Visualizing the Solubilization of Supported Lipid Bilayers by an

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Visualizing the Solubilization of Supported Lipid Bilayers by an Amphiphilic Peptide Shellie M. Rigby-Singleton,†,‡ Martyn C. Davies,† Helen Harris,§ Paul O’Shea,§ and Stephanie Allen*,† Laboratory of Biophysics and Surface Analysis, School of Pharmacy, and Cell Biology and Cell Biophysics Group, School of Biology, The UniVersity of Nottingham, Nottingham, NG7 2RD, UK ReceiVed January 12, 2006. In Final Form: April 19, 2006 The effect of the presequence peptide of cytochrome c oxidase subunit IV (p25) on supported phospholipid bilayers (SPBs) was visualized using atomic force microscopy (AFM). The presequence was found to cause the complete disruption of supported bilayers containing neutral lipids. At relatively low concentrations of presequence, the peptide was found to bind to the membrane, coalescing to form microdomains within the liquid-crystalline bilayer that were located predominantly at bilayer-mica boundaries. Further increases in peptide concentration resulted in the formation of holes within the SPB that were spanned by an interpenetrating network of narrower regions of the bilayer, which, at higher applied peptide concentrations, were observed to disappear through a budding process, ultimately leading to the formation of spherical structures at yet higher peptide concentrations. Within this paper, the impact the presequence has upon the structure and order of the membrane is discussed, as is the potential implication of this apparent solubilization process on the translocation of cytochrome c oxidase into the inner mitochondrial membrane.

Introduction Mitochondrial precursor proteins synthesized in the cytoplasm contain cleavable amino-terminal extensions, termed “pre”, “signal”, “leader”, or “transit” sequences. They encode the information required to target the precursors to their final intramitochondrial destination, and, in doing so, they must transport them across one or more membranes. Mitochondria contain two distinct import sites: one located in the outer membrane and the other in the inner membrane.1-3 These sites work cooperatively during the translocation of matrix proteins.2,4,5 The cytochrome c oxidase subunit IV presequence (p25) is thought to mediate the formation of these import sites,6-8 initiating hemifusion between the inner and outer mitochondrial membranes.8-9 p25 is known to adopt an amphiphilic R-helical secondary structure (a helix-break-helix motif) upon insertion into the membrane, in which two positive charges from the N-terminus helix are proposed to insert.10 The interaction of the presequence is dictated by the type of lipids present in the membrane. For example, enhanced peptide association has been observed with negatively charged phospholipids10 relating to an increase in the helical stability at the C-terminus.11 Cardiolipin (CL) has also been shown to enhance helical content, forcing the peptide to adopt a helix-turn-helix motif, with the N-terminal helix inserted * Corresponding author. E-mail: [email protected]. Phone: +44 (0)115 9515050. Fax: +44 (0)115 9515110. † School of Pharmacy. § School of Biology. ‡ Current address: Molecular Profiles, 8 Orchard Place, Nottingham Business Park, Nottingham, NG8 6PX. (1) Rassow, J.; Pfanner, N. FEBS Lett. 1991, 293, 85. (2) Mayer, A.; Lill, R.; Neupert, W. J. Cell Biol. 1993, 121, 1233. (3) Segui-Real, B.; Kispal, G.; Lill, R.; Neupert, W. EMBO J. 1993, 121, 2211. (4) Hwang, S.; Jascur, T.; Vestweber, D.; Pon, L.; Scatz, G. J. Cell Biol. 1989, 109, 487. (5) Kiebler, M.; Becker, K.; Pfanner, N.; Neupert, W. J. Membr. Biol. 1993, 135, 191. (6) Leenhouts, J. M.; de Gier, J.; de Kruijff, B. FEBS Lett. 1993, 327, 172. (7) To¨ro¨k, Z.; Demel, R. A.; Leenhouts, J. M.; de Kruijff, B. Biochemistry 1994, 33, 5589. (8) Colotto, A.; Martin, I.; Ruysschaert, J.-M.; Sen, A.; Epand, R. M. Biosci. Rep. 1998, 18, 251. (9) Mandieau, V.; Martin, I.; Ruysschaert, J.-M. FEBS Lett. 1995, 368, 15. (10) Golding, C.; Senior, S.; Wilson, M. T.; O’Shea, P. Biochemistry 1996 35, 10931.

