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J. Phys. Chem. C 2007, 111, 8988-8999
Asymmetric Distribution of Lipids in a Phase Segregated Phospholipid Bilayer Observed by Sum-Frequency Vibrational Spectroscopy† Jin Liu and John C. Conboy* Department of Chemistry, UniVersity of Utah, 315 S. 1400 E. RM 2020, Salt Lake City, Utah 84112 ReceiVed: December 30, 2006; In Final Form: January 31, 2007
The intrinsic symmetry constraints on sum-frequency vibrational spectroscopy (SFVS) have been exploited to measure the asymmetric distribution of lipid domains in the proximal and distal layers of a planar supported lipid bilayer (PSLB) in the gel-liquid-crystalline (l.c.) coexistence region of the phase diagram for several lipid systems. Four saturated phospholipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (DHPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and two equal-molar binary mixtures of DSPC with DMPC, and DSPC with a unsaturated lipid DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) were investigated. The destructive interference of the symmetric stretch (Vs) transition moments from the lipid fatty acid methyl groups (CH3) was used to monitor changes in the symmetry of the bilayer structure. A maximum in the CH3 Vs intensity is observed at the phase transition temperature (Tm) due to the break in the local symmetry of lipid bilayers caused by the dislocation of the gel and l.c. phase domains. The SFVS results were correlated to phase segregation in the membranes as measured by fluorescence microscopy. The SFVS measured Tm for DMPC, DPPC, DHPC, and DSPC were 23.4 ( 0.9 °C, 41.0 ( 0.4 °C, 52.4 ( 0.7 °C, and 57.5 ( 0.5 °C, respectively. These values correlate well with the literature values of 23.6 ( 1.5 °C, 41.3 ( 1.8 °C, 49 ( 3 °C, and 54.5 ( 1.5 °C for DMPC obtained by differential scanning calorimetry (DSC). In addition to providing a direct spectroscopic probe of the Tm of PSLBs, these studies also provide evidence for the delocalization of the gel and l.c. domain structures between the two layers of lipid bilayers.
Introduction The phase behavior of phospholipids dictates the motion of membrane constituents, influences the permeability of intra and extra cellular materials, and is even believed to be responsible for cell signaling by the formation of localized receptors in the membrane.1-5 The phase segregation of lipids in a membrane is governed by their phase transition temperature (Tm).6 Below the Tm, the lipids exist in a solid-like, gel phase. Above the Tm, the lipids are in a liquid or liquid-crystalline (l.c.) state. It is generally believed that the gel f l.c. phase transition is driven by an increase in lateral diffusion and decrease in conformational ordering of the lipids as the temperature of the system is increased.7-9 Methods such as differential scanning calorimetry (DSC) can be used to measure the change in heat capacity associated with the phase transition as a means to determine the Tm of lipid in solution-phase vesicles5,10 or for planar supported lipid bilayers (PSLBs) deposited on a high surface area material.11 Fluorescence spectroscopy can be used to infer the Tm based on changes in the emission characteristics of a fluorescent probe.12,13 Other analytical techniques, such as nuclear magnetic resonance (NMR), Raman, and Fourier transform infrared spectroscopy (FTIR) can be used to investigate the conformation of the lipid acyl chains to measure the phase transition.5,11,14-17 For a single component lipid system at the Tm, phase segregation of the gel and l.c. lipid states will occur due to the different mobilities and hydrophobic interactions between the lipids in each phase.7,18 In the gel phase, there is a relatively †
Part of the special issue “Kenneth B. Eisenthal Festschrift”. * To whom correspondence should be addressed.
low rate of lateral diffusion (2 × 10-10 cm2/s)19-22 and a strong interaction between neighboring phospholipids.7,18 In the l.c. phase, there is a relatively high rate of lateral diffusion (4 × 10-8 cm2/s)19-22 and a weak interaction between neighboring phospholipids.7,18 For a multicomponent or binary lipid system, phase segregation will occur over a temperature range defined by the Tm of the lipid components comprising the mixture.7,18,23-28 Fluorescence microscopy (FM) can be used to directly observe phase segregation by adding a fluorescently labeled lipid species which is soluble in a distinct lipid domain or by incorporating a fluorescence probe which has a specific lipid environmentdependent fluorescence response.23-29 Near field scanning optical microscopy (NSOM) has also been used to investigate phase segregated lipid films.30-32 NSOM can obtain topographic information using a fluorescent probe with sub-wavelength resolution, a distinct advantage over conventional fluorescence microscopy. Atomic force microscopy (AFM) has also been used to measure lipid domain growth and topology by examining variations in membrane thickness and frictional forces associated with the gel and l.c. domains.28,33-39 Fluorescence microscopy of large unilamellar vesicles and PSLBs reveals that the gel and l.c. domains in both leaflets of the bilayer are registered with each other. However, domain mismatch on length scales smaller than the optical resolution of the method (∼0.3 µm) cannot be ascertained. The “idea” of domain parity has important implications for cellular membranes where lipid rafts of different compositions are believed to exist on the cytosolic and extra-cellular leaflets of the membrane.40 Viral budding and intra- and extra-cellular vesicle fusion are also believed to require asymmetric sorting of lipid components
10.1021/jp0690547 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/20/2007
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in the membrane.40,41 Not surprisingly, the extent of lipid domain correlation has been the subject of some debate.41 Dislocated phase segregated domains have been prepared in PSLBs when the two leaflets of the bilayer are assembled via the Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS) method at a point in the phase diagram where domain segregation is present.19 Such films show domain disparity between the leaflets, however, when the samples are heated to a homogeneous l.c. state and then cooled the domains reform with complete parity between the two layers. Recent AFM studies have also investigated domain parity by examining changes in the thickness of the membrane film.28,33 These studies suggest that asymmetric lipid domains are present in phase segregated planer supported lipid bilayers. Giving credence to these findings are the results of nearest-neighbor recognition studies which suggested that asymmetry in fluid lipid membranes can be driven by differences in lipid chain lengths producing complementary structures in the two leaflets of the bilayer.42-44 These findings have also been supported by recent molecular dynamics calculations which show complimentary matching within segregated lipid bilayers.45 Few techniques can be used to directly measure the distribution of lipids or lipid domains between the two layers of a bilayer. One technique which is exquisitely sensitive to the molecular asymmetries of a lipid bilayer is sum-frequency vibrational spectroscopy (SFVS). SFVS is a nonlinear spectroscopy which is inherently sensitive to the arrangement of molecular species at an interface. SFVS is performed by overlapping both spatially and temporally a visible and tunable IR coherent light source at an interface where they combine to produce a photon at the sum of the incident frequencies. The sum-frequency intensity (ISF) is proportional to the square of the susceptibility tensor χ(2)46,47
