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Cholesterol Doping Induced Enhanced Stability of Bicelles Hirotaka Sasaki,† Seketsu Fukuzawa,*,† Jun Kikuchi,‡,§ Shigeyuki Yokoyama,‡ Hiroshi Hirota,‡,§ and Kazuo Tachibana*,† Department of Chemistry, Graduate School of Science, The University of Tokyo and CREST, Japan Science and Technology Corporation (JST) Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, Protein Research Group, Genomic Sciences Center, RIKEN Yokohama Institute, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan, and Graduate School of Integrated Science, Yokohama City University, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan Received March 26, 2003. In Final Form: August 6, 2003 To evaluate bicelles as a model membrane system, we examined the morphological changes of bicelles induced by the membrane-lytic peptide melittin. 31P NMR and dynamic light scattering experiments showed that melittin irreversibly disrupted the disk-shaped structure of bicelles and that the disrupted bicelles form giant spherical assemblies above the gel-to-liquid-crystalline transition temperature (Tm ) 24 °C). The melittin-induced disruption of bicelles was suppressed by the addition of cholesterol, suggesting that cholesterol effectively improved the stability of the bicelle membranes in the same manner as in vesicles. Furthermore, the location of bicelle-bound melittin and its role in bicelle assembly are also discussed.
Introduction A great number of membrane-active molecules, including membrane proteins, are known to play important roles in controlling cellular homeostasis in organisms.1 To understand the mode of action of such molecules, it is important to investigate these phenomena at the membrane water-lipid interface. Model membrane systems are ideal to gain insight into the interaction between the lipids and the membrane-active molecules. Vesicles are known to be useful as model membranes, and there have been many functional studies of membrane-active molecules where solid-state NMR methods were applied very successfully to vesicle systems.2,3 However, their long correlation times are unsuitable for analyses using solution NMR, and their large curvature makes them a less attractive system.4 Micelles have also served as a model membrane system in various studies on membraneactive molecules. In particular, micelles have been very fruitful in elucidating the structures of several membrane proteins by solution NMR.5,6 However, compared with * To whom correspondence should be addressed. S. Fukuzawa, Department of Chemistry, Graduate School of Science, The University of Tokyo and CREST, Japan Science and Technology Corporation (JST) Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel/ Fax: +81-3-5841-8059. E-mail:
[email protected]. † The University of Tokyo. ‡ RIKEN. § Yokohama City University. Abbreviations: DHPC, 1,2-dihexanoyl-3-sn-phosphatidylcholine; DMPC, 1,2-dimyristoyl-3-sn-phosphatidylcholine; DPPC, 1,2-dipalmitoyl-3-sn-phosphatidylcholine; EPR, electron paramagnetic resonance; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; PC, phosphatidylcholine; P/L, molar ratio of peptide to lipid; Tm, gel-to-liquid-crystalline transition temperature. (1) Stragljar, I.; Fields, S. Trends Biochem. Sci. 2002, 27, 559. (2) Montal, M.; Opella, S. J. Biochim. Biophys. Acta 2002, 1565, 287. (3) Naito, A.; Nagao, T.; Norisada, K.; Mizuno, T.; Tuzi, S.; Saitoˆ, H. Biophys. J. 2000, 78, 2405. (4) Baleja, J. D. Anal. Biochem. 2001, 288, 1. (5) Ferna´ndez, C.; Adeishvili, K.; Wu¨thrich, K. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2358. (6) Ma, C.; Marassi, F. M.; Jones, D. H.; Straus, S. K.; Bour, S.; Strebel, K.; Schubert, U.; Oblatt-Montal, M.; Montal, M.; Opella, S. J. Protein Sci. 2002, 11, 546.
