Article pubs.acs.org/Langmuir
Cytochrome c Complexes with Cardiolipin Monolayer Formed under Different Surface Pressure Margarita A. Marchenkova,*,†,‡ Yulia A. Dyakova,†,‡ Elena Yu. Tereschenko,†,‡ Mikhail V. Kovalchuk,†,‡,§ and Yury A. Vladimirov∥,⊥ †
Shubnikov Institute of Crystallography of Russian Academy of Sciences, 119333 Moscow, Russian Federation National Research Centre “Kurchatov Institute”, 123182 Moscow, Russian Federation § St. Petersburg State University, 199034 St. Petersburg, Russian Federation ∥ M.V. Lomonosov Moscow State University, 119991 Moscow, Russian Federation ⊥ Pirogov Russian National Research Medical University, 117997 Moscow, Russian Federation ‡
ABSTRACT: The formation of the complex of cytochrome c (Cytc) with a phospholipid cardiolipin (CL) in mitochondria is a crucial event in apoptosis development. There are two viewpoints on the structure of the complex. (1) Cytc is bound on the surface of the lipid bilayer. (2) The complex is a hydrophobic nanoparticle Cytc-CL formed by Cytc molten globule, covered by CL monolayer.1 In the present work, we attempted to bridge the gap between these two structures. We investigated the interaction between Cytc and Langmuir monolayers of CL. The surface pressure increase during incorporation of Cytc into CL monolayer obeys the equation: π = π0 + Δπ∞[1 − exp(−βt)], where β is pseudo-first-order rate constant of Cytc binding, directly proportional to the initial Cytc concentration c0. Parameters Δπ∞ and the rate β measured in different conditions were virtually equal for natural bovine CL and peroxidation-resistant tetraoleoyl CL in all experiments. Surface area−surface pressure isotherms of Cytc alone and in combination with a CL monolayer were similar in shape. Apparently, the protein exposes hydrophilic groups to the water phase and hydrophobic to the air or to the hydrocarbon chains of CL. The 30% ethanol dramatically accelerated the adsorption of Cytc on the water surface. The protein−lipid surface films showed, in compression−expansion cycles, that hysteresis loops were observed always when Cytc present, reproducible in repeating cycles. Taken together, our data show that when incorporated in a lipid monolayer or after adsorption on the water−air interface, Cytc undergoes conformational transition. In that, one part of the globule sphere becomes predominantly hydrophobic and the other, hydrophilic and charged (“stratified” Cytc). We hypothesize that in CL-containing bilayer membranes, Cytc incorporation into the lipid monolayer would result in membrane folding with subsequent formation of either catalytically reactive “bubbles” inside the bilayer, formed by Cytc-CL, or the appearance of hydrophilic pores. The role of lipid peroxidation catalyzed by Cytc-CL in the appearance of pores and apoptosis is also discussed.
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INTRODUCTION Cytc, a well-known electron carrier in the mitochondrial respiratory chain, plays at the same time a leading role in the initiation of apoptosis, a programmed cell death.2−4 It is now generally accepted that a key step in the apoptotic cascade involves the release of Cytc into the cytosol where it binds with apoptotic protease-activating factor 1 (Apaf-1) with the subsequent formation of apoptosome and development of a cascade of biochemical reactions terminated by cell death and its digestion by macrophages. It is shown in the experiments on cells in culture that the release of Cytc from mitochondria is preceded by peroxidation of CL, a phospholipid found in the cells almost exclusively in the inner mitochondrial membrane.5 Early in apoptosis, massive membrane translocations of CL take place resulting in its appearance in the outer mitochondrial membrane. Consequently, significant amounts of CL become available for interactions with Cytc, one of the major proteins of the intermembranous space.6 Cytc was shown to be necessary for both CL oxidation and apoptosis development in cells.5 © 2015 American Chemical Society
Although Cytc itself does not possess the peroxidase activity, it was shown to acquire that activity after forming a complex with CL or another anionic lipid.7,8 The structure of the complex is a matter of discussion (see review1). Since 1977,9 it has been considered well-known that Cytc is attached to the surface of the CL-containing lipid bilayer with electrostatic forces, hydrogen bonds, and hydrophobic interactions. Kinnunen and co-workers identified two Cytc binding sites on the CL-containing liposome surfaces termed A and C.10 Binding site A involving residues Lys72 and Lys73 is electrostatic by nature and the respective binding constant depends on the ionic strength of the solution. Binding via site C involving Arg52 has been suggested to occur mainly by means of hydrogen bonding and provides the ability of the complex to Received: August 23, 2015 Revised: October 20, 2015 Published: October 21, 2015 12426
