behavior of water near and inside the lipid headgroup. According to these experiments in bulk water, the orientation of water is isotropic. As water comes closer to the polar bilayer, it becomes more ordered and can be described as perturbed water by the bilayer. This ordering can be measured as first rank, hcos(βBp )i, or second rank, h 12 (3 cos2 (βBp ) − 1)i, water order parameters. In this case βBp is the angle between the principal frame, the O → H vector, and the director frame, the normal vector to the bilayer. 43 We calculated the average first rank and second rank order parameter, per water molecule, per frame, as a function of the distance form the bilayer. The average of these results for POPC (No Salt) can be seen in figure 4a for the first rank and figure 4b for the second rank. Similar to Äman et. al., the first rank order parameter exhibits a single negative region between 8 to 28 Å. This area also coincides with the sigmoidal drop in water density. The second rank 11 ACS Paragon Plus Environment

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order parameter has two regions. The first, from 8 to 19 Å, is negative and corresponds to the angle favoring a position parallel to the bilayer while the region between 19 and 28 Åis positive and denotes the angle is now perpendicular to the bilayer. To compute the amount of perturbed water by the bilayer, the number density of water was integrated, which gave ∼ 16.8 perturbed waters by the bilayer. This is consistent with the reported experimental and simulation findings. 43 Based on these perturbed waters one can introduce what we call “hydration boundary” where the water becomes isotropic. For POPC bilayer without any salt the hydration boundary is at 29.64 Å from the center of the bilayer.

Electrostatic properties Electrostatic potential is the solution of the Poisson equation with appropriate boundary conditions. Since we assume homogeneity in the xy-plane, we need only solve the Poisson equation in one dimension:

∂ 2 φ (z) ρ (z) . =− 2 ∂z ε0

(4)

In this equation φ (z) is the potential, and ρ (z) is the charge density as calculated using GROMACS g_density tool. Thus the general solution can be obtained by twice integrating the charge density of the system as,

φ (z) = −

1 ε0

Z z Z z′ 0

0

ρ (z′′ )dz′′ dz′ +C1 z +C2 .

(5)

To find the particular solution, two boundary conditions must be applied. First condition simply sets the potential at the boundary of the box. We set it to be zero at the center of the bulk water, which gives C2 = 0 in equation 5. The second conditions comes from the Gauss’s law. Since the system is electrostatically neutral electric field at the box boundary must be zero. The method 12 ACS Paragon Plus Environment

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of imposing the second boundary condition is more difficult as it can lead to a linear term in potentials. 18 On macroscopic scale the zero electric field condition is not a big issue, however microscopically slices in bulk water may still have small fluctuations in dipole moments which can lead to net non–zero (but very small electric field ∼ 3.1×105 V/m) electric field at the boundary. This small difference in electric field produces unrealistic second integral. To compensate for this, we averaged the electric field (the first integral of charge) in a region of bulk water, and subtracted that constant from the first integral in calculating potential. This procedure was repeated for the last 10, 10 ns runs and averaged. This produces C1 = 0. As you can see in figure 5a, this method produced accurate results with very small standard deviation in electrostatic potential. We note from the figure 5a the POPC bilayer without the salts produce a potential difference of 0.49 ± 0.03 V between the bulk water and the center of the bilayer, with a peak near the headgroup rising up to 0.76 ± 0.03 V. These numbers are consistent with the previously reported values of electrostatic potential. 8,16 In literature this potential difference is often referred as “dipole potential”. Another potential of importance in studying surface phenomena is the surface potential. This is an electrostatic potential difference between an arbitrarily chosen surface and the zero of the potential (usually in bulk water). In our case we identify the surface at two possible locations viz. hydrostatic boundary (accessible through electrophoretic mobility experiments 44 ) and hydration boundary (accessible through NMR experiments 43 ). We note that the surface potential is ∼ 0.01 V for POPC bilayer without salt, irrespective of the choice of the surface.