into the bilayer with its axis perpendicular to the membrane surface.12 It is probable that the insertion of the presequence peptide into the membrane causes a structural rearrangement of both lipids and proteins, facilitating the formation of import sites.9 When membranes undergo fusion, each element of the membrane begins to change shape, resulting in a tight membrane curvature.13,14 Since lipids are polymorphic, they are able to alter their critical packing shape depending on headgroup area, ionic strength, unsaturation, and temperature.15 For example, the introduction of divalent cations to anionic lipids increases the critical packing parameter (CPP) (given by V/aolc, where V is the hydrocarbon chain volume, ao is the headgroup area, and lc is the critical length) by reducing the headgroup area (ao). This leads to a molecular shape that favors a less positive membrane curvature. The introduction of unsaturation in the hydrocarbon chain decreases the critical length (lc) of the lipid, also resulting in an increase in the CPP,15,16 as does increasing the temperature. The CPP essentially describes the morphology of the lipid aggregates. Here we report p25-induced changes in the phase behavior and critical packing of supported phosphatidylcholine (PC) bilayers. Although the interaction of p25 with membranes has been extensively studied using monolayer techniques,7,17 nuclear magnetic resonance,11,12 circular dichroism,8,10 the stopped-flow mixing apparatus,10 and X-ray diffraction,8 the impact of the presequence on the structure and order of the membrane remains relatively unknown. The potential implications of these morphological changes upon the mediation of cytochrome c oxidase translocation are also discussed. (11) Chupin, V.; Leenhouts, J. M.; de Kroon, A. I. P. M.; de Kruijff, B. Biochemistry 1996, 35, 3141. (12) Chupin, V.; Leenhouts, J. M.; de Kroon, A. I. P. M.; de Kruijff, B. FEBS Lett. 1995, 373, 239. (13) Chemomordik, L. V.; Zimmerberg, J. Curr. Opin. Struct. Biol. 1995, 5, 541. (14) Chemomordik, L. V.; Frolov, V.; Leikina, E.; Bronk, P.; Zimmerberg, J. J. Cell Biol. 1998, 140, 1369. (15) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press, Inc. Ltd.: London, 1991. (16) Chupin, V.; Boots, J. W. P.; Killian, J. A.; Demel, R. A.; de Kruijff, B. Biophys. J. 2002, 82, 843. (17) Tamm, L. K. Biochemistry 1986, 25, 7470.

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Figure 1. AFM images of an SPB on mica. (A) An egg lecithin PC bilayer collapsed onto a mica substrate by vesicle fusion. The darkest areas represent holes in the bilayer in which regions of the underlying mica surface are exposed. (B) A cross-section of the surface topography presented in A (white line), illustrating a bilayer thickness of ∼ 4.4 nm. (C) The same region shown in A following the addition of 500 nM p25. (D) A cross-section of the surface topography presented in C (white line).

Experimental Section Materials. Egg lecithin PC was purchased from Avanti Polar Lipids (Alabaster, AL). The 25 amino acid presequence peptide of p25 (MLSLRQSIRFFKPATRTLCSSRYLL) was synthesized and purified by high-performance liquid chromatography (HPLC) in the School of Biomedical Sciences Analytical Unit (The University of Nottingham, UK) using standard solid-phase methods. The purity of the peptide was confirmed by subsequent mass spectroscopy analysis, and determined to be >99%. Preparation of Supported Phospholipid Bilayers (SPBs). The 100 nm unilamellar PC vesicles were prepared using the extrusion method previously described.18 PC was first dried down as a thin film from a solution of chloroform. The dried lipids were resuspended in 10 mM Tris buffer, pH 7.4, to give a 13 mM solution. The resuspended lipid then underwent five freeze/thaw cycles (by plunging the vessel containing the lipid suspension into liquid nitrogen and then thawing it in luke-warm water (∼40 °C)), and then was pressureextruded (at room temperature) through a 100 nm polycarbonate membrane using a hand-held system (Avanti Polar Lipids Inc., Alabaster, AL). Lipids were extruded a minimum of 10 times with the aim of producing vesicles with diameters as close to 100 nm as possible. SPBs were prepared using the vesicle fusion method.19,20 Freshly cleaved mica incubated in 10 mM MgCl2 provided the solid support. 30 µL of a 1.3 mM solution of 100 nm PC vesicles were allowed to adhere and collapse onto the mica surface for a period not less than 1 h, at ambient temperature. Once the lipid coverage had been (18) MacDonald, R. C.; MacDonald, R. I.; Menco, B. P.; Takeshita, K.; Subbarao, N. K.; Hu, L. R. Biochim. Biophys. Acta 1991, 1061, 297. (19) Seifert, U. AdV. Phys. 1997, 46, 13. (20) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806.