ISF ∝ |f˜SFfVisfIRχ(2)|2
(1)
where ˜fSF and fVis, fIR are the nonlinear and linear Fresnel coefficients for the generated SF and incident electric fields respectively.47,48 In the above equation χ(2) is the sum of a resonant (R) and nonresonant (NR) contributions given by
χ(2) ) χNR + χR
(2)
with the resonant contribution defined as
χR ) N
(
m
∑ x)1 ω
υx
〈AkMij〉x
)
-ωir - iΓυx
(3)
where N is the population density of molecules at the interface, m is the number of vibrational transitions, Ak and Mij are the IR and Raman transition probabilities respectively, ωir is the frequency of the input IR field, ωυx is the normal mode vibrational frequency of the transition being probed, and Γυ is the line width of the transition. A SFVS spectrum is obtained by tuning the IR frequency through the vibrational resonances of the molecules at the interface and measuring the intensity of the generated sum-frequency light. The symmetry constrains on χ(2) make SFVS forbidden in a medium with inversion symmetry, such as the bulk of most liquids and solids. SFVS is only active at an interface or surface where the inversion symmetry of the bulk is broken. This restriction makes SFVS the perfect spectroscopic technique for
probing molecules on a surface which cannot be obtained using traditional vibrational spectroscopies such as IR and Raman scattering.49 The susceptibility element χ(2) is also directly related to the sum of the individual molecular hyperpolarizabilities, βijk, through a statistical average over all molecular orientations
χ(2) )
N 〈β 〉 0 ijk
(4)
In addition to the interfacial symmetry requirements of SFVS, the symmetry of the molecular species comprising the interface also dictates the observed SFVS response, as illustrated in eq 4. For example, we have previously shown that the terminal CH3 groups of the lipid acyl chains can be used as an intrinsic probe of the symmetry of the bilayer.50 For a symmetric lipid bilayer, cancellation of the methyl symmetric stretch (CH3 Vs) transition dipoles from the termini of the lipid acyl chains in the upper and lower leaflets will occur resulting in a reduction in the measured SFVS CH3 Vs oscillator strength. An increase in membrane asymmetry will result in an increase in the intensity of the CH3 Vs as the local symmetry is relaxed.50 The intrinsic symmetry constraints on SFVS have been exploited here to measure the asymmetric distribution of lipid domains in the proximal and distal layers of a PSLB in the gell.c. coexistence region of the phase diagram for several lipid systems. Four saturated phospholipids, 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC), 1,2-diheptadecanoyl-sn-glycero-3phosphocholine (DHPC), and 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC), and two equal-molar binary mixtures of DSPC with DMPC, and DSPC with a unsaturated lipid DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) were investigated. These studies add new support for the presence of lipid asymmetry in phase segregated phospholipid bilayers and provide a spectroscopic tool for measuring the Tm of a PSLB membrane. Experimental Section Materials. DMPC, DPPC, DHPC, DSPC, 1,2-distearoyl-D70sn-glycero-3-phosphocholine-1,1,2,2-D4-N,N,N-trimethyl-D9 (DSPC-d83), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and the ammonium salt of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-DOPE) were purchased from Avanti Polar-Lipids. Methyltrimethoxysilane (TMMOS; 98%) was obtained from Sigma-Aldrich. D2O (99.9%) was purchased from Cambridge Isotope Laboratories. Gas chromatography grade CHCl3 was supplied from Mallinckrodt. All materials were used as received. The water used in these studies was obtained from a Nanopure Infinity Ultrapure water system with a minimum resistivity of 18.2 MΩ-cm. An IR/UV grade fused silica hemicylindrical prism (Almaz Optics, Marlton, NJ) was used as the substrate for all the SFVS experiments. Preparation of Symmetric Lipid Bilayers. Symmetric lipid bilayers were prepared on the surface of a fused silica prism using the LB/LS method.2 The prism and SFVS cell were cleaned by submerging them in a solution of 70% 18 M sulfuric acid and 30% H2O2 for at least 4 h and then rinsed with copious amounts of Nanopure water. The prism was then plasma cleaned (Harrick PDC-326) in Ar for 3 min just prior to use. A KSV Minitrough was used for the LB/LS film depositions. The lipids were dissolved in chloroform at a concentration of 1 mg/mL and spread at the water-air interface. The first layer was
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Figure 1. Pressure-area isotherms of DMPC (5 °C), DPPC, DHPC, and DSPC (22 °C).
deposited on the prism via a vertical pull from the aqueous subphase into air (LB transfer). The second layer was deposited by a horizontal dip into the subphase (LS transfer). The LB/LS film transfers were carried out at a surface pressure of 30 mN/ m, corresponding to 42 ( 1.1, 44 ( 1.5, 45 ( 1.0, and 46 ( 1.3 Å2/molecule for DMPC, DPPC, DHPC, and DSPC respectively. All depositions were carried out at 22 °C except for DMPC which was preformed at 5 °C. The pressure-area isotherms for the four lipids used in this study are presented in Figure 1. Preparation of a Hydrophobic Silica Surface and Hybrid Lipid Bilayer. A silica prism was cleaned in a solution of 70% 18 M sulfuric acid and 30% H2O2 and rinsed thoroughly by nanopure water. The prism was dried at room temperature, and treated with a 2% (v/v) TMMOS toluene solution for 24 h to obtain a hydrophobic surface. The prism was then rinsed with toluene and chloroform and dried in an oven at 120 °C for 2 h. A static contact angle with water was measured to be 140° for the freshly prepared TMMOS surface. A small drop of water was placed onto the TMMOS-coated substrate, an image was taken by a digital camera, and the contact angle was determined using the free image processing software Scion Image (http:// www.scioncorp.com/frames/fr_scion_products.htm). A monolayer of DSPC was then deposited on the hydrophobic surface prism by a horizontal dip into the subphase using the LS method. The LS film transfer was carried out at a surface pressure of 30 mN/m. Fluorescence Microscopy. Fluorescence images of the lipid bilayers were collected on an Olympus BX40 microscope with epi-illumination using a 40X-0.60 numerical aperture (NA) objective. Fluorescence micrographs were obtained by doping the PSLBs with 0.5 mol % Rh-DOPE. SFVS Experiments. Information on the SFVS instrument, experimental setup, and SFVS sample cell can be found elsewhere.51 Temperature control of the cell was accomplished by heating the Teflon cell block with an external circulating water bath (HAAKE PHOENIX II, Thermo Electron Corporation). A type K thermocouple with a resolution of 0.05 °C and an accuracy of 0.2 °C was used to measure the temperature of the solution above the bilayer. SFVS spectra were collected with s-polarized sum-frequency, s-polarized visible, and p-polarized IR (ssp). Data points were collected every 2 cm-1 by averaging 100 laser pulses. For the temperature scans, the temperature was increased at a rate of 0.24 °C/min unless otherwise stated. The data were obtained by continuously monitoring the CH3 Vs intensity at 2876 cm-1 with ssp. The CH3 Vs intensity was averaged for 5 s (50 laser pulses).
