Critical Temperature of 1-Palmitoyl-2-oleoyl-sn-glycero-3

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Critical Temperature of 1-Palmitoyl-2-oleoyl-Sn-glycero-3phosphoethanolamine Monolayers and Its Possible Biological Relevance Jordi Hernandez Borrell, and Oscar Domenech J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b04021 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 26, 2017

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The Journal of Physical Chemistry

Critical Temperature of 1-Palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine Monolayers and its Possible Biological Relevance

Jordi H. Borrell*1,2 and Òscar Domènech1,2

1

Department of Pharmacy, Pharmaceutical Technology and Physical Chemistry, Faculty of

Pharmacy and Food Sciences and 2Institute of Nanoscience and Nanotechnology (IN2UB), University of Barcelona (UB), E-08028-Barcelona, Spain

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ABSTRACT: Since transmembrane proteins (TMPs) can be obtained with sufficient purity for x-ray diffraction studies more frequently than decades ago, their mechanisms of action may now be elucidated. One of the pending issues is the actual interplay between transmembrane proteins and membrane lipids. There is strong evidence of the involvement of specific lipids with some membrane proteins, such as the potassium crystallographically sited activation channel (KcsA) of Streptomyces lividans and the secondary transporter of lactose LacY of Escherichia coli, the activities of which are associated with the presence of anionic phospholipids such as the phosphatidylglycerol (PG) and phosphatidyethanolamine (PE), respectively. Other proteins such as the large conductance mechanosensitive channel (MscL) of E.coli seem to depend on the adaptation of specific phospholipids to the irregular surface of the integral membrane protein. In this work we investigated the lateral compressibility of two homoacid phosphatidylethanolamines (one with both acyl chains unsaturated (DOPE), the other with the acyl chains saturated (DPPE)) and the heteroacid phosphatidyletanolamine (POPE) and their mixtures with POPG. The liquid expanded (LE) to liquid condensed (LC) transition was observed in POPE at temperature below its critical temperature (Tc=36 ºC). Because Tc lies below the physiological temperature, the occurrence of this phase transition may 2 ACS Paragon Plus Environment

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have something to do with the functioning of LacY. This magnitude is discussed within the context of the experiments carried out at temperatures below the Tc of POPE at which the activity of Lac Y and other TMPs are frequently studied.



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INTRODUCTION The influence of the physicochemical properties of phospholipids on the structure and activity of transmembrane proteins (TMPs) is poorly understood at the molecular level. The interplay between phospholipids and TMPs and the existence of a boundary or annular population of phospholipids surrounding the TMPs remain a matter of controversy and research because of the short duration of residence of these phospholipids in that region. However, the interplay between phospholipids located in the boundary region and some TMPs has been demonstrated for β-hydroxybutyrate dehydrogenase, an enzyme integrated into the mitochondrial inner membrane; the ion pump Ca+2ATPase, the large-conductance mechanosensitive channel (MscL) channel and secondary transporters such as melibiose permease and lactose permease (LacY)

of

Escherichia

coli;

and

the

homotetrameric

potassium

crystallographically sited activation channel (KcsA) of Streptomyces lividans.1 Although an extensive amount of work on TMP activity has been carried out using homoacid saturated phosphatidylcholines (PCs), a number of studies have

clearly

demonstrated

phosphatidylethanolamine (PE)

that 2,3

heteroacid

phospholipids

such

as

or phospahtidylglycerol (PG) are required

for folding, correct topology and physiological activity.

4

Remarkably, it has

been claimed that the hydrocarbon chains may also play a role in TMP 4 ACS Paragon Plus Environment

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activity, at least for LacY.

