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Phase Behavior of GM1 containing DMPC-Cholesterol Monolayer: Experimental and Theoretical Study Zarrin Shahzadi, Subhasis Das, Tanushree Bala, and Chaitali Mukhopadhyay Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02621 • Publication Date (Web): 03 Sep 2018 Downloaded from http://pubs.acs.org on September 3, 2018

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Phase Behavior of GM1 containing DMPC-Cholesterol Monolayer: Experimental and Theoretical Study Zarrin Shahzadi, Subhasis Das, Tanushree Bala, and Chaitali Mukhopadhyay* Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata – 700009, India * Address for Correspondence: Chaitali Mukhopadhyay Department of Chemistry, University of Calcutta 92, A. P. C. Road, Kolkata – 700 009, India. Email: [email protected], [email protected]

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Abstract Organization and distribution of lipids in cellular membranes play an important role in a diverse range of biological processes, such as membrane trafficking and signaling. Here we present the combined experimental and simulated results to elucidate the phase behavioral features of Ganglioside Monosialo 1 (GM1) containing mixed monolayer of the lipids 1, 2-Dimyristoyl-snglycero-3-phosphocholine (DMPC) and Cholesterol (CHOL). Two monolayers having compositions DMPC/CHOL and GM1/DMPC/CHOL are investigated at air-water and air-solid interfaces using Langmuir Blodgett experiments and Scanning Electron Microscope (SEM) respectively to ascertain the phase behavior change of the monolayers. Surface pressure isotherms and SEM imaging of domain formation indicate that addition of GM1 to the monolayer at low surface pressure causes a fluidization of the system but once the system attains the surface pressure corresponding to its liquid-condensed phase, the monolayer becomes more ordered than the system devoid of GM1 and interacts among each other more co-operatively. Besides, condensing effect of Cholesterol on the DMPC monolayer was also verified by our experiments. Apart from these, the effects induced by GM1 on the phase behavior of the binary mixture of DMPC/CHOL were studied with and without applying liquid expanded (LE)-liquid condensed (LC) equilibrium surface pressure using molecular dynamics simulation. Our Molecular dynamics (MD) simulation results give an atomistic level explanation of our experimental findings and furnish a similar conclusion. Introduction Lipids being the major components of the biological membrane have always been under great scrutiny.1-4 Lateral organization of lipids in cellular membranes and their interaction among each other play an important role in a diverse range of processes, such as membrane trafficking and


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signaling.5 The composition of lipid in the cellular membrane has a great impact on the features and function of the membrane.6 In order to understand the biological processes such as subcellular transport and invasion by infectious agent, a sound understanding of the physical and chemical properties of the membrane lipids is essential. Naturally occurring and mimic membranes have been studied extensively in the past and many physicochemical characteristics have been extrapolated by various physical methods, including differential scanning calorimetry7,

diffraction8,9,

X-ray

neutron

diffraction10,

optical

microscopy11,

IR

spectroscopy12and atomic force microscopy.13 Our current motive was to understand the principles behind phase behavior and dynamics of GM1 containing membrane by determining how these individual lipid molecules interact with each other before and after the liquidexpanded(LE)/liquid-condensed(LC) equilibrium. In order to mimic biological membrane for our study, we chose 1, 2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), Cholesterol (CHOL) and Ganglioside Monosialo 1 (GM1) as membrane components.

O

O

CH3 P H3C

N+

O

O O-

O H

DMPC O

CH3

O

OH

H

CHOL

H H


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OH CH2OH O OH O

OH O

OH CH2OH

CH3

N H

O

O O

H3C

O CH2OH

HO N H

O

O

OH

O

H

O HO OH

-

OH

OOC

NH

GM1

O

HO O CH2OH

H

OH

OH

Figure 1: Chemical structures of the lipids used in this work. (Prepared using ChemDraw Ultra 8.0)

DMPC was chosen for our experiments because over the years, DMPC membranes have been rigorously studied using a variety of physical techniques14,15 and as a result, they’ve been used to study problems regarding different biological systems like drug-membrane interactions16, cholesterol flip-flop17, membrane properties18 etc. DMPC contains phopsphocholine group and saturated lipid tail; phosphocholine is one of the major components of mammalian membranes and because of having fully saturated acyl chains DMPC monlayers and bilayers are robust and do not require special preparatory environments.19 Besides, from biophysical point of view it is 4 ACS Paragon Plus Environment

