Unbinding Transition in Lipid Multibilayers Induced by Copper(II) Ions

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2008, 112, 8430–8433 Published on Web 06/25/2008

Unbinding Transition in Lipid Multibilayers Induced by Copper(II) Ions Zolta´n Varga,†,§ Attila Bo´ta,*,† and Gu¨nter Goerigk‡,⊥ Institute of Nanochemistry and Catalysis, Chemical Research Center, Hungarian Academy of Sciences, Pusztaszeri u´t 59-67, H-1025 Budapest, Hungary, and Institute of Solid State Research, Forschungszentrum Ju¨lich, D-52425 Ju¨lich, Germany ReceiVed: March 20, 2008; ReVised Manuscript ReceiVed: May 08, 2008

We describe our observations on an unbinding transition in a multilamellar dispersion of phosphatydilcoline (PC) vesicles induced by copper(II) ions. The small-angle X-ray measurements clearly show that the increasing amount of CuCl2 in the millimolar concentration range continuously increases the amount of the unbound bilayers in the gel phase. Moreover, this phenomenon becomes more pronounced when the samples are heated above the so-called pretransition temperature between the gel and the ripple gel phase. The proposed reason for the latter is the increased repulsive electrostatic interaction due to the appearance of the surface modulation in the ripple gel phase. The observed effects reveal a new aspect of the unbinding phenomena since only the transition induced by the steric repulsion due to the layer fluctuations has been considered so far. Here, we show that the unbinding can also be triggered by the change in the electrostatic interactions. These findings are connected to the physical basis of the crucial role of copper(II) ions in biological processes such as neurodegenerative diseases and cell evolution. Introduction Multilamellar vesicles (MLVs) or liposomes serve as model systems for biomembranes since their basic building block, the phospholipid bilayer, is the same. In MLVs, the bilayers and the water layers form regular, centrosymmetric structures with a well-defined repeat distance, making possible the study of the physical properties of the bilayers with diffraction methods.1 However, the advantage of using MLVs is not only this property but the ability of studying the interactions between neighboring lipid bilayers, which is important in understanding many features of biomembranes. Besides the main components of the real cell membranes, there are many indispensable chemicals which are present in the millimolar range of concentrations, but they account for an important physiological function. Divalent cations fall into this group; therefore, their effect on biomembranes and also on model membranes attracted increasing attention in recent years.2,3 These ions can strongly affect the double-layer structure and the conformation of the embedded membrane proteins. For example the copper(II) ions under consideration are known to be released during synaptic transmission, which is in connection with the involvement of these ions in the pathology of neurodegenerative diseases such as Alzheimler’s, Parkinson’s, and Creutzfeldt-Jakob diseases.4,5 The unbinding is a feature of lipid multibilayers, which was described several years ago,6 but its experimental observation * To whom correspondence may be addressed. Tel.: +36 1 438 1100. Fax: +36 1 438 1164. E-mail: [email protected]; on leave from Budapest University of Technology and Economics, Departement of Physical Chemistry and Materials Science. † Hungarian Academy of Sciences. ‡ Research Centre Ju ¨ lich. § E-mail: [email protected]. ⊥ E-mail: [email protected].