Results With Salt Salt may affect lipid bilayer by, 1. binding to specific sites on the lipid, 2. altering the mechanical properties of the lipids and/or structural properties of water, and 3. changing the electrostatic properties viz. the surface potential and the dipole potential.


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Binding of Cations and Anions Positively charged cations solvate with the highly electronegative oxygens from the POPC lipid and water. Conversely, anions associate with positively charged atoms such as hydrogens and the choline group. We computed the radial distribution function (RDF) between ions and corresponding binding sites. We declared an ion to be bound to a particular binding site if and only if the site is in the first coordination shell of the ion RDF (Data not shown, please see supplemental material figures S1 through S4). A well equilibrated bilayer should reach a steady state, where the average number of cations bound to the bilayer does not change with time. Figure S5 is a plot of the number of cations bound to at least one lipid oxygen as a function time. We note that all the systems have reached, or were sufficiently close to, the steady state. Assuming first order reaction kinetic theory, off-rate constant Ko f f and on-rate constant Kon for each of the cations, was found by fitting to the data to the solution to the steady state equation,

n(t) =

NKon 1 − e−(Kon +Ko f f )t + n0 e−(Kon +Ko f f )t , Kon + Ko f f

(6)

Where, N is the total number of cations available, and n0 is the number of cations at the epoch point, t = 0. The best fit values are shown in figure S5. The quotient of the constants,

Kon Ko f f

, are

equal to the binding constant and are were consistent with expected values. 45 Figure 6a is a plot of the average number of cations bound to at least one binding site of a lipid. As previous works have shown, 16,17 the smaller the cation, the greater the affinity for binding. The figure clearly demonstrates this, however the trend appears to saturate below the ion size of Na+ . This clearly indicate that the binding properties of ions are not dependent on size for ions smaller or equal to the size of Na+ . Further, we investigate the preference of binding sites for various cations. We plot a histogram of fraction ions bound to binding sites (See Figure 7). We have three binding sites viz. the phos-


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phate group (denoted by PO4 in the figure), the carbonyl group on Sn–1 chain (denoted by CO-Sn1 in the figure), and the carbonyl group on Sn–2 chain (denoted by CO-Sn2). We count the number of ions bound only to one of the sites (first three groups of columns), number of ions simultaneously bound to a pair of sites (next three groups of columns), and number of ions simultaneously bound to all three sites. We note that more than 70% of bound ions are simultaneously bound to more than one sites. Further, those bound to only single site almost always exclusive prefer PO4 over other sites. Distinctive feature of Li+ binding is that Li+ overwhelmingly prefers simultaneous binding to PO4 and CO-Sn2. Perhaps due to very small ionic radius of Li+ the ion can penetrate deeper in headgroup region compared to other univalent ions and forms a bridge between one of phosphate oxygens and the carbonyl oxygen of Sn–2. The image over laid over figure 7 demonstrates a typical configuration of Li+ in the bilayer. Here, the left most lipid has bound to the Li+ with both the lower PO4 oxygen and an Sn–2 side carbonyl oxygen. Two other lipids have each contributing one additional binding site with the closer lipid bound with its PO4 oxygen and the farther lipid bound with its Sn–2 side carbonyl oxygen. Each ion prefers an ideal number of neighbors to bind with, or coordination number. Figure 8 shows the coordination number of each ion with each binding site and total number of bound sites. Smaller ions, have smaller coordination numbers. Ions inside the head group of the bilayer have a harder time finding locations that provide enough binding sites to fill the first coordination shell. This results in a lower total coordination number for ions inside the bilayer. This is especially the case for larger ions that have large shells to fill. This leads to larger ions retaining the water solvating in the head group where as smaller ions such as Li+ dehydrate almost completely. It is instructive to look at the locations of these binding sites within the bilayer structure. Figure 9 shows the number densities of phosphorus, nitrogen, carbonyl oxygens, the cations, and the anions. Clearly the anions associate with the choline head. The phosphate binding site of cations, is ∼5Å outward from the carbonyl sites. This reduces the accessibility of the carbonyl sites compared to the phosphate sites. Thus as seen in Figure 7 almost none of the cations bind to the carbonyl sites alone. Li+ with its small size fits well between the phosphate and the Sn–2 carbonyl


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sites and forms a bridge. It is such a small ion that even the Sn–1 carbonyl site stays outside its coordination first shell while it is bound to the phosphate.