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convenient to monitor phase transitions of saturated model lipids and for this fact DMPC has been widely used for Langmuir Blodgett experiments.20,21 Cholesterol influences individual molecular behavior thus regulating the overall mechanical behavior and eventually modulates biological membranes and their functions.22,23Glycolipids are basically lipid molecules which contains sugar groups, found in most animal cell plasma membranes, they regulate various physiological events at the cell surface.24,25 Gangliosides contain one or more negatively charged sialic acid groups and they are the most complex form of glycolipids.26 Gangliosides play a major role in a number of cellular functions, including cell recognition and adhesion, signal transduction and cell growth regulation.27 They constitute 5–10% of the total lipid mass in nerve cells. The external surfaces of certain cells contain10–20mol% gangliosides because they exist mostly on the outer leaflet of the cell membrane.28 One of the most commonly studied gangliosides is GM1, a member of the glycosphingolipid family that contains four neutral sugar groups and one sialic acid residue.29 The organization of gangliosides in model membranes has been broadly investigated with various methods, such as AFM,30 secondary ion mass spectrometry, 31 fluorescence, 32 NMR.33 Lee et al. in their work explain the effect of ganglioside on the phase behavior of dipalmitoylphosphatidlycholine (DPPC) lipid. With varying concentration of GM1 they found that at a lower concentration of GM1, it has a condensing effect on DPPC monolayer, whereas, at higher concentration, it has a fluidizing effect on the monolayer.28 Yuan and Johnston reported about the GM1 rich domain formation in DPPC/CHOL monolayer.34 It is identified that cholesterol promotes the formation of GM1 clusters.35 To begin with; we used Langmuir Blodgett isotherms to monitor the phase transitions in monolayer because pressure/area isotherm is an effective way to monitor the interfacial behavior of lipid molecules. The surface area isotherm provides information about phases of lipid molecules at the


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surface along with the corresponding changes in surface pressure.36 The surface pressure thus obtained corresponding to the LE/LC equilibrium phase of monolayers was used for further study. Besides, there are only a handful of computer simulation investigations to support a large number of existing experimental studies on Langmuir Blodgett (LB) isotherms.37-39 We used Molecular dynamics simulation to study the detailed structure of the monolayer under investigation. The chosen model of the monolayer of interest was observed at atomistic level to complement the experimental findings. Our current report provides a more realistic depiction of phase behavioral change of model membrane containing DMPC and Cholesterol in presence and absence of GM1 by both, experimental and theoretical studies. Experimental Section Materials DMPC and cholesterol were purchased from Sigma Chemical, USA and used without further purification. DEAE–Sephadex was obtained from Pharmacia. Ammonium acetate, KCl, KI, and Na2S2O3 were obtained from Merck and IR-120 cation exchange resin was from Mallinckrodt Chemical Works, New York. All the solvents were of the spectroscopic grade. Each experiment was performed at least in triplicate at 37°C. Methods Ganglioside Monosialo 1(GM1) extraction Ganglioside (GM1) was isolated and purified from goat brain following our published protocol.26 Briefly, brain tissue was homogenized and gangliosides mixtures were obtained from it. After extensive dialysis of the eluted mixture of gangliosides, it was lyophilized. The neuraminidase digestion removed sialic acid from polysialo-gangliosides and in the end gave the GM1. The


