Langmuir 2007, 23, 10929-10932
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Real-Time Atomic Force Microscopy Reveals Cytochrome c-Induced Alterations in Neutral Lipid Bilayers Sandrine Morandat† and Karim El Kirat*,‡ Laboratoire de Ge´ nie Enzymatique et Cellulaire, UMR-CNRS 6022, and Laboratoire de Biome´ canique et Ge´ nie Biome´ dical, UMR-CNRS 6600, UniVersite´ de Technologie de Compie` gne, BP 20529, 60205 Compie` gne Cedex, France ReceiVed July 18, 2007. In Final Form: September 7, 2007 The interaction of cytochrome c (cyt c) with supported lipid membranes was investigated on the nanoscale by real-time atomic force microscopy. Cyt c promoted the formation and the expansion of depressed areas in the fluid parts of the bilayer. When the depressions reached the gel domains, they induced the thickening of their edges. According to the step-height differences, cyt c was able to remove neutral lipids in the fluid phase and then to reside on the mica surface. Concerning gel phases, cyt c might insert between the two lipid leaflets, or it might intercalate between the mica and the bilayer.
Introduction Cytochrome c (cyt c) is a basic peripheral protein involved in the mitochondrial respiratory chain. This protein transfers electrons between two redox protein complexes integrated into the inner membrane, ubiquinol cyt c oxidoreductase (cytochrome bc1) and cyt c oxidase. Thus, to exchange electrons with the redox partners, cyt c is able to bind reversibly to the inner mitochondrial membrane that is particularly enriched in cardiolipin (CL), an anionic phospholipid.1,2 The cyt c binding to CL not only has a functional role but also triggers programmed cell death (apoptosis). Cyt c is able to catalyze CL peroxidation, leading to membrane permeation that is responsible for cyt c release into the cytosol. This event constitutes the earliest step in the apoptotic program.3,4 Therefore, the pivotal role of cyt c in apoptosis has attracted much interest during the past decade.5,6 It was shown that cyt c binding to CL-containing membranes requires both electrostatic and hydrophobic interactions.7-12 Indeed, when the protein is in the native state (i.e., it is able to transfer electrons in the respiratory chain), the cyt c/CL interactions involved are mainly electrostatic.13,14 When cyt c is unfolded, it is able to bury itself in the hydrophobic core of the bilayer.13,15,16 The biological significance of the cyt c bound to * Corresponding author. Phone: +33 (0)3 44 23 79 43. Fax: +33 (0)3 44 23 79 42. E-mail:
[email protected]. † UMR-CNRS 6022. ‡ UMR-CNRS 6600. (1) Fleischer, S.; Rouser, G.; Fleischer, B.; Casu, A.; Kritchevsky, G. J. Lipid Res. 1967, 8, 170-180. (2) Daum, G. Biochim. Biophys. Acta 1985, 822, 1-42. (3) Kagan, V. E.; Tyurin, V. A.; Jiang, J.; Tyurina, Y. Y.; Ritov, V. B.; Amoscato, A. A.; Osipov, A. N.; Belikova, N. A.; Kapralov, A. A.; Kini, V.; Vlasova, I. I.; Zhao, Q.; Zou, M.; Di, P.; Svistunenko, D. A.; Kurnikov, I. V.; Borisenko, G. G. Nat. Chem. Biol. 2005, 1, 223-232. (4) Iwase, H.; Takatori, T.; Nagao, M.; Iwadate, K.; Nakajima, M. Biochem. Biophys. Res. Commun. 1996, 222, 83-89. (5) Iverson, S. L.; Enoksson, M.; Gogvadze, V.; Ott, M.; Orrenius, S. J. Biol. Chem. 2004, 279, 1100-1107. (6) Gogvadze, V.; Robertson, J. D.; Zhivotovsky, B.; Orrenius, S. J. Biol. Chem. 2001, 276, 19066-19071. (7) Gorbenko, G. P.; Molotkovsky, J. G.; Kinnunen, P. K. Biophys. J. 2006, 90, 4093-103. (8) Snel, M. M.; Marsh, D. Biophys. J. 1994, 67, 737-745. (9) Bernad, S.; Oellerich, S.; Soulimane, T.; Noinville, S.; Baron, M. H.; Paternostre, M.; Lecomte, S. Biophys. J. 2004, 86, 3863-3872. (10) Rytomaa, M.; Kinnunen, P. K. J. Biol. Chem. 1995, 270, 3197-3202. (11) Tuominen, E. K.; Wallace, C. J.; Kinnunen, P. K. J. Biol. Chem. 2002, 277, 8822-8826. (12) Rytomaa, M.; Mustonen, P.; Kinnunen, P. K. J. Biol. Chem. 1992, 267, 22243-22248. (13) Salamon, Z.; Tollin, G. Biophys. J. 1996, 71, 848-857. (14) Kalanxhi, E.; Wallace, C.J. Biochem. J., in press, 2007.
