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Shape Changes of Giant Unilamellar Vesicles of Phosphatidylcholine Induced by a De Novo Designed Peptide Interacting with Their Membrane Interface Yuko Yamashita,†,§ Shah Md. Masum,‡,§ Tomoki Tanaka,‡ and Masahito Yamazaki*,†,‡ Department of Physics, Faculty of Science, Shizuoka University, Shizuoka, 422-8529, Japan, and Material Science, Graduate School of Science and Engineering, Shizuoka University, 422-8529, Japan Received September 5, 2002. In Final Form: October 14, 2002 We have designed and synthesized a peptide, WLFLLKKK (peptide-1), which has positive charges and a segment partitioned into the electrically neutral lipid membrane interface, and investigated its effect on the stability of the phosphatidylcholine (PC) membrane. Spacing of multilamellar vesicles of palmitoyloleoyl-PC increased greatly with an increase in peptide-1 concentration. The addition of 5 µM peptide-1 through a micropipet near the giant unilamellar vesicle (GUV) of dioleoylphosphatidylcholine induced several kinds of shape changes; for example, a discocyte was changed to two spheres connected by a neck, and small vesicles were budded into the outside of the spherical GUV. These results indicate that the de novo designed peptide-1 can be partitioned into the PC membrane interface and have a large effect on its structure and properties.
1. Introduction Interaction of proteins with lipid membranes plays important roles in static and dynamic structures of biomembranes and their functions. It is reported that many water-soluble proteins can be reversibly bound with lipid membrane regions in biomembranes and that their binding depends on their concentration in the aqueous phase, their conformation, and their local net charge. Especially, the electrostatic interaction between a cluster of basic amino acid residues of proteins and negatively charged lipid membranes and also the hydrophobic interaction between fatty acid covalently linked protein and lipid membranes are well understood, and they play an important role in the binding of proteins with biomembranes.1,2 For example, myristoylated alanine-rich C kinase substrate (MARCKS) and src (pp60src) can be bound with lipid membranes using both the electrostatic attractive interaction and the hydrophobic interaction due to the covalently linked fatty acid. Recent biophysical studies indicate that the lipid membrane interface is composed of hydrophilic segments (so-called headgroups), hydrophobic alkyl chains, and water molecules because of large thermal motions of the membranes such as undulation and protrusion.3,4 Peptides can be partitioned into the membrane interface or bound in the membrane interface. The thickness of the membrane interface of the dioleoylphosphatidylcholine (DOPC) mem* To whom correspondence should be addressed. Dr. Masahito Yamazaki, Department of Physics, Faculty of Science, Shizuoka University, 836 Oya, Shizuoka 422-8529, Japan. Tel and Fax: 8154-238-4741. E-mail:
[email protected]. † Department of Physics, Faculty of Science, Shizuoka University. ‡ Material Science, Graduate School of Science and Engineering, Shizuoka University. § These authors contributed equally. (1) Kim, J.; Blackshear, P. J.; Johnson, D.; McLaughlin, S. Biophys. J. 1994, 67, 227. (2) Buser, C. A.; Sigal, C. T.; Resh, M. D.; McLaughlin, S. Biochemistry 1994, 33, 13093. (3) Israelachvili, J. Intermolecular & Surface Forces, 2nd ed.; Academic Press: San Diego, 1992. (4) Kinoshita, K.; Furuike, S.; Yamazaki, M. Biophys. Chem. 1998, 74, 237.
brane was ∼1.5 nm, showing that an R-helix can be partitioned parallel to the bilayer plane into the membrane interface.5,6 Free energies of transfer of short peptides (Ac-WL-X-LL with X being any of the 20 amino acids) from the membrane interface of palmitoyloleoylphosphatidylcholine (POPC) to water were obtained experimentally, and from these data an interfacial hydrophobicity scale of amino acids was determined.6 According to these data, aromatic residues such as Trp (W) and Phe (F) have high interfacial hydrophobicity, meaning that partition of these residues into the lipid membrane interface is large. These data suggest that peptides containing amino acids with high interfacial hydrophobicity can be partitioned into the membrane interface of lipid membranes composed of electrically neutral lipids. In this report, we have synthesized a de novo designed peptide, which has positive charges and also can be partitioned into the lipid membrane interface composed of electrically neutral lipids such as phosphatidylcholine (PC). As the de novo designed peptide, we have designed and synthesized a peptide with the sequence WLFLLKKK (peptide-1). The N-terminal region (WLFLL) is the site (or the segment) of the peptide partitioned into the membrane interface, since an experimental result shows that the WLWLL peptide locates at the membrane interface and also F has a high interfacial hydrophobicity.6 The purpose of the design of this peptide is that it is partitioned into the membrane interface and gives positive charges in the membrane interface (Figure 1). In this report, we have investigated the effect of the de novo designed peptide-1 on PC membranes. First, we have investigated the effect of this peptide on the intermembrane interaction of multilamellar vesicles (MLVs) of POPC. We expect that the intermembrane distance would increase due to the electrostatic repulsive interaction between the positively charged membranes, and experimental results support this idea. Second, the effect of this peptide on the shape of giant unilamellar vesicles (GUVs) of DOPC has been investigated using phase-contrast (5) Wiener, M. C.; White, S. H. Biophys. J. 1992, 61, 434. (6) Wimley, W. C.; White, S. H. Nat. Struct. Biol. 1996, 3, 842.
