Retinoid Chromophores as Probes of Membrane Lipid Order

J. Phys. Chem. B 2007, 111, 10839-10848

10839

Retinoid Chromophores as Probes of Membrane Lipid Order Frida R. Svensson, Per Lincoln, Bengt Norde´ n,* and Elin K. Esbjo1 rner* Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers UniVersity of Technology, KemiVa¨gen 10, SE-41296 Gothenburg, Sweden ReceiVed: April 13, 2007; In Final Form: June 27, 2007

There is a great need for development of independent methods to study the structure and function of membraneassociated proteins and peptides. Polarized light spectroscopy (linear dichroism, LD) using shear-aligned lipid vesicles as model membranes has emerged as a promising tool for the characterization of the binding geometry of membrane-bound biomolecules. Here we explore the potential of retinoic acid, retinol, and retinal to function as probes of the macroscopic alignment of shear-deformed 100 nm liposomes. The retinoids display negative LD, proving their preferred alignment perpendicular to the membrane surface. The magnitude of the LD indicates the order retinoic acid > retinol > retinal regarding the degree of orientation in all tested lipid vesicle types. It is concluded that mainly nonspecific electrostatic interactions govern the apparent orientation of the retinoids within the bilayer. We propose a simple model for how the effective orientation may be related to the polarity of the end groups of the retinoid probes, their insertion depths, and their angular distribution of configurations around the membrane normal. Further, we provide evidence that the retinoids can sense subtle structural differences due to variations in membrane composition and we explore the pH sensitivity of retinoic acid, which manifests in variations in absorption maximum wavelength in membranes of varying surface charge. Based on LD measurements on cholesterol-containing liposomes, the influence of membrane constituents on bending rigidity and vesicle deformation is considered in relation to the macroscopic alignment, as well as to lipid chain order on the microscopic scale.

Introduction The lipid bilayer is the basis of cell membrane structures and constitutes an essential component of life by its compartmentalizing properties, serving as a highly selective hydrophobic barrier separating the cell interior from the exterior. The view of the lipid bilayer has evolved considerably since the proposal of the simple fluid mosaic model by Singer and Nicholson in 1972.1 Evidence for the complexity of lipid bilayers is emerging rapidly, with findings that their self-assembled macromolecular structures are able to form domains, so-called rafts, of differing fluidity and that lateral segregation of lipids may serve to functionalize the lipid portion of the membrane.2-4 In addition, from the Human Genome Project it has become clear that a significant number of the proteins encoded in our genome are in fact associated to cellular lipid membranes.5-7 The protein content of the plasma membrane may amount to as much as 50 wt %. These membrane-bound proteins perform vital functions including energy production via ATP synthesis, cell-cell communication, transmittance of extracellular signals to the cell interior, and controlled transport of specific molecules across lipid membranes.8 Yet, the advances in understanding biological function in membranes are held back, particularly due to the experimental difficulties associated with determining the structure and binding geometry of proteins in a lipid bilayer environment. The conventional techniques for high-resolution structural determination of biological macromolecules, NMR and X-ray crystallography, have severe limitations when it comes to membrane-bound proteins,9-12 and even though these * Corresponding authors. Telephone: +46 (0) 31 772 38 57 (E.K.E.); +46 (0) 31 772 30 41 (B.N.). Fax: +46 (0) 31 772 38 58 (B.N.). E-mail: [email protected] (E.K.E.); [email protected] (B.N.).

technologies are developing, the number of membrane protein structures deposited in the Protein Data Bank remains small, just a few hundred, compared to the nearly 40 000 structures of soluble proteins.13,14 In addition, we lack efficient technology for studying the structure of the membrane itself and how it is affected by the insertion of proteins. In conclusion, there is an urgent need for development of new techniques for in situ analysis of the structure and function of membrane proteins including development of probes for their interplay with the lipid matrixes they are imbedded in. Techniques based on fluorescence spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), electron paramagnetic resonance (EPR), and polarized light spectroscopy (linear dichroism, LD) cannot normally give information on three-dimensional (3D) structure but are useful for monitoring function and may often be applied to cell membrane models more realistic than those normally used for protein crystallography and solution NMR. In addition, it is often possible to obtain data over a wider range of biologically relevant conditions.15-18 We have in various contexts demonstrated the usefulness of polarized light spectroscopy in the UV/visible region (linear dichroism) for probing the orientation of organic molecules solubilized in the membrane of shear-aligned lipid vesicles.19,20 Recently we successfully employed this technique to study the binding and orientation of membrane-active peptides such as gramicidin,15 as well as wild-type and mutated versions of the cell-penetrating peptide penetratin.21,22 More detailed information about 3D structure can be gained by the SSLD technique (site specific linear dichroism by molecular replacement) developed for the study of RecA-DNA,23,24 but in principle applicable to any protein structure provided that the degree of

10.1021/jp072890b CCC: $37.00 © 2007 American Chemical Society Published on Web 08/21/2007

10840 J. Phys. Chem. B, Vol. 111, No. 36, 2007

Figure 1. (A) Molecular structures of retinoic acid (R1), retinol (R2), and retinal (R3). (B) van der Waals model of retinoic acid, with carbon atoms in black, hydrogen atoms in white, and oxygen atoms in red. The rodlike shape and potential for hydrogen bonding with polar head groups of lipids are the causes of a high degree of orientation of retinoic acid in the lipid bilayer membrane.

macroscopic orientation may be determined. Thus, the topic of the present study, a methodology to assess membrane orientation, is relevant in a general context for studying protein structure in membranes. Using linear dichroism, we early found that retinal and diphenylacetylene are valuable probes to monitor alkyl chain order in lamellar liquid crystalline phases,25 and recently Rajendra et al.26 have shown that an additional number of membrane-bound dyes, including retinol, have the potential of functioning as membrane probes in linear dichroism owing to their well-defined orientation within the membrane. To be used as an internal probe, together with, e.g., proteins, it is desirable that the membrane probe itself does not display significant absorption at wavelengths below 300 nm. Retinoids are promising candidates in this aspect, and therefore the applicability of retinoic acid, retinol, and retinal to function as membrane probes will be explored in this study. The hydrophobic retinoids all bind efficiently to lipid membranes and insert themselves parallel to the lipid chains with the bulky ring system toward the lipid chain ends (see Figure 1A for molecular structures of retinoic acid, retinol, and retinal and Figure 1B for a van der Waals model of retinoic acid).27 The main absorption of the retinoid chromophore has a transition moment oriented along the long axis of the molecule due to a single electronic transition in the near-UV/visible region of the spectrum. In this work these three retinoids will be characterized in terms of their orientations in lipid vesicles of varying composition and the results will be discussed in terms of a model for their interactions with the lipid membrane. The results will also be discussed in terms of lipid chain order and bending rigidity in membranes depending on their composition. In addition, the effects of solvent pH and polarity on the absorption characteristics of the retinoids will be explored in order to assess their value also as probes of the environment in the head group region of a membrane. Experimental Methods Materials. 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (POPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (POPG) were from Larodan (Malmo¨, Sweden). N-(1-(2,3-Dioleoyloxy)propyl)N,N,N-trimethylammonium chloride (DOTAP), 1,6-diphenyl1,3,5-hexatriene (DPH), all-trans-retinoic acid, all-trans-retinol, all-trans-retinal, and sucrose were from Sigma. Unless otherwise stated, the buffer was 10 mM potassium phosphate (pH 7.4). Deionized water from a Milli-Q system (Millipore) was used.

