Article pubs.acs.org/JPCB
Naphthalene Derivatives Induce Acyl Chain Interdigitation in Dipalmitoylphosphatidylcholine Bilayers Md. Arif Kamal*,†,‡ and V. A. Raghunathan† †
Raman Research Institute, C.V Raman Avenue, Sadashivanagar, Bangalore 560080, India S Supporting Information *
ABSTRACT: The interdigitated phase of the lipid bilayer results when acyl chains from opposing monolayers fully interpenetrate such that the terminal methyl groups of the respective lipid chains are located at the interfacial region on the opposite sides of the bilayer. Usually, chain interdigitation is not encountered in a symmetric chain phosphatidylcholine (PC) membrane but can be induced under certain special conditions. In this article, we elucidate the contribution of small amphiphatic molecules in altering the physical properties of a symmetric chain PC bilayer membrane, which results in acyl chain interdigitation. Using small-angle X-ray scattering (SAXS), we have carried out a systematic investigation of the physical interactions of three naphthalene derivatives containing hydroxyl groups: β-naphthol, 2,3dihydroxynaphthalene, and 2,7-dihydroxynaphthalene, with dipalmitoylphosphatidylcholine (DPPC) bilayers. On the basis of the diffraction patterns, we have determined the temperature−composition phase diagrams of these binary mixtures. The present study not only enables us to gain insight into the role played by small molecules in altering the packing arrangement of the acyl chains of the constituting PC lipids of the bilayer but also brings to light some important features that have not yet been reported hitherto. One such feature is the stabilization of the enigmatic asymmetric ripple phase over a wide temperature and concentration range. The results presented here strongly point toward a clear correlation between chain interdigitation and the stability of the ripple phase.
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INTRODUCTION Cellular membranes display an astonishingly rich diversity in their chemical composition. Membranes associated with different subcellular organelles exhibit an assorted mixture of various lipid classes and their subclasses.1,2 Out of the different lipid classes known, phospholipids (e.g., phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, etc.) occupy a special place as these are ubiquitous in cellular membranes.1 Interestingly, in spite of the aforementioned heterogeneity as well as complexity in their compositional makeup, all membranes exhibit the same universal morphology: a bilayer of lipid molecules. In an aqueous environment, a bilayer structure results from the joining of two phospholipid monolayers that contact each other at the terminal methyl group of their hydrophobic chains (Figure 1b−d), whereas their hydrophilic headgroups are in contact with water. Under normal conditions, the acyl chains from one monolayer generally do not penetrate the apposing monolayer. The hydrophobic thickness of the bilayer under this condition is approximately twice the length of the hydrophobic tails of the phospholipid. However, under certain special conditions, one does encounter an unusual situation where the acyl chains from opposing monolayers fully interpenetrate or interdigitate (Figure 1e) so that the terminal methyl groups of the lipid © 2015 American Chemical Society
chains are located at the interfacial region on the opposite sides of the bilayer. This unusual phase is referred to as the interdigitated phase, LβI. This distinctive packing arrangement of the acyl chain region in the interdigitated phase results in a considerable reduction in the bilayer thickness. Biologically, the substantial thickness change of the membrane that accompany the formation of LβI phase can in principle strongly affect the normal functions of membrane associated proteins due to the mismatch of the hydrophobic regions. In the present article, we investigate the efficacy of small amphiphatic molecules in inducing chain interdigitation in symmetric chain PC membranes. It is important to note that chain interdigitation is a phenomenon which is not usually encountered in these membranes. Rather, at high temperatures (i.e., above the main transition temperature Tm), these membranes exhibit the Lα phase or the f luid phase where the hydrocarbon chains show random chain order (Figure 1b), and at low temperatures, the gel phase Lβ′, with the hydrocarbon chains are predominantly in the fully stretched all-trans conformation and are tilted with respect to the bilayer normal (Figure 1c). Further, in an intermediate temperature range and Received: October 26, 2015 Revised: December 16, 2015 Published: December 20, 2015 164