10.1021/la0265124 CCC: $22.00 © 2002 American Chemical Society Published on Web 11/13/2002
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Figure 1. A schematic view of the membrane interface of the PC membrane and the partition of peptide-1 into the membrane interface. The lipid membrane consists of two membrane interfaces (MI) and the hydrophobic core (HC) (see details in the text).
microscopy, because the observation of shape changes of GUVs has been recently considered as a highly sensitive method detecting the interaction between substances and lipid membranes.7-9 2. Materials and Methods 2.1. Materials and Peptide Synthesis. 1,2-Dioleoyl-snglycero-3-phosphatidylcholine (DOPC) and 1-palmitoyl-2-oleoylsn-glycero-3-phosphatidylcholine (POPC) were purchased from Avanti Polar Lipids Inc. Peptides were synthesized by the FastMoc method using a peptide synthesizer 433A (PE Applied Biosystems, Foster City, CA). The sequence of peptide-1 is WLFLLKKK, and that of peptide-2 is LLKKK. These peptides have an amide-blocked C-terminus. Methods of purification and identification of peptides were almost the same as in the previous paper.10 The purified peptides were analyzed by ion-spray ionization mass spectrometry using a single quadrupole mass spectrometer (API 150EX, PE SCIEX, PE Applied Biosystems). The measured masses of peptide-1 and peptide-2 were 1074.2 ( 0.3 Da and 627.8 ( 0.1 Da, respectively. They correspond to the expected molecular masses in accord with their amino acid compositions. 2.2. Formation of GUVs of Phosphatidylcholine, Observation of GUVs under a Phase-Contrast Microscope, and Method of Addition of Peptides. GUVs of phosphatidylcholine were prepared by the standard method9 as follows. The phospholipid (DOPC or POPC) (100 µL, 1 mM) in chloroform in a small glass vessel was dried by N2 gas, and then the solvent was completely removed by placing the sample in a vacuum desiccator connected to a rotary vacuum pump for more than 12 h. Water (10 µL) was added into this vessel, and it was incubated at 45 °C for a few minutes (prehydration). Then, 1 mL of 0.1 M sucrose in water was added, and the sample was incubated at 37 °C for 2 h. GUVs were observed by a phase-contrast microscope using the standard method described in our previous paper.9 Various concentrations of peptides in 0.1 M glucose aqueous solution were added into the neighborhood of a GUV through a 10 µm diameter glass micropipet whose position was controlled by a micromanipulator (Narishige).9 2.3. Preparation of MLVs and Small-Angle X-ray Scattering (SAXS) Measurement. MLVs of a POPC/peptide mixture were prepared as follows.4 POPC dissolved in chloroform and various peptides dissolved in trifluoroethanol were mixed, dried by N2, and then kept under vacuum using a rotary vacuum pump for more than 12 h. An appropriate amount of 10 mM PIPES buffer (pH 7.0) containing a given concentration of NaCl was added to the dry powder of the mixture of POPC and peptide, in excess water (∼7 wt % lipids). Then, the suspensions were vortexed for about 30 s at room temperature (∼25 °C) several times and were incubated at room temperature for 30 min. For measurements of X-ray diffraction, pellets after centrifugation (13 000g, 30 min at 20 °C; Tomy, MR-150) of the lipid suspensions were used. X-ray diffraction experiments were performed by using nickel-filtered Cu KR X-rays (λ ) 0.154 nm) from a rotating anode (7) Farge, E.; Devaux, P. F. Biophys. J. 1992, 61, 347. (8) Yamashita, Y.; Oka, M.; Tanaka, T.; Yamazaki, M. Biochim. Biophys. Acta 2002, 1561, 129. (9) Tanaka, T.; Tamba, Y.; Masum, S. M.; Yamashita, Y.; Yamazaki, M. Biochim. Biophys. Acta 2002, 1564, 173. (10) Oblatt-Montal, M.; Yamazaki, M.; Nelson, R.; Montal, M. Protein Sci. 1995, 4, 1490.