established, increasing approximate concentrations (500 nM; 1 µM; 3 µM; 5 µM) of the presequence p25 were introduced directly to the SPB, assuming an imaging volume of approximately 20 µL. Instrumentation. Tapping-mode atomic force microscopy (AFM) was employed throughout these studies. Images of sample topography were obtained in an aqueous environment containing 10 mM Tris (pH 7.4), at a scan rate of 2 Hz using a multimode AFM with a Nanoscope IIIa Controller (Digital Instruments, Veeco Metrology Group, Santa Barbara, CA). V-shaped silicon nitride cantilevers (nominal spring constant of 0.06 Nm-1) with integrated pyramidal oxide-sharpened probes were employed (Digital Instruments, Veeco Metrology Group, Santa Barbara, CA). Images are displayed as gray-scale representations, where the higher features appear lighter in color. Measurements of the widths of features were taken at half of their observed height.

Results and Discussion The phase behavior, organization, and subsequent solubilization of PC SPBs by the peptide p25 were investigated using AFM. All studies were performed at ambient temperature, and hence the PC bilayers were present in a liquid-crystalline, LR phase. This phase is characterized by the hydrocarbon chains being in a fluid, disordered state.21 A representative topography image of a PC SPB on a mica support is shown in Figure 1A. In these studies, the concentration of the lipid required to almost completely cover the surface with bilayer was determined in each experiment, with the presence of small imperfections in the bilayer providing a means to measure and monitor changes in (21) Brown, D. A.; London, E. J. Membr. Biol. 1998, 164, 103.

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Figure 2. AFM images of the PC SPB following exposure to (A) 1 µM p25, where B is a cross-section of the surface topography presented in A (white line), and (C) 3 µM p25. Holes spanned by narrower regions (or branches) of the bilayer are evident in both images (indicated by the white arrows).

its thickness upon the addition of peptide. The height profile of the liquid-crystalline PC bilayer, including the thin water layer that exists between the lower leaflet and the mica surface,22 was always found to be around 4.4-4.5 nm (Figure 1B). Here it should be noted that a disadvantage of AFM imaging is that it is only able to resolve the topography of the uppermost surface presented, and thus it is not able to provide information on the complete cross-section of the features observed on the surface. However, because of the hydrophilic nature of the mica surface and the aqueous environment in which the images were obtained, it will be more energetically favorable for the lipids present in the lower leaflet of the bilayer (or the lower portions of the aggregates observed in Figures 2-4) to orient themselves with their headgroups toward the surface and/or aqueous environment (as described in ref 22 and the references therein). We therefore assume such behavior in our interpretation of the presented images, also taking into account any potential changes in membrane curvature induced upon peptide binding. For the sequence of images shown in Figures 1-4 the same SPB region was maintained throughout the addition of the p25 concentration range studied, with Figures 2 and 4 showing higher resolution views of a central region within the area presented in Figure 1. Although these images are from one experiment only, the experiment was repeated at least two or three times, and the same changes in the bilayer appearance were observed (with only slight variation in the applied peptide concentration, attributable to differences in bilayer coverage between samples). Upon the addition of 500 nM p25, marked changes can be