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final solution was again lyophilized and GM1 was dissolved in water. The solution was passed through a cation exchange IR-120 resin column so that any cation present in the mixture gets removed. After extensive dialysis followed by lyophilization, white powdered GM1 was obtained. The purity of GM1 was checked by TLC and 1H NMR spectroscopy. (Figure S1 and S2 of supporting information) Langmuir Blodgett Isotherms DMPC and cholesterol (10 mg/ml) were dissolved in chloroform. GM1 (4 mg/ml) was dissolved in chloroform/methanol (v/v, 80:20). DMPC/CHOL mixture was prepared in 2:1 molar ratio and GM1/DMPC/CHOL mixture was prepared with 10 mol% GM1 in 2:1 DMPC/CHOL. The monolayer of DMPC, GM1, DMPC/CHOL and GM1/DMPC/CHOL mixtures were prepared on Langmuir-Blodgett trough (KSV NIMA medium trough) using deionized water as the subphase. The sample solutions were spread on the sub-phase surface; after solvent evaporation (30 mins), the surface pressure was measured with a precision of 0.1 mN/m using a Wilhelmy balance. The compression rate of isotherm was 25cm2/min. To protect the experimental setup from dust it was placed in a laminar flow hood in which temperature was kept constant at 37°C. Surface pressure was recorded simultaneously as a function of the molecular area. The accuracy of measurements was 0.02 Å2 area per molecule. In order to monitor the LE/LC equilibrium surface pressure of binary (DMPC-CHOL) and ternary (DMPC-CHOL-GM1) mixtures and get a clear picture of phase transition, another set of LB-experiments was performed with lower concentrations of lipids: DMPC and cholesterol (1 mg/ml), GM1 (0.4 mg/ml). DMPC/CHOL mixture was prepared in 2:1 molar ratio and GM1/DMPC/CHOL mixture was prepared with 10 mol% GM1 in 2:1 DMPC/CHOL as used in our preceding experiment. The compression rate of isotherm was also lowered to 1.0 cm2/min. (Figure S3 of supporting information)


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Scanning Electron Microscope (SEM) SEM measurements were performed with a Zeiss EVO 18, Special Edition microscope. Triplicate samples were prepared for each monolayer composition at 37°C, lipid monolayers from the Langmuir trough were transferred onto Si (111) wafers at definite surface pressure (2mN/m, 7mN/m) during the compression by vertically dipping the Si (111) wafers into the trough and slowly pulling it up (2 mm/min). The Si (111) wafers with samples deposited onto it, were then thoroughly dried in air before imaging40 Theoretical Section System Setup Monolayer Model. Two monolayers were modeled maintaining the same composition as used in our experiments i.e. (i) 2:1 DMPC/CHOL (40 DMPC and 20 Cholesterol molecules) (ii) In order to closely mimic the 10% GM1 in 2:1 DMPC/CHOL solution as used in our experiments, 5 GM1 molecules were added to system (i), but to avoid any steric clashes 5 DMPC molecules were removed. Therefore, the final ratio of lipid components in the monolayer was 1:7:4 GM1/DMPC/CHOL. In order to stabilize each of the monolayer systems, an additional monolayer was constructed and rotated in such a way that the lipid head groups were facing each other. 4071 TIP3P water models were then added between the gap of the two monolayers to correspond to the system as lipid tail−lipid head−water−lipid head−lipid tail.37-41This was followed by a short energy minimization to release the stress of the system. As the gel-to-fluid transition temperature of DMPC is 23.9°C

42

, hence in order to maintain bilayer fluidity, the system was equilibrated for

50 ns at the target temperature of 310 K. The equilibrated system was used as the starting configuration for each simulation. Both the systems were simulated with and without LE/LC 8 ACS Paragon Plus Environment

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equilibrium surface pressure. The surface pressure Π is calculated as Π=γ0-γ, where γ0 is the surface tension of pure water and γ is the surface tension of the monolayer coated air-water interface.43 In order to attain the LE/LC equilibrium surface pressure obtained from our experimental data i.e. ∼4.5mN/m, surface tension of γ=45 mN/m was applied to both the system. Using γ0=49.5mN/m for TIP3P water at 310K 44, our target surface pressure was achieved and in order to attain zero surface pressure corresponding to the uplift region of experimental isotherm, surface tension of magnitude γ0 i.e. 49.5mN/m was applied to both the system. One thing to keep in mind is that despite the surface pressure of the system being zero there was interfacial surface tension acting between the lipid components which provided stability to the system. A 2fs time step was used to integrate the equation of motion and each simulation was run for 150 ns. Five Na+ ions were added to both systems to maintain electro neutrality. The system was prepared using CHARMM and was equilibrated using NAMD_2.10 package.45 with the standard CHARMM36 force field.46 Simulation protocol. The isobaric−isothermal (NPT) ensemble with imposed 3D periodic boundary conditions was used for all the MD simulations. The Langevin dynamics were used to maintain the temperature for all the simulations, while by using Nose−Hoover−Langevin piston the pressure was kept constant at 1 atm.47 Long-range electrostatic interactions were calculated using smooth Particle Mesh Ewald method calculations.48 Short-range interactions were computed with cut off at 10 Å. All bond lengths involving hydrogen atoms were constrained using the RATTLE49 and SETTLE50 algorithm. The trajectory analysis was performed with CHARMM 51(Chemistry at Harvard Macromolecular Mechanics). Results and Discussion Experimental


