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The Image-Based Observation of the LβI-to-Lβ′ Phase Transition in Solid-Supported Lipid Bilayers Robert L. McClain† and J. J. Breen* Department of Chemistry, 402 N. Blackford Street, Indiana University Purdue University Indianapolis, Indianapolis, Indiana 46202-3274 Received February 23, 2001. In Final Form: May 16, 2001
Introduction Phospholipids constitute one of the fundamental building blocks of nature. Their self-assembly into cell and organelle membranes not only forms a containment barrier but also provides a host matrix for membrane-associated proteins. The phospholipids in biological membranes are present as complex mixtures of lipids. The different lipids are not necessarily evenly distributed, which can lead to a considerable amount of lateral heterogeneity on the nanometer scale.1,2 The organization and arrangement of lipids in the membrane are also susceptible to changes in the physical and chemical environment that are triggered either by biochemical events in the cell or by extracellular events, making membranes dynamic structures.3 Thus, the phospholipids in the membranes can modify the functions of contained proteins and serve as messengers in cell-signaling processes.4,5 The most common organization of phospholipids in membranes (liquid, liquid-crystalline, or gel state) is the bilayer structure, which is held together by relatively weak intermolecular forces.6 The bilayer results from the joining of two phospholipid monolayers that contact each other with their hydrophobic alkyl chains while their hydrophilic headgroups contact water. For some time, it has been known that an alternative lipid packing arrangement, the interdigitated state, is also possible.7 In the case of lipids exhibiting little or no chain-length asymmetry, the fully interdigitated state exists with four acyl-chain-crosssection areas subtended by a single lipid headgroup. As depicted in Figure 1, the terminal methyl groups of the acyl chains extend beyond the bilayer midplane to interpenetrate into the opposing monolayer, thus spanning the entire width of the bilayer. The membrane thickness is significantly reduced, which has important implications for the rate of transport of ions across the barrier,8 as well as for the environment presented to transmembrane proteins. In addition, the presence of interdigitated domains is thought to stiffen the membrane structure, * Correspondence should be addressed to this author. † Permanent address Department of Chemistry, University of Indianapolis, 1400 E. Hanna Ave., Indianapolis, IN 46227. (1) Kusumi, A.; Sako, Y.; Yamamoto, M. Biophys. J. 1993, 65, 2021-. (2) Jacobson, K.; Sheets, E. D.; Simson, R. Science 1995, 268, 14411442. (3) Williams, E. E. Am, Zool. 1988, 38, 280-290. (4) Meer, G. v.; Holthuis, J. C. M. Biochim. Biophys. Acta 2000, 1486, 145-170. (5) Daleke, D. L.; Lyles, J. V. Biochim. Biophys. Acta 2000, 1486, 108-127. (6) Cevc, G.; Marsh, D. Phospholipid Bilayers: Physical Properties and Models; John Wiley and Sons: New York, 1987. (7) Slater, J. L.; Huang, C.-H. Prog. Lipid Research 1988, 27, 325359. (8) Komatsu, H.; Okada, S. Biochim. Biophys. Acta 1995, 1237, 169175.
Figure 1. Schematic illustration of the normal gel (Lβ′) and interdigitated (LβI) phases for DPPC bilayers.