the hydrophobic interior of the membrane is still unknown even if recent studies have suggested that it corresponds to a disabling sequestration of this protein for regulatory purposes.14 To date, the interaction of cyt c with model membranes was investigated with fluorescence spectroscopy,7,10,17 infrared spectroscopy,9,18,19 nuclear magnetic resonance,20-22 electron spin resonance,8,23 titration calorimetry,24 surface plasmon resonance (SPR),13,15 and atomic force microscopy (AFM).25-28 However, the exact real-time mechanism of cyt c interaction with biomembranes is still poorly documented. On the nanoscale, experiments were performed on anionic lipids that provided information about both the electrostatic and hydrophobic interactions with cyt c.25-27 In this context, because cyt c is a basic protein, the use of a zwitterionic lipid bilayer may permit the definition of the contribution of the hydrophobic driving force. AFM is a powerful technique that allows the imaging of the surfaces of samples in liquid environments with unprecedented resolution. This technique has already provided new nanoscale information concerning the interaction of supported lipid membranes with peptides,29-32 antibiotics,33 alcohols,34 proteins,35-37 polymers,38-41 and detergents.42-46 (15) Salamon, Z.; Tollin, G. Biophys. J. 1996, 71, 858-867. (16) Pinheiro, T. J. Biochimie 1994, 76, 489-500. (17) Gorbenko, G. P. Biochim. Biophys. Acta 1999, 1420, 1-13. (18) Choi, S.; Swanson, J. M. Biophys. Chem. 1995, 54, 271-278. (19) Bernabeu, A.; Lellys M. Contreras, A.; Villalain, J. Biochim. Biophys. Acta, in press, 2007. (20) Pinheiro, T. J.; Watts, A. Biochemistry 1994, 33, 2451-2458. (21) Brown, L. R.; Wuthrich, K. Biochim. Biophys. Acta 1977, 468, 389-410. (22) Kim, S. M.; Yamamoto, T.; Todokoro, Y.; Takayama, Y.; Fujiwara, T.; Park, J. S.; Akutsu, H. Biophys. J. 2006, 90, 506-513. (23) Heimburg, T.; Marsh, D. Biophys. J. 1995, 68, 536-546. (24) Heimburg, T.; Angerstein, B.; Marsh, D. Biophys. J. 1999, 76, 25752586. (25) Domenech, O.; Sanz, F.; Montero, M. T.; Hernandez-Borrell, J. Biochim. Biophys. Acta 2006, 1758, 213-221. (26) Choi, E. J.; Dimitriadis, E. K. Biophys. J. 2004, 87, 3234-3241. (27) Mueller, H.; Butt, H.-J.; Bamberg, E. J. Phys. Chem. B 2000, 104, 45524559. (28) Boussaad, S.; Dziri, L.; Arechabaleta, R.; Tao, N. J.; Leblanc, R. M. Langmuir 1998, 14, 6215-6219. (29) El Kirat, K.; Dufrene, Y. F.; Lins, L.; Brasseur, R. Biochemistry 2006, 45, 9336-9341. (30) El Kirat, K.; Lins, L.; Brasseur, R.; Dufrene, Y. F. J. Biomed. Nanotechnol. 2005, 1, 39-46. (31) El, Kirat, K.; Lins, L.; Brasseur, R.; Dufrene, Y. F. Langmuir 2005, 21, 3116-3121. (32) Zhong, J.; Zheng, W.; Huang, L.; Hong, Y.; Wang, L.; Qiu, Y.; Sha, Y. Biochim. Biophys. Acta 2007, 1768, 1420-1429. (33) van Kan, E. J.; Ganchev, D. N.; Snel, M. M.; Chupin, V.; van der Bent, A.; de Kruijff, B. Biochemistry 2003, 42, 11366-11372.