5

With the exception of the lung surfactant, in

which 1, 2-dipalmytoil-sn-3-glycero-phosphocholine (DPPC) is the major component, most of the naturally occurring membranes in which TMPs are embedded include mixed acyl chains (one saturated at the sn-1 position, the other unsaturated at the sn-2 position) linked to the glycerol backbone. Such is the

case

for

the

phosphoethanolamine

binary (POPE)

mixture1-palmitoyl-2-oleoyl-sn-glycero-3and

1-palmitoyl-2-oleoyl-sn-glycero-3-

phosphogycerol (POPG) that mixed at molar ratios of 3:1 mimics the inner membrane of E.coli where LacY resides. In this binary system we have demonstrated, using Förster resonance energy transfer (FRET) tools, that POPE resides at ∼ 3.4 nm from the LacY core 6 and appears to be selectively preferred to POPG by this protein.7 In this regard, the packing properties of the phospholipids, particularly their ability to provide the lateral force for an adequate hydrophobic match and lateral surface profile, seem to play an important role.

8

The lateral pressure

profile, p(z), is a theoretical parameter assumed to be constituted by three components: two repulsive and one cohesive (Figure 1). The first repulsive component has its origin in the electrostatic interactions occurring in the headgroup region (πHG), with the second arising from the steric interactions between the acyl chains (πCH). The third component (cohesive) is the pressure 5 ACS Paragon Plus Environment


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localized in the polar–apolar interface between the headgroups and the acyl chains (πIC). Since p(z) can only be inferred from theoretical studies,

9

the

most suitable experimental approach to infer the role of the lateral pressure exerted by phospholipids on membranes involves the lipid monolayer model. Therefore, in this work we investigated PE monolayers at different temperatures with four main purposes: (i) to investigate the isotherms of several PEs and PE:POPG (3:1, mol/mol) monolayers at 37 ºC; (ii) to observe the evolution of the LE to LC phase transition with temperature in the pure POPE monolayer; (iii) to calculate the differential molar latent heat of the transition (∆tQ) and the critical transition temperature (Tc) of POPE; and iv) to visualize the POPE monolayer at different temperatures using atomic force microscopy (AFM).

EXPERIMENTAL SECTION Materials. 1,2-palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2oleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine

(POPE),

and

1-palmitoyl-2-oleoyl-sn-

glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (POPG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). A table with the molecular structures is included as Supporting Information (S1). All other common 6 ACS Paragon Plus Environment

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chemicals (ACS grade) were purchased from SIGMA (St. Louis, MO). Buffer used throughout the experiments was 20 mM HEPES (pH 7.40) and 150 mM NaCl prepared in Ultrapure water (Milli-Q reverse osmosis system, 18.2 mΩ·cm resistivity). Methods. Monolayers differing in lipid composition were prepared in a 312 DMC Langmuir−Blodgett trough manufactured by NIMA Technology Ltd. (Coventry, England). The trough (total area, 137 cm2) was placed on a vibration-isolated table (Newport, Irvine, CA) and enclosed in an environmental chamber. The resolution of the surface pressure measurement was ± 0.1 mN·m-1. Temperature was maintained via an external circulating water bath (± 0.2 °C). Before each experiment, the trough was washed with ethanol and rinsed thoroughly with purified water. The corresponding aliquot of chloroform-methanol (2:1, v/v) lipid solution was spread onto the subphase using a Hamilton microsyringe. A 15 min period was required for solvent to evaporate before each experiment. The compression barrier speed was 5 cm2·min-1. All experiments were repeated at least three times. Langmuir monolayers were formed at the desired temperature and at a surface pressure of 30 mN·m-1. The Langmuir-Blodgett (LBs) films were then obtained by deposition of the Langmuir monolayers onto mica squares (0.40 cm2) at a constant vertical velocity of 1 mm·min-1 to assure the optimum 7 ACS Paragon Plus Environment


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transfer ratio values and allowed to dry overnight while protected from light and dust. Samples were glued onto steel discs prior to observation and directly mounted on top of the AFM scanner. Images were recorded in contact mode in air using TESPA cantilevers (Bruker, AXS Corporation, Santa Barbara, CA), with a nominal spring constant of 42 nN·nm−1. The spring constants of each cantilever were determined using the thermal noise method. Atomic force microscopy images were obtained using a Multimode AFM controlled by Nanoscope V electronics equipped with an “E” scanner (10 µm), and images were processed using the NanoScope software (Bruker AXS Corporation, Santa Barbara, CA). Temperature and humidity were maintained at 24 ºC and 30% respectively.