Svensson et al. Preparation of Large Unilamellar Vesicles (LUVs). Lipids dissolved in chloroform were mixed at the desired molar ratios, and the solvent was evaporated under reduced pressure using a rotary evaporator. To remove any remaining traces of solvent, the phospholipid film was put under vacuum for at least 2 h. Vesicles were prepared by dispersion of the lipid film in buffer under vortexing. Thereafter, the vesicles were subjected to five freeze-thaw cycles (liquid nitrogen/37 °C) before extrusion 21 times through polycarbonate filters with a pore diameter of 100 nm using a LiposoFast-Pneumatic extruder (Avestin, Canada). Flow Linear Dichroism (LD) Spectroscopy. Linear dichroism (LD) is defined as the differential absorption of linearly polarized light parallel and perpendicular to an orientation axis according to

LD ) A| - A⊥

(1)

The technique requires a macroscopically aligned sample which can be obtained by subjecting liposomes to shear flow in a Couette cell.20 Thus, molecules that associate to the liposome in a nonrandom fashion will also be aligned relative to the flow and exhibit linear dichroism. A quantitative assessment of orientation can be obtained by normalizing the LD with respect to the isotropic absorption of the sample. This quantity is referred to as the reduced LD (LDr), and for a nonoverlapping transition the LDr is wavelength-independent and directly related to the orientation of the transition according to

LDr )

LD 3 ) S(1 - 3〈cos2(R)〉) Aiso 4

(2)

S is the orientation factor determined by the macroscopic orientation of the liposomes as well as the stiffness and order of the lipid chains in each membrane, and 〈cos2(R)〉 is the ensemble average, where R represents the apparent angle between the transition moment and the membrane normal. LD spectra were recorded on a Jasco J-720 spectropolarimeter equipped with an Oxley prism to obtain linearly polarized light.28 The liposomes were oriented under a shear gradient of 3100 s-1 in an outer-rotating Couette cell with a total path length of 1 mm. Spectra were recorded between 250 and 450 nm in 1 nm increments using a scan speed of 500 nm/min and a bandwidth of 2 nm. Three spectra were accumulated and automatically averaged. Baselines were recorded without rotation of the Couette cell and subtracted from all spectra. The retinoids were dissolved at high concentration in ethanol and added to the liposome suspension so that the final ethanol concentration in the sample was less than 0.1%. The retinoidto-lipid molar ratio was approximately 1:200, and the lipid concentration was 1.25 mM. All samples were incubated for 2 h prior to measurements to allow the retinoids to adopt their final intercalated binding mode in the liposome membranes. The buffer used in all LD experiments was 5 mM potassium phosphate (pH 7.4) containing 50% (w/w) sucrose. Sucrose reduces light scattering from the liposomes by refractive index matching. In addition, the high sucrose content causes an increased viscous drag which results in better liposome deformation, and thus a higher degree of macroscopic orientation in the sample.15 Characterization of the Distribution of Insertion Angles. All three retinoids insert themselves into the lipid bilayer and orient with their long axes parallel to the lipid chains, so their average orientation is naturally along the membrane normal, which in a perfectly oriented sample would correspond to R ) 0° in eq 2. However, an angular distribution of configurations

Retinoid Chromophores as Membrane Probes

J. Phys. Chem. B, Vol. 111, No. 36, 2007 10841

around this mean orientation must inevitably exist since the lipid bilayer is a fluctuating structure. The information on this distribution that can be obtained from an LD experiment by the apparent insertion angle, R, is limited by the ensemble average 〈cos2(R)〉. The ensemble average may however be related to the physical distribution of insertion angles according to

∫0π/2F(R) cos2(R) sin(R) dR 〈cos (R)〉 ) ∫0π/2F(R) sin(R) dR 2

(3)

F(R) is a function describing the physical distribution of the polar angle R and sin(R) is a factor compensating for the number of possibilities to obtain a specific angle for a uniaxial distribution of angles around the membrane normal. From a physical statistical perspective it is reasonable to assume that the retinoid chromophores show a Gaussian distribution of insertion angles centered on the membrane normal and that this distribution is uniaxial (all azimuthal angle positions giving the same polar angle R relative to the membrane normal are equally probable). F(R) in eq 4 can then be described by

F(R) )

1

x2πσ

( )

exp -

R2 2σ2

(4)

σ is the angular standard deviation. In this study we used a multidimensional unconstrained nonlinear minimization algorithm (fminsearch) in the Matlab software to determine values of σ that define Gaussian functions for the angular distribution of retinol and retinal from the ensemble average 〈cos2(R)〉. To obtain a value of 〈cos2(R)〉 from a measured LDr value using eq 2, it is necessary to first estimate the orientation parameter S. As a first attempt, this was made by assuming that retinoic acid, which displays the best orientation yet obtained in our laboratory for any chromophore in an LD experiment using flow-oriented liposomes, is perfectly inserted (R ) 0°). Absorption Spectroscopy. Absorption spectra of retinoic acid in ethanol/buffer mixtures and in liposome suspensions with different head group charges and lipid chain saturation were measured as a function of pH using a Cary 4000 UV-vis spectrophotometer (Varian). The solvent pH was varied by titrating small aliquots of HCl or KOH to the solution, and the solution pH was measured with a calibrated pH meter (Metrohm). Absorption spectra were recorded in 1 nm increments between 200 and 500 nm in a quartz cell with 1 cm path length. Fluorescence Anisotropy. Fluorescence anisotropy measurements using the membrane-bound chromophore 1,6diphenyl-1,3,5-hexatriene (DPH) were performed in POPC lipid vesicles with 0, 20, or 40 mol % cholesterol, i.e., at a lipid-tocholesterol molar ratio of 100/0, 80/20, or 60/40. DPH has a total molecular length of approximately 14 Å and binds to lipid membranes preferentially parallel to the lipid acyl chains with one end close to the membrane surface.29 Changes in lipid chain order will affect the degree of DPH immobilization and thereby also its anisotropy.30 The experiments were performed on a SPEX Fluorolog-3 spectrofluorimeter (Jobin Yvon Horiba) equipped with Glan polarizers in a 1 × 1 cm quartz cell at 25 °C. The excitation monochromator was set to 358 nm, and the emission monochromator was set to 427 nm. The excitation bandpass and emission bandpass were 1 and 4 nm,

