Supported Lipid Bilayers on Mica and Silicon Oxide - American

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Supported Lipid Bilayers on Mica and Silicon Oxide: Comparison of the Main Phase Transition Behavior Heiko M. Seeger,†,‡ Alessandro Di Cerbo,†,‡,§ Andrea Alessandrini,‡,§ and Paolo Facci*,‡ Centro S3, CNR-Istituto di Nanoscienze, Via Campi 213/A, 41125 Modena, Italy and Department of Physics, UniVersity of Modena and Reggio Emilia, Via Campi 213/A, 41125 Modena, Italy ReceiVed: March 24, 2010; ReVised Manuscript ReceiVed: May 10, 2010

The usual biophysical approach to the study of biological membranes is that of turning to model systems. From these models, general physical principles ruling the lateral membrane structure can be obtained. A promising model system is the supported lipid bilayer (SLB) which could foresee the simultaneous investigation of the structure and physical properties of lipid bilayers reconstituted with membrane proteins. A complete exploitation of the model system to retrieve biologically relevant information requires an in-depth knowledge of the possible effect that experimental parameters could have on the behavior of the SLB. Here we used atomic force microscopy (AFM) to study the effect of different types of substrates on the behavior of SLBs as far as their main phase transition is concerned. We found that different substrates (mica and silicon oxide) can affect in dissimilar ways the interleaflet coupling of the bilayer, which might represent a sort of lipid signaling allowing communication between receptors on the extracellular leaflet and cytoplasmic components. By decreasing the interaction between the SLB and the substrate the interleaflet coupling is preserved independently of the bilayer preparation strategy. Moreover, we investigated by time-lapse AFM an isothermal phase transition induced by a pH change on a SLB. We established that the presence of a pH gradient across the bilayer can weaken the strength of the interleaflet coupling which is present in symmetrical pH conditions. Introduction Supported lipid bilayers (SLBs) represent a well-established model system to study physical principles underlying the behavior of biological membranes.1-5 Biological membranes are always in contact with other structures, and the mutual interaction between the membrane and other elements can give rise to particular structural or functional features. These features can be, to a certain extent, studied in SLBs. At the same time, SLBs have biotechnological relevance due to their possible exploitation in biosensors.1,3 They were initially developed by McConnell’s group to study the interaction of cells with lipid bilayers.6 They can be assembled mainly on hydrophilic surfaces (mica, glass, silicon oxide)7 by two different strategies: the LangmuirBlodgett/Schaefer approach8 and the vesicle fusion technique.6,9 The formation of SLBs on different surfaces and their consequent behavior depend on several parameters (surface chemistry, ionic strength of the solution, pH, and preparation temperature) which are often difficult to control and usually strictly interconnected.10,11 As a consequence, it is difficult to compare the behavior of SLBs prepared in different laboratories and sometimes there is no consensus on the physical properties of the lipid bilayers once on the solid support. Nonetheless, in order to fully exploit such a model system to retrieve biologically relevant information, it is important to understand as much as possible the influence of preparation conditions and substrate nature on the lipid bilayer. One of the main advantages of SLBs over other freely suspended systems is given by their stability and the possibility * To whom correspondence should be addressed. Fax: 0039 059 2055651. E-mail: [email protected]. † These authors contributed equally. ‡ CNR-Istituto di Nanoscienze. § University of Modena and Reggio Emilia.