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In order to obtain the pressure-area isotherms of the monolayers, we performed LangmuirBlodgett experiments and the results are as follow: Monolayers at air/water interface Pure lipids Figure 2 shows the surface pressure-area per molecule isotherm of the DMPC, GM1, 2:1 DMPCCHOL and 10% GM1 in 2:1 DMPC-CHOL at the air/water interface. The GM1 forms a stable monolayer at the air/water interface. The lateral distribution of the GM1 in the monolayer varies between (100-160Å2) in literature reports and depends on the ganglioside source, the content of unsaturated hydrocarbon chains and purity.34,52-54 As seen in inset of Figure 2, the lift-off area of the GM1 molecule in the Langmuir monolayer recorded at the air/water interface at temperature 37°C was close to ∼140.6Å2, which was in agreement with previously published result 140Å2.53 The slight discrepancy in the values reported here could be attributed to different experimental conditions and source of materials used.54-56 Isotherm did not show any plateau as such. Hence there was not any liquid-expanded (LE)/liquid-condensed (LC) phase equilibrium, representing that GM1 remains in LE phase till its collapse that was at 41.4mN/m also supported by the literature.53 Moreover, there was a kink at ∼69Å2 which might be due to the increased interaction between the hydrophobic tails of GM1.This observation was also supported by previous finding. 28


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Figure 2: Monolayer compression isotherms of 2:1 DMPC-CHOL and GM1 in 2:1 DMPCCHOL at 37°C. The inset curves are showing monolayer compression isotherm of pure DMPC and pure GM1 at 37°C. The isotherm for pure DMPC has uplift at ∼112.1 Å2and the isotherm collapsed at 36.4 mN/m. The lift-off area and the collapse pressure was in very good agreement with the literature results with reported values ∼115 Å2

57

and ∼37mN/m respectively.21,58 The isotherm of 2:1 DMPC-

CHOL mixture was shifted to a much lesser molecular area. Liftoff occurred at ∼73.4Å2 and it has a collapse pressure of 39.2mN/m, in agreement with published data for DMPC-CHOL20 and DPPC-CHOL.59 It signified a condensing effect induced by the cholesterol molecules to DMPC. The cholesterol molecule consists of higher hydrophobic structure and by the time it is added to the DMPC system, the molecules rearrange among their selves so that the contact between hydrophobic sterol ring and water gets minimized. This distribution causes energetically favored strong van der Waals cohesive interactions between sterol rings of cholesterol molecules and the


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alkyl chains of DMPC molecules.59 One more noticeable observation was that the collapse surface pressure of 2:1 DMPC/CHOL was 39.2 mN/m which was slightly higher than the collapse surface pressure of pure DMPC i.e. 36.4mN/m, which implied that the introduction of cholesterol molecules to DMPC monolayer increased the stability of the monolayer. Addition of GM1 to 2:1 DMPC-CHOL mixture could not change the nature of the isotherm of the lipid mixture as such but the uplift of the isotherm was shifted towards the right, supported by the literature.60 Addition of GM1 shifted the uplift to higher molecular area i.e. ∼78.5 Å2, while there was no change in the phase behavior. One notable observation in case of binary (DMPC-CHOL) and ternary mixture (DMPC-CHOL-GM1) was at lower surface pressure. At surface pressure∼4.5 mN/m we could see the two isotherms intersecting each other. At the beginning when the surface pressure was low (below 4.5 mN/m), addition of GM1 caused an increment in area per molecule of the system; hinting towards fluidizing effect of GM1 on the lipid monolayer but once the surface pressure was increased, the isotherm of ternary system (DMPC-CHOL-GM1) moved to the left of binary system (DMPC-CHOL) and exhibited lower area per molecule than the binary system. This suggested that after a certain surface pressure, GM1 might have a condensing effect on the lipid monolayer. Still it was too early to conclude anything and in order to further fortify our notion we performed other experiments. Moreover in order to monitor the LE/LC equilibrium plateau of the binary and ternary system, we performed LB-experiments with lower concentrations and lower compression rate (as mentioned in Methods section) and found the LE/LC equilibrium plateau exists at ∼4.5mN/m (Figure S3 in supporting information). SEM results