Figure 2. (A) Spacing of POPC-MLV in the presence of various concentrations of peptide-1 (O) and peptide-2 (b) in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C. R is the molar ratio of peptides to lipid (POPC). (B) Effect of NaCl concentration on the spacing of the POPC/peptide-1 membrane (R ) 0.030) in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C. (O) is represented as a normal scale of concentration (lower abscissa), and (2) is represented as a logarithmic scale of concentration (upper abscissa). type X-ray generator (Rigaku, Rotaflex, RU-300) at the operating conditions (40 kV, 200 mA). The detail was described in our previous papers.4,11
3. Results and Discussion 3.1. Interaction of Peptide-1 with POPC-MLV. POPC-MLV in excess water at 20 °C is well-known to be in the liquid-crystalline (LR) phase.4 We investigated the dependence of spacing of the POPC-MLV on the peptide concentration by SAXS. As shown in Figure 2A, the spacing (dl) of POPC-MLV gradually increased from 6.3 to 8.8 nm with an increase in peptide-1 concentration (R ) molar ratio of peptides to lipid (POPC)). On the other hand, in the case of peptide-2, the spacing did not change. The spacing is determined by the summation of the intermembrane distance (df) and the membrane thickness (dm); that is, dl ) df + dm. The values of df and dm can be determined by the electron density profile of the membranes. The POPC-MLV was selected for this experiment because its SAXS pattern has enough diffraction peaks to get an electron density profile of the membrane with sufficient resolution.4 However, in the presence of peptide-1 only two diffraction peaks were obtained, and thereby, it was difficult to obtain the electron density profile. Despite this result, we can get qualitative infor(11) Yamazaki, M.; Ohshika, M.; Kashiwagi, N.; Asano, T. Biophys. Chem. 1992, 43, 29.
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mation on the intermembrane distance (df) from Figure 2A. The membrane thickness of the LR phase membrane cannot change greatly.4 Thereby, the large increase in the spacing of POPC-MLV is due to the increase in the intermembrane distance. Figure 2B shows that the spacing of the POPC-MLV in the presence of peptide-1 (R ) 0.030) decreased with an increase in NaCl concentration in solution. This result indicates that the increase in the intermembrane distance of POPC-MLV by peptide-1 is due to electrostatic repulsive interaction. Based on these results, we can reasonably consider that the peptide-1 is partitioned into the membrane interface and gives positive charges to the membrane surface, resulting in the increase in electrostatic repulsive interaction between membranes in the MLV. In Figure 2A, the spacing shows saturation at a high concentration of peptide. The electric field due to the partitioned peptide-1 into the membrane decreases the partition of other peptide-1 molecules due to their electrostatic repulsive interaction, and thereby, the apparent partition coefficient of peptide-1 into membrane interface decreased with an increase in peptide-1 concentration, which is a phenomenon similar to the binding of La3+ to PC membranes.9 3.2. Shape Changes of DOPC-GUV Induced by Peptide-1. It is easy to prepare DOPC-GUV in water. In our previous paper, we investigated shape changes of DOPC-GUV induced by several substances and analyzed these phenomena.8,9 In this report, first we investigated the effect of peptide-1 on shapes of DOPC-GUV. Figure 3 shows two kinds of shape changes of a DOPC-GUV induced by addition of 5 µM peptide-1 through a 10 µm diameter micropipet near the GUV. At first (in the absence of peptide-1), the GUV was a discocyte. After the addition of peptide-1, the shape changed into a dumbbell (Figure 3A-(3)) and then into two spheres connected by a neck (n ) 5) (Figure 3A-(5)). In the case of 0.5 µM peptide-1, this type of shape change was not observed. When we added 5 µM peptide-1 near a cylindrical GUV (Figure 3B(1)), the shape changed into a GUV made of a series of many spherical vesicles connected by a narrow tube (socalled “pearl on a string”) (n ) 8) (Figure 3B-(4)). In the case of 0.5 µM peptide-1, this type of shape change was not observed. As a control experiment, peptide-2 was added near the GUV, but no shape changes of the GUV such as those in Figure 3A,B were observed. Figure 4A shows another type of shape change of a DOPC-GUV induced by addition of 5 µM peptide-1 near the GUV. At first (in the absence of peptide-1), the GUV was almost spherical. After the addition of peptide-1, the undulation motion of the membrane of the GUV largely increased and also the diameter of the GUV increased a little (Figure 4A-(2)). On further addition of peptide-1, small vesicles suddenly budded into the outside of the GUV (Figure 4A-(4)). These small vesicles were connected by a narrow tube, and thereby the appearance was similar to the pearl on a string. We observed this kind of shape change in five GUVs among five examined GUVs (n ) 5). In the case of 0.5 µM peptide-1, this type of shape change was not observed. As a control experiment, peptide-2 was added near the GUV, but no shape change of the GUV was observed. To determine the reversibility of the shape change induced by peptide-1, the addition of peptide-1 was stopped after the shape change of the GUV completed. Figure 4B shows the time course of the shape change of the GUV after the addition of peptide-1 was stopped. At first, the pearl on a string budded into the outside of the GUV changed into a tubular protrusion from the GUV (Figure 4B-(1)). Then, this tubular protrusion retreated (Figure
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Figure 3. Shape change of DOPC-GUV induced by the addition of 5 µM peptide-1 at 20 °C. (A) A discocyte was changed into a dumbbell and then into two spheres connected by a neck. (B) The cylinder to pearl on a string transformation. The time after the starting injection of peptide-1 solution is (1) 0 s, (2) 11 s, (3) 14 s, (4) 17 s, and (5) 19 s for (A) and (1) 0 s, (2) 23 s, (3) 27 s, and (4) 42 s for (B). The bar in the picture corresponds to 10 µm.