Figure 1. The amino acid sequence of melittin.
vesicles, the interpretations of the results using micelles as model membranes are not very straightforward, due to their lack of a lipid bilayer structure. Bicelles are bilayered discoidal lipid-detergent assemblies in which the lipid-rich bilayer planes are stabilized at their edges by the detergent component.7,8 The diameter of the bicelle bilayer plane changes according to the lipid/detergent molar ratio, or q value.9 The unique ellipsoidal shape of bicelles allows the solutes to align in a magnetic field to generate residual dipolar coupling, which provides the long-range orientational constraints that are important for a variety of biomolecular NMR applications.10 Bicelles also possess great potential as model membranes because of their planar lipid bilayer structure and relatively small size. The usefulness of bicelles as a model membrane system has recently been investigated by examining the functionality of a membrane protein reconstituted in bicelles.11-13 Melittin, a polypeptide of 26 amino acid residues, is the principal component of venom from the honey bee Apis mellifera and is known to interact with membranes (Figure 1).14,15 The peptide is known to bind spontaneously to the lipid bilayers16 and is also known to cause hemolysis of red blood cells and leakage of internal markers from pure lipid vesicles.17,18 Based on a large number of studies using phosphatidylcholine vesicles as a model membrane sys(7) Ram, P.; Prestegard, J. H. Biochim. Biophys. Acta 1988, 940, 289. (8) Sanders, C. R.; Hare, B. J.; Howard, K. P.; Prestegard, J. H. Prog. NMR Spectrosc. 1994, 26, 421. (9) Vold, R. R.; Prosser, R. S. J. Magn. Reson. 1996, 113, 267. (10) De Alba, E.; Tjandra, N. Prog. NMR Spectrosc. 2002, 40, 175. (11) Czerski, L.; Sanders, C. R. Anal. Biochem. 2000, 284, 327. (12) Faham, S.; Bowie, J. U. J. Mol. Biol. 2002, 316, 1. (13) Sanders, C. R.; Landis, G. C. Biochemistry 1995, 34, 4030. (14) Habermann, E. Science 1972, 177, 314. (15) Dempsey, C. E. Biochim. Biophys. Acta 1990, 1031, 143. (16) Dufourcq, J.; Faucon, J.-F. Biochim. Biophys. Acta 1977, 467, 1. (17) Sessa, G.; Freer, J. H.; Colacicco, G.; Weissmann, G. J. Biol. Chem. 1969, 244, 3575.
10.1021/la0345183 CCC: $25.00 © 2003 American Chemical Society Published on Web 10/15/2003
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tem, the current understanding of the mode of action of melittin is as follows: (i) at melittin-lipid molar ratios, P/L, lower than 3.3 mol %, melittin coexists with vesicles without perturbing the membranes; (ii) at P/L ratios greater than 3.3 mol % and up to 10 mol %, melittin induces a reversible transition from the disklike complexes to vesicular structures that takes place at about the gel-toliquid-crystalline transition temperature, Tm; and (iii) at higher doses (P/L > 10 mol %), melittin disrupts vesicles completely into micellar complexes, and the disk-to-vesicle transition is no longer observed.19,20 In this investigation, we have used the membrane-lytic peptide melittin and cholesterol as probes to evaluate bicelles as a model membrane system. Our results suggest that cholesterol doping effectively suppresses the melittininduced disruption of the bicelle structure. Materials and Methods Materials. Phospholipids and melittin were purchased from Sigma Chemicals (St. Louis, MO). Cholesterol was purchased from KANTO Kagaku Co. (Tokyo, Japan). Phospholipids were used as purchased without further purification. Prior to use, commercially available cholesterol was purified by recrystallization from methanol and melittin was purified by ODS-HPLC on a COSMOSIL 5C18-AR-II column, 20 × 250 mm (Nakalai Tesque, Kyoto, Japan), with elution at 8 mL/min over 30 min in a linear gradient of 30-100% aqueous acetonitrile with 0.1% TFA. Preparation of Cholesterol-Free Bicelles. Typically, a bicelle solution whose q value and total lipid concentration were 1.8 and 7.4% (w/v), respectively, was prepared as follows: a chloroform solution of 1,2-dihexanoyl-3-sn-phosphatidylcholine (DHPC) (5.0 mg) was dried in vacuo for 3 h and resuspended in 50 µL of water. 