RESULTS AND DISCUSSION The compression isotherms (π versus molecular area) of DPPE, POPE and DOPE recorded at 37 ºC are presented in Figure 2a. The π−molecular area curves show that the more expanded isotherm corresponds to DOPE and the more compacted one to DPPE. The features of the three monolayers are consistent with previously published data.

10,11,12

At 37 ºC POPE and DOPE

monolayers are in the LE phase while DPPE shows a LE–LC phase transition at ~ 4 mN·m-1. The LE to LC phase transition for POPE previously analyzed 8 ACS Paragon Plus Environment

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at 24 ºC 13 was not observed at 37 ºC. A visual delineation of the LE, LC and LE-LC regions can be found as Supporting Information (S2). However, the actual phase at a given surface pressure can be confirmed by calculation of the inverse of the isothermal compressibility or elastic modulus of area compressibility (Cs-1) 14 using -1

C s = ( − A)(

∂π ) T ,n ∂A

(1)

where A is the mean area per molecule at the indicated surface pressure and π is the corresponding surface pressure. Table 1 shows Cs-1 values at different surface pressures. The values conform the criterion of Davies and Rideal.14 While DOPE and POPE remain in the LE phase up to 30–35 mN m-1, DPPE is in the LC phase from approximately 20 mN m-1. Figure 2b presents the compression isotherms for DPPE:POPG, POPE:POPG and DOPE:POPG (3:1, mol/mol) monolayers at 37 ºC. This molar ratio mimics the lipid composition of the E.coli inner membrane, where LacY is embeded. Whilst POPE:POPG and DOPE:POPG showed a monotonic increase of π up to the collapse pressure, DPPE:POPG showed a plateau at approx. 46 mN m-1. This behavior may result from the immiscibility between DPPE and POPG, causing that POPG collapses as the first as reflected by the plateau, whilst further increase of the surface pressure results from compression of DPPE until it collapses at approx. 60 mN m-1. The values 9 ACS Paragon Plus Environment


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of Cs-1 confirmed that at 37 ºC, POPE:POPG and DOPE:POPG were in the LE phase and DPPE:POPG in the LC phase above 30 mN m-1 (see Table 1). Molecular areas, as obtained from monolayer isotherms at 30 mN m-1, can be used to fit the FRET data to a mathematical model specifically developed to evaluate the probability, µ, of finding a given phospholipid around the TMP. 7 The lipid-TMP selectivity depends upon the value of this probability. The term selectivity expresses the ability of a TMP for recruiting specific phospholipids for its own needs. According to the data in Figure 2, these areas were 0.68, 0.80 and 0.90 nm2·molecule-1 for DPPE, POPE and DOPE respectively; and 0.59, 0.68 and 0.79 nm2·molecule-1 for DPPE:POPG, POPE:POPG and DOPE:POPG mixed monolayers. The fitted values of µ show that the lipid selectivity of LacY is: DOPE ~ POPE > DPPE.

15,16,17

Besides, the DPPE:POPG mixture for which we have observed a double collapse in the monolayer seems not fulfill the requirements on surface lateral pressure of LacY. However, these data confirm that the molecular area occupied by a phospholipid, and hence the physical state of the phospholipid, is relevant in TMP–lipid interactions. Based on previously published information on lipid–TMP interactions,6,7 it seems that POPE and DOPE have their own specific physicochemical requirements for the cooperative interplay with many membrane proteins. PE 10 ACS Paragon Plus Environment

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seems to interact specifically with secondary transporters such as the multidrug transporter LmrP of Lactoccocus lactis, LacY.