observed in the bilayer phase behavior (Figure 1C,D). Raised domains begin to surround the bilayer-mica edges and also form discreet areas in the body of the bilayer. Higher regions, present only in the microdomains within the body of the bilayer (∼10 nm in height), can also be seen, which most likely represent either large aggregates of peptide or partially solubilized (or “budding”) regions of bilayer, which may be precursors to the holes observed at higher peptide concentrations (see Figure 2). The height difference between the lower regions of these domains or the domains observed at membrane edges and the surrounding bilayer was found to be ∼1.4 nm. The occurrence of these raised microdomains was accompanied by a decrease in bilayer coverage; for example, the bare mica region indicated by the × in Figure 1A expands by approximately 1 µm2 in Figure 1C (following exposure to 500 nM p25), while maintaining roughly the same overall morphology. This decrease in bilayer coverage is suggestive of an increase in the lipid packing density, possibly due to either a reduction in the steric repulsion between the phospholipid headgroups or the condensation (straightening/ordering) of the hydrocarbon chains. Both potential causalities would lead to alterations in the molecular shape of the phospholipids and, subsequently, a decrease in their CPPs. Table 1 summarizes the decreases in bilayer coverage observed upon peptide addition for the images presented in Figures 1-3. The observed increase in bilayer height is also indicative of the ordering of the hydrocarbon chains, as is the observed edge preference of these raised microdomains.23 The preference of long chain, saturated (ordered) phospholipids for membrane edges

(22) Shao, Z.; Mou, J.; Czajkowsky, D. M.; Yang, J.; Yaun, J.-Y. AdV. Phys. 1996, 45, 1.

(23) Tokumasu, F.; Jin, A. J.; Feigenson, G. W.; Dvorak, J. A. Biophys. J. 2003, 84, 2609.

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Figure 3. (A) AFM image of a PC SPB following the addition of 3 µM p25, in which the white line indicates an area of aggregates that appear to have deposited close to a thinning branched feature (as also observed in 2C). (B) Cross-section of a typical aggregate structure, with a width of ∼20 nm and a thickness of ∼10 nm (taken from the region marked with a circle in panel A). (C) AFM image of a PC SPB following the addition of 5 µM p25, illustrating the appearance of higher regions in the observed branched features, which typically occur prior to the formation or budding of aggregates (highlighted by circles). Table 1. Summary of the Changes in the SPB Surface Coverage Observed in Figures 1-3 upon the Application of Different Concentrations of p25 Peptidea change in surface coverage surface coverage decrease in applied p25 before after surface concentration concn increase concn increase coverage percentage 2 2 (µM) (µm ) (µm ) (µm2) decrease 0-0.5 1-3 3-5

19.36 1.90 1.07

18.72 1.73 1.00

0.97 0.17 0.07

5% 8.9% 6.5%

a Data corrected for any raised areas within the image and also any changes in the imaging position due to instrumental drift.

has been previously reported for binary phospholipid mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC, Tm ) -2 °C) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Tm ) 41 °C),23 and we have also observed this for 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC, Tm ) -22 °C) and sphingomyelin (Tm ) 35 °C) mixes (data not shown). Unsaturated hydrocarbons in the liquid-crystalline phase have increased CPPs, by means of a decreased critical hydrocarbon length, compared to their ordered counterparts.16 This suggests that more ordered (condensed), truncated cone-shaped phospholipids favor the formation of hemimicellar structures at the membrane edges to shield the hydrophobic moieties from the external aqueous medium. However, since p25 is a positively charged peptide, and considering the types of forces involved in maintaining a stable membrane on a mica support (electrostatic forces,24 hydrophobic and hydration forces, and also van der Waals attractive forces24-27), we cannot rule out the possibility that localized interactions between the p25-phospholipid microdomains and the bare mica surface may also contribute to the observed edge preference. (24) Egawa, H.; Furusawa, K. Langmuir 1999, 15, 1660. (25) Parsegian, V. A.; Evans, E. A. Curr. Opin. Colloid Interface Sci. 1996, 1, 53. (26) Ederth, T.; Claesson, P.; Liedberg, B. Langmuir 1998, 14, 4782. (27) May, S. Eur. Phys. J. E 2000, 3, 37.