Structural Changes Due to Salts Binding of ions to the lipid causes changes in the structure of the bilayer. Among these properties, area per lipid (Al ) is the most illustrative since many other properties mirror its changes. Additionally, a decrease in Al may correspond to an increase in surface tension if the area was held fixed. Figure 6b shows the Al for all five simulations. Rb+ , the largest cation, has no significant effect on Al compared to the simulations without salt. K+ and Na+ , both smaller ions, decrease Al . These results are in agreement with previous simulations that show smaller ions decrease area per lipid. 10,15,16,46 Most remarkably, we see this trend deviates for Li+ which has no significant difference in Al from POPC (No Salt). It has also been shown that a decrease in area should correspond to an increase in the ordering of the lipid hydrocarbon tails. 15 Figure 2a is the order parameter of the Sn–1 saturated hydrocarbon tail, while figure 2b is the order parameter of the Sn–2 mono-unsaturated hydrocarbon tail. For ions sizes of Na+ and larger, decrease in the size of the ion, increases the order of the hydrocarbon chain. This is not true for Li+ , where the average increase in order is between Rb+ and K+ . Furthermore Li+ sees a decrease in order for the first few hydrocarbons in each chain below that of Rb+ . This is especially clear in the Sn–1 chain, which does not frequently bind to Li+ . Another meaningful change in structure in the presence of salt, is the orientation of head groups. This change is important since head groups are believed to act as charge sensors for the cell. 47,48 Orientation is measured using the angle between the vector connecting the phosphate ~ and the bilayer plane. The average of this angle, for each simulation, is and nitrogen atoms (PN), plotted in Figure 6c. In all salt simulations the angle increases indicating a head groups becoming more parallel to the bilayer normal. Additionally, the smallest ions have the greatest effect on the angle. Li+ , with a similar number of bound ions as Na+ , still has a much larger angle. As Al decreases, the increase in hydrocarbon chain ordering causes 2Dc to increases. This com16 ACS Paragon Plus Environment

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~ results in thickening of the electron density (see figure S6). bined with the straightening of the PN These changes in electron density results in changes to the form factor shown in figure S7.

Changes in Water Order Ions are chaotropic agents and therefore disrupt water’s ability to solvate the lipid head groups. This disruption reduces the order of water in the head region of the bilayer. A plot of first rank and second rank water order is shown in figure 4. We clearly see from this figure that the order of water decreases with the addition of salt. Furthermore, smaller ions, such as Li+ , have the biggest reduction in water order and are therefore the most chaotropic. Thus Li+ expected to disrupt the structure of lipid bilayer more than any other monovalent cation in its class. This possibly destabilizes the bilayer. Figure 4 also shows an increase in the order of water outside of the bilayer. This increase in ordering introduces a new region in the water order parameter profile. The First rank order now has a second region which is positive, corresponding to the O → H vector pointing away from the bilayer. Similarly second rank order has a new third region which is negative and corresponds to the vector once again becoming parallel to the bilayer. It is interesting to note that, even if the ions reduce the number of water associated with the head group, we observe an overall increase in the number of perturbed waters around bilayer due to very long trailing density of Cl− ion. The transition from ordered water to bulk water is gradual in salt systems. it is therefore not reasonable to denote the hydration boundary at the point where order is absolutely zero. We instead, truncated the data where the curve demonstrated monotonic decay. Similar to the procedure used for hydrostatic boundary, again fitted equation 3 to the data by changing a, b, and c from their new start points of -0.1, 0.2, and 25 respectively. The length scale of the exponent was then used as the ”hydration boundary”.