19

18

or the above mentioned

Presumably in both cases PE increases magnitude of the πHG

component of the lateral pressure (Figure 1). However, in the case of the conductance mechanosensitive MscL channel it seems that the opening mechanism depends on the increase in gating tension induced by the presence of DOPE in the membrane rather than in a specific selectivity,

20

in this case

presumably by modifying the magnitude of πCH (Figure 1). Within the context of the fluid surface model of the membrane,

21

it is

accepted that the spontaneous curvature (co), of the phospholipid species

22

plays a crucial role in TMP incorporation and conformation in membranes. co is a measure of the tendency of the lipids to curve spontaneously leading to a negative or positive curvature. 1,21,22 Since it is assumed that the co of DOPE is similar to that for POPE, and as POPE was the only phospholipid in the present study for which the LE–LC phase transition has previously been reported, 13 we extended here the investigation of its thermal behavior. Figure 3 shows the compression isotherms of POPE at 11, 14, 19, 25, 29 and 37 ºC. Whilst the typical LE–LC phase transition is apparent between 11 and 25 ºC, it almost vanishes at 29 ºC and, as seen in Figure 2, was not observed at 37 ºC. Remarkably, a second transition, possibly between two 11 ACS Paragon Plus Environment


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liquid condensed phases (LC–LC’),

23

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appeared at high surface pressures (~

46–48 mN·m-1) and remained stable in the face of temperature changes. This was even more evident when we checked the compressibility modulus as a function of the different temperatures (Figure 4), which revealed the evolution toward higher surface pressures of the LE–LC transition as temperature increased. The second phase transition, highlighted with a black star, appeared as a shoulder before the isotherms collapsed. Above 25 ºC this second transition was hidden between the LE–LC transition and the collapse. Intriguingly at 37 ºC the isotherm collapsed at similar surface pressure values to those of the second phase transition. Furthermore, the lack of LE–LC phase transition at 37 ºC means that under biological conditions, POPE would exert a lateral pressure onto the surface of the TMPs, equivalent to 30 mN·m-1, 24 above its critical temperature (Tc) in monolayers. To estimate Tc, we used the two-dimensional Clausius-Clapeyron equation dπ tr ∆ tr S = dT ∆A

(2)

∆ tr Q T

(3)

and ∆ tr S =

where πtr is the plateau surface pressure at temperature T; and ∆A, ∆trS and ∆trQ are, respectively, the differences in molecular areas, molar entropy and


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latent heat associated with the LE–LC phase transition.

25

By plotting the

surface pressure of the phase transition versus temperature (Figure 5) a linear relationship was found with a slope of dπtr/dT, = 1.29 ± 0.09 mN·m-1·K-1. Meanwhile ∆A = ALC -ALE, the change in the molar area for the transition, was obtained by determination of ALE and ALC. Whilst ALE can be obtained by determination of the molecular area at πtr, the determination of ALC is more subtle. Some authors determine ALC as the maximum value when the phase transition coexistence region is fitted to a parabola; through Maxwell’s relationship

27

26

others evaluate ALC

or simply by extrapolation of the steepest

region of the isotherm to zero surface pressure. In our case the two phase transitions were so close that the use of Maxwell’s relation and the mathematical

adjustments

gave

inconsistent

results.

Similarly

the

extrapolation to zero surface pressure yielded larger values for ALC than for ALE. Hence we applied a straightforward method to our results. Since the derivation of the Clausius-Clapeyron equation requires the pressure be the same in any two phases at equilibrium, we took advantage of this condition in determining the value of ALC. Then ALC was calculated as the surface value when the steepest region of the isotherm in the LC state was extrapolated to the surface pressure at which the LE–LC transition started at each temperature (Figure 6). In fact, the phase transition does not occur at a constant surface 13 ACS Paragon Plus Environment