of being studied by several surface-sensitive techniques (e.g., scanning probe microscopy, ellipsometry, waveguide spectroscopies, X-ray and neutron reflectivity, quartz crystal microbalance).12-18 Much experimental evidence has established the presence of a thin water layer between the lipid bilayer and the solid substrate.19-21 The presence of this layer assures a certain degree of mobility to the lipid composing the leaflet nearer the substrate (proximal leaflet). In this context, the lipid diffusion coefficient in SLBs has been thoroughly studied by fluorescence recovery after photobleaching (FRAP)22 and fluorescence correlation spectroscopy (FCS).23 Even in this case the results of experiments performed in different laboratories are sometimes contradictory.24-26 In some cases, the diffusion is characterized by only one relaxation time, whereas in others, the presence of two distinct relaxation times has been found. The occurrence of two relaxation times is usually attributed to the different internal dynamics between the proximal leaflet and the leaflet facing the bulk water (distal leaflet).24 The observed discrepancies can be ascribed to intrinsic differences among samples prepared by different approaches or in different conditions (e.g., different substrate, ionic strength, pH, temperature).10,11 The occurrence of different dynamics between the two leaflets of a SLB is strictly connected with the possibility of observing two different phase transitions when the temperature is varied. Indeed, two different phase transitions attributed to separate transitions of the two leaflets have been observed by different techniques. In previous works, we and other groups demonstrated, by atomic force microscopy (AFM), the existence of two temperature-induced phase transitions in SLBs.27,10 In fact, the phase transition is characterized by variations in bilayer thickness which can be easily tracked by AFM.28 The two transitions have been assigned to the two leaflets which behave independently upon temperature variation.

10.1021/jp1026477  2010 American Chemical Society Published on Web 06/24/2010

Supported Lipid Bilayers on Mica and Silicon Oxide The presence of a substrate induces a vertical symmetry breaking on the two bilayer leaflets.29 This is even true in the case of a bilayer composed of only one type of lipid. In particular, recent simulation studies by Faller’s group demonstrated that in supported lipid bilayers the surface density of lipid molecules in the two leaflets differs, being higher in the proximal one.30,31 The substrate seems to act mainly on the proximal leaflet. In fact, the lipid density in the distal leaflet is very similar to what is expected for an unsupported bilayer. The lipid density asymmetry gives support to the interpretation of the lower temperature transition as due to the distal leaflet and the higher one as due to the proximal one.27,10 In fact, the higher surface density of the lipids in the proximal leaflet can explain the increased transition temperature according to the Clausius-Clayperon relation assuming a transition at constant area.32 Moreover, the more rigid solid ordered domains are entropically favored near the rigid substrate, where undulation suppression occurs.33 In a preceding paper we demonstrated that, by changing the preparation temperature of SLBs obtained by the vesicle fusion method on mica, it is possible to switch from separate transitions of the two leaflets to a single, coupled transition.10 We found that by increasing the preparation temperature of a particular lipid mixture assembled on mica it was possible to obtain a single-phase transition involving the two leaflets with the formation of in-register domains. In all these studies, the main aspect is the balance between the interaction of the support with the proximal leaflet and the presence of an interleaflet coupling force which enables interaction between the two leaflets.34,35 If the interaction between the substrate and the proximal leaflet is stronger than the interleaflet coupling, the two leaflets behave independently, whereas if the former interaction is weaker, the interleaflet coupling prevails and the two leaflets display a coupled behavior. The latter situation corresponds to perfect registry between domains of the same phase in both leaflets. The interaction between the substrate and the proximal leaflet is a complicated balance between different types of forces: van der Waals forces, entropic forces due to the undulation suppression exerted by the solid surface, and hydration forces.34 On the one hand, the magnitude of these forces depends on the properties of the substrate (from both a chemical and a physical point of view, e.g., its roughness), the thickness of the water layer sandwiched between the bilayer and the support, and the type of lipid bilayer studied. On the other hand, the interleaflet coupling represents a sort of lipid signaling across the bilayer and is at present the focus of many experimental and theoretical studies aimed at elucidating it in light of its possible biological relevance.36 As an extension of our previous work, here we studied the role of two different substrates in determining the behavior of POPE/POPG SLBs prepared by vesicle fusion. We compared the temperature-induced main-phase transition of a SLB prepared on silicon oxide with previous results on mica (ref 10). We demonstrated that, in contrast to the behavior on mica, the temperature-induced phase transition on silicon oxide proceeds always with the two leaflets in a coupled configuration, independently of the bilayer preparation conditions, highlighting the prevalence of the interleaflet coupling over the substrate/ proximal leaflet interaction. Moreover, here we also studied the isothermal transition induced by pH. Due to the presence of charged lipid molecules in the bilayer, the phase state of the lipids is strongly dependent on pH.37,38 We induced a phase transition by exchanging the pH of the imaging buffer and followed the domain evolution in situ by time-lapse AFM. The