Figure 2. SFVS spectra of symmetric DSPC, DHPC, DPPC, and DMPC bilayers obtained below, near, and above the Tm for each lipid.
Results and Discussion Lipid Bilayer Structure. The SFVS spectra of DMPC, DPPC, DHPC, and DSPC lipid bilayers were taken below, near and above the Tm for each lipid (Figure 2) using the ssp polarization combination which probes transition normal to the surface. Five CH vibrational stretching modes are observed in the frequency region of 2800-3050 cm-1. The frequencies at 2848, 2876, and 2935 cm-1 are assigned to the CH2 symmetric stretch (Vs), CH3 Vs and CH3 Fermi resonance (FR) from the fatty acid chains, respectively.14,17,52 The peak centered at 2974 cm-1 is a combination band of the CH3 antisymmetric stretch (Vas) at 2960 cm-1 from the fatty acid chains and the CH3 Vs at 2975 cm-1 originating from the choline head group. These vibrational assignments have been identified previously.53 At all temperatures, a SFVS spectrum is obtained from the symmetric bilayers. However, it is important to note that the magnitude of the SFVS signal is an order of magnitude smaller than that measured for an asymmetric bilayer, in which one leaflet is perdeuterated.51,53 The small but measurable SFVS signal measured from the symmetric bilayer suggests that there is a small local break in symmetry. Since the lower layer is supported on a fused silica surface and the upper layer is in contact with D2O, a dielectric disparity between the top and bottom layers of the bilayer exists. This disparity breaks the “pure” symmetry of the PSLB, which most likely gives rise to the measured SFVS intensity. It is also possible that defects in the film introduced during the deposition process give rise to the measured intensity. However, we have previously shown that the structure of the lipids in the distal and proximal leaflets is identical with regards to the orientation of the headgroup and tail. In addition, the gauche defect content as measured by SFVS is nearly identical for both leaflets.53
Asymmetric Distribution of Lipids
Figure 3. Schematic representation of an all-trans (a), single gauche defects (b), adjacent gauche defects (c), and nonadjacent gauche defects (d). Also shown are the transition moments of the CH2 Vs.
In order to interpret the spectra in Figure 2, the symmetry of the lipid acyl chains must be considered. For an all-trans conformation of the alkyl chains (Figure 3a), the SFVS CH2 Vs mode will not be seen due to the cancellation of the methylene transition dipole moments.54 There is a decrease in the CH2 Vs intensity with increasing temperature for all the lipids examined, which is counter to the expectation that the l.c. state should contain more gauche defects than the gel state as previously demonstrated by NMR, IR, and Raman studies of similar systems.11,15,16,55-60 However, the reduction in the CH2 Vs measured in our study is consistent with previous SFVS studies of lipid monolayers at the CCl4/D2O interface in which a lower CH2 Vs intensity for DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine, Tm ) -1 °C) in the l.c. phase was observed compared with that of DSPC in the gel phase.61 The lower CH2 Vs intensity of DLPC was interpreted as a higher degree of interfacial order (less gauche defects) due to the greater interfacial concentration of DLPC versus DSPC. It was concluded by the authors that longer chain phosphocholine lipids form more disordered monolayers than shorter chain lipids at equivalent interfacial coverages.61 This explanation is obviously in conflict with previous studies of lipid chain ordering conducted by NMR, IR, and Raman.11,15,16,55-60 It is important to note that this type of behavior is not observed in SFVS studies of monolayers of single chain amphiphiles in which an increase in the CH2 Vs intensity is seen with increasing temperature.54,62,63 Compared to the more loosely packed single chain amphiphiles, the close proximity of the acyl chains in lipids will influence the structure of these assemblies. The number of gauche defects and the geometric relationship between the gauche defects in the acyl chains of the lipid will also have a dramatic effect on the SFVS signal observed from the CH2 Vs resonance. In the gel phase, the alkyl chains of a pure lipid bilayer are tightly packed in an all-trans conformation with the lipids oriented along the surface normal.53 As the temperature of the system approaches the Tm, gauche conformers are introduced into the alkyl chains. Initially, a single gauche defect is present located near the terminus of the alkyl chains, as shown in Figure 3b.57 As more energy is supplied to the system, the gauche conformers propagate further along the chain. NMR studies have shown that the gauche defects adopt a pairwise conformation, Figure 3c, in order to reduce the lateral pressure
J. Phys. Chem. C, Vol. 111, No. 25, 2007 8991 in the film and maintain the orientation of the acyl chains along the surface normal.57 If a single gauche defect is introduced, as illustrated in Figure 3b, the all-trans conformation is broken resulting in an observable CH2 Vs resonance by SFVS. If two adjacent gauche defects, g+ and g- or g- and g+ (g+ and g- refer to the rotation of carbon-carbon bonds in the gauche conformers as illustrated in Figure 3c57) are present in the alkyl chains no SFVS signal from the CH2Vs mode should be observed for the ssp polarization combination due to cancellation of the normal components of the transition dipole moments of the adjacent methylene groups. However, if two nonadjacent gauche defects are introduced into the alkyl chain, such as the g+ and g+ or g- and g- pairs shown in Figure 3d, the CH2 Vs intensity will be larger than that of a single gauche defect. NMR studies indicate that pairwise gauche conformations are likely to occur as the alkyl chains melts, resulting in a decrease in the SFVS CH2 Vs intensity as seen in Figure 2. In addition, the smaller CH2 Vs intensity of DLPC in the l.c. phase compared to that of DSPC in the gel phase measured in the study by Walker et al.61 is more likely due to the formation of pairwise gauche conformers in the alkyl chains rather than a decrease in the number of gauche defects. Therefore, the conclusion made by Walker et al. that longer chain lipids have a more disordered conformation than shorter chain lipids, may not be entirely accurate. It is important to stress that a comparison with linear spectroscopic methods, such as IR, Raman, or NMR is needed to fully explain the structure of the lipid acyl chains. By examining the SFVS results in conjunction with IR, Raman, and NMR data, the alkyl chain structure of DMPC, DPPC, DHPC, and DSPC PSLBs in the gel and l.c. phases (Figure 2) can be summarized as follows: In the gel phase, the alkyl chains are predominantly in an all-trans conformation as demonstrated by NMR, FTIR, and Raman. A small CH2 Vs intensity is measured by SFVS, which is consistent with the nearly alltrans conformation of the alkyl chains. In the l.c. phase, previous NMR studies indicate there are more gauche defects (3-6% gauche defects).57 However, a decrease in the CH2 Vs intensity in the l.c. phase is observed by SFVS. The observed decrease in the CH2 Vs measured can be reconciled with the NMR data if the presence of pairwise gauche conformers is considered. It is important to note that care must be taken when using SFVS alone for the determination of the gauche content in lipid systems as the technique is not capable of distinguishing between the all-trans conformation and pairwise gauche conformers. Asymmetric Distribution of Lipid Domains at the Tm. In addition to the changes in the CH2 Vs intensity with temperature, the most dramatic change in the SFVS spectra of DMPC, DPPC, DHPC, and DSPC is observed for the CH3 Vs at 2876 cm-1. For temperatures below the Tm, the CH3 Vs is relatively modest. However, as the temperature is increased to the Tm a marked increase is observed. Above the Tm, the measured intensity returns to that measured below the Tm or drops slightly lower. We have demonstrated previously that the methyl resonance is extremely sensitive to the symmetry of a PSLB.50 The nearly normal orientation of the CH3 Vs transition moment coupled with the antiparallel arrangement of the lipids in the bilayer makes the CH3 resonances sensitive to the compositional and structural asymmetries of the membrane. This sensitivity to the membrane asymmetry can be described using eq 5, in which the secondorder hyperpolarizability is expressed in terms of the molecular
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Figure 4. Representation of gel (black) to l.c. (gray) phase transition illustrating (a) domain dislocation and (b) domain size disparity which could give rise to membrane asymmetry.