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Changes to Pressure Profile Changes in the structure of the lipid, and the molecules that bind to it, affect the pressure profile of the system. Plots of Pn − Pl , for the four salt simulations, are shown in figure 3. The first distinction we can make about the profiles in the lipid region, is the decrease in Pn − Pl between 10 and 15 Å. This reduction corresponds to a increase in lateral pressure 2-3 times that of POPC (No Salt), in the region around the backbone carbonyl groups. Li+ in particular shows the greatest increase the lateral pressure (∼ 50 bars more than Na+ ). Work by Cantor et. al. has shown that lateral pressure could be a mechanism for regulating protein functions. 49 If so, the increase in lateral pressure in the presence of Li+ could be the mechanism for the its effect on physiology.

Changes in Electrostatic Potential The cations and anions in solution bind to different binding sites and these sites are separated by ∼ 5 Å. Effect of such charge separation of bound ions is twofold 1. The cations and anions together form dipole potential in addition to the dipole potential formed by lipids and water, and 2. Electrostatic surface potential due to ions is formed by superposition of cationic and anionic double layer potential which is displaced by ∼ 5 Å. Figure 5 shows the electrostatic potential for the 4 salt simulations. In these simulations, the total electrostatic potential, between the bulk water and the center of the bilayer (table 3 row 1), increased ∼ 0.2 V from the base case of POPC (No Salt). There is little difference in the electric potential among salt simulations despite the difference in the number of ions bound to the bilayer. This is primarily due to the dipole like structure formed by cations and anions at different surfaces. Cordomi et. al. has previously demonstrated, that the large increases in electrostatic potential due to ions are balanced by opposite changes to water and lipid head group. 16


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Surface Charge The surface charge density is important as it relates to ζ -potential which is measured in electrophoretic mobility experiments. 13,45 Surface charge density was calculated from the equation

σ (z) =

Z z 0

ρ (z′ )dz′ ,

(7)

where ρ (z′ ) is the charge density, and z is the distance from the center of the bilayer. The results of these calculations can be seen in figure 10. These figures contain vertical dashed lines indicating the location of the previously discussed, “hydration boundary” and “hydrostatic boundary”. We note that irrespective to which boundary we choose, the surface charge density is effectively zero. This result suggests that surface potential of a lipid membrane is minimally altered by monovalent salts including even Li+ . Primarily the ionic effects are through modification of water and lipid structure and the lateral pressure profile.

Summary and Conclusions In this work we simulated POPC bilayers in salt solutions with various cations. Additionally, we simulated a POPC bilayer without salt as a control. This POPC (No Salt) bilayer matches very well with experimental data. This fit is due to long simulation times, larger bilayer size, and accurate force field. In the salt simulations, we found that accurate results require additional water so that a realistic bulk phase is produced. Use of new ionic force fields from Joung et. al. has been important in enabling accurate (and novel) results in our simulations. 19 As in earlier simulations, smaller ions are bound to the bilayer in greater numbers than larger ions. But, this effect saturated with ions size Na+ and smaller. Another difference was larger ions, such as K+ , bound in greater numbers than with other force fields. As monovalent ions bound to the bilayer, the structure of the bilayer changed. Hydrocarbon


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chain order increased. The thickness of the bilayer increased. And, in some cases, the area per lipid Al decreased. In the lipid head region, ions straitened the P → N dipole. Bound cations also dehydrated the lipid head, reducing both the number of water molecules and the order of those remaining. Outside the head group, ions increased the order of water molecules, even creating new regions of oppositely orientated water outside the peak in Cl− density. Cations bind to a different, deeper region than anions. This difference in charge distribution created an increase in electrostatic potential between the bilayer and bulk water. This increase is balanced by changes in water order, and an increase in the P → N dipole. thus resulted in very little change to the electrostatic potential between cations. We therefore conclude that physiological changes due to Li+ are unlikely to be caused by changes in surface potential of the membrane. Structural changes to the bilayer previously mentioned, were proportional to the size of the ion with the frequent exception of Li+ . Li+ increased the average area per lipid almost to that of the salt free bilayer size. hydrocarbon chain ordering of Li+ was between that of Rb+ and K+ , or even below them for the upper most carbons in the chains. These complicated structural effects to the membrane, could be a driver to the changes to endothelium-dependent blood vessel relaxation, since Endothelial cells are composed of 36.3% Phosphatidyl choline. 50 Li+ bound deeper into the bilayer than any other ion. It favored binding to a Sn–2 carbonyl over Sn–1 carbonyl, sometimes not even needing phosphate to fill its coordination shell. Although we did not study the effects of these bindings on membrane proteins directly, It could be that these deeper binding locations, or even structural changes, would modulate protein functions. These functions could even be related to enzymes inhibition. This will be the topic of future work.