With the addition of 1.0 µM p25, an increase in the overall bilayer height, from 4.4 ( 0.2 to 6.0 ( 0.3 nm, was observed (Figure 2A,B). A series of holes penetrating the bilayer also started to appear in roughly the same location as the microdomains observed in the previous images at 500 nM (Figure 1C), which were spanned by an interpenetrating network of narrower “branching” regions of bilayer. It should be noted that the height of the branches was similar to that of the larger bilayer regions, and thus it was assumed that they were of the same composition, and that they presented at this concentration with an average lateral width of 40.7 ( 4.4 nm (Figure 2B). With a further increase in p25 (3.0 µM) (Figure 2C), the lateral width of these branches was found to decrease to an average of 20.4 ( 3.6 nm, and their appearance was also observed to become more irregular. At this higher concentration an increasing number of “spherelike” features began to appear, located close to the branches on the bare regions of mica (with heights between 6 and 10 nm) (Figure 3A,B). Interestingly, upon increasing the applied concentration to 5 µM, the network branches were found to become even more irregular in appearance and constricted in locations that were very close to regions of increased height (from 6.0 ( 0.3 to 10.6 ( 2.7 nm) (indicated by the circles in Figure 3C). The occurrence of such features prior to the appearance of, and in close proximity to, the sphere-like features suggests a possible budding mechanism and that the increasing peptide-to-lipid ratio induces a further decrease in the lipid packing parameter, leading to more highly curved aggregates. At an applied p25 concentration of 5 µM, an increasing number of larger/higher “spherical” structures were also found to emerge (Figure 4A), which had an average lateral diameter of 24.1 nm (SD: 9.4 (n ) 35)) and a height of 16 nm (SD: 3 (n ) 25)) (Figure 4B). It should be noted that this larger type of aggregate can also be observed at the preceding concentrations, but at a much lower frequency. The observed gradual increase in the height/width of the budded features may also suggest a mechanism

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Figure 5. AFM image of 100 µM p25 dried onto a mica substrate and imaged in 10 mM Tris, pH 7.4.

Figure 4. (A) AFM image of a PC bilayer with the addition of 5 µM p25. The arrows indicate spherical aggregates. (B) Cross-section of a representative “spherical” feature with a diameter of ∼20 nm and height of >10 nm (taken from the region marked with a dotted line).

in which they evolve from “discoidal” to “spherical” micellar/ vesicular types of lipid-peptide aggregates.28 From the images, it is difficult to ascertain the composition of the observed aggregates, that is, whether they are purely lipid or peptide or mixed lipid-peptide aggregates. However, it should be noted that using aqueous imaging conditions and peptide concentrations similar to those employed in Figures 1-4, we were unable to obtain control the images of just the peptide in the absence of an SPB (when these samples were dried, p25 was found to form aggregates of 40 nm (SD: 5.6 (n ) 20)) on the mica surface (Figure 5)). Taking our difficulties in imaging the peptide into account, together with the fact that the features observed in Figures 1-4 will most likely exist in equilibrium with all of the involved monomeric counterparts, we suggest that the structures observed at the highest lipid-peptide ratio will be predominantly mixed p25-lipid aggregates. It is therefore likely that the insertion of increasing amounts of the p25 peptide into PC SPBs induces a modification in the phase behavior of the phospholipids, resulting from changes in the dynamic lipid shape and, hence, the lipid packing densities and structural aggregates. The molecular shapes of PC lipids in a liquid-crystalline state resemble truncated cones (0.5-1 CPP), (28) Edwards, K.; Johnsson, M.; Karlsson, G.; Silvander, M. Biophys. J. 1997, 73, 258.