hyperpolarizability (β) and the orientation averages for the CH3 Vs in the distal and proximal leaflets of the bilayer
χ(2) )
Ndistal CH3νs Nproximal CH3νs 〈βijk 〉 〈βijk 〉 0 0
(5)
The following convention was used to determine the sign of the molecular hyperpolarizabilities in eq 5. Assuming an isotropic distribution of lipids in the surface plane, angles of the CH3 Vs transition moment ranging from 0° to 90° to 180° are negative. (Note: the signs of the hyperpolarizabilities are arbitrary and may not reflect their true nature.) For a bilayer in which the composition and structure are identical in both leaflets of the bilayer, the summation over all possible orientations of the CH3 Vs in eq 5 returns a net zero macroscopic hyperpolarizability or a minimum in the CH3 Vs resonance as measured by SFVS. This condition is exactly what is expected for a PSLB below the Tm. In this temperature range, the membrane is in the gel state characterized by a well-ordered, all-trans arrangement of the lipid acyl chains. Near complete interference of the terminal methyl symmetric stretch is observed due to the antiparallel orientation of the transition dipole moments of the CH3 Vs in this homogeneous phase. As the membrane passes through the gel to l.c. phase transition, there is a marked increase in the CH3 Vs intensity. At the Tm, there is a coexistence of both gel and l.c. domains in the membrane. Discontinuities between the gel and l.c. domains in the two layers of the bilayer (illustrated in Figure 4a) could give rise to an increase in the CH3 Vs resonance. Another possible mechanism for the introduction of membrane asymmetry is that the two layers of the bilayer undergo the gel to l.c. phase transition separately, Figure 4b, which has been suggested by recent DSC measurements of a PSLB on a mica surface.35 Such an arrangement will also break the local symmetry of the bilayer and increase the SFVS signal. Domain discontinues will result in both structural and compositional asymmetry between the two leaflets of the bilayer. The structural asymmetries are due to the difference in the acyl chain conformations between the gel (all-trans) and l.c. (gauche) phases. In conjunction with the structural changes, the composition of the phases is also different. The packing density of the
lipids in the gel state (∼45 Å2/molelcue) is greater than that found in the more fluid l.c. phase (∼54-62 Å2/molecule).64,65 Changes in the mean molecular area will affect the local values of Ndistal and Nproximal in eq 5, which are averaged over the coherence length of the measurement, which is on the order of several hundred nanometers. In addition, the combination of structural and compositional difference also affects the relative permittivity () of each phase. The permittivity of the l.c. phase is greater than that of the gel phase due to the more fluid nature of the lipids, changes in packing density, the reorientation of the headgroup and water penetration into the membrane.66 Misalignment of the gel and l.c. domains would therefore give rise to a dielectric discontinuity in the membrane. As the CH3 Vs oscillator strength and frequency are sensitive to its local environment, change in the surrounding dielectric would give rise to a change in β, reflecting a change both in the structure and packing density between the two layers of the bilayer. The mobility of the terminal CH3 group will also affect the measured CH3 resonance. In the gel state, the average angular distribution is smaller than that in the l.c. state.57 Equation 5 shows that the orientation distribution will also scale the effective molecular hyperpolarizabilities. The disparity in the mean and distribution of the orientation angles of the CH3 Vs between the gel and l.c. phases will therefore give rise to an asymmetry in the membrane which can be detected by SFVS. As the DMPC, DPPC, DHPC, and DSPC bilayers are heated above their respective transition temperatures, the CH3 Vs intensity decreases in all cases. Based on the arguments presented above, this is exactly what would be expected for a completely homogeneous l.c. phase in both leaflets of the bilayer. The slightly lower CH3 Vs observed for the l.c. phase versus the gel phase may be due to the more rapid diffusion of the lipid species which will average out any local structural inhomogenaeties between the distal and proximal leaflets on the time scale of the experiment. In addition, the added thermal energy increases the motion of the lipid chain terminus57 which will result in a boarder distribution of orientation angles for the CH3. The increased distribution width will decrease the normal component of the CH3 resonance, leading to a reduction in signal intensity.54,63 This behavior has been seen in monolayers of lipids,61,67 as will be discussed below. The CH3 Vs intensity from the fatty acid chains was measured continuously as a function of temperature for the four lipids,
Asymmetric Distribution of Lipids
Figure 5. CH3 Vs (2876 cm-1) intensity as a function of temperature for a DMPC (Tm ) 23.4 °C), DPPC (Tm ) 41.0 °C), DHPC (Tm ) 52.4 °C), and DSPC (Tm ) 57.8 °C), and a DOPC bilayer (top) and the SFVS spectrum of a DOPC bilayer (bottom).