DOI: 10.1021/acs.jpcb.5b10478 J. Phys. Chem. B 2016, 120, 164−172
Article
The Journal of Physical Chemistry B
Water-soluble organic solvents such as acetone, acteonitrile, propionaldehyde, and tetrahydrofuran are also known to induce chain interdigitation in PC membranes.33 Other known factors that are responsible for the induction of interdigitation are changes in the environment, like a change in the hydrostatic pressure,34,35 the pH of the solution,36,37 or the molecular structure of the lipid, for example, by introducing an etherlinkage in the headgroup of the phospholipids.38,39 A classic example is bilayers formed by dihexadecylphosphatidylcholine (DHPC), which exhibit a fully interdigitated structure in excess water.40−42 The bilayer interdigitation in the case of etherlinked PCs is attributed to the weaker interaction between polar head groups as compared to the ester-linked PCs, such as, dipalmitoylphosphatidylcholine (DPPC). All these results demonstrate that specific interactions do not seem to play a critical role in the formation of LβI phase. It has been proposed that the main driving force behind interdigitation is an increase in the headgroup surface area, which results in an energetically unfavorable packing of the hydrocarbon chains. Interdigitation of the chains decreases the average separation between the chains, thus increasing the van der Waals attraction between them. The main aim of the present work is to elucidate the contribution of small amphiphatic molecules in altering the physical properties of a symmetric chain PC bilayer membrane, the effective headgroup surface area in particular, which has been proposed to be responsible for chain interdigitation in these systems. Using small-angle X-ray scattering (SAXS), we have carried out a systematic investigation of the physical interactions of three naphthalene derivatives containing hydroxyl groups, namely, β-naphthol (Figure 2b), 2,3-
Figure 1. (a) Cartoon of a lipid molecule. Schematic of the lipid bilayer in the (b) f luid phase Lα; gel phase (c) Lβ′ − acyl chains are tilted w.r.t the bilayer normal or (d) Lβ − acyl chains lie along the bilayer normal, and (e) interdigitated phase, LβI. Note in (b), (c), and (d) that the acyl chains from one monolayer do not penetrate the apposing monolayer, whereas in (e), the acyl chains from opposing monolayers fully interpenetrate.
at high water content, PCs also exhibit a modulated (Pβ′) phase between the Lα and the Lβ′ phases,3−17 which is characterized by a one-dimensional height modulation of the bilayers. Existence of the L βI phase was first experimentally demonstrated by Luzzati et al.18 in the gel phase of dipalmitoylphosphatidylglycerol (DMPG). Later the interdigitated phase was also shown to manifest in membranes composed of phosphatidylcholines (PCs). Although, symmetric chain PC membranes do not spontaneously exhibit LβI phase, it has been shown that presence of additional driving forces leads to chain interdigitation. Interdigitation in PC membranes can be induced by the incorporation, at the membrane interface, of small amphiphilic molecules, like glycerol,19 anesthetics,20 or alcohols.21−28 Short-chain alcohols (methanol through heptanol) are widely known to induce interdigitation.21−26,29 These alcohols exhibit a biphasic melting behavior on heating in PCs;23,24 that is, the main transition temperature of PCs is reduced at low concentrations of alcohol but is increased at high concentrations. It was subsequently shown by Simon et al.29 that the alcohol-induced biphasic behavior observed in PCs is a consequence of acyl chain interdigitation. At low concentrations of alcohol, the disorder of the lipid tails increases, leading to a lower transition temperature. At high concentrations, the more tightly packed interdigitated phase is formed, resulting in an increase of the transition temperature. Therefore, the study of the LβI phase is of particular interest while dealing with alcohol toxicity, alcohol tolerance, general anesthesia, cell viability, food sterilization, membrane fusion, and metabolic changes,30−32 because the normal functioning of the membrane embedded proteins can in principle be modified by the change in membrane thickness associated with this phase.