Figure 2. (A) Absorption spectra (upper panel) and linear dichroism spectra (lower panel) of retinoic acid (black), retinol (red), and retinal (green) bound to POPC lipid vesicles at a retinoid-to-lipid molar ratio of 1:200. LD is expressed in absorbance units, and both LD and absorbance was measured in cells with a total path length of 1 mm. (B) Reduced linear dichroism (LDr) curves of retinoic acid (black), retinol (red), and retinal (green) in DOPC/DOTAP (10 mol % positively charged species) (solid lines), DOPC (dotted lines), and DOPC/DOPG (40 mol % negatively charged species) (dashed lines) lipid vesicles. The LDr curves have been truncated at wavelengths where the absorbance was less than 5% of the maximum value.

respectively. The fluorescence anisotropy was calculated from emission data according to

r)

IVV - GIVH IVV + 2GIVH

(5)

IVV is the fluorescence intensity measured with vertically polarized excitation and emission, and IVH is the fluorescence intensity measured with vertically polarized excitation and horizontally polarized emission. G is defined as IHV/IHH and is an instrument correction factor giving the ratio of the sensitivity of the detection system for vertically and horizontally polarized light. The lipid concentration was 200 µM, and samples were incubated with 1 µM DPH added from a stock solution in ethanol for at least 2 h prior to measurements, to ensure that all added DPH had associated with the lipid vesicles. Results Binding Geometry of the Retinoid Chromophores in Lipid Vesicle Membranes. The absorbance and LD spectra of retinoic acid, retinol, and retinal bound to POPC LUVs in pH 7.4 buffer at a chromophore-to-lipid molar ratio of 1:200 are presented in Figure 2A. The retinoid chromophores absorb light in one single broad absorption band in the near-UV/visible region of the spectrum. The absorption maximum wavelength (λmax) varies


Svensson et al.

TABLE 1: Parameters Describing the Distribution of Configurations around the Membrane Normal That the Retinoids Adopt in a Lipid Bilayer retinoic acid retinol retinal

LDr a

Rapparentb (deg)

〈cos2(R)〉c

σd (deg)

-0.0495 -0.0375 -0.017

0 24 35

1 0.84 0.68

not defined 18 28

a Reduced linear dichroism calculated according to eq 2 (see text) from LD and absorbance data for the retinoids bound to DOPC vesicles. b The apparent angle of insertion calculated from LDr. The LDr for retinoic acid has been used to determine the orientation parameter S, assuming that all retinoic acid chromophores align perfectly parallel to the membrane normal. c The ensemble average expressed in the parameter that is linear to LDr calculated from the apparent insertion angle. d The standard deviation of a Gaussian function centered around 0° describing the uniaxial distribution of retinoid chromophores around the membrane normal. See text for further information on the evaluation of this parameter.

for the three retinoids, between 329 nm for retinol and 379 nm for retinal. In POPC the absorption for retinoic acid is centered on 350 nm. When bound to lipid vesicles all three retinoid chromophores show negative LD, proving their preferred alignment parallel to the lipid chains, i.e., perpendicular to the flow orientation axis according to eq 1. When present in lipid vesicles at the same concentration, retinoic acid has the most negative LD of the three retinoids, as shown by the normalized spectra in Figure 2A. The differences indicate that retinoic acid is significantly better oriented in the lipid membrane than retinol and retinal. We have examined the distribution of insertion angles for retinol and retinal compared to retinoic acid as described under Experimental Methods, assuming that the insertion angle, R, relative to the membrane normal follows a Gaussian distribution and that retinoic acid, being by far the best oriented chromophore that we have yet encountered in an LD experiment, is perfectly oriented. The LDr value for retinoic acid in DOPC liposomes was used to determine the macroscopic orientation parameter S: A LDr of -0.050 for retinoic acid in DOPC lipid vesicles results in a S of 0.033. This value of S was used to calculate apparent angles of insertion (Rapparent) for retinol and retinal from their respective LDr values as shown in Table 1. The result of our attempt to determine the standard deviation, σ, of a Gaussian angular distribution function using eqs 3 and 4 and the experimentally obtained ensemble average, 〈cos2(R)〉, from the apparent insertion angles of retinol and retinal are also given in Table 1. Effect of Lipid Composition on the Retinoid LDr Values. To assess how the linear dichroism of the retinoids is affected by variations in lipid composition, the three retinoids were incubated with liposomes composed of lipids with different head group charges and different degrees of lipid chain unsaturation. LDr curves for the three retinoids in three selected types of lipid vesicles, shown in Figure 2B, were obtained by normalizing the LD with respect to the isotropic absorbance spectrum of each sample according to eq 2. To quantify the LDr from different measurements, the value at the wavelength of maximum absorption was used. The LDr values for the three retinoids in vesicles of varying surface charge or lipid chain composition are given in Figure 3. The first column in each group (colored in black) shows the LDr in DOPC vesicles. These columns should be compared to the following two which represent LDr in vesicles having the same lipid chains but different surface charges. Incorporation of 40 mol % negatively charged DOPG obviously increases the LDr of all three retinoids, whereas incorporation of 10 mol % positively charged DOTAP decreases the LDr. In contrast to liposomes with two oleoyl chains, introduction of 40 mol % negative charge in lipid vesicles with palmitoyl/oleoyl lipid chains (light gray and white columns in Figure 3) has a relatively modest effect on the LDr. For retinoic acid and retinol a small decrease is in fact observed. There is a difference in LDr between the retinoids measured in liposomes composed of lipids with two oleoyl chains (DOPC and DOPG)

Figure 3. LDr for retinoic acid, retinol, and retinal bound to zwitterionic (DOPC and POPC), negatively charged (DOPC/DOPG and POPC/ POPG, both with 40 mol % negatively charged species), and positively charged (DOPC/DOTAP, with 10 mol % positively charged species) lipid vesicles with varying lipid chain composition (dioleoyl (DO) or palmitoyl/oleoyl (PO)), calculated from LD and absorbance data, as described in the text. The retinoid-to-lipid molar ratio was approximately 1:200, and all samples were incubated for 2 h prior to measurements.