DMPC, DPPC, DHPC, and DSPC (Figure 5). Above a scan rate of 0.24 °C/min, an irreproducible temperature-dependent response was observed, presumably due to the time required for the lipid film and domain structures to reach equilibrium in the sample. No variation in the temperature-dependent SFVS data was observed for scan rates at or below 0.24 °C/min. A scan rate of 0.24 °C/min was used to obtain the data shown in Figure 5 which allowed for an optimal acquisition time and sample equilibrium to be reached. Maxima in the CH3 Vs intensity are observed at 23.4 ( 0.9, 41.0 ( 0.4, 52.4 ( 0.7 and 57.5 ( 0.5 °C for DMPC, DPPC, DHPC, and DSPC, respectively. These values are correlated with the Tm values obtained by DSC, 23.6 ( 1.5, 41.3 ( 1.8, 49 ( 3, and 54.5 ( 1.5 °C for DMPC, DPPC, DHPC, and DSPC, respectively.5,68-70 The broad response observed in the temperature-dependent CH3 Vs signal is not due to instrumental error but rather reflects the change in membrane asymmetry as a function of temperature, suggesting that phase segregated domains are present before and after the Tm. This observation can be supported by fluorescence microscopy results which show the presence of lipid domains well before and after the onset of the lipid phase transition.71,72 Similar changes are also seen by dielectric spectroscopy, where the permittivity of lipid vesicles changes over a much wider range (5-10 °C) than typically measured by DSC.73 As a control experiment, the CH3 Vs intensity was also measured continuously as a function of temperature for an unsaturated phospholipid DOPC shown in Figure 5a. The SFVS spectrum of the DOPC bilayer was also recorded at 22 °C and is shown in Figure 5b. No change in the CH3 Vs intensity is observed for the DOPC bilayer in the temperature range of 2470 °C. Because the phase transition temperature of DOPC (Tm ) -18 °C)74 does not lie within the temperature range examined and no phase segregation occurs, the SFVS intensity of the CH3
J. Phys. Chem. C, Vol. 111, No. 25, 2007 8993 Vs does not change with temperature. These results strongly suggest that the CH3 Vs intensity does measure membrane asymmetry associated with domain segregation and mismatch between the leaflets of the bilayer near the Tm. In order to correlate the changes in the SFVS spectra with the phase behavior of the lipid bilayer, fluorescence images of a pure DPPC bilayer labeled with 0.5 mol % Rh-DOPE recorded below, near, and above the Tm were obtained (Figure 6). Below the Tm of DPPC (T ) 24 °C), a uniform gel state is observed (Figure 6a). Rh-DOPE is not entirely miscible in the ) ∼6 °C)75 of DPPC resulting in segregation gel phase (TDOPE m of the lipid probe into small domains which are visible in the fluorescence image (Figure 6a). The CH3 Vs intensity at this same temperature is weak, consistent with DPPC residing in a single homogeneous gel phase. Figure 6b shows the fluorescence image of DPPC at 40 °C which is near the Tm. Bright spots, due to the incorporation of the Rh-DOPE probe into the l.c. phase and gel phase (dark background) domains, are visible. Near the Tm, a maximum in the SFVS intensity is observed (Figure 5), which is correlated, to the phase segregation of the gel and l.c. domains observed by fluorescence microscopy. It should be noted that domain mismatch is not visible in the fluorescence images due to the limited optical resolution of ∼480 nm (based on the image magnification of 40×, 0.6 NA and a detected wavelength of 580 nm). At T ) 50 °C (Figure 6c), a uniform fluorescence image is obtained indicating that DPPC is in a homogeneous l.c. phase. Above the Tm, the lowest SFVS intensity is observed which is consistent with the homogeneous l.c. phase seen in the fluorescence image of Figure 6c. The fluorescence micrographs provide visual evidence that phase segregation of the gel and l.c. phase domains is correlated to the rise to the SFVS increase of the CH3 Vs resonance at the phase transition. However, these experiments cannot address the role of domain dislocations on the observed SFVS response due to the inability to observe such structures by fluorescence microscopy. Although not directly observable by fluorescence microscopy, the extent of membrane asymmetry can be estimated from the change in the CH3 Vs intensity. Figure 7 shows the square root of the CH3 Vs intensity as a function of temperature for an asymmetric lipid bilayer of DSPC/DSPC-d83 and a symmetric lipid bilayer composed of premixed 1:1 mole ratio of DSPCDSPC-d83. The intensities of the asymmetric and symmetric lipid bilayers have been normalized with respect to the maximum CH3 Vs intensity from the asymmetric bilayer for comparison. The maximum intensity for the asymmetric bilayer is measured at 25 °C.76 As the temperature is increased, membrane asymmetry decreases due to lipid flip-flop and the CH3 Vs intensity decreases to a minimum just before the Tm is reached, at which point complete mixing of the distal and proximal leaflets has occurred.76 At the Tm (54.8 °C), an increase in the CH3 Vs intensity is observed due to the dislocation of the gel and l.c. domains between the two leaflets of the bilayer as discussed previously. The lower Tm measured for the mixed DSPC + DSPC-d83 bilayer compared to pure DSPC is a consequence of the perdeuterated DSPC.76 The intensity of the asymmetric lipid bilayer is the same as that of the symmetric lipid bilayer at the Tm, indicating that DSPC and DSPC-d83 molecules have symmetrically distributed in the bilayer, but the gel and l.c. phase domains are asymmetrically distributed between the two leaflets. Above the Tm, the intensity of the initially prepared symmetric and asymmetric bilayers is identical, consistent with the asymmetric bilayer undergoing complete lipid exchange to produce a symmetric bilayer.
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Figure 6. Fluorescence images of a DPPC bilayer labeled with 0.5 mol % Rh-DOPE recorded below, near and above the Tm of DPPC (Tm ) 41.3 ( 1.8 °C).
Figure 7. CH3 Vs intensity as a function of temperature for an asymmetric lipid bilayer of DSPC/DSPC-d83 (black) and a symmetric lipid bilayer of premixed 1:1 DSPC-DSPC-d83 (gray). The inset shows the extent of overlap of two hypothetical domains based at the CH3 Vs intensity measured at the Tm.
Since the SFVS intensity is directly proportional to the square of the population difference of lipids in the distal and proximal leaflets, eqs 1 and 5, the percent asymmetry (% AS) between the two leaflets of the bilayer at the Tm can be calculated using the following equation:
xI % AS )
CH3νs Tm
ν xICH max
× 100%
(6)
3 s
3νs where ICH max is the maximum CH3 Vs intensity measured at 25 CH3νs °C and ITm is the intensity measured at the Tm. Using the data presented in Figure 7, the % AS is 27% for DSPC at the phase transition. The previously reported domain size in similar phase segregated lipid films ranges from 0.1 to 1.0 µm in diamater.29-32,39,77,78 Assuming an ideal circular domain with 1 µm diameter, the upper limit on the nonoverlapping area, or asymmetry, is about 0.2 µm2 with a distance of ∼300 nm from one domain boundary to the other (see Figure 7). Based on this simplistic model of domain mismatch, the dislocation would not be visible by fluorescence microscopy. It is important to note, that domain mismatch could be more nonuniform in nature and may even involve the mismatch of patches within the large scale domains observed by fluorescence microscopy. Structure of a Hybrid Lipid Bilayer. In the absence of direct visual evidence of domain mismatch, the temperature-dependent
Figure 8. Schematic of the molecular arrangement of the lipid monolayer.