10.1021/la702158j CCC: $37.00 © 2007 American Chemical Society Published on Web 09/22/2007
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Figure 1. Cyt c interaction with supported 1:1 mol/mol DOPC/DPPC membranes. An AFM deflection image (50 µm × 50 µm) of a mixed 1:1 DOPC/DPPC bilayer was first recorded in Tris buffer (A). After the addition of 4 µg/mL of cyt c, images of the same area were acquired at different incubation times: (B) 5, (C) 8, (D) 12, (E) 17, (F) 22, (G) 32, and (H) 48 min. The asterisks correspond to the same fixed point on a gel domain to evaluate the progression of the depressed areas. Please note that deflection mode images do not contain useful height information.
This work presents the first real-time imaging of cyt c interaction with a zwitterionic-supported model membrane on the nanometer scale. The model bilayers were composed of 1:1 mol/mol dioleoylphosphatidylcholine (DOPC)/dipalmitoylphosphatidylcholine (DPPC). These neutral membranes were chosen to favor the hydrophobic mode of interaction with cyt c, and they exhibited phase segregation to mimic cell membranes. Therefore, these model membranes were used to determine the influence of the lipid molecular packing on cyt c behavior.
Results and Discussion The mechanism of cyt c interaction with membranes was investigated by using 1:1 mol/mol mixed DOPC/DPPC lipid bilayers. In situ real-time AFM experiments were carried out to visualize the morphological changes of this model membrane in the presence of cyt c at different concentrations. Contrary to (34) Mou, J.; Yang, J.; Huang, C.; Shao, Z. Biochemistry 1994, 33, 99819985. (35) Saslowsky, D. E.; Lawrence, J.; Ren, X.; Brown, D. A.; Henderson, R. M.; Edwardson, J. M. J. Biol. Chem. 2002, 277, 26966-26970. (36) Geisse, N. A.; Wasle, B.; Saslowsky, D. E.; Henderson, R. M.; Edwardson, J. M. J. Membr. Biol. 2002, 189, 83-92. (37) Alattia, J. R.; Shaw, J. E.; Yip, C. M.; Prive, G. G. J. Mol. Biol. 2006, 362, 943-953. (38) Hong, S.; Leroueil, P. R.; Janus, E. K.; Peters, J. L.; Kober, M. M.; Islam, M. T.; Orr, B. G.; Baker, J. R., Jr.; Banaszak Holl, M. M. Bioconjugate Chem. 2006, 17, 728-734. (39) Mecke, A.; Majoros, I. J.; Patri, A. K.; Baker, J. R., Jr.; Holl, M. M.; Orr, B. G. Langmuir 2005, 21, 10348-10354. (40) Hong, S.; Bielinska, A. U.; Mecke, A.; Keszler, B.; Beals, J. L.; Shi, X.; Balogh, L.; Orr, B. G.; Baker, J. R., Jr; Banaszak Holl, M. M. Bioconjugate Chem. 2004, 15, 774-782. (41) Mecke, A.; Uppuluri, S.; Sassanella, T. M.; Lee, D. K.; Ramamoorthy, A.; Baker, J. R., Jr.; Orr, B. G.; Banaszak, Holl, M. M. Chem. Phys. Lipids 2004, 132, 3-14. (42) El Kirat, K.; Morandat, S. Biochim. Biophys. Acta 2007, 1768, 23002309. (43) Morandat, S.; El Kirat, K. Langmuir 2006, 22, 5786-5791. (44) Morandat, S.; El Kirat, K. Colloids Surf., B 2007, 55, 179-184. (45) Milhiet, P. E.; Gubellini, F.; Berquand, A.; Dosset, P.; Rigaud, J. L.; Le Grimellec, C.; Levy, D. Biophys. J. 2006, 91, 3268-3275. (46) Rinia, H. A.; Snel, M. M.; van der Eerden, J. P.; de Kruijff, B. FEBS Lett. 2001, 501, 92-96.