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is quite rare,7–13 although its biological significance is beyond doubt if one considers, for example, the fission and the fusion of cells. Recently, it was reported that some cations can cause a transition from a bound to an unbound state.10–12 However, the changes in the electrostatic interactions and the formation of surface modulation in the so-called ripple gel phase were not considered as the reason for the observed effects so far. In this letter, we present our small-angle X-ray scattering results on the effect of CuCl2 on dipalmitoyl-phosphatidylcholine MLVs as a representative lipid of the human cell membranes, at different concentrations and temperatures. Materials and Methods Synthetic high-purity 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and copper chloride (CuCl2) were obtained from Avanti Polar Lipids (U.S.A.) and from Merck (Germany), respectively. The chemicals were used without further purification. Twenty wt% MLV dispersions were produced by the simple mixing of the pure lipid with a 50 mM TRIS buffer solution (pH ) 7.4) containing the proper amount of CuCl2. The studied nominal concentrations in Cu(II)/DPPC ratio were 0:100, 2:100, 3:100, and 5:100 corresponding to 0, 6.8, 10.2, and 17 mM CuCl2, respectively. The samples were kept at 50 °C for 10 min and then vortexed intensively and quenched to 4 °C. This process was repeated at least 20 times to get a homogeneous liposome system. For the X-ray measurements, the samples were transferred into thin-walled (Plexiglas without significant small angle scattering) sample holders and placed into a thermostatted aluminum block with beryllium windows for precise incubation. Small-angle X-ray scattering experiments were carried out at the JUSIFA (B1) synchrotron beamline at HASYLAB (DESY, Hamburg)14 in the regime of the scattering variable  2008 American Chemical Society

Letters

Figure 1. SAXS patterns of DPPC MLV dispersions at T ) 25 °C having different amounts of CuCl2. Squares (0), circles (O), triangles (∆), and upturned triangles (3) correspond to 0, 6.8, 10.2, and 17 mM CuCl2, respectively. The curves were shifted for clarity.

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Figure 2. DSC heating thermograms of DPPC MLV dispersions having different amounts of CuCl2; (a-c) correspond to 0, 6.8, and 10.2 mM CuCl2, respectively.

q ) 4π/λ sin θ from 0.02 up to 0.5 Å-1, which corresponds to the size range in the real space of ≈300-13 Å. The scattering curves were calibrated to absolute units of macroscopic cross sections (cm2/cm3 ) cm-1). Scanning microcalorimetric measurements were made on a MicroDSCIII apparatus (SETARAM). Approximately 20 mg of liposome sample was placed into the measurement cell and was incubated for 1 h at T ) 20 °C. The heating scans were executed between 20 an 50 °C with a scan rate of 0.5 °C/min. Results and Discussion The SAXS curves of the pure DPPC/water system exhibit Bragg peaks up to 4 or 5 orders due to the ordered layer structures in all three of its phases, that is, in the gel (Lβ′), ripple gel (Pβ′), and the liquid crystalline (LR) phase. Uppon the addition of CuCl2 in the millimolar range of concentrations, the well-ordered structure of the MLVs disappears, as can be seen on Figure 1, on which the SAXS curves of the systems doped with different amounts of CuCl2, measured at 25 °C (which corresponds to the Lβ′ phase), are shown. The SAXS curve of the system having 17 mM CuCl2 resembles the typical shape of the scattering curve of the uncorrelated bilayers, while the curve of the sample having 6.8 mM CuCl2 resembles that of a well-ordered MLV system. One can see from these results that the system having 10.2 mM CuCl2 represents a transition state between the fully bound bilayers, characteristic of the pure system, and the fully unbound bilayers, characteristic of the system having 17 mM CuCl2. To reveal the thermotropic behavior of the systems under consideration and to rule out the possibility of the inhomogeneous distribution of the CuCl2, we have performed DSC measurements, the results of which are shown in Figure 2. From these, we can conclude that the samples were homogeneous, that is, there were no Cu2+-free and rich bilayer stacks in the MLVs, and systematic changes occur with increasing CuCl2 concentration. The most significant changes are in the measured pretransition (the phase transition between the Lβ′ and Pβ′ phase) temperature (35.7, 33.6, and 32.2 °C corresponding to 0, 6.8, and 10.2 mM CuCl2, respectively). The enthalpy change of this transition is also decreasing (1.3, 1.2, and 1 kcal/mol for the increasing concentrations). While there is little difference in the main

Figure 3. The scattering curves of the system having 6.8 (top) and 10.2 mM (bottom) CuCl2 at different temperatures. Squares (0), circles (O), and triangles (∆) corresponds to the Lβ′, Pβ′ and the LR phase, respectively. The fitted model functions are drawn with solid lines.