1,2-Dimyristoyl-3-sn-phosphatidylcholine (DMPC) (13.5 mg) was also suspended in 200 µL 12.5% (v/v) of a waterdeuterium oxide mixture. Each solution was homogenized by vortexing followed by several freeze (-30 °C)/thaw (40 °C) cycles. Two dispersions were then warmed to 40 °C and mixed by vigorous vortexing. After cooling to 0 °C, the phospholipid mixture was vigorously vortexed until the mixture turned transparent. Preparation of Cholesterol-Doped Bicelles. Cholesteroldoped bicelle solutions (6.7 mol % vs DMPC) whose q value and total lipid concentration (DMPC + DHPC) were 1.8 and 7.4% (w/v), respectively, were prepared as follows: a chloroform solution of DHPC (5.0 mg) was dried in vacuo for 3 h and resuspended in 50 µL of water. DMPC (13.5 mg) and cholesterol (0.5 mg) were then mixed in the chloroform solution. This solution was dried in vacuo for 3 h followed by the addition of 200 µL of a 12.5% (v/v) water-deuterium oxide mixture. Each solution was homogenized by vortexing, followed by several freeze (-30 °C)/thaw (40 °C) cycles. Two dispersions were then warmed to 40 °C and mixed by vigorous vortexing. After cooling to 0 °C, the phospholipid mixture was vigorously vortexed until the mixture turned transparent. Dynamic Light Scattering Experiments. Dynamic light scattering experiments were performed on a Protein Solutions DynaPro-MS/X dynamic scattering instrument (Charlottesville, VA). The measurement range for dynamic light scattering was set from 1 to 10 000 Å. The average hydrodynamic radius was determined using DYNAMICS operating software. Samples for dynamic light scattering experiments were incubated in the presence or absence of melittin at 25 °C for 24 h prior to making measurements. Both samples were allowed to equilibrate at the desired temperature for at least 15 min prior to the acquisition of light scattering data. NMR Spectroscopy. 31P NMR was carried out on a Bruker DRX-500 NMR spectrometer with a 5-mm tunable multinuclear probe. All spectra were collected with both the deuterium field (18) Benachir, T.; Monette, M.; Grenier, J.; Lafleur, M. Eur. Biophys. J. 1997, 25, 201. (19) Dufourcq, J.; Faucon, J.-F.; Fourche, G.; Dasseux, J.-L.; Maire, M. L.; G.-Krzywicki, T. Biochim. Biophys. Acta 1986, 859, 33. (20) Pott, T.; Dufourc, E. J. Biophys. J. 1995, 68, 965.
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Figure 2. 31P NMR spectra of bicelles before (A) and after (B) disruption with melittin. The concentration of melittin was 1.0 mM. Both samples contained 7.4% (w/v) total lipid (DMPC + DHPC), and the DMPC/DHPC molar ratio was 1.8 (13.5 mg of DMPC and 5.0 mg of DHPC in 250 µL of water). NMR spectra were recorded at 293 K (a) and 303 K (b). frequency lock and sample spinning turned off. 31P (202.46 MHz) spectra were acquired using a 90° 31P pulse (13 µs), WALTZ 1H decoupling (1H 90° pulse, 78 µs), and a relaxation delay of 2.0 s between scans. Spectra were collected with 8192 points, 128 scans, and a sweep width of 40 ppm centered at -5 ppm. Deuterium (D2O) lock was used, and 31P chemical shifts were referenced to 85% H3PO4 (H3PO4 ) 0 ppm). In the case of the NMR experiments using cholesterol-doped bicelles, the temperature of the sample was raised to the desired value at the rate of 1 °C/min. Samples were allowed to equilibrate at the desired temperature for at least 15 min prior to the acquisition of NMR data.