affecting the translocation of phospholipids within the membrane.9,10 Whereas the presence of the interdigitated state in fully saturated, like-chain phophatidylcholine (PC) lipids can be induced by increasing the hydrostatic pressure, a wide variety of small amphiphilic molecules are also capable of inducing the interdigitated state in PC and phophatidylglycerol (PG) bilayers.7 These amphiphilic molecules include ethanol and other simple alcohols, glycerol, ethylene glycol, thiocyanate, and Tris, as well a number of common anesthetics. The most extensively investigated inducing agent-lipid system is ethanol-PC because of its once proposed relevance to the problems of alcohol intoxication, dependence, and tolerance. Experiments with ethanol and other alcohols suggest that the primary effect of the inducing agent in causing interdigitation is in the interfacial region where the alcohol hydroxy replaces water.11-13 The alcohol brings with it a hydrophobic moiety that disrupts the interfacial water, which, in turn, favors an increase in area per lipid headgroup that is accommodated by the interdigitated state. In addition to small amphiphilic molecules, the interdigitated state can also be induced by short peptides such as polymixin B14 and the Met f Nle variant of the N-terminal domain of the capsid protein cleavage product of the flock house virus.15,16 In this latter case, recently reported by Ghadiri and co workers, the 21-residue oligopeptide cleavage product is shown to be a very potent inducing agent relative to ethanol, and the authors propose an alternative mechanism for interdigitation involving the insertion of the helical peptide into the hydrophobic regions of the bilayer. Because the peptide is one of five such units attached to the virus, it is proposed that the formation of the interdigitated state is crucial in making biological membranes permeable for RNA translocation in the host cell and might have further implications for the infection strategy adopted by simple RNA viruses. In this work, we report AFM images and structural information pertaining to the interdigitated state in solidsupported dipalmitoylphosphatidylcholine (DPPC) bilayers. Since its invention is 1987, the atomic force microscope (AFM) has proven to a be a powerful and versatile tool for studies of surface structures, material properties, and (9) Moss, R.; Battacharya, S. J. Phys. Org. Chem. 1992, 5, 467-470. (10) Boon, J. M.; R. L, M.; Breen, J. J.; Smith, B. D. J. Supramol. Chem. 2001, 1, 17-21. (11) Rowe, E. S. Biochemistry 1983, 22, 3299-3305. (12) Rowe, E. S. Effects of Ethanol on Membrane Lipids; Rowe, E. S., Ed.; CRC Press: Boca Raton, FL, 1992; pp 239-267. (13) Rowe, E. S.; Campion, J. M. Biophys. J. 1994, 67, 1888-1895. (14) Boggs, J. M.; Tummler, B. Biochim. Biophys. Acta 1993, 1145, 42-50. (15) Janshoff, A.; Bong, D. T.; Steinem, C.; Johnson, J. E.; Ghadiri, M. R. Biochemistry 1999, 38, 5328-5336. (16) Bong, D. T.; Steinem, C.; Janshoff, A.; Johnson, J. E.; Ghardiri, M. R. Chem. Biol. 1999, 6, 473-481.
10.1021/la010295+ CCC: $20.00 © 2001 American Chemical Society Published on Web 07/07/2001
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molecular interactions.17-21 AFM images of interdigitated domains in solid-supported membranes of DPPC and DSPC were first reported in 1994 by Shao and coworkers.22 Their experiments showed that stable interdigitated domains can be recognized as depressed areas in a membrane surface. In addition, they reported threshold concentrations of ethanol and 2-propanol required for observing the interdigitated state that were much lower than those previously reported from calorimetry and X-ray experiments with multilayers of the same lipids. AFM images of interdigitated domains have also been reported for the interactions of the cleaved peptides from the flock house virus with DPPC15 and for the interactions of atropine with dipalmitoylphophatidylglycerol (DPPG).10 Our AFM images reveal the changes in the solidsupported bilayer structure following the transition from the interdigitated state to the normal gel state. It is known that alcohols reversibly induce the interdigitated state in DPPC bilayers, but the transitions in either direction are slow (hours to days) for membranes in the gel phase. From our imaging