transition (the phase transition between the Pβ′ and the LR phase) temperatures, ∆Tc ) 0.7 °C, the enthalpy change of this transition is increasing with the higher amount of CuCl2 (7.4, 8.1, and 8.7 kcal/mol). On the basis of our DSC results, we performed SAXS measurements on the systems with 6.8 and 10.2 mM CuCl2 at three different temperatures corresponding to the three phases, that is, at T ) 25 (Lβ′), 38 (Pβ′), and 46 °C (LR), and the results are shown in Figure 3. In order to interpret the curves of these measurements, we recall the expression of the scattered intensity from such MLV systems. In general, the latter can be written as the product of the structure factor (S(q)), which describes

8432 J. Phys. Chem. B, Vol. 112, No. 29, 2008

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TABLE 1: The Ratio of the Unbound Bilayers at Different Concentrations and Phases temperature [°C]/ CuCl2 conc. [mM]

0

6.7

10.2

17

25°C(Lβ′) 38°C(Pβ′) 46°C (LR)

0 0 0

0 65.5 83.9

84.9 100 100

100 100 100

the one-dimensional order of the bilayers, and the square of the form factor (|F(q)|2), which is the Fourier transform of the bilayer electron density profile.

I(q) ) (1 - xdiff) · S(q)|F(q)|2/q2 + xdiff · |F(q)|2/q2

(1)

The last term in the expression represents the scattering contribution from uncorrelated bilayers, with xdiff being the ratio of the diffuse scattering due to the latter structures. To analyze the scattering patterns from the systems, which represent a transition state between the fully bound and the fully unbound states, we have performed a model fitting. We used the structure factor obtained from the paracrystalline theory (PT), while for the form factor, we applied the 1G model as follows:15–17 N-1

SPT(q) ) N + 2

∑ (N - k)cos(kqd)exp(-k2q2∆2/2)

(2)

k)1

where N is the number of the layers, d is the repeating unit, and ∆ is the mean-square fluctuation of the bilayers, and

F(q) ) √2π(2σH exp(-σH2q2/2)cos(qzH) - FCHσCH 2 exp(-σCH q2/2)) (3)

where σH and zH are the width of and the position from the bilayer center of the headgroup Gaussian functions, while σCH and FCH are the width of and the relative ratio of the Gaussian function representing the chain region, respectively. Here, we will restrict our discussion to the value of xdiff, but all of the fitted parameters can be found in the Supporting Information. The determination of the ratio of the unbound bilayers was trivial at the pure system (bound), the sample with 17 mM CuCl2 (unbound), the sample with 6.8 mM in the Lβ′ phase (bound), and the sample with 10.2 mM in the LR phase (unbound). In the other cases, the model fitting was applied to reveal the degree of the unbinding. The fitted model functions are also shown in Figure 3, and the corresponding values of the ratio of the unbound bilayers18 are summarized in Table 1. On the basis of the latter results, we have drawn the corresponding phase diagram, which is shown in Figure 4. Turning to the explanation of the observed effects, we should start considering the interactions of divalent cations with phospholipid bilayers. Some experimental3 and simulation studies19,20 show that these ions bind tightly to the headgroup region of the bilayers, causing a surface charge on the lipid/ water interface. However, generally, one cannot observe an increment in the electrostatic repulsion, unless the concentration is in the millimolar range, where the Debye length (κ-1) is relatively large. Beyond the electrostatic interactions, the osmotic effect can also be considered, but it cannot account for our observations since the lipid systems consisting of PC lipids doped with divalent cations show an ordered multilamellar structure if one increases the salt concentration further than the millimolar range due to the decrement in the Debye length.11,21,22 The experimental observations of the unbinding transition reported so far consider the enhancement in the steric repulsion

Figure 4. The phase diagram of the DPPC/CuCl2 system constructed from the investigated systems. The colorbar represents the ratio of the unbound bilayers. Colorcoding was made by using linear interpolation between the measured data points from Table 1. The transition temperatures measured by DSC are also shown.