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SEM images were taken at two different surface pressures concurrently to image the effect of GM1 on the phase behavior of the monolayer. In order to do this, the binary and ternary lipid monolayers were transferred to the silicon wafer at two different surface pressures, low and high. Low surface pressure (~2mN/m) was corresponding to the LE phase of the monolayer and relatively high surface pressure (∼7mN/m) corresponding to the beginning of LC phase of the monolayer. The surface pressure was obtained from the corresponding isotherm of that respective system. The first image taken was of 2:1 DMPC-CHOL at lower surface pressure (Figure 3a). The image obtained, provided us the picture of the beginning of phase separation and majorly exhibited LE domains along with few LC domains.

Figure3: SEM image of (a) 2:1 DMPC-CHOL monolayer at lower surface pressure.(∼2mN/m) (b) 2:1 monolayer DMPC-CHOL at higher surface pressure.(∼7mN/m) (c) GM1 added to 2:1 DMPC-CHOL monolayer at lower surface pressure.(∼2mN/m) (d) GM1 added to 2:1DMPCCHOL monolayer at higher surface pressure.(∼7mN/m)


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On imaging the same mixture at the surface pressure corresponding to the beginning of the LC phase (Figure 3b), it showed condensed domains of diameter 9.7±0.01µm. A significant increase in the amount of the condensed domain with many of the smaller domains now having coalesced into a larger one was observed. Addition of GM1 to 2:1 DMPC-CHOL changed the morphology of monolayer significantly. At lower surface pressure, the image showed that GM1 preferentially remained in a more ordered phase and it distributed the whole lipid monolayer into small clusters, forming roughly circular smaller domains of average diameter 0.55±0.001 µm. On comparing this image (Figure 3c) with the image of 2:1 DMPC-CHOL without GM1 (Figure 3a), we could say that GM1 chopped the condensed domains and distributed the whole system evenly into smaller domains. On taking the image of the same system at relatively higher surface pressure, i.e. surface pressure corresponding to the beginning of the LC phase of the system, we found that system attained condensed domains of size of 20.7±0.01µm which was much larger than the condensed domains of monolayer without GM1. Up to this, it was reflected from the pressure-area isotherms and SEM images that at the beginning when the DMPC-CHOL monolayer remained mainly in the LE phase, addition ofGM1 to the system had a fluidizing effect on the monolayer but once the monolayer crossed the surface pressure corresponding to the LE/LC equilibrium, the system was more compact than the system without GM1 and GM1 caused the formation of condensed domains of larger dimensions. Simulation Results So far from our experimental observations, we found that GM1 exhibits contrary effects to the phase behavior of the monolayer in the absence and in presence of surface pressure. In order to


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analyze this at the atomistic level, we performed MD simulation of the concerned monolayer with and without LE/LC equilibrium surface pressure applied to it and from the simulation trajectories we carried out the following analyses. Preferential partitioning of lipid components SEM images indicated that from an initial homogeneous distribution, upon achieving the LE/LC equilibrium, GM1 molecules promoted the formation of large condensed domains. To specify the extent of mixing of monolayer components in presence and absence of LE/LC equilibrium surface pressure, we studied the preferential partitioning of GM1 along with other components in the monolayer. The preferential partitioning of membrane components is defined as the relative number of contacts of a lipid species with each of the other constituents, corrected for the total number of lipids in the system61: PA=

∑

Where, PA is the preferential partitioning of membrane component A to another component B, CA the number of contacts of component A and nA the number of molecules of component A. The data were averaged over the last 10 ns of simulation trajectory.