Figure 2. Molecular structure of (a) naphthalene, and the naphthalene derivatives, (b) β-naphthol, (c) 2,3-dihydroxynaphthalene, and (d) 2,7-dihydroxynaphthalene.
dihydroxynaphthalene (Figure 2c), and 2,7-dihydroxynaphthalene (Figure 2d) with DPPC bilayers. On the basis of the diffraction patterns, we have also determined the partial phase diagrams (Figure 4) of the binary mixtures consisting of different naphthalene derivatives and DPPC. We show that all the naphthalene derivatives stabilize the ripple phase over a wide temperature and concentration range. We further show that with the exception of β-naphthol, other naphthalene derivatives induce chain interdigitation when present at high concentration in the DPPC membrane.
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MATERIALS AND METHODS β-naphthol, 2,3-dihydroxynaphthalene, and 2,7-dihydroxynaphthalene (Figure 2) were procured from Sigma-Aldrich while 165
DOI: 10.1021/acs.jpcb.5b10478 J. Phys. Chem. B 2016, 120, 164−172
Article
The Journal of Physical Chemistry B
Figure 3. Characteristic diffraction patterns of the different lamellar phases. (a) Fluid phase Lα, (b) Ripple phase Pβ′. Inset: additional satellite reflections in the small-angle region of the diffraction pattern correspond to an oblique two-dimensional lattice. (c) Gel-phase Lβ′. Presence of two sharp wide-angle reflections, one on-axis and the other off-axis, indicates the presence of chain tilt. (d) Interdigitated phase LβI. Absence of chain tilt is indicated by the presence of only one on-axis peak in the wide-angle region.
transition temperature of DPPC, and the diffraction patterns were recorded during cooling from the Lα phase. Samples were equilibrated for at least 45 min at each temperature. A typical exposure lasted for about 15 min. Small-angle scattering techniques can in principle detect microscopic phase separation in the plane of bilayers, if there is sufficient contrast in the scattering densities of the two phases. However, even in the absence of such contrast, macroscopic phase separation can easily be detected from nonoverlapping reflections in the diffraction pattern coming from the individual phases. Intensities of the Bragg peaks obtained in the smallangle region in SAXS experiments give information on the various structural parameters of the membranes. Lattice parameter(s) of the different structures formed by the bilayers can be directly determined from the positions of the peaks in the small-angle region of their diffraction patterns. The wideangle region of these patterns contains information about the conformation of the hydrocarbon chains. Different phases observed in our experiments were identified on the basis of their characteristic diffraction patterns. The lamellar phases Lα, Lβ′, and LβI all gave rise to a set of peaks in the small-angle region corresponding to a one-dimensional lattice (Figure 3). Owing to the differences in chain packing, the wide-angle patterns of these lamellar phases were markedly different. In the Lα phase, we found only a diffuse wide-angle peak. The gel-phase Lβ′ was identified from the presence of two or three sharp wide-angle reflections, in the diffraction pattern,5 one on-axis and the other off-axis, depending on the direction of chain tilt with respect to the chain lattice. Only one on-axis (qz = 0, z being the direction of the bilayer normal) pair was seen in the LβI phase, indicating that the chains in this phase are not tilted (Figure 3d). Further, the lamellar periodicity of the