Supporting Information Available Electron densities and cosine transform of those densities for salt simulations; Radial Distribution Functions for each ion and potential binding site. This material is available free of charge via the Internet at http://pubs.acs.org/.


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Author Information Corresponding Author Email: [email protected], [email protected]

Notes The authors declare no competing financial interest.

Acknowledgement Author SAP thanks NIH for the partial support under the grant 1R01GM086707-01A1. The Authors would like to thank Paul Tatasciore and Andrew P. Stevens for helping monitor simulations and prepare preliminary data.


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Table Captions Table 1: The name of each simulation used throughout this paper, composition of the system, simulated duration, Effective Bulk Concentration. The Effective Bulk Concentration was determined from the concentration of cations in a 10 to 20 Å region of neutral bulk water far from the bilayer. Table 2: Physical properties of simulated bilayer averaged over the last 100 ns. Vc and VHG are the volumes per lipid of the carbon chains, and head group and back bone respectively. Vl is the volume per lipid of the entire lipid. 2Dc and Db are the thicknesses of the carbon chains and entire lipid respectively. Al is the area per lipid calculated from Vc and 2Dc . DHH is the peak to peak distance in the electron density. These properties compare well with reported experimental values. 31,36 P-N angle is the average angle between the vector from the lipids phosphate to nitrogen atom and the bilayer plane. The uncertainty of the angle is taken as the standard deviation of means. Number of lipids is the average number of lipids an ion is bound to if bound to at least one lipid. Table 3: Electrostatic Properties


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Figure Captions Figure 1: Electron density as a function of the z-coordinate for POPC (No Salt) (a). Cosine transform of the symmetrized electron density from POPC (No Salt) compared to experimental x-ray diffraction data taken from Kuvcerka et. al. 31 (b). Figure 2: Order parameters for Sn–1 (a) and Sn–2 (b) hydrocarbon tails of each system. Figure 3: Plot of PN - PL as a function of distance from the center of the bilayer for each salt system: POPC (No Salt) (a), POPC-LiCl (b), POPC-NaCl (c), POPC-KCl (d), POPC-RbCl (e). Each plot has an exponential curve fit to the section of the plot that exhibits monotonic decay. The red dashed line in each plot denotes the length scale of the exponential that was fit and is taken as the hydrostatic boundary. Figure 4: First Rank (a) and second rank (b) water orientational order parameter as a function of z-coordinate for all 5 systems. Figure 5: Electrostatic potential of as a function of z-coordinate for each salt system: POPC (No Salt) (a), POPC-LiCl (b), POPC-NaCl (c), POPC-KCl (d), POPC-RbCl (e). Figure 6: Average number of cations bound to the bilayer (a). Average area per lipid, Al , (b) and angle between P → N vector and bilayer plane in degrees (c), for each system. Figure 7: Average number of cations bound to each possible combination of lipid binding locations. Figure 8: Average number of coordinated atoms by type (water in purple, A phosphate oxygen in green, Sn2 side carbonyl in light blue, Sn1 side carbonyl in orange, and the total of the 4 groups in dark blue) for Li+ (a), Na+ (b), K+ (c), Rb+ (d) vs the distance from the center of the bilayer. Figure 9: Number density of phosphorus (P), nitrogen (N), Sn–2 carbonyl (O16), Sn–1 carbonyl (O37), cations, and anions as a function of z-coordinate for four salt systems; POPC-LiCl 23 ACS Paragon Plus Environment