TABLE 2: Absorption Maximum Wavelengths (λmax) for the Retinoids Bound to Liposomes of Different Compositionsa retinoic acid (nm) retinol (nm) retinal (nm) DOPC DOPC/DOPG (60/40) DOPC/DOTAP (90/10) POPC POPC/POPG (60/40) a

348 352 344 350 354

329 329 329 329 329

380 380 380 379 381

The retinoid to lipid molar ratio is approximately 1:200.

compared to liposomes composed of lipids with one palmitoyl and one oleoyl chain (POPC and POPG) (compare the first two columns to the last two columns within each group). The LDr values are larger in dioleoyl liposomes than in palmitoyl/oleoyl liposomes, and the differences are much more marked in liposomes with negative charge (compare column 2 and column 5 for the three retinoids in Figure 3). Apart from showing varying degrees of orientation in different types of liposomes, retinoic acid displays significant absorption shifts in different membrane environments as can be seen in Table 2, where the absorption maximum wavelengths (λmax) for retinoic acid, retinol, and retinal are listed. The retinoic acid absorption is apparently sensitive to the surface charge of the lipid vesicle, and λmax is blue-shifted in the presence of positively charged lipids (DOTAP) and red-shifted in the presence of negatively charged lipids (DOPG or POPG) compared to when in the presence of zwitterionic lipid vesicles (DOPC or POPC). By contrast, λmax values for retinol and retinal are not significantly affected by variations in lipid head group composition. Effect of Solvent Polarity on Absorption Characteristics. The observed shifts in absorption maximum wavelength of



Figure 5. Absorption maximum wavelengths (λmax) for retinoic acid as a function of pH in ethanol/water (50/50) (-O-), POPC/POPG (60/ 40) lipid vesicles (-9-), DOPC vesicles (-0-), and DOPC/DOTAP (90/10) vesicles (-2-).

Figure 4. Effect of solvent polarity on absorption maximum of retinoic acid (b, O), retinol (2, 4), and retinal (9, 0) at pH 3 (filled symbols) and pH 11 (open symbols). For easier comparison of data from different measurements, the absorption maxima on the y-axis are given in wavenumbers to provide a scale that is linear in energy. The straight lines are linear fits to the experimental data.

retinoic acid depending on the surface charge of the lipid vesicles made us study how the retinoid chromophores are affected by solvent polarity. In Figure 4 the position of the absorption maximum of retinoic acid, retinol, and retinal is shown as a function of ethanol content in ethanol/water mixtures. The absorption maxima have been plotted using wavenumbers to obtain a linear energy scale. Retinoic acid is estimated to have a pKa of approximately 6 in an ethanol/water (50/50) solution (see Figures 5 and 6) and is thus found to be fully deprotonated above pH 8 and fully protonated below pH 5. As can be seen in Figure 4, there is a marked difference (approximately +1600 cm-1 corresponding to -22 nm) in the positions of the absorption maximum between the protonated (filled circles) and deprotonated (open circles) forms, with the deprotonated form being higher in energy. In addition to pH sensitivity, the absorption of retinoic acid is sensitive to the polarity of the solvent. Increasing the ethanol content, i.e., decreasing the polarity, thus results in a shift in absorption maximum of retinoic acid to higher energies (approximately 400 cm-1 corresponding to a blue shift of approximately 8 nm). This characteristic is seemingly independent of the protonation of the carboxylic group on retinoic acid since the approximately linear increase in wavenumber position of the absorption maximum with decreasing solvent polarity has the same slope at pH 3 as at pH 11. Also, the retinal absorbance depends on the solvent polarity in a manner similar to that of retinoic acid (squares in Figure 4). Retinol absorption characteristics are virtually not affected at all by either polarity or pH, as can be seen in Figure 4 (triangles), where the absorption maximum wavenumber remains constant with increasing solvent polarity and changing pH. Neither retinol nor retinal displays any pHdependent characteristics in the relevant pH interval, as expected from their lack of titratable proton acceptor or donor moieties. By contrast, retinoic acid has a carboxylic acid end group and is highly sensitive to pH, which will be further addressed below.

Effect of Solvent pH on Retinoic Acid Absorption Characteristics. Since retinoic acid was shown to display pHdependent absorption characteristics (Figure 4), its behavior was further characterized by monitoring the absorption maximum wavelength as a function of pH in samples containing lipid vesicles of different compositions and in a water/ethanol (50/ 50 v/v) mixture (see Figure 5). At low pH retinoic acid is protonated and it can be observed in Figure 5 that λmax is rather similar in all lipid vesicle types. By contrast, at high pH retinoic acid is negatively charged and λmax is more sensitive to the lipid head group composition in the membrane, as inferred by a greater spread of data obtained from measurements in different types of lipid vesicles (also in Figure 5). Single value decomposition (SVD) of the absorption spectra of retinoic acid collected during the pH titrations was performed to analyze the number of components responsible for the spectral shape and pH-dependent shift. The analysis showed that two components are sufficient to describe the variations in absorbance due to pH since the third singular value was less than 1% of the first in all data series. Thus, the measured absorption spectra at different pH values can be accurately described as a linear combination of two spectral components, which we assign to the protonated and deprotonated forms of retinoic acid. Reference absorption spectra were recorded for the protonated and deprotonated forms, and the degree of ionization as a function of pH was estimated by projecting absorption spectra from the pH titration onto these reference spectra. The results are shown in Figure 6, where the degree of ionization (calculated as the fraction of deprotonated retinoic acid relative to the total concentration of retinoic acid) has been plotted as a function of solvent pH for the same lipid vesicle types and solvents in Figure 5. In addition, pH titration curves were fitted to experimental data using the following equation (derived from the Henderson-Hasselbalch equation):

[A-] -

[HA] + [A ]

)

1 1 + 10(pKa+pH)

(6)

where [HA] is the concentration of the protonated form and [A-] is the concentration of the deprotonated form of retinoic acid. The fits are shown as solid lines in Figure 6. The effective pKa for retinoic acid in lipid vesicles is dependent on the liposome surface charge density, and a higher pKa (8.1) is observed in negatively charged lipid vesicles (POPC/POPG (60/ 40), filled squares in Figure 6) compared to a pKa of 7.2 in zwitterionic vesicles (DOPC, open squares). This is in agreement with earlier reports on the ionization behavior of retinoic acid