DPPC was obtained from Avanti Polar Lipids. All of these were used as received without any further purification. SAXS experiments on different lipid−naphthalene derivative systems were carried out on samples which consisted of a periodic stack of bilayers aligned on a substrate. Details about the experimental setup and data acquisition are provided elsewhere.43,44 In short, aligned multibilayers were prepared as follows: A concentrated solution of DPPC−β-naphthol (or 2,3dihroxynaphthalene or 2,7-dihroxynaphthalene) binary mixture (dissolved in chloroform in designated molar ratio) was deposited on the outer surface of a clean cylindrical glass substrate (radius of curvature ∼9 mm). After deposition, the samples were placed overnight under vacuum to remove all traces of the solvent. Subsequently, they were kept in a watersaturated atmosphere and were hydrated for a couple of days to obtain a stack of bilayers oriented parallel to the surface. Cu Kα (λ = 1.54 Å) radiation from a rotating anode X-ray generator (Rigaku, Ultra X18) operating at 48 kV and 70 mA and rendered monochromatic by a multilayer mirror (Xenocs) was used to illuminate the hydrated sample kept inside a sealed chamber with two mylar windows. The chamber temperature was controlled using a circulating water bath to an accuracy of ±0.1 °C and the relative humidity (Rh) inside it was maintained at 98 ± 2%, by keeping a reservoir of water. The axis of the cylindrical substrate was oriented perpendicular to the incoming X-ray beam, such that the beam is incident tangentially to the sample. Diffraction patterns were recorded on a 2D image plate detector of 345 mm diameter and 0.1 mm pixel size (Marresearch). The sample temperature and the Rh close to the sample were measured with a thermo-hygrometer (Testo 610) inserted into the chamber. Oriented multilayers were first heated to a temperature well above the main 166
DOI: 10.1021/acs.jpcb.5b10478 J. Phys. Chem. B 2016, 120, 164−172
Article
The Journal of Physical Chemistry B
Figure 4. Partial Phase diagrams determined at 98 ± 2% relative humidity of (a) DPPC−β-naphthol, (b) DPPC−2,3-dihydroxynaphthalene, and (c) DPPC−2,7-dihydroxynaphthalene mixed bilayers. The Lα phase is represented by ⧫, Lβ′ by ▲, Pβ′ by ● and LβI by ▼. Points on the phase diagram correspond to conditions for which diffraction data were collected.
Figure 5. Variation of the wavelength λ of the ripple phase at 25 °C with increasing concentration of (a) β-naphthol, (b) 2,3-dihydroxynaphthalene, and (c) 2,7-dihydroxynaphthalene in the DPPC membrane. The typical error in λ is ±0.5 Å. The smooth lines are merely guides for the eye.
LβI phase is lower than that of the Lβ′ phase as the bilayer thickness is almost half in the former (Figure 3). The ripple phase Pβ′, was identified from the presence of additional satellite reflections in the small-angle region, which correspond to an oblique two-dimensional lattice (Figure 3b). Presence of crystallites was inferred from the appearance of a large number of sharp peaks both in the small-angle and the wide-angle regions of the diffraction pattern.
practically no effect on the phase behavior of the pure lipid. Both the main and the pretransition temperatures remain unaffected. However, behavior of the mixture starts to deviate from that of the pure lipid when the concentration of naphthol in the mixture exceeds 30 mol %. Between 30−40 mol %, we find that the rippled Pβ′ phase coexists with the lowtemperature gel Lβ′ phase down to 10 °C. Between 40 and 70 mol % of β-naphthol, the Lβ′ vanishes, and the whole region of the phase diagram is dominated by two phases, the hightemperature fluid phase Lα, and at low temperatures by the Pβ′ phase, which seems quite surprising. Plot of the wavelength λ of the ripple phase as a function of β-naphthol concentration is shown in Figure 5a. With increasing concentration of β-naphthol in the membrane, the wavelength decreases at first until 50 mol %, beyond which it increases. However, the variation of ξ (where ξ = (π − γ; Figure S1a (Supporting Information (SI)), γ being the unit cell parameter), which is a measure of