DOI: 10.1021/acs.langmuir.5b03155 Langmuir 2015, 31, 12426−12436
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In the present paper, we tried to build up the sequence of events during complex formation of Cytc-CL, starting from the electrostatic binding of Cytc to the surface of the lipid monolayer in aqueous phase and terminated by the formation of nanospheres of Cytc-CL and hydrophilic megapores. With this purpose, we have performed the systematic investigation of the two-dimensional Cytc-CL surface complex by measuring the kinetics of its formation and surface pressure−area isotherms, with particular emphasis on the comparison of the data obtained, under different initial conditions, separately for CL and Cytc films with those of the complexes. Non-oxidizable tetraoleoyl cardiolipin (TOCL) was used in the majority of the experiments and the results obtained with TOCL were, in most cases, compared with those observed using natural CL for a better comparison of our own and literature data.
initiate lipid peroxidation. In addition, hydrophobic forces participate in binding (see reviews1,6,7,11,12 for more detail). The data have recently appeared showing that the Cytc-CL complex may have a completely different structure than that in the form of membrane-bound CL, namely, in the form of nanospheres consisting of a molten Cytc globule surrounded by a CL monolayer with the polar head of the lipid attached to the protein surface and hydrophobic hydrocarbon tails facing out.13 It was shown that in the mixture of Cytc with an excess of CL a water-insoluble complex was formed with a certain CL/protein molar ratio. In small-angle X-ray scattering diagrams, regularly spaced reflections were observed showing the lattice structure of the complex with a cell size of 11.2 nm.13 This implies that the Cytc-CL complex is a nanosphere with a swollen (molten) protein molecule inside covered with a CL monolayer. The scheme of evolution of the membrane-bound Cytc to a Cytc-CL nanosphere included into the mitochondrial membrane lipid bilayer can be understood in light of the experiments with giant liposomes.14,15 By using fluorescent markers, Cytc was shown to penetrate to the membrane. The authors supposed that opening of the lipid pores formed by the Cytc-CL conjugates took place. They offered a model in which the formation of toroidal lipid pores was driven by the initial Cytc-induced negative spontaneous membrane curvature. We may hypothesize that the first step of this evolution is a perturbation of the local CL monolayer by a Cytc molecule absorbed on the membrane surface (ref 1, Figure 7c). The mechanism of this perturbation remains yet obscure. One possible approach to the problem of the evolution from Cytc binding on the surface of CL-containing bilayer to formation of hydrophobic Cytc-CL nanospheres or hydrophilic channels could be experiments with lipid monolayers onto which Cytc is absorbed from the water subphase. Several authors used measurements of the parameters of Langmuir films to investigate which CL monolayer properties are modified under the action of Cytc added to the water subphase.16−18 Quinn and Dawson showed in their pioneer work that Cytc injection in the subphase under the phospholipid monolayer brought about a rapid increase of the surface pressure and surface potential followed by a slower increase of the protein amount in the layer.16 The concentration of Cytc, at which the maximal effect was achieved, was several times lower for anionic phospholipid CL monolayers as compared to those of neutral phospholipids due to an essentially higher affinity of negatively charged CL to a positively charged Cytc globule. Two indices (Δπ and ΔV) were taken as a measure of Cytc penetration into the lipid monolayer, while the amount of protein in the film (mol/cm2) was assumed to be a measure of adsorption of the protein on the monolayer. Although the description of the phenomena in this and other relevant papers19−21 was very detailed, there was no satisfactory physical interpretation. In addition, many conclusions are questioned, because the authors did not take into account that natural CL (containing 90% of readily oxidizable linoleic acid) used in the experiments is rapidly oxidized by air oxygen in the presence of Cytc.22 Cytc is destroyed under lipid peroxidation,8 and the physical properties of lipid membranes change dramatically.23−26 In quoted papers,16−18 there was no data showing that this was not the case in CL monolayers. Similary, in the experiments by Beales and co-workers with giant liposomes showing changes in the membrane morphology and the appearance of large pores, natural CL was used14,27 that could be oxidized during the experiment.