J. Phys. Chem. B, Vol. 114, No. 27, 2010 8927 pH-induced transition, which is here reported for the first time as observed by AFM, proceeds always in a decoupled manner on mica, even if the sample has been prepared at high temperature. We discuss some peculiar aspects of the isothermal phase transition induced by pH on a SLB which may be relevant for freely suspended bilayers. Materials and Methods Sample Preparation. The lipids 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (POPG) were purchased from Avanti Polar Lipids (Alabaster, USA). Stock solutions (in CHCl3) were mixed to obtain the desired lipid molar ratio of 3:1 (POPE:POPG). Then the chloroform was evaporated under a flow of nitrogen while heating the sample in a water bath at 50 °C. Thereafter, the sample was kept under vacuum (10-2 mbar) for at least 4 h to remove the remaining chloroform molecules. Afterward, the lipids were rehydrated in a buffer solution of 450 mM KCl, 25 mM Hepes at a pH of 7. The sample was stirred at about 30 °C for 1 h. During this time the sample was vortexed at least two times. The transition midpoint temperatures of pure small unilamellar liposomes of POPG and POPE at neutral pH are -5 and 25 °C, respectively. Preparation of Silicon Oxide Supports. A silicon oxide support was cleaned with water and detergent. Then it was thoroughly washed with bidistilled water (18 MΩ cm) and dried under N2 stream. Afterward, the support was exposed to a piranha solution (H2O2:H2SO4 1:3), washed extensively in bidistilled water, dried under N2 stream, and exposed to an oxygen plasma discharge in a plasma cleaner (Diener Electronic GmbH, Germany). The preparation procedure produced a silicon oxide surface with a rms roughness of 0.20 nm compared to the rms roughness of 0.05 nm for mica. Preparation of Supported Lipid Bilayers. The SLBs were prepared by the vesicle fusion technique. Either a freshly cleaved piece of mica (SPI Supplies/Structure Probe, Inc., USA) or a cleaned silicon dioxide support was placed on the AFM stage (see below). The support was fixed using a ring of polydimethylsiloxane (PDMS) and polytetrafluoroethylene (Teflon). Then we added 500 µL of a 450 mM KCl, 25 mM Hepes, pH 7 buffer. The lipid suspension was sonicated for 30 s in an ultrasonic bath to obtain small unilamellar vescicles (SUVs). We equilibrated the vesicle solution at the temperature of interest together with the excess buffer in the sample cell. Then we injected the SUVs in order to obtain a final lipid concentration of either 0.25 (mica) or 0.5 mg/mL (silicon dioxide). The lipid suspension was incubated for 15 min. Then the incubation buffer was exchanged for the desired imaging buffer (150 mM KCl, 10 mM potassium dihydrogen citrate at pH of 7, 5.6, or 3). We adjusted the sample temperature to the desired starting temperature of 32-34 °C, and the lipid bilayer system was allowed to equilibrate. Temperature-Controlled AFM Measurements. Atomic force microscopy experiments were performed with a Bioscope microscope equipped with a Nanoscope IIIA controller (Veeco Metrology, USA). We constructed a temperature-controlled stage based on a circulating water bath on which we could mount the Bioscope head. Imaging was performed in tapping mode at a scan rate of 1-2 lines/s using triangular silicon nitride cantilevers (Olympus OMCL-TR400PB-1, Japan) with a nominal spring constant of 0.09 N/m and a resonance frequency in liquid between 8 and 9 kHz. The force applied to the membrane

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Figure 1. Melting transition behavior of SLBs on silicon oxide and comparison with SUVs. (a-d) Sequence of AFM images (image size: 7.5 µm × 7.5 µm) on the same area showing the temperature-induced phase transition of a SLB on silicon oxide. Down to a temperature of 22 °C no domain formation was visible. Domains started to appear at 21 °C and extended upon further cooling of the sample. (e) The line section along the solid line of image b reveals a step height between the two domains of 1.4 ( 0.2 nm. (f) The black solid line, representing the inverse transition enthalpy, is compared to the solid ordered fraction (open circles, dashed curve is a guide for the eye) of the SLB. The transition on the silicon oxide support occurs at a slightly higher temperature than the one of the SUVs. (Inset) Excess heat capacity of SUVs of POPE:POPG 3:1 sample at pH 7 obtained by DSC.