the obliqueness of the unit cell in the reciprocal space, as a function of β-naphthol concentration reveals an interesting feature, Figure S2a (SI). It is seen that with increasing concentration of β-naphthol in the ripple phase, the obliqueness of the unit cell decreases. Thus, increasing amounts of β-naphthol in the membranes makes the ripples more symmetric. Beyond 70 mol % naphthol crystallizes out of the membranes. This was identified by the presence of very sharp crystalline peaks in the wide-angle as well as the small-angle region of the SAXS pattern. Interestingly, β-
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RESULTS The thermotropic phase behavior of different PCs in general and DPPC in particular has been well characterized (see ref 45 for a review). As mentioned earlier, pure DPPC exhibits three different lamellar phases at high hydration, consisting of stacks of bilayers separated by water: the Lα phase above the main transition (Tm ≈ 42 °C), the gel phase (Lβ′) below the pretransition (≈ 36 °C), and the ripple phase (Pβ′) between. DPPC−β-Naphthol Binary Mixtures. Naphthols are naphthalene homologues of phenol, with the hydroxyl group being more reactive than in phenols. β-naphthol is a colorless crystalline solid with the formula C10H7OH. The structure of βnaphthol is shown in Figure 2b. It consists of a pair of fused benzene rings, to which a lone hydroxyl group is attached at the carbon position 2. Figure 4a represents the partial phase diagram of DPPC-βnaphthol binary mixtures at 98 ± 2% relative humidity (Rh). We find that addition of β-naphthol in very small quantities has 167
DOI: 10.1021/acs.jpcb.5b10478 J. Phys. Chem. B 2016, 120, 164−172
Article
The Journal of Physical Chemistry B
Figure 6. Trans-bilayer electron density profile of the bilayer in the Lα phase at 45 °C as a function of (a) β-naphthol, (b) 2,3-dihydroxynaphthalene, (c) 2,7-dihydroxynaphthalene concentration in the membrane at 45 °C. Trans-bilayer EDP of the bilayer in the LβI phase as a function temperature at (d) 60 mol % of 2,3-dihydroxynaphthalene and (f) 50 mol % of 2,7-dihydroxynaphthalene. (e) Trans-bilayer EDP of the bilayer in the LβI phase at 10 °C as a function of 2,7-dihydroxynaphthalene concentration in the membrane.
Figure 7. Plots of the bilayer thickness dpp and the d-spacing of the DPPC bilayer as a function of the naphthalene derrivative concentration in the Lα phase at 45 °C. (a, b) β-naphthol, (c, d) 2,3-dihydroxynaphthalene, and (e, f) 2,7-dihydroxynaphthalene. The smooth lines are merely guides for the eye.
naphthol was unable to induce the interdigitated phase at high concentrations. From the diffraction data we have calculated the trans-bilayer electron density profile (EDP) of the bilayer using the procedure described in the SI. The trans-bilayer EDP of the bilayer in the L α phase, with increasing β-Naphthol concentration in the membrane at 45 °C, is shown in Figure 6a. The peak-to-peak separation dpp in these profiles is a good measure of the bilayer thickness. Incorporation of β-naphthol in
the DPPC membrane does not have any significant effect on either the bilayer thickness or the d-spacing as shown in Figure 7a,b. DPPC−2,3-Dihydroxynaphthalene Binary Mixtures. 2,3-Dihydroxynaphthalene is a beige-colored crystalline solid with the chemical formula C10H6(OH)2. The molecular structure of 2,3-Dihydroxynaphthalene is similar to β-naphthol but with an extra OH− group at carbon position 3 (Figure 2c). The presence of this additional OH− group gives 2,3168
DOI: 10.1021/acs.jpcb.5b10478 J. Phys. Chem. B 2016, 120, 164−172
Article
The Journal of Physical Chemistry B