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(a), POPC-NaCl (b), POPC-KCl (c), POPC-RbCl (d). A portion of the density is zoomed in the window of the upper right of each window. Figure 10: Surface charge density, σ , as a function of z-coordinate for each salt system: POPC (No Salt) (a), POPC-LiCl (b), POPC-NaCl (c), POPC-KCl (d), POPC-RbCl (e). The green dashed line denotes the ”hydration boundary” while the blue dotted line denotes the ”hydrostatic boundary”.


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Table 1 System Name

Composition

Run Length

POPC (No Salt) POPC-LiCl POPC-NaCl POPC-KCl POPC-RbCl

200 POPC, 10,000 SPC/E 200 POPC, 29,784 SPC/E, 108 Li+ , 108 Cl+ 200 POPC, 29,784 SPC/E, 108 Na+ , 108 Cl+ 200 POPC, 29,784 SPC/E, 108 K+ , 108 Cl+ 200 POPC, 29,784 SPC/E, 108 Rb+ , 108 Cl+

0.5 µ s 0.5 µ s 0.5 µ s 0.5 µ s 0.5 µ s


Effective Bulk Concentration – 70 mM 70 mM 90 mM 120 mM

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Table 2 Properties Vc (Å3 ) VHG (Å3 ) Vl (Å3 ) 2Dc (Å) Db (Å) Al (Å2 ) DHH (Å) P-N angle (◦ ) No. of lipids

POPC (No Salt) 902.9 ± 0.7 313.5 ± 0.5 1216.4 ± 0.6 26.89 ± 0.52 35.80 ± 0.72 67.18 ± 1.33 37.01 ± 0.38 14.45 ± 1.45 –

POPC-LiCl 925.8 ± 1.1 312.0 ± 0.7 1237.8 ± 1.1 27.61 ± 0.30 39.75 ± 0.40 67.07 ± 0.78 38.07 ± 0.70 36.33 ± 1.76 3.2 ± 0.6

POPC-NaCl 924.8 ± 1.3 310.7 ± 0.6 1235.6 ± 0.7 28.55 ± 0.30 40.96 ± 0.44 64.80 ± 0.73 40.32 ± 0.74 31.36 ± 1.53 3.5 ± 0.7


POPC-KCl 923.9 ± 1.2 316.4 ± 0.6 1240.3 ± 1.1 28.05 ± 0.23 40.16 ± 0.31 65.87 ± 0.58 40.19 ± 0.78 29.46 ± 1.45 3.7 ± 0.8

POPC-RbCl 926.5 ± 0.8 315.3 ± 0.6 1241.8 ± 0.9 27.40 ± 0.27 39.07 ± 0.38 67.62 ± 0.69 38.94 ± 0.74 29.63 ± 1.50 3.8 ± 0.8

Page 33 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Table 3 Properties Dipole potential (V) Surface potential (V) Hydration Boundary (Å) Hydrostatic Boundary (Å)

POPC (No Salt) 0.49 ± 0.03 0.01 ± 0.01 29.64 28.14

POPC-LiCl 0.72 ± 0.07 0.01 ± 0.03 36.63 37.4

POPC-NaCl 0.72 ± 0.10 0.02 ± 0.04 37.59 38.5


POPC-KCl 0.69 ± 0.09 0.02 ± 0.05 36.05 39.5

POPC-RbCl 0.65 ± 0.09 0.01 ± 0.04 35.05 37.9

Langmuir

Figure 1

(a)

400 Density (e Å−3)

325

250

−30 −15

20

(b)

0 z (Å)

15

30

Experimental Data POPC (No Salt)