was adjusted to the lowest possible value, allowing reproducible imaging. In this paper we present data corresponding to cooling scans of the lipid bilayers always starting from a temperature of 32-34 °C, which assures good lipid mixing for our lipid mixture. The determination of the sample temperature was based on a calibration scan obtained by a digital thermometer Fluke 16 (Fluke, Italy) equipped with a small K-thermocouple probe (Thermocoax GmbH, Germany) in direct contact with the imaging buffer. The sample temperature was changed in steps of 1.4 °C if not otherwise specified. After each temperature step, the lipid bilayer was allowed to equilibrate before acquiring an image at a constant temperature. To assess the equilibrium condition, consecutive AFM scans were performed until, when possible, no appreciable changes were observed in two subsequent images. pH Exchange at Constant Temperature. To study the pHinduced isothermal phase transition of the SLB, the system was initially equilibrated at the desired temperature and pH. We used a syringe pump (PHD 2000, Harvard Apparatus, USA) to perform buffer exchange in the sample cell. We established a flux rate of around 150 µL/min and stopped after a 10-fold exchange of the initial buffer volume. The slow exchange rate ensured a constant buffer temperature during the buffer exchange. A calibration measurement without the Bioscope head allowed estimating the buffer’s pH value during the exchange. Image Analysis. For quantitative image analysis we exploited height images. We converted the AFM images into graphical files using WSxM (Nanotec Electronica, Spain).39 Then we opened them in Adobe Photoshop C3 (Adobe, USA), marked the different lipid domains manually, and calculated the relative areas to determine the extent of each fraction with respect to the total bilayer area. Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) measurements were performed with a VPDSC from Microcal (Northhampton/MA) using high feedback mode at a scan rate of 5 °C/h. Suspensions of POPE:POPG 3:1

at concentrations of 10 mg/mL were sonicated for 30 s in an ultrasonic bath prior to measurements. Excess heat capacity profiles of the sample were directly measured in the calorimeter cell. Integrating the resulting curve allowed for determination of the transition enthalpy. Results and Discussion Temperature-Induced Main Phase Transition. In a previous work we showed that POPE:POPG (3:1) SLBs on mica undergo a split temperature-induced phase transition when they are prepared at incubation temperatures lower than that of their main phase transition, whereas they undergo a coupled phase transition with domains in register when they are assembled at a temperature higher than that of their main phase transition.10 Here, to better understand the role of the substrate in the SLB behavior, we studied the same lipid mixture on a different support. We assembled a POPE:POPG (3:1) bilayer on a silicon oxide surface by the vesicle fusion technique, incubating the sample at 33 °C. Figure 1 shows a sequence of AFM images (from Figure 1a to 1d) of a SLB measured at different temperatures on the same sample area. Starting from a temperature of 30 °C, upon decreasing the temperature, at 21 °C a transition from the liquid disordered to the solid ordered phase starts. This is characterized by the growth of domains in the lipid bilayers thicker than the surrounding lipid disordered membrane. Small areas of the lipid bilayer remain in the liquid disordered phase even at a temperature of 16 °C. By analyzing the line section of an image in the transition region it is possible to measure the difference in height between the two phases (Figure 1e). A figure of 1.4 ( 0.2 nm results, which can be attributed to both leaflets undergoing a coupled phase transition to the solid ordered phase, according to ref 10. In Figure 1f the inverse transition enthalpy (a measure of the solid ordered fraction) calculated from the heat capacity profile obtained using differential scanning calorimetry of small unila-