which commonly aggregate to form flexible bilayers15 (such as those observed in Figure 1). Upon increasing the p25-tophospholipid ratio, a sequential series of transient mixed aggregates is observed from a flexible bilayer (Figure 1), through branched structures (Figures 2 and 3) that bud to ultimately form spherical structures (Figure 4). In order for the bilayer to curve and form the observed aggregates, the phospholipids must be able to pack into increasingly truncated cones. Thus, a reduction in the CPP is necessary for the progression of each structural aggregate from one configuration to the next. Following the assumption that the lipid hydrocarbon volume is incompressible,29 the reduction in the CPP of the phospholipids can be attributed to changes in the headgroup area and/or critical length. From the AFM studies, it is difficult to specify whether p25 induces modifications in the headgroup area or in the critical length of the phospholipids, or both. However, the initial increase in bilayer thickness and the decrease in surface area accommodated by the phospholipid bilayer (Figure 1C,D), when p25 is introduced, suggests an increase in the critical length, which could allow the phospholipids to pack into a more dense configuration. It is also feasible that the increasing positive membrane curvature of the transient aggregates with the increasing p25-to-lipid ratios may also be attributable to the increasing positive charge from the peptide. Early work on micelle formation showed that increasingly charged micelles shrink in radius.30 An increase in steric repulsion associated with the increase in charge results in an increase in the headgroup area and, thus, a decrease in the CPP. Interestingly, increasingly nonionic micelles grow and begin to take a more cylindrical configuration.31,32 The solubilization of lipid membranes by detergents (for detailed reviews, see refs 33 and 34) has also been found to give rise to transient intermediate structures with characteristics similar to those observed here. The basis of detergent-governed (29) Muresan, A. S.; Lee, K. Y. C. J. Phys. Chem. B 2001 105, 852. (30) Missel, P. J.; Mazer, N. A.; Benedek, G. B.; Young, C. Y.; Carey, M. C. J. Phys. Chem. 1980 84, 1044. (31) Kato, T.; Seimiya, T. J. Phys. Chem. 1986, 90, 3159. (32) Herrington, T. M.; Sahi, S. S. J. Colloid Interface Sci. 1988, 121, 107. (33) Litchenberg, D.; Opatowski, E.; Kozlov, M. M. Biochim. Biophys. Acta. 2000, 1508, 1. (34) Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146.

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solubilization is the destabilization and transformation of the membrane into mixed micelles composed of detergent, phospholipids, and membrane-bound proteins.35 Transient intermediate structures, such as open vesicles, cylindrical micelles,36,37 and circular “disk-like” micelles have been postulated to occur,28 although flexible cylinders are described as the more dominant intermediate.38 From these AFM studies, it remains unclear as to whether the final spherical aggregate is of a micellar or vesicular structure. However, considering that the maximum radius for a pure micelle composed of PC lipids will be approximately 2-3 nm15 and that the resulting spherical structures described have a lateral radius of ∼12 nm, the likelihood of the final aggregate being micellar is minimal. In AFM imaging studies, we must also consider the effect of feature broadening due to tip convolution.39 Such an effect would mean that the real dimensions of the observed spherical structures are less than the lateral dimensions recorded. However, when the vertical heights of the observed features are also taken into account (∼16 nm), and considering that they are also likely to suffer some degree of compression due to the forces imposed by the AFM tip during imaging, a final vesicle structure again seems more plausible. The observed dimensions are, in addition, closer to the predicted critical radius of 11 nm for a pure PC vesicle.15 If the structure of these aggregates is micellar, the process of solubilization of the bilayer most likely follows a “carpet-like” mechanism,40,41 similar to that reported for antimicrobial peptides. In this mechanism, amphiphilic antimicrobials initially bind onto the surface of the target membrane, in a random conformation, covering it in a carpet-like manner. The reorientation of the hydrophobic amino acid moieties toward the hydrophobic core of the membrane follows, causing a disruption in the bilayer curvature and the subsequent encapsulation of a phospholipid micelle,42 as illustrated in Figure 6A. In previous studies, the solubilization of SPBs with the protein melittin has been visualized using AFM and proposed to occur via this route, although highresolution images of transient structures formed during that process were not observed.43 Alternatively, if the final spherical features are vesicular, evolving from the preceding discoidal aggregates, they most likely form as a result of the increasing positive curvature of the upper membrane leaflet with increasing peptide concentration. Figure 6B illustrates a tentative model of the formation of such a mixed vesicle. In this model, the upper leaflet of the bilayer forms the outer monolayer of the vesicle, which is comprised of a phospholipid-peptide mix. The inner monolayer, formed from the lower bilayer leaflet, however, contains only phospholipids, as a result of packing constraints. It is postulated that, in vivo, the structural rearrangement of the phospholipids to form positive membrane curvature upon the association of the presequence peptide is likely to play a facilitative role in the formation of a fusion intermediate between (35) Stubbs, G. W.; Litman, B. J. Biochemistry 1978, 17, 220. (36) Vinson, P. K.; Talmon, Y.; Walter, A. Biophys. J. 1989, 56, 669. (37) Edwards, K.; Almgren, M. J. Colloid Interface Sci. 1991, 147, 1. (38) Kozlov, M. M.; Lichtenberg, D.; Andelman, D. J. Phys. Chem. 1997, 101, 6600. (39) Magonov, S. N.; Whangbo, M.-H. Surface Analysis with STM and AFM: Experimental and Theoretical Aspects of Image Analysis; Wiley-VCH: Weinheim, Germany, 1995. (40) Pouny, Y.; Rapaport, D.; Mor, A.; Nicolas, P.; Shai, Y. Biochemistry 1992, 31, 12416. (41) Gazit, E.; Boman, A.; Boman, H. G.; Shai, Y. Biochemistry 1995, 34, 11479. (42) Oren, Z.; Shai, Y. Biopolymers 1998, 47, 451. (43) Steinem, C.; Galla, H.-J.; Janshoff, A. Phys. Chem. Chem. Phys. 2000, 2, 4580.