15 | F(q) |

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 54

10 5 0 0.1

0.3 q (Å−1) 34

ACS Paragon Plus Environment

0.5

Page 35 of 54

Figure 2

POPC (No Salt) POPC−LiCl POPC−NaCl

POPC−KCl POPC−RbCl

(a) Sn−1

0.25

−SCD

0.2

0.15

0.1

(b) Sn−2

0.25 0.2 −SCD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

0.15 0.1 0.05

2

4

6

8 10 C number

12


14

16

18

Langmuir

Figure 3

600

(a) POPC (No Salt)

28.14 Å

300 0 −300 600

37.37 Å

(b) POPC−LiCl

300 0 −300 PN − PL (bar)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 54

600

38.50 Å

(c) POPC−NaCl

300 0 −300 600

39.49 Å

(d) POPC−KCl

300 0 −300 600

37.92 Å

(e) POPC−RbCl

300 0 −300 0

10

20

30

40 z (Å)

50


60

70

80

Page 37 of 54

Figure 4

POPC−LiCl POPC−NaCl POPC−KCl

0.1

POPC−RbCl POPC(No Salt)

(a) P1

P1(cos

H)

0

−0.1

−0.2

−0.3 0.04 (b) P2

H)

0

P2(cos

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

−0.04

−0.08

−0.12

10

20

30 z (Å)

40


50

Langmuir

Figure 5

1

(a) POPC (No Salt)

0.5 0 1

(b) POPC−LiCl

0.5 Electrostatic Potential (V)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 54

0 1

(c) POPC−NaCl

0.5 0 1

(d) POPC−KCl

0.5 0 1

(e) POPC−RbCl

0.5 0 5

10

15

20

25

z (Å) 38 ACS Paragon Plus Environment

30

35

Page 39 of 54

Figure 6

Number Bound

80 75

(a)

70 65 60 55

+

+

Rb

K

Na

+

+

Li

P−N Angle (degrees)

Area per Lipid (Å2)

Cation 69

(b)

67 65 63 35

(c)

25

15 PO

PO

PO

PO

PO

System 39 ACS Paragon Plus Environment

iCl

−L

PC l

aC

−N

PC

)

alt oS

Cl −K

PC

(N

l bC −R

PC

PC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Langmuir

Figure 7

Average Number of Instances per Frame

70 Li Na K Rb

60 50 40 30 20 10 0

d an

d an

n1

n1 −S

CO

n2 −S

CO 2

Sn

O−

dC

an


All

PO 4

PO 4

−S

CO n1

−S

CO n2

−S

CO

PO 4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 54

Page 41 of 54

Figure 8

OW PO4

5

CO−Sn2 CO−Sn1

Total

(a) LiCl

3 1 7

(b) NaCl

5 Number of Neighbors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

3 1 7

(c) KCl

5 3 1 9

(d) RbCl

7 5 3 1 25

50 z (Å) 41 ACS Paragon Plus Environment

75

Langmuir

Figure 9 P N

O16 O37

Cation Anion

(a) LiCl

3

1

2 1 10

20

30

10

20

30

10

20

30

10

20

30

(b) NaCl

3

1

2 Density (Å−3)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 54

1 (c) KCl

3

1

2 1 (d) RbCl

3

1

2 1

0

10

20

30

40

50 z (Å)

60

70


80

90

100

Page 43 of 54

Figure 10

(a) POPC (No Salt)

1.5 0.5 −0.5 (b) POPC−LiCl

Surface Charge Density (X 10−2 C/m2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1.5 0.5 −0.5 (c) POPC−NaCl

1.5 0.5 −0.5 (d) POPC−KCl

1.5 0.5 −0.5 (e) POPC−RbCl

1.5 0.5 −0.5 10

20

30 z (Å)


40

Langmuir


PO 4 CO

−S

n2

CO

−S

n1

CO

−S

n1

an

dC

PO 4

O−

Sn

an

dC

O−

PO 4

Sn

2

an

dC

Li Na K Rb

O−

Sn

All ACS Paragon Plus Environment

70

60

50

40

30

20

10

0

1

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 44 of 54

Page 45 of 54

(a)