Figure 6. Fraction of deprotonated retinoic acid (degree of ionization) as a function of pH in ethanol/water (50/50) (O), DOPC/DOTAP (90/ 10) lipid vesicles (2), DOPC vesicles (0), and POPC/POPG (60/40) vesicles (9). The solid lines represent theoretical curves, fitted to the experimental data using eq 6. The effective pKa values for retinoic acid in the above-mentioned environments are respectively 6.1, 6.1, 7.2, and 8.1.

in lipid bilayers.31 Further, the opposite effect is observed in positively charged liposomes (DOPC/DOTAP (90/10), filled triangles in Figure 6), where retinoic acid has an effective pKa around 6.1. It should be noted that only 10% positively charged lipids is needed to decrease the effective pKa with one pH unit whereas as much as 40% negatively charged lipids will increase the effective pKa with less than one pH unit. The effective pKa in a water/ethanol mixture (50/50 (v/v)) is the same as for retinoic acid in positively charged vesicles (6.1). Figure 6 clearly reveals that the measured pH titration curves for retinoic acid in lipid vesicles are not as steep as the theoretically fitted curves, although the experimental data in POPC/POPG (60/40) vesicles (filled squares) can be better described by the HendersonHasselbalch equation than data in DOPC or DOPC/DOTAP (90/ 10) lipid vesicles. A theoretical titration curve could, on the other hand, be excellently fitted to titration data obtained in the water/ethanol mixture (open circles). This indicates that the pKa of retinoic acid in lipid vesicles is not as well-defined as in a homogeneous solvent. This is an interesting observation that may be explained by the heterogeneity in location of the chromophore with respect to the lipid membrane environment or by the microscopic heterogeneity of the membrane itself, or by the presence of additional titratable groups in the head group region, or by a combination of them all. Effect of Cholesterol. Linear dichroism and absorption spectra were recorded for the three retinoids bound to POPC lipid vesicles with 0, 20, or 40 mol % cholesterol. The LDr values at the maximum absorption wavelengths are given in Figure 7A. All three retinoids exhibit a significant increase in LDr upon incorporation of 20 mol % cholesterol in the lipid vesicles, whereas further addition of cholesterol (40 mol %) cholesterol has seemingly no additional effect on LDr. The increase in LDr due to 20 mol % cholesterol is more pronounced for retinol and retinal than for retinoic acid. Retinoic acid shows an increase in LDr of about 25%, while the orientation factor for retinol and retinal are increased by as much as 50 and 100%, respectively. Thus, incorporation of cholesterol in the lipid vesicle membranes apparently reduces the systematic variation in LDr between the three retinoids that we observed for several different phospholipid compositions (Figures 2 and 3). Incorporation of cholesterol had little effect on the absorption maximum wavelengths for the three retinoids. The only observ-

Svensson et al.

Figure 7. (A) LDr of retinoic acid, retinol, and retinal bound to POPC LUVs containing 0 (white bars), 20 (light gray bars), or 40 (dark gray bars) mol % cholesterol. (B) Fluorescence anisotropy of DPH (1,6diphenyl-1,3,5-hexatriene) in POPC vesicles containing 0, 20, or 40 mol % cholesterol.

able shift was for retinoic acid in lipid vesicles containing 40 mol % cholesterol where the increase in maximum absorption wavelength was 4 nm compared to pure POPC vesicles (data not shown). To further assess the effect of cholesterol on lipid chain order, fluorescence anisotropy measurements using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed in POPC lipid vesicles containing 0, 20, or 40 mol % cholesterol. The anisotropy, r, was calculated using eq 5 as described under Experimental Methods, and the data obtained are shown in Figure 7B. There is clearly an increase in DPH polarization with increased cholesterol content, which is to be expected since cholesterol imposes a higher degree of order on the hydrocarbon chains. This is in agreement with the observation that incorporation of cholesterol increased the LDr for the retinoids as shown in Figure 7A. However, the monotonic increase in lipid chain order with increasing cholesterol observed from the DPH anisotropy experiments could not, as mentioned above, be seen in the LD experiments, suggesting that cholesterol affects not only lipid chain order but also somehow the deformability and orientability of the lipid vesicles. Discussion In this study we address the potential of three retinoid chromophores, retinoic acid, retinol, and retinal, to function as probes of the microscopic order of the bilayer membranes of flow-oriented lipid vesicles, including the macroscopic orientation parameter, S, of the linear dichroism experiment. We will from now on differentiate between microscopic and macroscopic ordering. The microscopic ordering refers to the degree of lipid chain order within a small section of a perfectly oriented membrane and is related to the ensemble average 〈cos2(R)〉 in eq 2 but also to the distribution of orientations through the standard deviation σ in a Gaussian distribution function. The macroscopic ordering refers to the degree of deformation of the lipid vesicles and their alignment in the direction of the flow and is governed by the orientation factor S. By thoroughly examining the reduced linear dichroism (LDr) in liposome membranes of different lipid compositions, we have also been able to draw conclusions about how different constituents of the lipid membrane affect both the macroscopic and microscopic orientation in lipid vesicles aligned by shear flow under the conditions used in a typical LD experiment. The results in this study will be discussed in terms of models for the membrane interaction of the three retinoids, and their individual assets and drawbacks as membrane orientation probes