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Figure4: Preferential partitioning matrix of the monolayer. (a) Without GM1 without any surface pressure (b) without GM1 with LE/LC equilibrium surface pressure (c) with GM1 without any surface pressure (d) with GM1with LE/LC equilibrium surface pressure. If a lipid has more than one contact with another lipid this interaction is only counted once. Two lipids are defined as being in contact if the distance between the molecules is less than 6 A˚.

From figure 4(a), it was evident that when there was no surface pressure DMPC has a strong preference for DMPC molecules, whereas cholesterol exhibited the preference for both DMPC and other cholesterol molecules. In presence of LE/LC equilibrium surface pressure, it was found that slowly the preferential partitioning of individual components among themselves was decreased whereas the same was increased for cross components indicating the mixing of monolayer components. From SEM image of DMPC-CHOL monolayer we have seen that after attaining the LC surface pressure, the monolayer forms condensed domains. The increase in preferential partitioning for cross components observed here could be attributed to this condensed domain formation at high surface pressure. Considering the GM1 containing 16 ACS Paragon Plus Environment

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monolayers, there was an increase in preferential partitioning of cholesterol and GM1 for each other at LE/LC equilibrium surface pressure. This was in accordance with the previous reports where it was shown that GM1 has a high affinity for cholesterol molecules.62,63 and GM1 forms micro domains in presence of cholesterol.64 However, GM1 has high preference for other GM1 molecules to form self-aggregates was also reflected in this data.63,65,66Overall, we could also say that after applying the LE/LC equilibrium surface pressure, the mixing of lipid components was increased thus favoring the formation of condensed domains as imaged by SEM. Area per lipid Average area per lipid from the last 10 ns of the trajectory was calculated for lipid molecules and graphs are shown in figure 5. As shown in Table 1, in absence of any surface pressure, the average area per lipid attained by the GM1 containing monolayer at the end of the trajectory was higher than that for the system without GM1. The system with GM1 reached a value of area per lipid of 70.4±0.2 Å2, whereas the same in absence of GM1 was 66.1±0.2 Å2. This observation also supported our findings from experiments where it was found that GM1 has a fluidizing effect on monolayer before the monolayer attains surface pressure corresponding to its LE/LC equilibrium. While on applying the LE/LC equilibrium surface pressure to the monolayer, addition of GM1 increased the compactness of the monolayer. On applying LE/LC equilibrium surface pressure, the area per lipid of the monolayer without GM1 was found to be 65.0±0.2 Å2, whereas the same for the monolayer containing GM1 was 61.2±0.3 Å2. The SEM images showed that once the system attains LE/LC equilibrium, afterward the monolayers forms condensed domains, size of the condensed domain being larger in presence of GM1. Here also the GM1 containing


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monolayerhaving LE/LC equilibrium surface pressure applied to it, exhibited the least area per lipid thus reflecting condensed state of the monolayer Table 1: Area per lipid (Å2) of different systems.

System

Area per lipid (Å2)

Without Surface pressure, without

66.1±0.2

GM1 With

LE/LC

equilibrium

surface

65.0±0.2

pressure, without GM1 Without Surface pressure, with GM1 With

LE/LC

equilibrium

70.4±0.2

surface 61.2±0.3

pressure, with GM1


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Figure5: Area per lipid of the monolayer. (a) without GM1 without surface pressure (b) without GM1 with LE/LC equilibrium surface pressure (c) with GM1 without surface pressure (d) with GM1 with LE/LC equilibrium surface pressure. Lateral diffusion coefficient The lateral diffusion coefficient of different component of the binary (DMPC-CHOL) and ternary (DMPC-CHOL-GM1) systems in presence and absence of LE/LC equilibrium surface pressure are given in Table 2. Table 2: Diffusion coefficient of different lipid components of different systems.(10-7cm2s-1) Systems (a)Without Surface pressure, without GM1 (b)With LE/LC equilibrium surface pressure, without GM1 (c)Without Surface pressure, with GM1 (d)With LE/LC equilibrium surface pressure, with GM1