4B-(2),(3)) into a spherical GUV. In this experiment, 5 µM peptide-1 was added to the vicinity of the GUV through the micropipet, and thereby, as the addition was stopped, peptide-1 diffused away from the vicinity of the GUV into the bulk solution, inducing the decrease in peptide-1 concentration near the GUV and then the desorption of peptide-1 from the membrane interface. Therefore, the result of Figure 4B indicates that this shape change was almost reversible, indicating that no vesicle fission occurred. We also investigated the reversibility of the shape changes shown in Figure 3A,B, and the results indicate that these shape changes were also reversible. Compared with the formation of DOPC-GUV, it is a little difficult to prepare many POPC-GUVs in water, but possible. So, in the next step, we investigated the effect
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Figure 4. (A) Shape change of a spherical DOPC-GUV induced by the addition of 5 µM peptide-1 at 20 °C. The time after the starting injection of the peptide solution is (1) 0 s, (2) 62 s, (3) 72 s, and (4) 80 s. (B) The reversibility of the shape change of DOPC-GUV induced by the addition of peptide-1. After the addition of peptide was stopped, the shape change was reversed. The time after stopping the injection is (1) 40 s, (2) 44 s, (3) 47 s, and (4) 58 s. The bar in the picture corresponds to 20 µm.
of peptide-1 on shapes of POPC-GUV. The shape changes of POPC-GUV induced by peptide-1 were almost the same as those of DOPC-GUV, and they were reversible (data not shown). What kind of effects of the peptide on the PC membranes can induce such shape changes of the GUV? Several experimental results indicate that the bilayer-coupling model can reasonably explain shape changes of the GUV under several conditions.7-9,12 In the bilayer-coupling model, the shape of the GUV is determined by the minimization of the bending energy of the membrane of the GUV for a given area A of the GUV, a given volume V of the GUV, and also a given area difference between (12) Ka¨s, J.; Sackmann, E. Biophys. J. 1991, 60, 825.
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the two monolayers in the bilayer membrane ∆A (i.e., ∆A ) Aex - Ain, where Aex and Ain are the area of the external monolayer and that of the internal monolayer of the GUV, respectively). In the experiments of the shape change of GUVs, peptide-1 was added into the neighborhood of GUVs from the outside of the GUVs and was partitioned into the membrane interface of the external monolayer membrane of the GUV. The partition of peptide-1 into the membrane interface increases the area of the membrane due to the partition itself and also the electrostatic repulsive interaction between the peptides partitioned into the membrane interface. Therefore, only the area of the external monolayer membrane of the DOPC-GUV, Aex, increases due to the partition of peptide into the membrane interface, while the area of the internal monolayer, Ain, does not change. Hence, the interaction of peptide-1 with the GUV induces an increase in ∆A without the change of the volume of the GUV. On the other hand, the simulation based on the bilayer-coupling model13-15 shows that under the condition of the constant volume of the GUV, the shape transformations such as the prolate via pear to a sphere connected with small vesicles (budding transition), the prolate to two spheres connected by a neck,13,14 and also the cylinder to the pearl on a string15 occur with an increase in ∆A. Therefore, the several types of shape changes of the DOPC-GUV induced by peptide-1 can be explained reasonably by the increase in ∆A. All the results in this report clearly showed that the de novo designed peptide-1 is partitioned into the membrane interface of the electrically neutral PC membranes. From this conclusion, we expect that the segment of proteins composed of amino acids with high interfacial hydrophobicity such as WLFLL can play an important role in the binding of proteins with lipid membranes in cells. Acknowledgment. This work was supported in part by a Grant for Basic Research Projects from the Sumitomo Foundation (Japan) to M.Y. LA0265124 (13) Svetina, S.; Zeks, B. Eur. Biophys. J. 1989, 17, 101. (14) Seifert, U.; Berndl, K.; Lipowsky, R. Phys. Rev. A 1991, 44, 1182. (15) Iglic, A.; Kralj-Iglic, V.; Majhenc, J. J. Biomech. 1999, 32, 1343.