SFVS response of a hybrid lipid bilayer was used to support the hypothesis that the local break in symmetry of the pure bilayer due to the dislocation of gel and l.c. domains is the source of the increase in the CH3 Vs intensity near the Tm. A hybrid lipid bilayer, composed of a DSPC monolayer and a TMMOS layer covalently attached to the silica substrate, was prepared (see Figure 8). As there is only a monolayer of lipid, dislocation of the gel and l.c. domains is not possible. Figure 9a shows the SFVS spectra of a DSPC monolayer at 23, 55, and 65 °C. Five CH vibrational modes are observed between 2800 and 3050 cm-1. The peaks at 2876, 2903, and 2936 cm-1 can be assigned to the CH3 Vs, CH2 FR, and CH3 FR, respectively.14,17,52 The peaks at 2854 and 2955 cm-1 are assigned to CH2 Vs and CH3 Vas, respectively.14,17,52 These frequencies are identical to those observed from a symmetric DSPC bilayer discussed previously. In order to examine the effect of TMMOS on the spectrum of DSPC, especially at the CH2 Vs and CH3 Vs frequencies, the spectrum of the SiO2/ TMMOS/air interface was obtained and is shown in Figure 9b. Only two small peaks at 2910 and 2970 cm-1 are observed which are assigned to the CH3 Vs and CH3 FR, respectively.79 This experiment confirms there is no spectral interference resulting from the underlying TMMOS layer on the spectra of DSPC, particularly in the frequency range of 2800-2900 cm-1.
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Figure 9. (a) SFVS spectra of a DSPC monolayer on a TMMOS treated surface recorded at 23, 55, and 65 °C. (b) SFVS spectrum of the air/TMMOS/SiO2 interface recorded at 23 °C.
In Figure 9a, the intensity of the CH3 Vs is large and then decreases as the temperature is increased. This observation is in contrast to the result seen for a symmetric DSPC PSLB where the largest SFVS signal is observed at the Tm. However, the intensity of the CH2 Vs decreases as the temperature increases which is consistent with the symmetric bilayer system. In order to investigate the temperature dependence of the CH3 Vs at 2876 cm-1 and CH2 Vs at 2854 cm-1, the SFVS intensities at these frequencies were continuously recorded in the temperature range of 38-65 °C and are shown in Figure 10a. The CH3 Vs intensity does not change in the range of 38-56 °C. Between 56 and 60 °C, a sharp reduction in the CH3 Vs intensity is observed. A similar reduction has been seen for related monolayer systems by SFVS and attributed to an increase in the orientational disorder of the terminal methyl group.54,63 For temperatures above 58 °C, the intensity of the CH3 Vs remains constant. In order to determine the temperature region in which the maximum change in the SFVS signal occurs, the first derivative of the CH3 Vs response was computed and is shown in Figure 10b. A minimum in the derivative is seen at 58.6 °C, and this correlates extremely well with the Tm of DSPC as measured by DSC.5 For comparison, the CH3 Vs intensity as a function of temperature for a symmetric DSPC bilayer (Figure 5) is shown in Figure 10c. The maximum intensity observed for the CH3 Vs in the DSPC bilayer correlates extremely well with the first derivative of the monolayer response (58.6 °C), indicating that the largest change in the SFVS response is observed at the Tm in both systems. Unlike the hybrid bilayer, the pure DSPC bilayer has little SFVS response below and above the Tm, illustrating the cancellation effect of the CH3 transition moments. These results support the hypothesis that the large intensity variation observed in the pure bilayers of DMPC, DPPC, DHPC, and DSPC at the Tm is due to the dislocation of the gel and l.c. domains between the top and bottom layers of the bilayer, resulting in a local break in the symmetry of the bilayer. In addition to changes in the CH3 Vs intensity with temperature, the intensity of the CH2 Vs at 2854 cm-1 was also monitored in the temperature range of 38-65 °C (Figure 10a) for the hybrid bilayer. The CH2 Vs intensity is weak with little change from 38 to 53 °C. When the temperature is increased to
Figure 10. (a) Change in the CH3 Vs intensity (forward temperature scan in black and reverse scan in gray), CH2 Vs intensity (black), and CH3 Vs/CH2 Vs intensity ratio (gray) for a DSPC monolayer deposited on a TMMOS/SiO2 surface. (b) The first derivative of the CH3 Vs intensity from the DPSC monolayer as a function of temperature. (c) Change in the CH3 Vs intensity, CH2 Vs intensity, and CH3 Vs/CH2 Vs intensity ratio (gray) for a DSPC bilayer deposited on SiO2.