DMPC

CHOL

1.77±0.20

1.43±0.22

1.30±0.35

1.26±0.20

2.63±0.31

1.83±0.20

1.21±0.34

0.50±0.18

The lateral diffusion coefficient of components of system (a) was in well agreement with the litrature. Czub et al. in their work report lateral diffusion coefficient of DMPC and CHOL as 0.50±0.73 cm2/s and 0.6±1.79 cm2/s respectively at 300K.67 Our value was slightly higher than the reported value which could be because of the higher temperature applied in our simulations. On considering the binary system first, it was observed that on applying LE/LC equilibrium surface pressure, the lateral diffusion coefficient values for both the components were decreased to some extent signifying slight condensation of the system. However, for the system containing GM1 devoid of any surface pressure, the lateral diffusion coeffiecint values were increased


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convincingly. Persuing our idea of fluidization effect of GM1 previous, we could say that addtion of GM1 increased the lateral motion of other lipid components. For system (d) there was a remarkable decrease in lateral diffusion coefficeint of DMPC and CHOL. This might be due to the condesing effect induced by the GM1 molecules in presence of LE/LC equilibrium surface pressure. The newly formed condensed domains provide very less scope to the lipid molecules to move freely which eventually decreases the lateral diffusion coefficient. Radial distribution

Figure6: Radial distribution function g(r) profile of lipid components in DMPC-CHOL system (a,b,c) and DMPC-CHOL-GM1 system (d,e,f), without any surface pressure. Red (i) and blue (f) lines indicate the beginning and the end of the simulation, respectively. As concluded from our previous results, before the monolayer attained specific surface pressure corresponding to LE/LC equilibrium, the addition of GM1 to the monolayer caused a fluidization of the system; this was further fortified with our radial distribution function (RDF) calculations. The data were averaged over initial and final 10 ns of simulation trajectory. In case of the system


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where there was no GM1 or surface pressure applied, with the progress of simulation time, it was observed that the peak height for DMPC-CHOL at 6Å was increased significantly i.e. from 1.3 to 1.9 (Figure6c), indicating a mixing between DMPC and CHOL. The same DMPC-CHOL peak for the monolayer containing GM1 was however lowered. Moreover, GM1 also caused the decrement in the peak values for DMPC-DMPC and CHOL-CHOL. As depicted in the figure, Peak for CHOL-CHOL system within the first shell was unaltered but for second shell it was lowered from 2.9 to 2.1 (Figure6d). Also, for the DMPC-DMPC system, the peak within the first shell was lowered from 5.1 to 4.1 (Figure6e). In addition to this, the peak heights of RDF for individual lipids coupled with GM1 did not increase significantly. (Figure S4 of supporting information). These results indicated that addition of GM1 to the system, in absence of any surface

pressure,

caused

a

fluidization


of

the

monolayer.

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Figure7: Radial distribution function g(r) profile of lipid components in DMPC-CHOL system (a,b,c) and DMPC-CHOL-GM1 system (d,e,f), with LE/LC equilibrium surface pressure. Red (i) and blue (f) lines indicate the beginning and the end of the simulation, respectively.

On calculating the RDF of monolayer devoid of GM1 having LE/LC equilibrium surface pressure applied to it, it was observed that the peak height was increased for all three pairs i.e. CHOLCHOL, CHOL-DMPC, DMPC-DMPC, increase being maximum for DMPC-CHOL pair (Figure7a,b,c). Now, this was in agreement with our LB isotherm and SEM images. LB isotherm revealed that after a certain surface pressure which is mainly the LE/LC equilibrium surface pressure (∼4.5 mN/m in our experiments) the 2:1 DMPC-CHOL monolayer started attaining LC phase and this result was also reflected in SEM image of 2:1 DMPC-CHOL monolayer taken at ∼7mN/m showing the formation of the condensed domains. Now as we stated above that on applying LE/LC equilibrium surface pressure to the monolayer containing GM1, it was found that peak heights for all three pairs i.e. CHOL-CHOL, CHOL-DMPC and DMPC-DMPC increased remarkably. In addition to this the peaks values for individual components of monolayer paired with GM1, also increased significantly (Figure S5 of supporting information). One noticeable thing here, though there was an increase in the peak values in presence of LE/LC equilibrium surface pressure for all the lipid pairs for both the systems i.e. with and without GM1, this increase was much greater in magnitude in case of the system where GM1 was present in the system. These high increments in peak values were also reflected in our SEM image where it was observed that GM1 containing monolayer at relatively high surface pressure formed larger domain. GM1-GM1 RDF peaks also increased remarkably (from 2.2 to 7.2) near 7Å pointing