height mode images that give quantitative information about the sample topography, deflection mode images do not contain useful height information. However, deflection mode images highlight edges where drastic height changes occur and therefore, in some instances, provided a better qualitative visualization of the cyt c-induced alteration of membranes. (Height images are available in Figure 1 of Supporting Information.) Figure 1A corresponds to the deflection image of a DOPC/ DPPC bilayer exhibiting two lipid phases. As previously described, the DPPC gel phase was 1.1 ( 0.1 nm higher than the DOPC fluid phase.43,44 Cyt c was then injected into the bulk phase at a concentration of 4 µg/mL, and a new image was recorded 5 min after protein addition (Figure 1B). Within the first minutes after cyt c addition, a new level appeared in the DOPC fluid phase in areas previously presenting no visible defects. After 8 min (Figure 1C), this new level was depressed with a step height difference of 2.2 ( 0.3 nm with respect to the surrounding DOPC fluid phase (Figure 2Aii). These depressed domains expanded in a centrifugal way. Furthermore, another new structure was visible at the edge of the expanding depressions: it corresponds to a new flat level higher than DOPC (step height of about 2.1 ( 0.3 nm). The radial progression of the depressed areas continued, and they finally reached the DPPC domains (Figure 1D-H). After 48 min, the edges of the DPPC patches were thicker (Figure 1H). At the end of the incubation (150 min), a complete thickening of the DPPC patches was observed, and no further alteration occurred on these domains (data not shown). Other cyt c concentrations ranging from 1 to 8 µg/mL were also tested on the mixed model bilayers. For all concentrations, the bilayers were profoundly modified by the formation of depressions and by the thickening of some areas in the bilayer (data not shown). The velocity of propagation of the depressed areas was estimated on low-magnification images (50 × 50 µm2) for each cyt c concentration. These velocities were measured during the first 30 min, and the obtained values are summarized in Table 1. According to these results, the velocity of propagation
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Langmuir, Vol. 23, No. 22, 2007 10931 Table 1. Velocity of Expansion of the Depressed Areas as a Function of Cyt c Concentrationa cyt c concentration (µg/mL) velocity of expansion (µm2/min)
1 1
2 6.8
4 28.3
6 41.7
8 61.6
a Cyt c concentrations ranging from 1 to 8 µg/mL were tested on mixed 1:1 mol/mol DOPC/DPPC bilayers. The velocity of propagation of the depressed areas was estimated during the first 30 min on lowmagnification images (50 × 50 µm2) from two independent experiments.
Figure 2. Progression of cyt c-induced defects in fluid and gel phases. Higher magnification (20 µm × 20 µm; Z-scale ) 10 nm) of the AFM height images corresponding to the deflection images presented in Figure 1I-L. Images Ai and Bi were acquired 25 and 90 min, respectively, after cyt c injection in the bulk phase of a mixed 1:1 mol/mol DOPC/DPPC bilayer. The different height levels are denoted as follows: R, DPPC; β, newly formed depressed areas; and γ, DOPC. Cross-section analyses Aii and Bii correspond to the white lines in panels Ai and Bi, respectively.