(known as Helfrich repulsion23), due to the fluctuations, as the reason for the unbinding. However, that cannot be the explanation for our results since we have observed the unbinding transition in the gel phases (Lβ′ and Pβ′). In the Lβ′ phase, the bending rigidity (wherewith the Helfrich repulsion is inversely proportional) is about 1 order of magnitude larger than that in the fluid, LR phase; therefore, the fluctuations in the Lβ′ phase can be neglected related to those in the LR phase. In the ripple Pβ′ phase, the bending rigidity becomes anisotropic, and its value parallel to the wave vector of the surface ripples remains nearly the same, while in the perpendicular direction, it increases;24 therefore, the previous statement holds for the Pβ′ phase too. We propose that the changes in the electrostatic interactions serve as a reason for the unbinding transition in our experiments. In the case of the measurements in the Lβ′ phase, the increasing concentration of CuCl2 caused an enhancement in the electrostatic repulsion, and at a critical concentration, the repulsive interactions overcome the van der Waals attraction, and the system becomes unbound. The reason for the observation, that the unbinding appeared (6.7 mM) or the layered structures disappeared (10.2 mM) when the samples were heated above the pretransition, roots in the formation of the surface ripples. It was shown by Goldstein et al.25 that the electrostatic repulsion between two charged layers increases if the surfaces of them are modulated, or in other words, the critical repulsion can be reached earlier, when the modulation appears. According to ref 25, the increment in the electrostatic repulsion at small undulations can be approximated by

1 3 (Vmod - Vflat)/Vflat ) (κa)2 + (ap)2 2 4

(4)

where a modulation in the form of a · cos(px) was assumed. Using the characteristic parameters of the Pβ′ phase of DPPC bilayers (a ≈ 1 nm, p ≈ 2π/14 nm-1), and κ-1 ≈ 3 nm), one gets ≈20% for the quantity described by eq 4, which corresponds to a relative high increment in the repulsion. Since this jump in the electrostatic interactions accompanies the pretransition, the possibility of an unbinding transition in this temperature region (below the main transition), the value of which does not coincide with the pretransition temperature, is negligible. The validity of eq 4 for our case is also supported by our DSC measurements, namely, the theoretical considerations leading to this formula imply that the average free energy of

Letters such a system becomes more negative with the appearance of the modulation, which can be traceable on the decreasing pretransition temperature with the increasing concentration. In other words, the rippled structure is stabilized by the appearing surface charge, the result of which is that the Pβ′ appears at smaller temperatures. Conclusions Our results can be summarized as follows: (i) The increasing concentration of the Cu(II) ions causes an unbinding in the Lβ′ phase of the bilayers. (ii) The unbinding appears at lower concentration, when the sample is heated above the pretransition temperature. Ruling out the other possibilities, we came to the conclusion that the reason for the unbinding at both cases is the change in the electrostatic repulsion, that is, the increment of the latter with the concentration (i) and with the formation of the surface ripples in the Pβ′ phase (ii). The observations presented here about the structural changes in multibilayers doped with copper(II) ions show a new aspect of the unbinding phenomena; moreover, they reveal the crucial role of divalent cations in complex biological systems. Acknowledgment. We are grateful to R. Lipowsky for critical reading of the manuscript and for giving us valuable suggestions. We wish to thank U. Vainio at HASYLAB for her kind help with the scattering measurements and M. Zrı´nyi for giving the opportunity for the calorimetric measurements. This work was supported by the Hungarian Scientific Funds OTKA (Bo´ta, T 43055) and by the Contract RII3-CT-2004-506008 of the European Community at DESY/HASYLAB Supporting Information Available: The model parameters obtained from the model fitting for the curves from the 6.8 and 10.2 mM CuCl2/DPPC systems shown in Figure 3. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) See, for example: Katsaras, J., Gutberlet, T., Eds. Lipid Bilayers; Springer: Berlin, Germany, 2000.

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