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pressure but increases following a curve that indicates the existence of a work term in the process. This has been evaluated as contributing less than 10% of the total heat involved in the transition. 28 The values of ALE and ALC obtained at the different temperatures are listed in Table 2. Then, inserting dπtr/dT and ∆A values into eq. 2, ∆trS values were obtained. Afterwards ∆trQ was obtained by substitution of ∆trS at each temperature in eq. 3. The outcome of these calculations and the other thermodynamic parameters for the compression process are listed in Table 2. Finally, the critical temperature, Tc, was obtained on extrapolation of ∆trQ to zero in the plot of ∆trQ versus T (Figure 7). This value was calculated as 36 ± 2 ºC and came from fitting the data to a linear function that yielded a correlation coefficient of 0.997. This means that above this temperature, the LC phase cannot be achieved by compression of the monolayer. It is noteworthy that at Tc and above, the LC and LE phases become indistinguishable. As discussed elsewhere, 29 systems at critical points display a universal behavior described by scaling invariance that develops strong fluctuations in monolayers in terms of density and composition, on scales from molecules to the size of the entire system. Indeed, at the critical point there is no characteristic length scale that may have biological meaning. To shed some light on the lateral organization of POPE molecules at the different temperatures we used AFM to observe Langmuir-Blodgett films 14 ACS Paragon Plus Environment

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extracted at 30 mN·m-1

24

onto mica surfaces (Figure 8). Notice, that the

extraction has been carried out at the nominal temperature of the monolayers whilst the AFM observation is carried out at 24ºC.We have assumed that these changes would affect all the LBs in the same way so the AFM observation provide valuable comparative information. Therefore the structures observed may not reflect the exact organization At 11 ºC the substrate (mica) was fully covered by the lipid monolayer, exhibiting two different structures: the more extensive domain covered 80% of the surface (lighter in the image) and presented a step height difference of 0.51 nm (Table 3) over a lower domain (darker in the image) formed by round structures with diameters below 100 nm. When the temperature was increased to 19 ºC the step height difference between the two domains remained stable but changes in the size of the round structures were observed. Although the coverage of the substrate by the higher domain remained almost unchanged, wider ellipsoidal structures appeared that may be attributed to the coalescence of the small round lower domains. At 25 ºC the arrangement of the monolayer changed drastically but not the step height difference between the two domains, which fell within the error values at 11 and 19 ºC. At 25 ºC the more extensive domain was the lower domain, with the higher domain representing 20% of the covered surface. Round structures were not observed at this temperature but irregularly shaped islands 15 ACS Paragon Plus Environment


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of the higher domain were seen on a background constituted by the lower domain. Increasing the temperature up to 29 ºC did not modify the coverage of the surface but the islands became elongated in one direction, maybe due to the scanning force exerted by the AFM tip. Possibly this effect will be not observed by using the AFM in Tapping® mode. At this temperature the step height difference between the domains increased to a mean value of 0.66 nm (Table 3). At 37 ºC, the highest temperature reached (just one degree over the calculated Tc), the taller domain vanished and only isolated patches were observed. Among the isotherms in Figure 2 only the one at 14 ºC was close to the LE–LC phase transition at 30 mN·m-1. However, we observed the coexistence of LE and LC domains at all temperatures investigated, even at 37 ºC, at which no phase transition was observed in the isotherm. Regarding the AFM observations a note of caution needs to be sounded. Thus, during the extraction of the LBs, changes in the structure of the film may occur either by concurrent adsorption of ions present in the buffer or by uncontrolled small changes in the surface pressure. Whether the Tc of a defined phospholipid may have biological implications will be a matter of discussion depending on each particular species. Thus at 37 ºC, the relevant temperature for many microorganisms, POPE would be close to its Tc, therefore there is no point considering the LE–LC transition under 16 ACS Paragon Plus Environment

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these conditions. Thus, the polymorphic behavior as observed at the air–water interface should not have a defined meaning in biological terms in POPE. Conversely, similar studies carried out with DPPC, using similar methodologies to those in the present paper, yielded a Tc for this saturated phospholipid of 32 ºC. 29 This confirms that DPPC, the main component of the lung surfactant, conserves the polymorphic property for undergoing the LE– LC transition under relevant biological conditions, providing a means for the continuous expansion-compression cycles occurring in the alveoli during respiration. In fact it is well known that this physical mechanism forms the basis of the physiological activity of the lung surfactant.