of the depressions increased linearly with increasing concentration of cyt c. Higher-magnification images corresponding to the set presented in Figure 1A-H were acquired (Figure 2). Figure 2Ai shows one DPPC domain surrounded by DOPC and four depressed areas expanding in the fluid phase. The step heights between these depressions and DOPC or DPPC were estimated to be 2.2 ( 0.3 and 3.3 ( 0.4 nm, respectively (Figure 2Aii). The thickness of
a gel DPPC bilayer supported on mica is known to be about 5.5 ( 0.1 nm.44,47 Moreover, the height difference on DPPC and DOPC is 1.1 ( 0.1 nm, and one can then deduce that the thickness of a DOPC fluid bilayer is 4.4 ( 0.1 nm. Two hypotheses could explain the thicknesses observed between the depressed domains and lipid surfaces: either (i) an interdigitation of the two lipid leaflets or (ii) the desorption of DOPC and the simultaneous coating of the mica surface by cyt c. In a previous study, Choi and Dimitriadis26 showed that cyt c molecules are spherical (mean diameter of 2.4 nm) and are inserted into the hydrophobic tails of an anionic lipid membrane. According to the literature, cyt c-bilayer interaction results from two main driving forces: hydrophobic and electrostatic.10-12 On the basis of our results, cyt c might coat the mica substrate as attested by the step height differences between depressions and DOPC and DPPC. In fact, cyt c may be able to cross and destabilize the neutral DOPC membrane via hydrophobic interactions, and then it adsorbs onto the mica substrate. It is worth noting that when cyt c was added to a membrane presenting performed holes (partially uncovered mica), cyt c was able to coat the areas of bare mica substrate immediately after injection. (For details, see Figure 2 in Supporting Information.) Interestingly, the holes appeared in areas of the fluid phase where no pre-existing defects were visible. This result is surprising because the gel/fluid phase boundaries exhibit a molecular packing disorder that is usually more favorable to the insertion of external agents in bilayers.29,43,48 Pioneering studies on polycationic polymers described the formation of large defects in a PC membrane that was initially intact.38-41 The polycationic dendrimers were able to cross the bilayer, leading to the removal of membrane parts, but the polymers did not remain adsorbed on the mica surface. Therefore, contrary to the behavior of polycationic polymers, cyt c would be able to provoke DOPC desorption by crossing the fluid phases and subsequently adsorbing onto the mica. The thickening at the edge of the expanding domains was clearly visible in Figure 2Ai, and the height difference with DOPC was 2.1 ( 0.3 nm (Figure 2Aii). One may also notice the formation of branched structures in the defects that reinforce the impression of centrifugal progression (Figures 2Ai,Bi and 3). They might correspond to residual DOPC bilayers as attested to by the step height difference of 2.4 ( 0.3 nm with the surrounding depression (Figure 3). Ninety minutes after cyt c addition, the DPPC patch was considerably modified, essentially by the thickening of its edges (Figure 2Bi). Indeed, the step height difference between the DPPC and the thickened edges was 3.4 ( 0.2 nm (Figure 2Bii). This thickening corresponds to the largest dimension of cyt c.49,50 In contrast to DOPC, the DPPC thick areas were stable over time because no desorption or further (47) Shao, Z.; Mou, J.; Czajkowsky, D. M.; Yang, J.; Yuan, J.-Y. AdV. Phys. 1996, 1, 1-86. (48) Milhiet, P. E.; Giocondi, M. C.; Baghdadi, O.; Ronzon, F.; Roux, B.; Le Grimellec, C. EMBO Rep. 2002, 3, 485-490. (49) Takano, T.; Kallai, O. B.; Swanson, R.; Dickerson, R. E. J. Biol. Chem. 1973, 248, 5234-5255. (50) Bushnell, G. W.; Louie, G. V.; Brayer, G. D. J. Mol. Biol. 1990, 214, 585-595.