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MATERIALS AND METHODS
Materials/Chemicals. 1,1′,2,2′-Tetraoleoyl cardiolipin (TOCL) was purchased from Avanti Polar Lipids (115404−77−8). Bovine heart Cardiolipin (MFCD00071040) (BCL) and horse heart cytochrome c (MFCD00130890) were purchased from Sigma-Aldrich, USA. The concentration of spreading phospholipid solutions was 1.5 mg/mL in chloroform (Sigma-Aldrich, USA). The buffer 10 mM NaH2PO4− Na2HPO4 (PBS) (pH = 7.4) was prepared in ultrapure water (Milli Q, Millipore, 18 MΩ*cm). Preparation of Monolayers and Kinetic Measurement of the Protein−Lipid Interaction. The monolayers were formed in a KSV 5000 Langmuir−Blodgett trough (KSV Instruments, Finland). The working surface area of the trough was 750 cm2. The surface pressure (π) was measured by the Wilhelmy balance method using a platinum plate with a resolution of ±0.1 mN m−1. The trough was placed on an isolated table and enclosed in an environmental chamber. All monolayer experiments were carried out at T = 18.5 ± 0.5 °C. Before each experiment, the trough was washed with ethanol and rinsed thoroughly with purified water. In the experiments, 27 ± 3 nmol (if other is not indicated) lipid dissolved in chloroform was spread on a Cytc-free subphase by using a microsyringe (Hamilton), and following 10 min the lipid film was compressed with a 10 mm/min barrier speed to a certain surface pressure (π0). The refinement of the lipid amount in the film was performed by comparison of the isotherm obtained in each experiment with that for a CL solution of the known concentration. Since all four fatty acids in TOCL are unsaturated, the phase transition temperature was far below the room temperature at which the experiments were performed so that the monolayer was in the liquid state. After 10 min (the time necessary for monolayer stabilization) Cytc was injected with a microsyringe beneath the monolayer to a certain final concentration (c0) and gently stirred. Then a change in the surface pressure with time was measured at a fixed barrier position. Measurement of Surface Pressure−Area Isotherms. When the maximal surface pressure of the monolayer was reached (after kinetic measurement), the surface pressure−area isotherm of the lipid−protein monolayer was recorded. As the interactions between the lipid and protein in the monolayer may suggest some kinetically dependent response (nonequilibrium phase transitions, relaxation, etc.), a series of experiments was carried out with various barrier speeds. (A slight alteration was demonstrated with changing scan rate.) The forward and backward barrier speed rates were equal. These rates are given in the figure captions. In order to check the reversibility of the lipid−protein system, at least 5 “compression-expansion” cycles were performed.