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towards a localized domain of GM1 within the monolayer (Figure S5 of supporting information) and reassured the formation of GM1 aggregates.63,65,66 Therefore, the results obtained from our experiments and simulations reveal that when GM1 is added to DMPC-CHOL membrane, it has a unique mixing behaviour upon the monolayer. At lower surface pressure i.e. surface pressure corresponding to the LE phase, GM1 has a fluidizing effect on the system, whereas as soon as the system reaches LE/LC equilibrium surface pressure, the GM1 imposes a condensing effect to the system and makes it more compact. This could be explained if we have a closer look at the geometry of GM1 (Figure1). GM1 itself has a fluid nature due to large headgroup consisting of four sugar groups coupled with the negatively charged sialic acid. This large head group of GM1 is the reason behind the loose packing of GM1 molecules and thus causes fluidity of the GM1.28 Therefore, the structural feature of GM1 could be the probable reason behind the monolayer fluidization at low surface pressure. On the other hand, when LE/LC equilibrium surface pressure is applied to the system, the GM1 molecules come closer and start to form aggregates with the help of cholesterol molecules. The hydrophobic interaction between the sterol ring of cholesterol molecules and tail region of GM1 promotes the co-operative packing.65,68 Moreover, due to compression, the lipid molecules come closer and intermolecular hydrogen bonding between DMPC molecules and head groups of GM1 forms. As represented in figure 7, one of the terminal glycans i.e. galactose forms the hydrogen bond with the phosphorus attached oxygen of the DMPC molecule. Earlier this kind of intermolecular hydrogen bonding has been reported between 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) and GM1 molecules and are found to be responsible for the condensed domain formation.35 Altogether these interactions i.e. hydrogen bonding and hydrophobic


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interaction (shown in figure 7) cause the tight packing of lipid molecules and formation of larger LC domains at high surface pressure.

Figure8: Snapshot of a condensed domain showing hydrogen bonding between DMPC-GM1 and hydrophobic interaction between CHOL-GM1, taken at the end of simulation trajectory of the system having LE/LC equilibrium surface pressure applied to it. GM1, DMPC and CHOL are represented in yellow, red and blue color respectively. Conclusions: Effect of GM1 on the phase behavior of monolayer DMPC-CHOL was studied. Our experimental results suggested that initially, addition of GM1 has a fluidizing effect on the monolayer and GM1 distributes the DMPC-CHOL monolayer into small circular domains but eventually when the system attains LE/LC equilibrium, afterward GM1 aids the monolayer to bind more co-operatively. The system becomes more compact with higher stability and forms larger condensed domains. These findings were also supported by our theoretical results. Supporting Information:


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TLC plate of purified ganglioside GM1 1

H NMR spectra of purified ganglioside GM1

Monolayer compression isotherms of 2:1 DMPC-CHOL and GM1 in 2:1 DMPC-CHOL at 37°C with lower concentrations and lower compression rate. Radial distribution function g(r) profile of GM1 paired with lipid components in DMPC-CHOLGM1 system, without any surface pressure and with LE/LC equilibrium surface pressure. Acknowledgements Z.S. and S.D. thank the University Grants Commission (UGC), New Delhi, for the award of a Junior Research Fellowship. This work is partially funded by the Center for Advanced Studies, Department of Chemistry, University of Calcutta. References: (1)Koldsø, H.; Shorthouse, D.; Hélie, J.;Sansom, M. S. P. Lipid Clustering Correlates with Membrane Curvature as Revealed by Molecular Simulations of Complex Lipid Bilayers. PLoS Comput. Biol .2014, 10,10. (2) van Meer, G.; Voelker D. R.; Feigenson G. W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 2008, 9, 112-124. (3) van Meer, G.; de Kroon, A. I. P. M. Lipid map of the mammalian cell. J. Cell Sci. 2011, 124, 5–8. (4) Shevchenko, A.; Simons, K. Lipidomics : coming to grips with lipid diversity. Nat. Rev. Mol. Cell Biol. 2010, 11, 593-598. (5) Simons, K.; Toomre, D. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 2000, 1, 31-39.


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