Supported Lipid Bilayers on Mica and Silicon Oxide mellar vesicles of the same lipid composition (see inset to Figure 1f) is reported along with the fractional occupancy of the solid ordered phase obtained from the AFM images as a function of temperature. The shapes of the inverse enthalpy and the solid ordered fraction obtained by AFM images agree well. They both show a cooperative transition with a half width of 3 °C. Still, the bilayer/substrate interaction shifts the SLB transition temperature by 2.5 °C to higher temperatures. It is to be stressed that the fractional occupancy of the solid ordered phase can have a thermodynamic sense only if it represents a series of equilibrium states. The possibility of observing equilibrium states by AFM is strictly connected with the kinetics of lipid domain formation, which, in turn, depends on the diffusion properties of the lipids.40 In the case of a coupled phase transition of a SLB on mica we were not able to produce a plot of the fractional occupancy because the kinetics of domain formation was very slow (on the order of hours).10 In the case of SLBs on silicon oxide, equilibrium states upon a temperature change were obtained for time intervals on the order of minutes. This behavior allowed obtaining a plot like the one shown in Figure 1f. The different kinetics on the two substrates could be related to the different diffusion coefficient of the lipids in the bilayer. In favor of this hypothesis, comparing results from the literature on similar systems (SLBs of DPPC), it can be found that the diffusion coefficient of the lipids on mica is significantly lower than that on silicon oxide.4,41 In order to understand the effect of the incubation temperature on the SLB phase transition behavior on silicon oxide with respect to mica, we incubated a new SLB on silicon oxide at 10 °C. Then we increased the temperature to 33 °C and monitored the phase transition upon a temperature decrease. Also, in this case the main phase transition induced by temperature occurred in a coupled manner between the two leaflets, and the fractional occupancy of the solid phase as a function of temperature is very similar to the previous case (data not shown). This means that at variance with what happens for SLBs on mica,10 the temperature-induced phase transition for a SLB on silicon oxide does not depend upon the preparation temperature of the membrane. As pointed out above the main transition temperature obtained from the supported lipid bilayer is very similar to what has been obtained by DSC on small unilamellar vesicles. This result agrees with those obtained by Tamm and McConnell for a DPPC bilayer supported on oxidized silicon by measuring the lateral diffusion of a fluorescent lipid probe.4 In the case of the same lipid system on mica the starting of the main transition occurred at a temperature significantly higher than that obtained by DSC. The observed behavior on mica10 can be attributed to a modulation of the interleaflet coupling strength upon variation of the incubation temperature. An increased incubation temperature leads to a decrease of the lipid density in the proximal leaflet which becomes similar to the lipid density in the distal leaflet. On the contrary, the low incubation temperature gives rise to a strong difference in the lipid densities of the two leaflets, preventing the dynamic interdigitation mechanism of the lipid hydrophobic tails. Indeed, the dynamic interdigitation of the lipid chains is considered one of the main phenomena causing interleaflet coupling.35 This interdigitation could be considered as a phenomenon which increases the entropy of the lipid chains, and the entropy loss due to a restricted dynamic interdigitation could be the driving force for maintaining a coupling between the two monolayers. In the specific case of a SLB, Merkel et al. found that on increasing the packing density of the proximal layer an increase in the diffusion coefficient for the distal layer