Retinoid Chromophores as Membrane Probes will be pointed out. In relation to this, the effect of pH and solvent polarity on the absorption characteristics of the retinoids will also be discussed. Binding Geometry and Insertion Depth of Retinoid Chromophores in Lipid Membranes. We showed explicitly in Figure 2, and again in Figure 3, that the LDr in a lipid membrane varies significantly between the three retinoids and that retinoic acid has an LD that is nearly twice that of retinal, whereas retinol has an LD between the other two. The extended molecular shape and the hydrophobicity of the retinoids strongly suggest that they intercalate into the membrane and align along the lipid chains, as was early concluded for retinal and other rodlike probes25 and repeatedly confirmed for retinol, by linear dichroism experiments on lipid vesicles.26 Thus, interaction with a fluid lipid bilayer will result in that the retinoid is, on average, oriented perpendicular to the lipid surface (the liposome orientation axis) and hence parallel to the membrane normal. The actual meaning of different degrees of orientation for the three retinoids is best interpreted in terms of how much they diverge from this ideal orientation (which will be discussed in detail below), and refers to how well they can sense the microscopic ordering in their lipid environment. The difference in LD can be explained by a model of interaction based on variations in insertion depth between the retinoids related to the chemical characteristics of their end groups (the part of the molecule that faces the membrane head group region; see Figure 1A). Retinoic acid is expected to be firmly anchored with its hydrophilic carboxylic group in the head group region due to nonspecific electrostatic interactions as well as hydrogen bonding with lipid head groups, water, and ions in the membrane head group region. Its hydrophobic tail will consequently be forced into a position within the upper part of the hydrocarbon chain region of the lipid bilayer. The anchoring in combination with that lipid chain order is largest just below the lipid head groups can thus explain the high degree of orientation that we observe for retinoic acid. Retinol, with an alcohol end group, is less hydrophilic than retinoic acid and may therefore penetrate somewhat deeper into the membrane, resulting in worse ordering due to greater motional freedom of its surroundings, but still preferentially well anchored at the head groups. It may be appropriate to compare retinol to cholesterol since this steroid resides at a relatively specific longitudinal position within the membrane due to its alcohol moiety, which is found close to the lipid glycerols.32 A reasonable conclusion would thus be that also retinol will position its alcohol group at the membrane interface, a positioning that may, in analogy with cholesterol, be stabilized by hydrogen bonding. From the evaluation of the distribution of configurations for retinol (Table 1) a standard deviation of 18° was obtained, indicating that 67% of the retinol molecules have an angle relative to the membrane normal that is smaller than this value. Retinal, with its aldehyde end group, does not participate in hydrogen bonding with the lipid head groups. In addition, it is the least polar of the three retinoids. Also, it cannot hydrogen bond in the lipid head group region. Along the lines of the above argumentation, it is thus probable that retinal can penetrate deeper into the lipid bilayer and its lower degree of orientation may be explained by that it lacks as firm an anchoring to the head group region as retinoic acid or retinol. As a result, it will experience greater motional freedom and may be expected to adopt a wider distribution of insertion angles relative to the membrane normal (we estimated the standard deviation to 28°; see Table 1). This conclusion is further supported by the sloping LDr curves for retinal in Figure 2B which indicate that retinal

J. Phys. Chem. B, Vol. 111, No. 36, 2007 10845 displays a more heterogeneous depth distribution than retinoic acid and retinol (which both display a practically constant LDr over a large range of wavelengths). If a recorded LD spectrum stems from a homogeneous population and the absorption in the region of interests is, as in our case, due to one single transition, the normalized LD spectrum (the LDr) should be constant over the absorption band. As mentioned above, this is true for retinoic acid and retinol (black and red lines in Figure 2B), at least in the region where absorption is relatively intense. Retinal, on the other hand, displays a continuously sloping LDr, implying that several populations exist and that these have both different absorption and orientation characteristics. The LDr amplitude increases with increasing wavelength, indicating that the red-shifted population is the best oriented one. We show in Figure 4 that retinal is sensitive to solvent polarity and that an increase in solvent hydrophobicity is concomitant with an increase in absorption maximum wavenumber and thus with a decrease in absorption maximum wavelength. Therefore, it is reasonable to conclude that the red-shifted population of retinal molecules is in a more hydrophilic environment, i.e., closer to the lipid head group region than the blue-shifted population. This is also in sound agreement with that we in general observe better orientation for chromophores with anchoring in the head group region (retinoic acid and retinol), but also indicates that retinal experiences a depth distribution within the bilayer and, as a consequence, also a wider angular distribution. Additional information on the penetration depth and motional freedom experienced by the different retinoids can be deduced from Figure 7A, in which we show that incorporation of cholesterol leads to increased LDr values and that the effect is largest for retinal, which has the lowest degree of orientation in the absence of cholesterol. First, if any of the retinoids partitioned to a measurable extent into the central part of the bilayer, it is likely that incorporation of cholesterol would result in a reduction of LDr amplitude since the incorporation of cholesterol can create space for a wide range of movement at the terminal end of the lipid chains.30 Second, a greater increase in LDr for retinol and especially retinal compared to retinoic acid is expected because the motional restriction obtained in the upper part of a cholesterol-containing membrane should have a greater effect on these two than on retinoic acid, which is already very well oriented in pure POPC membranes. From the discussion above we can now propose a model for the insertion of the retinoids, involving their longitudinal as well as angular distribution. We suggest that retinoic acid is firmly anchored in the head group region; retinol penetrates somewhat deeper, suggestively with its alcohol moiety at the level of the lipid glycerols. Retinal will most likely experience a wider distribution of insertion depths, and the deeper it penetrates the less will the motional restriction be. From the evaluation of the angular distribution around the membrane normal for retinol and retinal compared to retinoic acid, we obtained the standard angular deviation (that is, the angle of a uniaxial cone around the membrane normal within which 67% of the molecules will reside). In Figure 8 is a cartoon model of the binding of the three retinoids in a lipid bilayer. The widths of the cones for retinol and retinal are set to the standard deviation, and the insertion depth is indicated. Our model is in qualitative agreement with previous observations on the positioning of retinoids from electron paramagnetic spin resonance (EPR) studies.33 The superior alignment of retinoic acid compared to the other retinoids indicates that it is indeed the best choice for accurate assessment of the orientation parameter S in eq 2.


Figure 8. Cartoon model of the binding geometry distribution of (A) retinoic acid, (B) retinol, and (C) retinal in a lipid bilayer. The horizontal line is at the level of the lipid glycerols and marks the membrane interface. The widths of the cones in (B) and (C) correspond to the standard deviation of a Gaussian distribution of configurations around the membrane normal (see also Table 1).

Retinoid Chromophores as Membrane Probes. Figure 3 shows, importantly, that the difference in LDr among the three retinoids is essentially independent of the lipid vesicle composition. Retinoic acid always displays the best orientation, followed by retinol and then retinal. This strongly suggests that the orientation within the membrane of these three molecules is not dependent on specific interactions with the membrane lipids. Therefore, even though we have concluded that the retinoids reside at varying depths within the membrane, they do indeed probe the lipid chain order in their immediate respective environment, which is an unconditional requirement for a molecule to function as a probe of the microscopic ordering in a shear-deformed liposome membrane. We can thus expect that if the lipid membrane experiences perturbations, for example due to binding of a protein or peptide, the retinoids will respond to these perturbations in the same way as the lipid chains. However, we have concluded that the retinoids bind in the upper part of the lipid bilayer with the end group in or close to the lipid head group region, and this implies that they will preferentially sense the order in this region and hence probably not be as sensitive in probing changes in the center of the bilayer, where nonpolar rodlike probes such as diphenylacetylene25 are more appropriate to use. The retinoids have a molecular length of approximately 14 Å, and given that their end groups are positioned approximately at the level of the lipid glycerols (at the membrane interface), they will cover approximately carbons 1-12 in the hydrocarbon core region of the membrane (see the cartoon model in Figure 8). In addition to sensitively probing membrane order in lipid vesicles composed of different membrane constituents (see Figures 2, 3 and 7), we have shown that retinoic acid displays pH-sensitive absorption characteristics (Figures 5 and 6) and that the absorptions of both retinoic acid and retinal are sensitive to solvent polarity (Figure 4). The absorption maximum of retinal does not change with lipid composition (Table 2), even though we have proven that retinal itself is sensitive to solvent polarity (Figure 4). This suggests that the longitudinal distribution of this probe is affected neither by lipid head group charge nor by the degree of lipid chain saturation. Therefore, the observed variation in LDr values in membranes of different compositions (Figure 3) can hardly be an effect of the lipid composition changing the longitudinal distribution of retinal, again emphasizing that retinal senses the true lipid chain order in the membrane in a nonspecific way. Since the absorption maximum wavelength of retinoic acid changes with both solvent pH and solvent polarity, as well as with membrane composition, it is not straightforward to separate the influence on the absorption maximum of these three