Results Monitoring the Morphological Changes of Bicelles Induced by Melittin. Morphological changes in bicelles were monitored by 31P NMR. Generally, two anisotropic singlet peaks (i.e., ca. -0.5 ppm for DHPC and ca. -8.5 ppm for DMPC) are good indicators for bicelle formation.21 As shown in Figure 2A, curve b, the 31P NMR spectra showed the two typical bicelle-associated signals recorded at 303 K before the addition of melittin. However, immediately after addition of melittin, these two anisotropic peaks disappeared completely and isotropic peaks whose line shapes were narrow and broad appeared at 1.6 and 0.7 ppm, respectively (Figure 2B, curve b). These results implied that the lipid bilayer structure of bicelles was disrupted and was followed by formation of small and large lipid assemblies that tumbled isotropically in solution. In the absence of melittin, lowering the temperature from 303 to 293 K disrupted the bicelle structure, as evidenced by the disappearance of the two anisotropic peaks and the appearance of isotropic peaks that seemed to be derived from DHPC micelles and DMPC vesicles (Figure 2A, curve a). This change was found to be reversible, as the two anisotropic peaks at -0.5 and -8.5 ppm reappeared when the temperature was raised to 303 K (results not shown). When the structure of the bicelles was disrupted by melittin, narrow and broad isotropic peaks were also observed at 293 K (Figure 2B, curve a). However, we did not observe reappearance of any bicelleassociated anisotropic peaks in the 31P NMR spectra when the temperature was varied between 298 and 318 K (results not shown). Taken together, these results imply that melittin irreversibly changed the bicelle structure to another type of lipid assembly. Dynamic light scattering experiments revealed that before the addition of melittin the phospholipid mixture was made of two components (Figure 3A). The smaller (21) Picard, F.; Paquet, M. J.; Levesque, J.; Be´langer, A.; Auger, M. Biophys. J. 1999, 77, 888.
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Figure 3. Normalized histograms from dynamic light scattering experiments using bicelles that were incubated in the absence (A) and presence (B) of melittin for 24 h. The concentration of melittin was 1.0 mM. Experiments were performed at 303 K as described in Materials and Methods. Both samples contained 5.0% (w/v) total lipid (DMPC + DHPC), and the DMPC/DHPC molar ratio was 3.2 (4.1 mg of DMPC and 0.86 mg of DHPC in 100 µL of water).
Figure 5. 31P NMR spectral changes of bicelles at different concentrations of melittin in the absence and presence of cholesterol. In panel A, 31P NMR spectra of bicelles without cholesterol were recorded at 303 K: (a) 0 mM, P/L ) 0 mol %; (b) 0.11 mM, P/L ) 0.14 mol %; (c) 0.22 mM, P/L ) 0.28 mol %; (d) 0.55 mM, P/L ) 0.69 mol %. In panel B, 31P NMR spectra of bicelles with cholesterol (6.7 mol % vs DMPC) were recorded at 308 K: (a) 0 mM, P/L ) 0 mol %; (b) 0.80 mM, P/L ) 1.0 mol %; (c) 1.2 mM, P/L ) 1.5 mol %; (d) 1.6 mM, P/L ) 2.0 mol %. Both samples contained 7.4% (w/v) total lipid (DMPC + DHPC), and the DMPC/DHPC molar ratio was 1.8 (13.5 mg of DMPC and 5.0 mg of DHPC in 250 µL of water). Sample B also contained 0.5 mg of cholesterol.
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Figure 4. P NMR spectral changes of bicelles mixed with different concentrations of melittin at 303 K. The P/L ratio for each concentration of melittin used is also indicated. Panel A: (a) 0 mM, P/L ) 0 mol %; (b) 0.22 mM, P/L ) 0.36 mol %; (c) 0.30 mM, P/L ) 0.50 mol %. The DMPC/DHPC molar ratio was 3.0, and the total lipid (DMPC + DHPC) concentration was 5.0% (w/v) (10.2 mg of DMPC and 2.3 mg of DHPC in 250 µL of water). Panel B: (a) 0 mM, P/L ) 0 mol %; (b) 0.11 mM, P/L ) 0.14 mol %; (c) 0.22 mM, P/L ) 0.28 mol %. The DMPC/DHPC molar ratio was 1.8, and the total lipid concentration was 7.4% (w/v) (13.5 mg of DMPC and 5.0 mg of DHPC in 250 µL of water).
component consisted of residual micelles with an average hydrodynamic radius, Rh, of ca. 20 Å, and the larger component consisted of bicelles with an average Rh of ca. 100 Å. In contrast, incubation with melittin at 25 °C transformed bicelles into some tremendously large assemblies, whose average Rh was not less than 1 µm (Figure 3B). Relationship between the Size of Bicelles and Their Stability against Melittin. Next, we investigated how much melittin could be added to the bicelles without perturbing their structure. The 31P NMR spectra revealed the relationship between the size of the bicelles and their stability against melittin (Figure 4). In the case of larger bicelles (q value of 3.0), the two typical signals for bicelles were observed at melittin-DMPC molar ratios (P/L) of