J. Phys. Chem. B, Vol. 114, No. 27, 2010 8929 was obtained, probably due to a decreased interdigitation effect.26 In the case of silicon oxide, a lower interaction between the proximal leaflet and the substrate could result in a lipid density in the proximal leaflet not significantly higher than the lipid density in the distal leaflet. In this case an increase in the preparation temperature is not required to make the two leaflets’ density similar and the lipid bilayer is always coupled. Figure 2 reports a summarizing scheme based on what is reported in our previous study for the phase transition behavior of the mica support10 and of the behavior of the SLBs on silicon oxide as a function of the SLB incubation temperature. Each of the possible situations reported in Figure 2 are visualized by a sketch, a representative AFM image, and a line section along the solid line drawn in the corresponding AFM image. In Figure 2a it is shown that if a SLB is prepared on mica at an incubation temperature lower than its melting transition, three phases for the bilayer appear. We called these phases liquid disordered, intermediate, and solid ordered phase (throughout the paper we will use for the lipid monolayer the same well-established nomenclature of the lipid bilayer relative to the adopted phases). This behavior is a consequence of the absence of interleaflet coupling in the bilayer. The kinetics of the SLB in Figure 2a (presence of intermediate phase) is on the minutes time scale, and the reported AFM image is representative of an equilibrium state (as far as it can be established from consecutive AFM images). If the bilayer is assembled on mica at a temperature higher than its melting transition (Figure 2b), the presence of interleaflet coupling prevents the formation of the intermediate phase and only domains in perfect registry occur. For the system reported in Figure 2b, the SLB is not at equilibrium (equilibrium is not reached within a time scale compatible with the experiment) but evolution of the domains does not involve a variation of the height difference between the two observed phases. If a SLB is assembled on silicon oxide (Figure 2c), independently from the incubation temperature, the bilayer leaflets are always coupled. In this case preparing the SLBs at different temperatures does not appreciably affect the lipid density in the proximal leaflet. Several studies by atomic force microscopy on the phase transition behavior of SLBs established that the conversion from the liquid disorder to the solid ordered phase occurred at significantly higher temperature with respect to freely suspended bilayers, at least for one of the two leaflets if two separate transitions are observed.10,27,32 This aspect can be ascribed to the interaction between the bilayer, especially the proximal leaflet, and the substrate. The behavior observed on silicon oxide can be interpreted by assuming that the interleaflet coupling is stronger than the interaction of the proximal leaflet with the support. The mica and silicon oxide surfaces have similar charge in the conditions used for assembling and studying the SLB. A different electrostatic force between the substrate and the bilayer cannot be evoked to explain the observed differences. The overall interaction between the substrate and the membrane is the result of many types of forces.34 The main contributions come from the van der Waals attractive interaction and the repulsive interactions resulting from both the undulation forces and the hydration forces. The balance between these forces produces a minimum in the potential energy, which assures the stability of the lipid bilayer. All involved forces have a strong dependence on the separation distance between the two interacting bodies. Of special interest to our case is the effect of the substrate surface topography on the interaction potential energy. The silicon oxide surface is characterized by a higher roughness than mica (rms roughness of 0.20 nm against 0.05 nm). A higher

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Figure 2. Sketch of the behavior observed for SLBs on mica and silicon oxide substrates. We show a cartoon of the situation, a representative AFM image (image size: 10 µm × 10 µm) of the SLB, and a line section along the solid line of the corresponding image. In the line sections the different phase are specified. (a) Preparing a SLB on mica at a temperature below the melting transition regime leads to a decoupling of the two leaflet’s transitions. The step heights between different domains are on the order of 0.7 ( 0.2 nm each. (b) Increasing the incubation temperature above the main phase transition temperature couples the transitions of the two bilayer leaflets. The step height between the domains is 1.4 ( 0.2 nm. The lowest level in the line section represents the mica level. (c) On silicon oxide, independently of the incubation temperature, there is always a coupled transition. The step height is again on the order of 1.4 ( 0.2 nm.

roughness is related to a higher effective surface area, which could in principle lead to increased adhesion energy if the bilayer were able to exactly follow the contour of the surface. However, this latter aspect would require a high bending of the lipid bilayer, resulting in an increased energy for the bilayer. Due to this higher energetic cost and the nonlinear dependence of the interaction forces on the distance between the substrate and the membrane, the adhesion energy of the membrane to the substrate decreases in the case of a rough surface.42 The presence of a rough surface resembles the occurrence of fluctuations in the bilayer, reducing the probability for the bilayer to lie close to the substrate and, consequently, decreasing the adhesion energy between the membrane and the support per unit of projected area. Furthermore, a possible increased alkyl chain disorder related to a larger roughness could increase the hydrophobic exposed surface and, hence, the interleaflet hydrophobic interaction. These considerations agree with experimental results in which it is clearly observed that SLBs on silicon oxide are less stable than on mica and the borders between the SLB and the bare substrate are easily disturbed by the scanning AFM tip.

Main Phase Transition of SLBs as a Function of pH. In bilayers composed of charged lipids, the main phase transition results in a variation of the electrostatic free energy due to a difference in the surface charge density following expansion or contraction of the bilayer surface. By varying pH, it is possible to modify the surface charge of the bilayer and alter the electrostatic free energy. If the charge density increases, a decrease of the main phase transition temperature is expected.37 The main phase transition temperature of the PE:PG mixture has a strong dependence upon pH due to the presence of the charged PG headgroup.43 Due to the fact that biological systems are remarkably constant in temperature and a phase transition induced by a pH variation has a biologically relevant interest,37,44 we decided to study by AFM the behavior of a SLB on mica as a function of pH. All the supported bilayers were prepared at high temperature in order to obtain a single transition. Figure 3a shows the DSC measurements over the phase transition region of POPE:POPG (3:1) small unilamellar vesicles for different pH values (7 and 3). An increase of the transition temperature is observed by decreasing pH as expected from the