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Figure 3. High magnification of the branched structures left inside the cyt c-induced defects. AFM height image (10 µm × 10 µm; Z scale ) 10 nm) acquired 92 min after cyt c injection in the bulk phase of a mixed 1:1 mol/mol DOPC/DPPC bilayer. The different height levels are denoted as follows: R, DPPC; β, newly formed depressed areas; and γ, DOPC. Cross-section analysis ii corresponds to the white line depicted in panel i.
modification occurred, even for long observation times. Finally, DPPC thickening reached the center of the patch at the end of the incubation time by centripetal progression (data not shown). This progressive thickening from the edges to the center of DPPC patches suggests that cyt c was able to diffuse between the bilayer and the mica without destabilizing the DPPC because this lipid phase presents a high molecular packing. As a comparison, the thickening of DOPC areas never exceeded 2.1 ( 0.3 nm, thus revealing the destabilization of the fluid phases as confirmed by their subsequent desorption. In a previous study, steady-state fluorescence resonance-energy transfer (FRET) was employed to investigate cyt c interaction with liposomes of PC and CL or phosphatidylglycerol (PG).51 The authors have observed that the extent of protein penetration into the bilayer was modulated by the ionic strength of the solution and the membrane anionic lipid content. This suggests that both electrostatic and hydrophobic forces contribute to cyt c interaction with biomembranes. Similar investigations with the quartz crystal microbalance (QCM) and SPR confirmed the important role (51) Domanov, Y. A.; Molotkovsky, J. G.; Gorbenko, G. P. Biochim. Biophys. Acta 2005, 1716, 49-58.
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played by negatively charged lipids.13,52 It is noteworthy that the prior insertion of cyt c oxidase into the lipid bilayer leads to the suppression of the hydrophobic interaction between cyt c and the membrane.15 Several studies conducted by Kinnunen’s group10-12 have indicated the existence of two distinct sites in cyt c (A and C sites) that are responsible for the association with lipid bilayers. With the help of fluorescence spectroscopy and steady-state absorbance spectroscopy, they have shown that the negative surface charge density, pH, and ionic strength together determine whether cyt c is bound via its A site (electrostatic bond) or C site (hydrophobic interaction). Most importantly, they have identified a significant change in the conformation of cyt c provoked by C-site binding. Under these conditions, one of the acyl chains of anionic phospholipid was accommodated within a hydrophobic channel in the cyt c structure. Some AFM studies provided evidence of the dual interaction of cyt c with biomembranes. Boussaad et al.28 studied the interaction of monolayers of PC and CL with cyt c by AFM and cyclic voltammetry. They have observed that cyt c was able to cover the CL lipids whereas no protein layer was observed on the PC surface. With punch-through experiments on PG bilayers, Choi and Dimitriadis26 have demonstrated that the bilayer becomes more permeable and more fluidlike because of cyt c insertion. More recently, Domenech et al.25 have shown that cyt c was not visible on 4:1 mol/mol phosphatidylethanolamine/CL bilayers but the roughness was modified. This suggests that cyt c may be inserted within the membrane. Accordingly, the influence of lipid composition may also constitute an important parameter to take into account when studying cyt c behavior. Indeed, cyt c was able to form small aggregates on top of the neutral bilayers containing cholesterol.27 In the present study, real-time AFM provided yet unobserved results of the ability of cyt c to alter neutral bilayers. Moreover, the use of a membrane presenting phase segregation permitted us to show that cyt c behavior can be strongly modulated by lipid molecular packing. Indeed, cyt c promoted the desorption of fluid phases whereas the gel domains were not disorganized. Acknowledgment. The support of the Centre National de la Recherche Scientifique (CNRS) of the French Research Ministry and of the Universite´ de Technologie de Compie`gne (UTC, Plan de Pluriformation , PPF Nanobiotechnologies .) is gratefully acknowledged. We thank the Service d’Analyses PhysicoChimiques of the UTC for the use of the atomic force microscope. Supporting Information Available: Experimental section, deflection and height images of cyt c interaction with DOPC/DPPC bilayers, and cyt c interaction with a membrane partially covering the mica surface. This material is available free of charge via the Internet at http://pubs.acs.org. LA702158J (52) Glasmastar, K.; Larsson, C.; Hook, F.; Kasemo, B. J. Colloid Interface Sci. 2002, 246, 40-47.