53 °C, a decrease in the CH2 Vs resonance is observed. Upon increasing the temperature further from 55 to 65 °C, the CH2 Vs intensity does not change and stays at a minimum. For comparison, the CH2 Vs intensity from a pure DSPC bilayer was monitored in the temperature range of 38 to 65 °C (Figure 10c). A minimum CH2 Vs intensity is also observed above the Tm. The decrease in the CH2 Vs intensity above the Tm in both the hybrid and pure bilayers is not in agreement with the results of NMR, FTIR, and Raman which measured more gauche defects in the fatty acid chains with increasing temperature.11,15,16,55-60 Hence, the decrease of the CH2 Vs intensity is most likely due to the formation of gauche defect pairs in the lipids as discussed previously. The CH2 Vs intensity measured for the monolayer is larger by 0.7 a.u. compared to the bilayer, suggesting that there is also cancellation of the CH2 Vs transition dipoles between the proximal and distal leaflets of the bilayer, in addition to cancellation within the alkyl chain. The ratio of the CH3 Vs/CH2 Vs intensities has been used previously to determine the conformation of the alkyl chain and the Tm of Langmuir monolayers by SFVS.54,61-63,80-82 For example, the CH3 Vs/CH2 Vs intensity ratio was previously used to measure the Tm of octadecylamine (ODA) at the liquid/vapor interface by SFVS.63 A significant decrease in the CH3 Vs/CH2 Vs ratio was observed at the Tm of ODA.63 For comparison, the CH3 Vs/CH2 Vs intensity ratios for the hybrid and pure DSPC bilayers as a function of temperature are shown in panels a and c of Figure 10, respectively. A CH3 Vs/CH2 Vs maximum is seen near the Tm of DSPC for both the symmetric DSPC bilayer and
8996 J. Phys. Chem. C, Vol. 111, No. 25, 2007 the hybrid bilayer. These results are contrary to those observed for ODA at the air/water interface. This disparity is most likely due to the difference in molecular structures of ODA versus DSPC. ODA has a single alkyl chain, whereas lipids are composed of two alkyl chains. The close proximity of the alkyl chains in the lipid affects the structure of these assemblies compared to the more loosely packed single chain amphiphiles. One consequence of this structural difference, as discussed previously, is the fact that the lipid alkyl chains easily form pairwise gauche conformers in the l.c. phase as demonstrated by NMR.57 These conformers are silent in SFVS. In addition, there appears to be cancellation of the CH2 Vs transition dipole moments between the two leaflets of the bilayer. Hence, the CH3 Vs/CH2 Vs intensity ratio cannot be used to measure the Tm for lipid system by SFVS as the change in the CH2 Vs intensity with temperature is not correlated to the change in the relative number of gauche defects in the alkyl chains. It is noted that the sharp reduction in the CH3 Vs intensity measured for the hybrid bilayer (Figure 10a) could be caused by desorption of the lipid film from the surface or possibly lipid inversion. In order to investigate these possibilities, the CH3 Vs intensity as a function of temperature was monitored from 38 to 65 °C, and then from 65 to 38 °C. A nearly reversible change in the CH3 Vs intensity with temperature is observed (see Figure 10a). For either lipid desorption or inversion the CH3 Vs intensity would not be reversible with temperature. The measured decrease in the CH3 Vs is therefore, most likely due to an increase in the orientational disorder of the terminal methyl groups with increasing temperature.54,63 Phase Behavior of Binary Lipid Mixtures. The phase behavior of the binary mixtures DSPC + DOPC and DSPC + DMPC were investigated to further support the hypothesis that gel and l.c. domain dislocation between the two layers of the bilayer results in the measured increase in the CH3 Vs intensity. Figure 11a shows the intensity of the CH3 Vs at 2876 cm-1 as a function of temperature for the bilayers containing 1:1 mole ratios of DSPC + DOPC and DSPC + DMPC. For the bilayer composed of DSPC + DOPC, the maximum intensity in the CH3 Vs is observed at the lowest temperature examined, 10 °C, when DOPC is in the l.c. phase and DSPC is in the gel phase. The intensity decreases slightly until the temperature is increased to 42 °C. When the temperature is increased further from 42 to 50 °C, the intensity sharply decreases to a minimum value. No further changes in the intensity of the CH3 Vs are observed above 50 °C, further suggesting a homogeneous l.c. state in the binary mixture. In order to clearly see the change in the CH3 Vs intensity with temperature, the first derivative of the SFVS response was calculated (Figure 11b). A minimum value of 47.5 °C is observed in the derivative curve. This value corresponds with the Tm of 46 °C as measured by DSC for the same binary mixture.83 This indicates that the CH3 Vs intensity of the terminal alkyl chains is directly related to the phase behavior of the membrane which is consistent with the analysis of the single lipid component system. This result also supports the hypothesis that the dislocation of the gel and l.c. domains between the top and bottom layers breaks the local symmetry of lipid bilayer leading to an increase in the CH3 Vs measured by SFVS. For the bilayer composed of DSPC + DMPC, there is a small CH3 Vs intensity in the temperature range from 10 to 20 °C due to the fact that both lipids are in the gel state in this temperature range (Figure 11a). With increasing temperature, a large and broad SFVS signal is observed between 30 and 50 °C. The broad peak represents a continuously changing ratio of gel phase DSPC and l.c. DMPC. Upon increasing the temperature from 52 to
Liu and Conboy
Figure 11. (a) CH3 Vs intensity as a function of temperature for a premixed 1:1 mole ratio of DOPC + DSPC bilayer (gray) and a premixed 1:1 mole ratio of DMPC + DSPC bilayer (black). (b) The first derivative of the CH3 Vs intensity for DOPC + DSPC (gray) and DMPC + DSPC (black).
70 °C, the intensity decreases as both DSPC and DMPC are in the l.c. phase. Upon taking the first derivative of the CH3 Vs intensity with temperature, a maximum at 27.1 °C and a minimum at 50.5 °C are obtained (Figure 11b). The SFVS results correlate extremely well with published DSC results for the same system, which show a broad peak with maxima at 30 and 45 °C.37 These experiments demonstrate that the CH3 Vs intensity is very sensitive to the phase segregation in binary lipid mixtures. In order to visualize the phase segregation and support the SFVS observations, fluorescence images of a premixed 1:1 mole ratio of DOPC + DSPC bilayer labeled by 0.5 mol % of RhDOPE were taken at different temperatures and are illustrated in Figure 12. Phase segregation is clearly observed at 23 °C (Figure 12a) due to the coexistence of the immiscible gel (DSPC) and l.c. (DOPC) phases. The maximum in the SFVS CH3 Vs intensity is correlated to the segregation of the gel and l.c. domains. When the temperature is increased to 46 °C (the Tm as measured by SFVS and DSC)80 a coexistence of DSPC in the gel phase and DOPC + DSPC in the l.c. phase is observed (Figure 12b). At 53 °C a nearly uniform l.c. phase with a small population of gel phase lipid is observed (Figure 12c). The disappearance of phase segregation in the bilayer is correlated to a decrease in the SFVS signal in this same temperature range (46-53 °C). At 60 °C, the fluorescence image (Figure 12d) reveals a homogeneous l.c. phase. Correlated to the homogeneous l.c. phase is a minimum in the measured SFVS intensity. Although SFVS is not capable of directly visualizing membrane asymmetry in phase segregated lipid films, the symmetry constraints imposed on SFVS lead to the conclusion that such asymmetry must be present in the membrane in order for the
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Figure 12. Fluorescence images of a premixed 1:1 DOPC-DSPC bilayer labeled with 0.5 mol % DOPE-Rh at 23 (a), 46 (b), 53 (c), and 60 °C (d).