400 Density (e Å−3)

325

250

−30 −15

20

(b)

0 z (Å)

15

30

Experimental Data POPC (No Salt)

15 | F(q) |

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

10 5 0 0.1

0.3 q (Å−1)


0.5

Langmuir

POPC (No Salt) POPC−LiCl POPC−NaCl

POPC−KCl POPC−RbCl

(a) Sn−1

0.25

−SCD

0.2

0.15

0.1

(b) Sn−2

0.25 0.2 −SCD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 54

0.15 0.1 0.05

2

4

6

8

10

12

C number ACS Paragon Plus Environment

14

16

18

Page 47 of 54

600

(a) POPC (No Salt)

28.14 Å

300 0 −300 600

37.37 Å

(b) POPC−LiCl

300 0 −300 PN − PL (bar)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

600

38.50 Å

(c) POPC−NaCl

300 0 −300 600

39.49 Å

(d) POPC−KCl

300 0 −300 600

37.92 Å

(e) POPC−RbCl

300 0 −300 0

10

20

30

40

50

z (Å) ACS Paragon Plus Environment

60

70

80

Langmuir

POPC−LiCl POPC−NaCl POPC−KCl

0.1

POPC−RbCl POPC(No Salt)

(a) P1

P1(cos ΒH)

0

−0.1

−0.2

−0.3 0.04 (b) P2

0 P2(cos ΒH)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 54

−0.04

−0.08

−0.12

10

20

30

40

z (Å) ACS Paragon Plus Environment

50

Page 49 of 54

1

(a) POPC (No Salt)

0.5 0 1

(b) POPC−LiCl

0.5 Electrostatic Potential (V)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

0 1

(c) POPC−NaCl

0.5 0 1

(d) POPC−KCl

0.5 0 1

(e) POPC−RbCl

0.5 0 5

10

15

20 z (Å)

25


30

35

Langmuir

Page 50 of 54

(a)

75 70 65 60 55

Rb+

K+

Na+

Li+

P−N Angle (degrees)

Area per Lipid (Å2)

Cation 69

(b)

67 65 63 35

(c)

25

15 PO

PO

PO

PO

PO

l

aC

System ACS Paragon Plus Environment

iCl

−L PC

−N PC

Cl

−K PC l

bC

)

alt

oS

(N

−R PC

PC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Number Bound

80

Page 51 of 54


PO 4 CO

−S

n2

CO

−S

n1

CO

−S

n1

an

dC

PO 4

O−

Sn

an

dC

O−

PO 4

Sn

2

an

dC

Li Na K Rb

O−

Sn

All ACS Paragon Plus Environment

70

60

50

40

30

20

10

0

1

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Langmuir

Langmuir

OW PO4

5

CO−Sn2 CO−Sn1

Total

(a) LiCl

3 1 7

(b) NaCl

5 Number of Neighbors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 52 of 54

3 1 7

(c) KCl

5 3 1 9

(d) RbCl

7 5 3 1 25

50 z (Å) ACS Paragon Plus Environment

75

Page 53 of 54

P N

O16 O37

Cation Anion

(a) LiCl

3

1

2 1 10

20

30

10

20

30

10

20

30

10

20

30

(b) NaCl

3

1

2 Density (Å−3)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1 (c) KCl

3

1

2 1 (d) RbCl

3

1

2 1

0

10

20

30

40

50 z (Å)

60


70

80

90

100

Langmuir

(a) POPC (No Salt)

1.5 0.5 −0.5 (b) POPC−LiCl

Surface Charge Density (X 10−2 C/m2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 54 of 54

1.5 0.5 −0.5 (c) POPC−NaCl

1.5 0.5 −0.5 (d) POPC−KCl

1.5 0.5 −0.5 (e) POPC−RbCl

1.5 0.5 −0.5 10

20

30 z (Å)


40

Effects of Lithium and Other Monovalent Ions on Palmitoyl Oleoyl

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