30

Obviously, when

either POPE or POPG is predominant, the lung surfactant will be rendered inoperative in any case. One of the aims of this work was to provide information on how physical properties of POPE may influence the physiological activity of LacY. As a member of the major facilitator superfamily, LacY is considered a paradigm for secondary transporters transport, the structure and function of which have been well established.

31,32

We have demonstrated previously that POPE lipid

molecules are in close contact with LacY

17

and the requirement of PEs for

the correct folding of LacY in proteoliposomes has also been reported. 5 The mechanism of action of LacY involves several steps31 between an initial 17 ACS Paragon Plus Environment


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inward-facing (∨ shape facing the periplasm) to an outward-facing (∧ shape facing the cytoplasm) conformation. Since our results indicate that POPE is close to its Tc at 37 ºC, we tentatively propose that these conformational changes may occur by coordinate changes in both the inner and the outer lipid monolayer of the membrane. Thus, it is possible that when the substrate binds to LacY the lateral stress in the outer monolayer increases whilst the inner monolayer remains in the fluid phase. POPE in the outer monolayer may then undergo a transition towards a more condensed phase (LC or LC’). At the same time POPE would expand to the LE phase in the inner monolayer in order to facilitate the inward conformational change and the release of the substrate. Molecular dynamic (MD) simulations, in turn, have shown that LacY undergoes different conformational changes during the transport of substrate when reconstituted in POPE or in DMPC.32 Despite the differences between this theoretical study and our experimental approach it has emerged from MD simulations that the lipid requirement of LacY can not only be ascribed to the PE headgroup but also to the plasticity of the lipid–protein interaction.33 It is noteworthy that these MD simulations were performed at 27 ºC, at which the bilayers modeled were in fluid phase but below the Tc of both POPE and DMPC monolayers,

34

such that there was an equilibrium between

the LE and LC phases. Furthermore, there is clear evidence from ATR-FTIR 18 ACS Paragon Plus Environment

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spectroscopy that the lipid mixture POPE:POPG (3:1, mol/mol) protein ratio (mol/mol) should be around 750 in order to provide the ideal lateral surface pressure for adequate LacY tilting and activity at 25 ºC.35 Both theoretical and experimental studies were carried out with liposomes in fluid phase, normally named Lα. Although Lα in bilayers is usually assimilated to the LE phase in monolayers, it remains unclear how the boundary phospholipids may behave. These phospholipids are in close-transient contact with the hydrophobic surface of the TMPs. Whether they conserve such properties, and therefore whether the transitions have any real meaning within different biological contexts, is debatable. Based on the information obtained in the present work we suggest that all experiments should be performed with lipids in Lα phase and above the corresponding Tc of the monolayers. Under these conditions, it is likely that phospholipids will adopt the adequate co for an ideal interplay with the TMPs.

CONCLUSIONS Among the compression isotherms of PE:POPG mixtures investigated at 37 ºC, only DPPE:POPG (3:1, mol/mol) was in the LC phase above 30 mN m-1. Under the same conditions, DOPE:POPG and POPE:POPG were in the LE phase. POPE is the only specie studied that presents a characteristic LE-LC 19 ACS Paragon Plus Environment


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phase transition at 24 ºC. Then, by using the two-dimensional ClausiusClapeyron equation it is possible to establish the ∆tQ and the Tc of POPE from isotherms recorded at different temperatures. AFM imaging of LBs of POPE evidenced the existence of two different domains that evolve from 11 ºC up to 37 ºC, close to the Tc of POPE, where no phase transition was observed in the isotherm. The information gathered indicates that all experiments dealing with LacY reconstituted in binary PE:POPG mixtures should be performed with lipids in fluid phase and above the corresponding Tc of the lipid monolayers.

AUTHOR INFORMATION *Corresponding Author Phone: +34 93 403 59 86, Fax: +34 93 403 59 87. E-mail: [email protected]

ACKNOWLEDGEMENTS This work was supported by Grants Nº TEC2016-79156-P from the Ministry of Economy and Competivity of the Spanish Government and Nº 2014SGR1442 from the Generalitat of Catalonia.