Supported Lipid Bilayers on Mica and Silicon Oxide

Figure 3. pH dependence of the transition behavior. (a) Comparison of the excess heat capacity curves of POPE:POPG 3:1 SUVs measured at pH 3 and 7. Increasing pH leads to a shift of the transition region to higher temperatures. (Inset) Linear dependence of the transition midpoint temperature on pH. (b and c) AFM images of a SLB of POPE: POPG 3:1 incubated at 34 °C on mica (image size: 10 µm × 10 µm). Formation of solid ordered domains was induced by lowering the temperature. At a pH of 3 domains started to form at 27.0 ( 0.5 °C. The time lapse between images b and c was 15 min. (d) The line section along the solid line of image c reveals that the transition of the two leaflets is coupled. The step height has a figure of 1.4 ( 0.2 nm.

protonation of the PG headgroup. Figure 3b and 3c shows two images of a SLB on mica obtained by varying the temperature at pH 3. The solid ordered phase starts appearing at 27.0 °C at pH 3 instead of 22.0 °C at pH 7. This behavior confirms the dependence of the transition temperature on pH for a POPE: POPG 3:1 SLB on mica. The obtained results clearly highlight that SLBs mirror, as long as the main phase transition is concerned, the behavior observed for liposomes of the same lipid composition. pH-Induced Phase Transition. After establishing the dependence of the phase transition temperature in SLBs upon pH variations, we studied the possibility of observing by time-lapse AFM an isothermal phase transition induced by a change in pH. We prepared a SLB on mica at pH 7 using an incubation temperature of 33 °C and adjusted the temperature at 27 °C, a value intermediate between the two transition temperatures at pH 7 and 3 (see Figure 4a). In these conditions the bilayer was in the liquid disordered phase. After that, we exchanged the imaging buffer for the buffer at pH 3 while imaging the bilayer by AFM. Figure 4b-4f shows a sequence of images obtained while the pH was changed from 7 to 3 and then back to 7. After an exchange of buffer which assures a pH of 4 in the sample cell, we observed the formation of domains which initially were in the coupled solid ordered phase and progressively changed to the intermediate state. It has to be noted that during the transition from the coupled solid domains to the intermediate domains only the domains which undergo this transition grow significantly. In the case of domains remaining in the solid ordered phase, their size does not appreciably change between two consecutive scans. Moreover, the total solid area of the domain 1 of Figure 4c (considering the sum over the two leaflets) is similar to the area in the solid ordered phase of the same domain in Figure 4d (14 vs 15 µm2). This behavior can be ascribed to a lipid flip-flop movement which brings

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Figure 4. pH-induced domain formation. (a) Traces of the heat capacity of POPE:POPG 3:1 SUVs at pH values of 7 and 3. The curves have been corrected for the effect of the mica support which leads to an increase of the melting temperature by about 5 °C. (b) The AFM image (image size: 10 µm × 10 µm) at a pH of 7 does not show any lateral heterogeneity. The membrane was prepared at high temperature to ensure a coupling of both leaflets. (c) Starting the buffer exchange, at some point (after reaching a pH of about 4) domains started to form. The different height levels in domain 1 indicate that the transition was not fully coupled. (d) Following the evolution of domain 1, it becomes clear that a flip-flop mechanism was present. The initial solid ordered domain changed to an intermediate domain. (e and f) After finally reaching a pH of 3, the buffer was again exchanged for a buffer at pH 7. The intermediate domains vanished, demonstrating the reversibility of the transition.