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Figure 6. (A) Proposed model for a p25-PC mixed micelle as would be formed by the carpet-like mechanism of solubilization. (B) Tentative model of a vesicle (C) budding from a raised region of bilayer, where the upper leaflet forms the outer monolayer of the vesicle and is peptide rich, and the lower bilayer leaflet forms the inner monolayer and is absent of peptide.

the two mitochondrial membranes.8 Such fusion is required for the translocation of the nuclear encoded subunits of cytochrome c oxidase into the inner mitochondrial membrane. Previous studies have reported the ability of the peptide to induce lipid mixing between CL-containing unilamellar liposomes, without mixing of the liposome aqueous phases, suggesting the formation of a continuous import channel through the mitochondrial membranes.9 Although, CL is not present in this investigation, it is important to note that only the inner mitochondrial membrane is rich in CL.6 Thus, it is feasible that positive curvature of the outer mitochondrial membrane is required to initiate the formation of the mitochondrial import site in the outer membrane. Significant further investigations incorporating multiple bilayers that mimic the core lipid and biomolecular components of both the outer and inner mitochondrial membranes are, however, required to clarify the proposed role of the presequence as a facilitatory unit for the formation of membrane fusion sites.

Conclusions Lipid solubilization is a phenomenon commonly associated with detergents33,34 and antimicrobials.42 Here we have demonstrated that, at high concentrations, amphiphilic R-helical presequence peptides are also capable of inducing the solubilization of phospholipid membranes. The precise mechanism by which this solubilization process occurs remains unresolved, but it is clear that the presequence peptide, p25, induces positive

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curvature and the subsequent solubilization of the membrane via a sequential series of transient mixed peptide-phospholipid aggregate structures. Because of the dimensions of the final spherical aggregate, relative to micellar structures, it is also most likely that membrane solubilization by p25 ultimately results in the formation of mixed lipid-peptide vesicles. Importantly, since membrane fusion is thought to proceed via a structural re(44) Yang, L.; Huang, H. W. Science 2002, 297, 1877.

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arrangement of lipids into intermediates with high local curvature,13,14,44 the induced positive membrane curvature upon p25 association may be relevant in the mediation of fusion sites between the outer and inner mitochondrial membranes. Acknowledgment. We are grateful to the Engineering and Physical Sciences Research Council (EPSRC) for supporting this work. LA060114+