Svensson et al. individual parameters. It is, however, important to ask whether the observed variations in absorption maximum wavelength with lipid composition for retinoic acid could be due to changes in longitudinal positioning, which would result in changed polarity in the surroundings of this probe. The argument that depth variation with lipid composition can be ignored also with retinoic acid is that the relative degree of orientation (LDr) between the different retinoids is the same regardless of lipid composition, and since we have experimental evidence that the depth distribution of retinal is not significantly affected, it is justifiable to assume that the situation is similar for the other retinoids. It is possible that shifts in maximum absorption wavelength of the pH-insensitive retinal may be used to probe, for example, changes in the access of water in the membrane head group region. Retinoic acid, on the other hand, can most likely never be used to probe such changes even though it is clearly sensitive to solvent polarity in much the same way as retinal (see Figure 4). There are simply too many other parameters that influence the absorption of retinoic acid that may change concomitantly. The shifts in absorption maxima for retinoic acid (see Table 2) are clearly related to the degree of ionization of this pHsensitive chromophore. The increase in effective pKa when passing from DOTAP-containing to POPG-containing lipid vesicles suggests that the protonated form of retinoic acid is stabilized by negatively charged species in the head group region of the membrane and vice versa (see Figures 5 and 6). This effect is expected for electrostatic reasons both because carboxylic acids are preferentially more protonated in less polar solvents than in polar solvents and because of the repulsion that the carboxylic anion will experience from negative lipid groups. The divergence between the fitted titration curves and experimental data in Figure 6 also indicates that the environment in the head group region is heterogeneous, so different retinoic acid molecules have different probabilities of being protonated. Thus, in addition to probing the membrane order, retinoic acid may in fact also be used to probe how the local environment in a membrane is affected by electrostatic interactions with polar or ionic species such as peptides. We have, for example, observed that the absorption maximum wavelength for retinoic acid is gradually blue-shifted when the cationic peptide melittin is titrated to a POPC/POPG lipid vesicle suspension (unpublished observations). Effects of Lipid Composition and Cholesterol on Macroscopic and Microscopic Order Parameters. The variations in LDr between different types of lipid vesicles in Figures 3 and 7A may have essentially three origins: they may be due to differences in lipid chain order, they may be due to differences in membrane bending rigidity and thus in liposome deformability, or they may be due to differences in interactions of the orientation probe (the retinoid chromophore) with membrane components. We have already dismissed the last alternative since specific interactions between the retinoids and any lipid membrane constituents have not been found (vide supra). The effect of cholesterol on lipid chain order has been touched upon above: incorporation of cholesterol increases the LDr. Additionally, we have shown that the DPH anisotropy, and thus the motional restriction in the membrane, increases almost linearly as a function of cholesterol content in POPC lipid vesicles (see Figure 7B). By contrast, the LDr values for all three retinoids are essentially the same at 20 and 40 mol % cholesterol (Figure 7A). In the DPH anisotropy experiment the microscopic ordering in the membrane will be selectively probed, whereas in the linear dichroism experiment the retinoids probe both the microscopic and macroscopic ordering. Incor-



poration of cholesterol in a membrane undoubtedly increases the lipid chain order in the upper part of the hydrocarbon chains, resulting in enhanced microscopic ordering, but in addition cholesterol will affect the membrane bending rigidity. The membrane rigidity in turn affects the deformability of lipid vesicles and thus the degree of alignment of the flow-oriented sample and therefore the macroscopic ordering. The deformability of a lipid vesicle in a shear flow can be described by a dimensionless shear rate χ (also denoted capillary number) according to

γ˘ ηoutR3 χ) κ

(7)

where γ˘ is the shear rate, ηout is the viscosity of the solvent that the vesicles are submerged in, R is the radius of the perfectly spherical vesicle, and κ is the bending rigidity.34 Henriksen et al.35 have shown, using vesicle fluctuation analysis on giant unilamellar vesicles, that the membrane bending rigidity (κ) increases linearly with the molar concentration of cholesterol in a POPC lipid membrane. κ is increased by as much as 82% upon incorporation of 20 mol % cholesterol and by 125% upon incorporation of 30 mol % cholesterol. Thus, even if cholesterol initially increases LDr by restricting motion in the upper part of the membrane, it will also limit the deformability of the lipid vesicles thereby decreasing the macroscopic alignment. We have also been able to observe a reduction in LDr in POPC membranes compared to DOPC membranes (see Figure 3). As pointed out under Results, there is only a minor difference in the chemical nature between these two lipids, but one may envisage that the mixture of saturated and monounsaturated lipid tails in POPC prevents efficient lipid packing, which could explain why the LDr is somewhat lower in this membrane type. Importantly, this difference in the degree of orientation is most likely only a microscopic effect and the fact that the retinoids can sense such small environmental changes points toward their great sensitivity as probes of membrane order. Differences in membrane leakiness between membranes composed of dioleoyl lipids and oleoyl/palmitoyl lipids has been observed in studies on induced leakage by the bee venom peptide melittin. This study proved POPC to be the leakier of these two membrane types, which is in agreement with that we observe less regular lipid packing in this study.36 There is an apparent difference in retinoid LDr between lipid vesicles with varying lipid head group charge (Figure 3). The origin of this effect is somewhat difficult to apprehend since differences in head group charge have no obvious effect on lipid chain order and since the possible effect of membrane charge on bending rigidity remains inconclusive. Some studies have indeed indicated that incorporation of anionic phosphatidic acid lipids can reduce membrane bending rigidity and that addition of cationic surfactants, even at rather low concentrations, increases membrane binding rigidity. This would be in agreement with the observed differences in LDr (better ordering for negatively charged membranes and less ordering for positively charged membranes), but no physical explanation for these observations has been provided and other studies have produced contradicting results regarding the effect of membrane surface charge on membrane rigidity.37 Thus, we can at this point neither confirm nor exclude that charged lipid species affect bending rigidity and thereby the macroscopic orientation. Conclusions We have shown that retinoic acid, retinol, and retinal are useful as probes of the macroscopic orientation of lipid vesicles