The trans-bilayer electron density profile (EDP) of the bilayer in the interdigitated phase was calculated from the diffraction data. The EDP of the bilayer in the interdigitated phase LβI as a function of temperature at a fixed concentration of 60 mol % of 2,3-dihydroxynaphthalene is shown in Figure 6d, whereas Figure 6e shows the EDP of the bilayer in the LβI as a function of concentration at a fixed temperature of 10 °C. Figure S4a (SI) shows the EDP at 10 °C when the concentration of 2,3-dihydroxynapthalene in the membrane is 60 mol %. For comparison, we have also calculated the EDP in the Lβ′ phase, which is shown in Figure S4b (SI). DPPC−2,7-Dihydroxynaphthalene Binary Mixtures. 2,7-Dihydroxynaphthalene is an isomer of 2,3-dihydroxynaphthalene, where the hydroxyl groups reside at carbon positions 2 and 7, respectively (Figure 2d). As a result, 2,7-dihydroxynaphthalene has the largest effective headgroup area in comparison to both β-naphthol and 2,3-dihydroxynaphthalene. Partial phase diagram of DPPC−2,7-dihydroxynaphthalene binary mixtures at 98 ± 2% Rh is shown in Figure 4c. Addition of 2,7-dihydroxynaphthalene in DPPC membranes has a very profound effect on the main transition of the pure lipid. With increasing concentration, the main transition was found to decrease from ∼42 °C for the pure DPPC to around 30 °C at 40 mol % of 2,7-dihydroxynaphthalene. Concentration of 2,7dihydroxynaphthalene below 2.5 mol % in the membrane does not seem to have much effect on the phase phase behavior of DPPC. Within the concentration range 5−20 mol %, 2,7dihydroxynaphthalene stabilizes the Pβ′ phase to very low temperatures. Variation of the wavelength λ of the ripple phase and ξ with respect to 2,7-dihydroxynaphthalene concentration are shown in Figure 5c and Figure S2c (SI), respectively. Both these figures indicate that there is a drastic change in the values of these parameters in the concentration range 10−15 mol %. λ decreases by around 40 Å, whereas ξ increases by 15°, Figure S2c (SI). Over a very broad concentration range, 20−30 mol %, this ripple phase coexists with the interdigitated phase. Beyond 30 mol %, only the interdigitated phase was found to exist below the main transition. Interestingly between 70 to 75 mol %, a two-phase coexistence region was observed over a temperature range 15−30 °C. This region was found to consist of the interdigitated LβI phase and a fluid phase rich in 2,7dihydroxynapthalene. The interbilayer spacing of this fluid phase is around 11 Å less than the usual Lα phase. At 75 mol %, 2,7-dihydroxynaphthalene crystallizes out of the membranes. Figure 6c shows the trans-bilayer EDP of the bilayer in the Lα phase, with increasing 2,7-dihydroxynaphthalene concentration in the membrane at 45 °C. Bilayer thickness dpp and the dspacing as a function of 2,7-dihydroxynaphthalene in the DPPC membrane in the Lα phase are shown in Figure 7e,f. We find that with increasing concentration of 2,7-dihydroxynaphthalene in the membrane the d-spacing decreases by 11 Å, Figure 7e. Further, the bilayer thickness decreases by 7 Å at high concentration of 2,7-dihydroxynaphthalene, Figure 7f. Using the diffraction data, we have calculated the transbilayer EDP of the bilayer in the interdigitated phase. The plot of the bilayer thickness dpp (Figure S3a (SI)) as well as the dspacing (Figure S3b (SI)) as a function of 2,7-dihydroxynaphthalene concentration in the membrane in the LβI at 10 °C reveals that this molecule does not have any effect on these quantities. Both dpp and d-spacing shows a marginal increase with increasing 2,7-dihydroxynaphthalene concentration. The plot of the EDP of the bilayer in the interdigitated phase LβI as a function of temperature at a fixed concentration of 2,7-
dihydroxynaphthalene an effective headgroup area, which is larger than that of β-naphthtol. The partial phase diagram of DPPC−2,3-dihydroxynapthalene binary mixtures at 98 ± 2% Rh is shown in Figure 4b. Addition of 2,3-dihydroxynaphthalene in very small quantities (