SUPPORTING INFORMATION 20 ACS Paragon Plus Environment

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Molecular structures of the phospholipids investigated and visual delineation of the LE, LC, LE-LC, and collapse regions of POPE isotherms at different temperatures. This material is available free of charge via the Internet.

REFERENCES (1)

Borrell, J. H.; Domènech, O.; Keough, K. M. W. Membrane proteinlipid interactions: physiscs and chemistry in the bilayer. 1st. ed. Springer International Publishing: Amsterdam, 2016.

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Figure 1. Theoretical lateral pressure profile p(z) with distance z from the bilayer midplane in a membrane of DPPC.


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Figure 2. Surface pressure-area isotherms at 37 °C of A) pure DPPE (□), pure POPE (○) and pure DOPE (∆); B) DPPE:POPG (3:1, mol/mol) (□), POPE:POPG (3:1, mol/mol) (○) and DOPE:POPG (3:1, mol/mol) (∆).



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Figure 3. Surface pressure-area (π-A) isotherms of pure POPE at 11 ºC (■), 14 ºC (●), 19 ºC (▲), 25 ºC (□), 29 ºC (○) and 37 ºC (∆).


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Figure 4. Cs-1 as a function of surface pressure (π) of pure POPE at different temperatures.



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Figure 5. Surface pressure (πtr) (A) and molecular area (Atr) (B) in the transition phase LE– LC as a function of temperature.


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Figure 6. Schematic representation of the procedure used to determine ALE and ALC.



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Figure 7. Variation of the molar entropy (∆Str) (A) and latent molar heat (∆Qtr) (B) of the transition phase LE–LC as a function of temperature.


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Figure 8. AFM height images of pure POPE monolayer transferred at 30 mN·m-1 onto mica surface at different temperatures.



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Table 1. Compressibility Modulus (Cs-1) of the Different Isotherms Shown in Figure 2 at Different Surface Pressure (π π) Values Cs-1 (mN·m-1) -1 π (mN·m ) DPPE POPE DOPE DPPE-POPG POPE-POPG DOPE-POPG 5

13.2

37.1

24.1

36.5

28.1

26.0

10

35.9

45.3

33.5

31.4

23.8

26.5

15 20

47.0 104.6

56.1 89.7

44.8 47.7

46.4 70.9

34.5 49.9

36.3 63.5

25

107.6

91.9

88.6

75.9

62.7

89.5

30

127.8

83.1

125.0

104.4

74.7

95.1

35 40

174.4 339.8

106.0 107.5

87.3 83.9

151.0 131.8

68.8 58.0

77.2 64.6

45

245.2

59.9

-

66.7

30.8

-

50

69.0

-

-

26.2

-

-


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Table 2. Thermodynamic Parameters Describing the LE–LC Phase Transition of Pure POPE Monolayers at Different Temperatures ALE ALC ∆A π ∆S ∆Q T tr

tr

2

tr

(K) (mN·m-1) (Å2·molec-1) (Å2·molec-1) (Å ·molec-1) (J·K-1·mol-1) (KJ·mol-1) 284

21.0

74.82

61.77

-13.05

-101.48

-28.82

287 292

27.0 34.0

69.82 65.79

58.75 57.64

-11.07 -8.15

-86.08 -63.38

-24.71 -18.51

298

41.5

62.45

56.74

-5.71

-44.40

-13.23

302

44.3

59.33

55.77

-3.56

-27.66

-8.35



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Table 3. Step Height Values of the Lipid Domain and High Domain Percentage Cover Values from AFM Images in Figure 8 T Domain height Covering (K) (nm) (%) 284

0.51 ± 0.07

80

292

0.53 ± 0.05

77

298

0.59 ± 0.13

20

302

0.66 ± 0.19

22

310

0.73 ± 0.09

3


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TOC IMAGE


Critical Temperature of 1-Palmitoyl-2-oleoyl-sn-glycero-3

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