in flow linear dichroism experiments and, in addition, that these chromophores can also sense subtle variations in the microscopic ordering of the lipid membrane. We provide evidence that variations in the orientation of these chromophores, whose positioning within the membrane is mainly governed by nonspecific electrostatic interactions, may be used for studying the internal order in the lipid membrane, virtually independent of membrane constituents. A model for the insertion of the three retinoids in a phospholipid bilayer including the insertion depth as well as the angular distribution of configurations relative to the membrane normal has been presented. Retinoic acid exhibits superior alignment within the membrane compared to its sister retinoids and can be used as a standard to estimate the macroscopic orientation parameter S, which is a characteristic of the alignment in a lipid vesicle sample subjected to shear flow. In addition, compared to retinol, the second best oriented chromophore, retinoic acid displays significantly less spectral overlap with the absorbance from aromatic amino acids and DNA, simplifying measurements in which proteins or DNA are present. In addition, the wavelength of maximum absorption of retinoic acid is sensitive to solvent pH and the wavelength shift depending on the net surface charge of the membrane suggests that retinoic acid may be used to gauge electrostatic effects in the lipid membrane, including interactions with solute molecules. Since the probe orientation reflects the lipid chain order due to short-range steric forces, the ability of the retinoids to sensitively respond to small changes in lipid chain packing makes our LD technique a promising tool for future studies on lipid membrane characteristics. In particular, we believe that this technique could contribute to understanding how lipid chain order in membranes composed of varying lipids is affected by, for example, ionic strength and lipid head group hydration. Acknowledgment. This work was funded by grants to B.N. from the Swedish Cancer Foundation (Cancerfonden) and the European Commission’s Sixth and Seventh Framework Programmes (project references: AMNA Contract No. 013575 and ZNIP Contract No. 037783). References and Notes (1) Singer, S. J.; Nicolson, G. L. Science 1972, 175, 720. (2) Hanzal-Bayer, M. F.; Hancock, J. F. FEBS Lett. 2007, 581, 2098. (3) Jacobson, K.; Mouritsen, O. G.; Anderson, R. G. Nat. Cell Biol. 2007, 9, 7. (4) Simons, K.; Ikonen, E. Nature 1997, 387, 569. (5) Fleishman, S. J.; Unger, V. M.; Ben-Tal, N. Trends Biochem. Sci. 2006, 31, 106. (6) Liu, J.; Rost, B. Protein Sci. 2001, 10, 1970. (7) Venter, J. C.; et al. Science 2001, 291, 1304. (8) Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D. Molecular biology of the cell; Garland Publishing, Inc.: New York, 1994. (9) Arora, A.; Tamm, L. K. Curr. Opin. Struct. Biol. 2001, 11, 540. (10) Torres, J.; Stevens, T. J.; Samso, M. Trends Biochem. Sci. 2003, 28, 137. (11) Wiener, M. C. Methods (San Diego, CA) 2004, 34, 364. (12) Opella, S. J.; Marassi, F. M. Chem. ReV. 2004, 104, 3587. (13) Tusnady, G. E.; Dosztanyi, Z.; Simon, I. Bioinformatics 2004, 20, 2964. (14) White, S. H. Protein Sci. 2004, 13, 1948. (15) Ardhammar, M.; Lincoln, P.; Nordeń, B. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15313. (16) Johnson, A. E. Traffic 2005, 6, 1078. (17) Tatulian, S. A. Biochemistry 2003, 42, 11898. (18) Vigano, C.; Manciu, L.; Buyse, F.; Goormaghtigh, E.; Ruysschaert, J. M. Biopolymers 2000, 55, 373. (19) Ardhammar, M.; Lincoln, P.; Nordeń, B. J. Phys. Chem. B 2001, 105, 11363. (20) Ardhammar, M.; Mikati, N.; Nordeń, B. J. Am. Chem. Soc. 1998, 120, 9957.

10848 J. Phys. Chem. B, Vol. 111, No. 36, 2007 (21) Caesar, C. E.; Esbjo¨rner, E. K.; Lincoln, P.; Nordeń, B. Biochemistry 2006, 45, 7682. (22) Brattwall, C. E.; Lincoln, P.; Nordeń, B. J. Am. Chem. Soc. 2003, 125, 14214. (23) Frykholm, K.; Morimatsu, K.; Nordeń, B. Biochemistry 2006, 45, 11172. (24) Morimatsu, K.; Takahashi, M.; Nordeń, B. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11688. (25) Nordeń, B.; Lindblom, G.; Jonas, I. J. Phys. Chem. 1977, 81, 2086. (26) Rajendra, J.; Damianoglou, A.; Hicks, M.; Booth, P.; Rodger, P. M.; Rodger, A. Chem. Phys. 2006, 326, 210. (27) Ortiz, A.; Aranda, F. J.; Gomez-Fernandez, J. C. Biochim. Biophys. Acta 1992, 1106, 282. (28) Nordeń, B.; Kubista, M.; Kurucsev, T. Q. ReV. Biophys. 1992, 25, 51.

Svensson et al. (29) Kaiser, R. D.; London, E. Biochemistry 1998, 37, 8180. (30) New, R. R. C. Liposomes a practical approach; Oxford University Press: Oxford, 1990. (31) Noy, N. Biochim. Biophys. Acta 1992, 1106, 159. (32) Ohvo-Rekila, H.; Ramstedt, B.; Leppimaki, P.; Slotte, J. P. Prog. Lipid Res. 2002, 41, 66. (33) Wassall, S. R.; Phelps, T. M.; Albrecht, M. R.; Langsford, C. A.; Stillwell, W. Biochim. Biophys. Acta 1988, 939, 393. (34) Kantsler, V.; Steinberg, V. Phys. ReV. Lett. 2005, 95, 258101. (35) Henriksen, J.; Rowat, A. C.; Ipsen, J. H. Eur. Biophys. J. 2004, 33, 732. (36) Rex, S.; Schwarz, G. Biochemistry 1998, 37, 2336. (37) Rowat, A. C.; Hansen, P. L.; Ipsen, J. H. Europhys. Lett. 2004, 67, 144.

Retinoid Chromophores as Probes of Membrane Lipid Order

Recommend Documents