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RESULTS AND DISCUSSION In the present paper, we used CL monolayers as a model of part of lipid bilayer in the mitochondrial inner membrane. Such a system seems to be adequate because the initial interaction of Cytc dissolved in an aqueous medium with the charged surface of the lipid monolayer will be the same in biological membranes or 12427
DOI: 10.1021/acs.langmuir.5b03155 Langmuir 2015, 31, 12426−12436
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Langmuir liposomes and in Langmuir films. In both cases, the hydrophobic surface of the monolayer with adsorbed or incorporated Cytc will be in contact with nonpolar substrate: hydrocarbon molecules or the air. The presence of neutral phospholipids in the membrane or monolayer is not critical, since Cytc binds strongly only to CLrich domains14 and neutral phospholipids are out of the game. Indeed, Beales and co-workers observed in the membranes, containing CL and noncharged phospholipids, the lipid separation induced by addition of Cytc, with the protein binding exclusively to the CL domain. All further events occurred exclusively within this domain (ref 14, Figure 1). Kinetics of Cytochrome c Adsorption: The Effect of the Initial Protein Concentration in the Subphase. In a series of experiments, Cytc was added to the subphase under the lipid monolayer previously formed and compressed to the initial surface pressure π0 = 5 mN m−1. The concentration of the protein under the monolayer varied in the range of 1 to 250 nM. Figure 1 shows the changes in the surface pressure with time. According to a pseudo-first-order model of adsorption kinetics28
π = π0 + Δπ∞[1 − exp( −βt )]
(1)
where π0 is the selected initial surface pressure, Δπ∞ is the final increment of the surface pressure at infinite time, β is the pseudofirst-order rate constant of Cytc binding with the lipid monolayer, and t is the time after the beginning of Cytc−CL interaction. For surplus of Cytc, β equals kc0, and for excess of lipid, β equals kl0, where c0 is the initial Cytc concentration in the aqueous phase, l0 is the initial lipid amount in the monolayer, and k is a bimolecular constant of the reaction. To determine the adsorption parameters, we fitted the kinetic adsorption data to eq 1 by using OriginPro program standard algorithms. We may conclude that the simplest adsorption kinetics model describes the experimental curves good enough for adequate estimation of the rate constants. The value dπ/dt in the initial part of the curve (see Figure 1) characterizes the rate of interaction of Cytc with the CL monolayer. In the course of time, the rate of the pressure growth decreases, as the binding sites for Cytc are gradually occupied. The kinetic curves, similar to that shown in Figure 1, were obtained in a series of experiments where we varied the protein concentration in the subphase (c0). Figure 2 demonstrates the parameters of the adsorption kinetics of Cytc at the TOCL and BCL monolayers obtained by fitting the experimental data to eq 1 for different protein concentration in the subphase. The maximum increase in the surface pressure in all the experiments did not depend on the protein concentration and was about the same for BCL and TOCL: 18.6 ± 1.1 mN m−1 and 18.1 ± 1.8 mN m−1, respectively. This may evidence that, for a given initial pressure, the final amount of Cytc adsorbed on the monolayer does not depend on the amount of the injected protein. Hence, the amount of binding sites (i.e., lipid amount) in the lipid monolayer was the only limiting factor during protein binding, but unlike protein concentration, because Cytc was present in excess in all the experiments. It is notable that this amount was the same for natural and synthetic CL. On the other hand, the rate of protein binding (β) was proportional to its concentration in the subphase, at least up to concentrations 100 nM. β values were similar for TOCL resistant to oxidation and for those obtained in case of BCL containing unsaturated fatty acid
Figure 1. Time dependence of the surface pressure change during the adsorption of Cytc with the TOCL monolayer. c0 = 50 nM, l0 = 27.4 nmol. Solid line is the experimental curve; dashed line shows the curve calculated by eq 1. Here, c0 is Cytc initial concentration and l0 is the TOCL amount in the monolayer.
and assuming that the increment of the surface pressure is proportional to the amount of the protein penetrating to the lipid monolayer, the following equation can be obtained:
Figure 2. Parameters of the adsorption kinetic curves obtained by fitting the experimental data to eq 1 for different protein concentration in the subphase. A protein solution was injected under the lipid monolayer at the initial surface pressure π0 = 5 mN m−1. (a) 26.4 ± 0.5 nmol BCL monolayer; (b) 28.4 ± 1.0 nmol TOCL monolayer. Here, Δπ∞ is the final increment of the surface pressure at infinite time (■), and β is the pseudo-first-order rate constant (●) of Cytc binding with the lipid monolayer. 12428
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Figure 3. (a) Increase of the surface pressure with time for a TOCL monolayer (27 ± 3 nmol) compressed up to different initial surface pressure π0 followed by the injection of Cytc into the subphase (the final concentration is 50 nM). (b) Dependence of the pressure increment (Δπ∞, ■) and pseudofirst-order rate constant (β, ●) at the Cytc penetration into the TOCL monolayer at the initial surface pressure (π0).