phospholipids which are in the solid ordered configuration to the opposite leaflet while being substituted for lipids in the liquid disordered phase in analogy to what has been observed by Lin et al.33 However, in contrast to ref 33, considering our data we cannot conclude whether lipid flip-flop occurred at the domain boundaries or in the center of the domains. The new solid ordered domains, occupying only one leaflet, grow until they reach an equilibrium state. If the solution pH is changed back to 7, the previously formed domains disappear. This behavior assures that the domain effectively had formed as a consequence of the change in pH. We attempted to establish which one of the two leaflets was involved in the observed phase transition. Thus, at first we exchanged the pH of the solution from a pH of 7 to a pH of 5.6. This resulted in the formation of domains of intermediate characteristics. Then, keeping the pH constant at 5.6, we decreased the sample temperature in order to further follow the phase transition. After completion of the phase transition of one of the two leaflets (the same involved in the pH-induced phase transition), upon further cooling, we observed a lower temperature phase transition. This lower temperature phase transition occurred at the same temperature at which it is possible to observe the lower phase transition for a bilayer at pH 5.6 without leaflet coupling (Figure 5 and ref 10). This lower transition temperature was attributed to the distal leaflet, and it occurred at a temperature similar to that measured for liposomes of the same lipid composition in DSC measurements.10,27,32 In light of this behavior, it can be argued that the first transition observed by varying the pH occurred in the proximal leaflet and the pH variation caused the loss of interleaflet coupling in the bilayer. The flip-flop phenomenon in lipid bilayers is a fundamental process whose study is unfortunately hampered by many technical problems. In particular, its rate has been determined with contradictory results.45 It is believed that it is essentially a slow process unless it is catalyzed by proteins, but, under

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Seeger et al. formed, even if the bilayer has been assembled to undergo a coupled phase transition in register, the pH-induced phase transition involves only one leaflet. This could be the behavior also of a freely suspended bilayer when it is exposed to a transmembrane pH gradient. The biological relevance of this finding is connected to the possibility, experienced by many biological membranes, to be exposed to a pH gradient. The consequences of this fact could also be a loss of the interleaflet coupling in the lipid bilayer. Acknowledgment. Financial support by the Italian MIUR FIRB project Italnanonet is acknowledged. References and Notes

Figure 5. pH and temperature change. Sequence of AFM images of a SLB of POPE:POPG 3:1 incubated at a temperature of 34 °C on mica (image size: 20 µm × 20 µm). (a) At a temperature of 27.5 °C at pH 7 the bilayer is in its liquid disordered phase. (b) At a pH 5.6 intermediate domains started to appear (c-e). After equilibration, we started to decrease the temperature from 27.5 to 19.5 °C. The intermediate domains enlarged until they almost completely covered a leaflet. (f) By further decreasing the temperature a second transition started. This transition occurred at a temperature as expected for the distal leaflet at a pH of 5.6.

some circumstances, it can greatly increase its rate. One of these circumstances is related to the lipid phase transition. It has been demonstrated that at the main phase transition the flip-flop rate strongly increases.46 Another case in which flip-flop movement can be enhanced is the presence of a pH gradient across the lipid bilayer.47 The exchange of pH on the SLB can result in a pH gradient across the bilayer. At pH 3 the phosphodiester group of PG starts to be protonated (pKa ≈ 2.9) and the PG headgroup turns out to be neutral. As a consequence, POPG in the distal leaflet could move to the proximal leaflet where it can release protons and contribute to reduce the pH gradient. Moreover, lipids in the solid order phase are entropically favored in the proximal leaflet due to undulation suppression near the substrate. The flip-flop mechanism could modify the lipid density in the two leaflets, destroying the interleaflet coupling which was initially assured by the high incubation temperature of the SLB. Conclusions Here we demonstrated that the substrate supporting a lipid bilayer can have a strong influence on the behavior of the bilayer. In particular, increasing the roughness of the substrate reduces the interactions between the support and the membrane. The reduced interaction modifies the phase transition behavior of the SLB, making it more similar to a freely suspended bilayer, as confirmed by DSC measurements. The lower interaction makes the interleaflet coupling stronger, as deduced from the observed formation of domains in register between the two leaflets on silicon oxide independently of the incubation temperature. These results make the SLB on silicon oxide a more exploitable substrate to study protein/lipid interactions due to increased lipid mobility with respect to the mica support and lower probability for the membrane proteins to get stacked on the underlying solid surface. A pH difference shifts the transition temperature of SLBs as expected from DSC experiments. In contrast, if the pH is changed once the lipid bilayer is already

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