experiments, it appears that the transition from the interdigitated state to the normal gel state can be accelerated through the interactions of the AFM probe tip with the membrane sample. Analysis of the domain and defect areas present in the two-image sequence leads to information about the packing of the lipids in the interdigitated domains. Materials and Methods Solid-supported unilamellar DPPC bilayers were prepared using the vesicle fusion method introduced by McConnell and co-workers23,24 as described by Shao and co-workers.22 Briefly, a DPPC solution was prepared by combining dry lipid powder (0.5 mg/mL from Avanti Polar Lipids, Alabaster, AL) in a 170 mM NaCl solution. The solution was agitated under argon using a bath sonicator (Cole Parmer, Vernon Hills, IL) at 60 °C for multiple periods of up to 1 h until the solution was no longer cloudy. When the solution became transparent, it was considered to contain unilamellar vesicles 30-50 nm in diameter. Approximately 60 µL of the vesicle suspension was deposited on a 3 mm by 3 mm piece of freshly cleaved mica that had been glued to a Teflon disk. After incubation overnight at 4 °C, the specimen was heated to temperatures above the melting temperature (60 °C) for 2 h to allow the vesicles to fuse into the membrane, and then, it was allowed to cool to room temperature. Interdigitated domains were created by replacing the solution covering the membrane (3 times) with a 10% 2-propanol/water solution while taking care to keep the sample hydrated at all times. The sample was again heated at 60 °C for various times up to 2 h and then allowed to cool to room temperature. AFM imaging experiments were conducted using a Nanoscope III atomic force microscope, AFM liquid cell, and either standard or oxide-sharpened SiN cantilevers, all from Digital Instruments (Santa Barbara, CA). The images of gel-phase-supported membrane specimens were obtained at room temperature in unbuffered 0.10 or 0.010 M NaCl solutions using a variety of cantilevers at scan rates of less than 10 lines per second (512 × 512 image (17) Binnig, G.; Quate, C. F.; Gerber, C. H. Phys. Rev. Lett. 1986, 56, 930-933. (18) Hansma, P. K.; Ealings, V. B.; Marti, O.; Bracker, C. E. Science 1988, 242, 209-216. (19) Engel, A. Annu. Rev. Biophys. Biophys. Chem. 1991, 20, 79108. (20) Heinz, W. F.; Hoh, J. H. Trends Biotechnol. 1999, 17, 143-150. (21) Janshoff, A.; Neitzert, M.; Oberdorfer, Y.; Fuchs, H. Angew. Chem., Int. Ed. Engl. 2000, 39, 3212-3237. (22) Mou, J.; Yang, J.; Huang, C.; Shao, Z. Biochemistry 1994, 33, 9981-9985. (23) Bain, A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6159-6163. (24) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105-113.
Figure 2. (a) 1.5 µm by 1.5 µm image of a solid-supported DPPC bilayer revealing normal, interdigitated, and defect regions. The image was captured in the contact mode with a 0.06 N/m cantilever in 100 mM NaCl at room temperature. The image size is 512 × 512 and has been flattened (0 order). (b) Cross-sectional plot along the line in a indicating the three predominant surface heights present for the sample. size). Further details about the imaging conditions and image processing are given in the figure captions.
Results and Discussion Pictured in Figure 2a is a typical image of a DPPC membrane following treatment with 10% 2-propanol/water at 60 °C for 2 h. Visible in the image are light gray regions indicative of normal bilayer regions of the membrane, darker gray regions indicative of interdigitated domains, and black regions indicative of imperfections in the solidsupported membrane (holes). A cross-sectional plot with the relative depth scale of the image is presented in Figure 2b. From this and other similar images, we measure the thickness of the membrane to be 5.8 ( 0.4 nm, corresponding to a single bilayer on the mica surface and a thin layer of water between the bilayer and the membrane. This depth is in good agreement with previously reported AFM measurements for DPPC membranes.15,22,25 The depth of the darker gray regions in the image relative to the normal membrane surface height is 2.0 ( 0.2 nm. These depressed regions correspond to the interdigitated domains in the membrane sample. This reported depth (25) Mou, J.; Yang, J.; Shao, Z. Biochemistry 1994, 33, 4439-4443.