Figure 4. TOCL isotherms before (1) and after (2) the injection of different amounts of Cytc into the subphase at a different initial surface pressure. (a) c0 = 10 nM, l0 = 29.4 nmol, π0 = 5 mN m−1; (b) c0 = 50 nM, l0 = 27.4 nmol, π0 = 5 mN m−1; (c) c0 = 100 nM, l0 = 27.7 nmol, π0 = 5 mN m−1; (d) c0 = 250 nM, l0 = 29.4 nmol, π0 = 5 mN m−1; (e) c0 = 50 nM, l0 = 27.4 nmol, π0 = 5 mN m−1; (f) c0 = 50 nM, l0 = 26.9 nmol, π0 = 15 mN m−1; (g) c0 = 50 nM, l0 = 29.5 nmol, π0 = 25 mN m−1; (h) c0 = 50 nM, l0 = 26.1 nmol, π0 = 35 mN m−1. Here, l0, lipid amount; c0. protein concentration; π0, initial surface pressure.
Kinetics of Pressure Changes at Different Initial Pressure of the Lipid Monolayer. The data above were obtained for small values of the initial surface pressure in a lipid monolayer. At higher initial pressures, the feature of the kinetic curves is essentially changed (Figure 3). First of all, Δπ∞ (the final increment of the surface pressure at infinite time) decreases linearly with increasing initial pressure of the lipid monolayer (π0). Earlier, such a dependence was
chains and subject to oxidation by atmospheric oxygen. This indicates that the data obtained using natural CL in our experiments and in the experiments by other authors16 are not complicated by changes in the monolayer properties resulted from a spontaneous peroxidation of natural CL (Figure 2). Equation 1 also describes well the pressure kinetics of Cytc-CL films with various lipid amount in the monolayer (the data are not shown). 12429
DOI: 10.1021/acs.langmuir.5b03155 Langmuir 2015, 31, 12426−12436
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Figure 5. Isotherms of 50 nmol TOCL after the injection of Cytc (250 nM) into the subphase at the initial surface pressure π0 = 5 mN m−1. The forward and backward barriers speed rates were (a) v = 20 mm/min; (b) v = 10 mm/min; (c) v = 5 mm/min; (d) v = 2 mm/min.
Figure 6. (a) Isotherm of TOCL surface films (13.5 nmol) before (1) and after (2) the injection of Cytc (50 nM) under the monolayer at the initial surface pressure π0 = 5 mN m−1. Inset: the dependence of Δπ on π0. (b) Comparison of the surface pressure−area isotherms of Cytc alone and that in the complex with TOCL. Curves 1 and 2, as in (a); 3, Cytc contribution calculated as the difference between curve 2 and curve 1 at the same surface pressure; red, isotherm of Cytc film after the injection of Cytc (250 nM) under the lipid-free surface; π0, surface pressure; Δπ, an increment of the surface pressure from lipid monolayer without protein to the lipid monolayer after the protein injection at the same area.
observed by Quinn in the experiments on the phospholipid monolayers29 including BCL,16 but a quantitative explanation was not received. In fact, Δπ∞ (N m−1) is numerically equal to the change of the surface energy density ΔW (J m−2) at the Cytc incorporation into the lipid film, so that one may consider Δπ∞ as the protein binding energy (PBE). This energy decreases at an increased surface pressure because fewer protein molecules are incorporated. The π−A isotherm study of CL and Cytc-CL films is necessary to better understand this situation. Pressure versus Area Compression−Expansion Isotherms. The surface pressure−area isotherm was obtained for all the experiments described above, after the plateau on the adsorption kinetic curve was reached, to study the elastic properties of the protein−lipid film formed. The π−A isotherms for the lipid monolayer before (1) and after (2) Cytc injection in all the experiments are presented in Figure 4. It should be noted that the repeatedly obtained “compression-expansion” cycles have the same form, suggesting the reversibility and repeatability of the process; Figure 4 shows only the first cycle of the record. In all the experiments, the isotherms of CL monolayers were identical when the films were gradually compressed or expanded
(left (1) curves in Figure 4a−h). It is typical of phospholipids.19,30 The isotherms of the Cytc−CL films formed as described above had essentially different shapes, namely, they exhibited a shoulder that implies a kind of phase transition at 16 mN m−1. These data are in good agreement with those obtained by SaintPierre-Chazalet et al. in the films of Cytc adsorbed on other phospholipids.31 It is interesting that this phase transition pressure, 16 mN m−1, is the same as that obtained in a pure Cytc monolayer, though in this case the equilibrium state of the monolayer was reached after a long period of time (15 h), and the stability of the monolayer was highly dependent on the surface pressure.32 The control experiment where Cytc surface film was formed on the lipid-free surface is shown in Figure 6b for monolayers prepared as described32 and isotherms obtained were of the same character, except the pressure phase transition were lower by 4 mN m−1 (Figure 6b). Another important peculiarity of the isotherms of the monolayers containing both lipid and protein (Figure 4) is the fact that in all the cases the expansion curve (the lower part of the loop) did not coincide with the compression curve (the upper 12430
DOI: 10.1021/acs.langmuir.5b03155 Langmuir 2015, 31, 12426−12436
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protein molecule to penetrate into the lipid monolayer, and the equilibrium shifts to the left with the growing surface pressure.