Figure 13. Representation of (a) domain mismatch with gel (black) and l.c. complimentary structures and (b) illustration of complete gel and l.c. domain parity.
changes in the CH3 Vs signal to be observed. One plausible explanation for the asymmetry measured by SFVS is the presence of complimentary lipid structures in the distal and proximal leaflets of the bilayer near the domain edges, as illustrated in Figure 13a. The gel and l.c. phases have different effective thickness in the membrane, with the l.c. state being slight more compressed. For example the thickness of a DPPC bilayer in the l.c. phase is 3.7 nm84 compared to 4.6 nm in the gel phase.85 Nearest-neighbor recognition measurements have suggested that lipids of different alkyl chain lengths adopt complementary structures in the membrane due to the more favorable hydrophobic interactions.42-44 Such a structure maximizes lipid-lipid hydrophilic interaction and minimizes lipidwater interaction by reducing the amount of exposed lipid which lowers the interfacial energy between the gel and l.c. phases. This arrangement is thermodynamically more stable compared to the configuration in which the gel and l.c. domains have complete parity, Figure 13b. Complimentary matching within lipid bilayers, such as that illustrated in Figure 13a, has also been observed by molecular dynamics simulations.45
The SFVS results obtained here are consistent with the growing number of studies which suggest that lipid domains in the opposing leaflets of a bilayer may not possess complete registry. Although not capable of directly visualizing such structures, the SFVS results do provide strong evidence that complimentary structures are present in phase segregated lipid bilayers. Conclusions SFVS was used for the first time to directly measure the asymmetric distribution of lipid domains in phase segregated PSLBs. The destructive interference of the symmetric stretch transition moments from the fatty acid methyl groups (CH3) was used to monitor changes in the symmetry of the bilayer structure. Four saturated phospholipids, DMPC, DPPC, DHPC, and DSPC, and two equal-molar binary mixtures of DSPC + DMPC and DSPC + DOPC were investigated. A maximum value of the CH3 Vs SFVS intensity was observed due to the break in the local symmetry of lipid bilayers as caused by the dislocation of the gel and l.c. phase domains at the Tm. The SFVS measured Tm for DMPC, DPPC, DHPC, and DSPC were 23.4 ( 0.9, 41.0 ( 0.4, 52.4 ( 0.7, and 57.5( 0.5 °C, respectively. These values correlate well with the literature values of 23.6 ( 1.5, 41.3 ( 1.8, 49 ( 3, and 54.5 ( 1.5 °C for DMPC, DPPC, DHPC, and DSPC, respectively obtained by DSC of lipid vesicles in solution.5,68-70 Two phase segregated binary lipid systems were also examined, DOPC + DSPC and DMPC + DSPC over an extended temperature range. A minimum in the first derivative of the CH3 Vs intensity as a
8998 J. Phys. Chem. C, Vol. 111, No. 25, 2007 function of temperature was observed at 47.5 °C for the binary mixture of DOPC + DSPC, whereas a maximum at 27.1 °C and a minimum at 50.5 °C for the DMPC + DSPC system were measured. These values correlate extremely well with the DSC results of 46 °C for the DOPC + DSPC, as well as 30 and 45 °C for DMPC + DSPC mixtures. The high degree of correlation between the SFVS spectroscopic measurements of phase segregation in these lipid systems and the previously reported DSC results suggests the Tm of these lipids is not significantly altered upon immobilization on a surface. Fluorescence microscopy was used to correlate the SFVS results with the state of the lipid bilayers. A direct correlation exists between the observed increase in the CH3 Vs intensity and the phase segregation within the membranes. Although SFVS is not capable of directly visualizing the membrane asymmetry at the phase transition, the symmetry constraints imposed on SFVS lead to the conclusion that such asymmetry must be present in the membrane in order for the changes in signal to be observed. These results therefore provide important evidence for the delocalization of the gel and l.c. domain structures between the two layers of lipid bilayers. Studies are being extended to investigate effects of cholesterol on the phase transitions and structures of lipid membranes, as well as the influence of proteins and other exogenous lipid components. Acknowledgment. This work was supported by funds from the National Institutes of Health (GM068120) and from the National Science Foundation (CHE 0515940). References and Notes (1) Sackmann, E. Science (Washington, DC) 1996, 271 (5245), 43-8. (2) Thompson, N. L.; Palmer, A. G., III. Comments Mol. Cell. Biophys. 1988, 5 (1), 39-56. (3) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47 (1), 10513. (4) Devlin, T. M., Ed.; Textbook of Biochemistry: with Clinical Correlations, 4th ed.; Wiley: New York, 1997. (5) Koynova, R.; Caffrey, M. Biochim. Biophys. Acta 1998, 1376 (1), 91-145. (6) Chapman, D.; Williams, R. M.; Ladbrooke, B. D. Chem. Phys. Lipids 1967, 1 (5), 445-75. (7) Wu, S. H. W.; McConnell, H. M. Biochemistry 1975, 14 (4), 84754. (8) Komura, S.; Shirotori, H.; Olmsted, P. D.; Andelman, D. Europhys. Lett. 2004, 67 (2), 321-327. (9) Borden, M. A.; Martinez, G. V.; Ricker, J.; Tsvetkova, N.; Longo, M.; Gillies, R. J.; Dayton, P. A.; Ferrara, K. W. Langmuir 2006, 22 (9), 4291-4297. (10) Rinia, H. A.; Boots, J.-W. P.; Rijkers, D. T. S.; Kik, R. A.; Snel, M. M. E.; Demel, R. A.; Killian, J. A.; Van der Eerden, J. P. J. M.; de Kruijff, B. Biochemistry 2002, 41 (8), 2814-2824. (11) Brown, K. G.; Peticolas, W. L.; Brown, E. Biochem. Biophys. Res. Commun. 1973, 54 (1), 358-64. (12) Smith, L. M.; Weis, R. M.; McConnell, H. M. Biophys. J. 1981, 36 (1), 73-91. (13) Von Tscharner, V.; McConnell, H. M. Biophys. J. 1981, 36 (2), 409-19. (14) Snyder, R. G.; Strauss, H. L.; Elliger, C. A. J. Phys. Chem. 1982, 86 (26), 5145-50. (15) Yan, W.-H.; Strauss, H. L.; Snyder, R. G. J. Phys. Chem. B 2000, 104 (17), 4229-4238. (16) Mendelsohn, R.; Moore, D. J. Chem. Phys. Lipids 1998, 96 (1-2), 141-57. (17) Tamm, L. K.; Tatulian, S. A. Q. ReV. Biophys. 1997, 30 (4), 365429. (18) Webb, S. J.; Greenaway, K.; Bayati, M.; Trembleau, L. Org. Biomol. Chem. 2006, 4 (12), 2399-2407. (19) Seul, M.; Subramaniam, S.; McConnell, H. M. J. Phys. Chem. 1985, 89 (17), 3592-5. (20) Hac, A. E.; Seeger, H. M.; Fidorra, M.; Heimburg, T. Biophys. J. 2005, 88 (1), 317-333. (21) Von Tscharner, V.; McConnell, H. M. Biophys. J. 1981, 36 (2), 421-7.
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