part of the loop). This effect could be repeated several times using the same film and remained at a very low scanning rate (Figure 5), so it could not be explained by the fact that the equilibrium in the system did not have time to set. The time-independent hysteresis is known to be a consequence of dissipation of part of the energy, when the monolayer is being stretched or expanded. Since the hysteresis occurs only in the protein-containing films, one may suggest conformational changes in Cytc during its incorporation into and/or release from the CL monolayer. However, we cannot rule out the possibility of restructuring of the lipid/protein packing at the surface being responsible for rapid protein incorporation or release. A quantitative analysis of the surface-area isotherms enable to estimate the amount of the protein incorporated into the lipid monolayer at each given surface pressure. First of all, the areasurface pressure dependence for a Cytc-containing lipid monolayer (Figure 6b, curve 3) could be drawn from the experimental data (Figure 6a). According to this curve, the sum of Cytc and CL isotherms does not fit exactly to isotherm obtained for Cytc-CL film only until the pressure is raised to about 14 mN m−1. After that, the lipid and protein isotherms are additive. The surface pressure−area isotherms for Cytc dissolved in PBS and Cytc dissolved in PBS and ethanol (2:1 v/v) were similar. Under the compression−expansion, π−A isotherms were identical when being repeated. Protein molecules undergoing some conformational changes form a monolayer as shown in Figure 8b. It was much easier to obtain monolayer when a Cytc solution was premixed with ethanol. The protein alters its conformation during the interaction with ethanol. Already “prepared” protein with clearly distinguished hydrophilic and hydrophobic parts readily forms a monolayer. To produce the same π−A isotherm described in both cases, the protein amount required difference by almost 30 times: 5.7 nmol is necessary in case of Cytc dissolved in PBS and ethanol and 165 nmol in case of Cytc dissolved in PBS. From the experimental data (Figure 6b, 3), the amount of Cytc molecules per one lipid molecule in the film can be found if we know the mean molecular surface area of Cytc Ap:
Free Cytc + lipid monolayer ⇄ membrane bound Cytc
We made the calculation under the assumption that a Cytc molecule, after being incorporated, does not change its conformation and shape in the film upon the compressions (much as in ref 17). The complex features of the surface-area isotherms, hysteresis, and similar behavior of a pure protein monolayer and that of the lipid−protein films suggest that the protein does change its conformation upon surface pressure variation. The vertical arrows in Figure 6a show the pressure increment Δπ resulting from Cytc penetration into the lipid monolayer from the aqueous subphase. The dependence of Δπ on the initial surface pressure π0 was linear (Figure 6a, inset) as well as in the direct measurements shown in Figure 4b and obtained earlier in monolayers of Cytc with different phospholipids by Peter Quinn et al.16,29 Before starting the final discussion, one can ask, however, whether our results tell us anything interesting about what might be happening physiologically. It is a matter of fact that the lateral pressure believed to exist in bilayers is in the range 47−50.21 This is much above values, obtained in refs 16, 17, and 20 and the present study, permitting the binding of soluble protein to lipid monolayer (