Notes
Figure 3. Two 5 µm by 5 µm images of a solid-supported DPPC bilayer exposed to a 10% 2-propanol solution revealing (a) normal, interdigitated, and defect regions and (b) only normal gel-phase domains and defects. The images were captured in the contact mode with a 0.32 N/m cantilever in 100 mM NaCl at room temperature. The images are 512 × 512 and flattened (0 order).
is the average of 20 different measurements on the membrane shown in Figure 3a and is consistent with the 1.9 ( 0.2 nm depth previously reported from AFM measurements,22 as well as the 1.6 nm decrease in the repeat lamellar periods reported from X-ray diffraction experiments with multilayer DPPC samples.26,27 Pictured in Figure 3 are two 5 µm by 5 µm images of a DPPC membrane sample obtained in sequence (3a before 3b) using a 0.32 N/m cantilever (manufacturer’s suggested value). This sample had been treated with the 10% (26) McIntosh, T. J.; McDaniel, R. V.; Simon, S. A. Biochim. Biophys. Acta 1983, 731, 109-114. (27) Simon, S. A.; McIntosh, T. J. Biochim. Biophys. Acta 1984, 773, 169-172.
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2-propanol/water in the same way as the sample depicted in Figure 1a. Clearly evident in Figure 3a are regions of normal membrane, defect regions (or holes), and large expanses of interdigitated domains (dark gray regions). In Figure 3b, which was obtained immediately after the image in Figure 3a, the dark gray regions of the image indicating interdigitated domains have disappeared. The interdigitated domains have relaxed and been converted into more normal membrane regions, leading, as a consequence of the conservation of mass, to the creation of more defect areas. Careful examination of the two images, considering the presence and location of the characteristic defects common to both, supports the assertion that the images are of the same region in the membrane sample. The main difference is that the image in Figure 3b was taken after that in Figure 3a and while the instrument was drifting in terms of increasing the applied force for the AFM probe. Additional experiments aimed at purposely erasing the interdigitated domains and triggering the relaxation of these domains to the normal gel phase by scanning a sample with increasing amounts of applied force were pursued. An example of these results is depicted in Figure 4a-d. In these imaging experiments, performed using a 0.58 N/m oxide-sharpened cantilever (nominal tip radius of curvature 5-40 nm), the same region of a sample was repeatedly imaged for approximately 45 min while the applied force was gradually increased from 3 to 7 nN. Figure 4a shows the initial scan, which exhibits mainly regions of normal gel-phase membrane and interdigitated domains, with a small amount of defect regions. Following the series 4b through 4d, scans 17, 31, and 47, respectively, the area of interdigitated domains decreases, while the areas of normal membrane and surface defects increase. In Figure 4d, very little of the interdigitated domains remains, and new regions of normal gel-phase membrane are evident. The conversion occurs in every scan. In addition, the experiment was reproducible a number of times with different sections of the same sample. The observation of the LβI f Lβ′ phase transition in DPPC bilayers treated with alcohols was also reported in an earlier AFM study.22 In those experiments, it was reported that, upon removal of ethanol from the buffer solution in which the sample is immersed, the interdigitated domains remained stable at room temperature for a period of days. This suggests that either the agent inducing interdigitization lingers at the membrane/water interface or the movement of the lipids is sufficiently hindered in the gel state. In addition, as in the formation process, Lβ′ f LβI, the reverse process, LβI f Lβ′, is also accelerated by raising the sample temperature above the melting temperature. The evidence of the LβI f Lβ′ phase change was the appearance of more defects in the bilayer sample. Although the erasure of the interdigitated domains is an impediment to our studies of interdigitated lipid domains, it does allow us to measure the relative area occupied by the headgroups in the interdigitated domain compared to that in the normal membrane. Using the AFM’s image-analysis software, we measured the areas of the normal membrane regions, interdigitated regions, and holes in a series of well-defined subregions in Figure 3a, as well as the areas of the membrane and holes in the same subregions in Figure 3b. In each analysis of the image, the sum of the area of new membrane formed plus the area of new membrane defects formed was equal to the area of interdigitated membrane lost to within (5%. Assuming that no lipid is removed from the system and that the edge effects associated with the membrane defects
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Figure 4. Four 3 µm by 3 µm images of a solid-supported DPPC bilayer prepared with exposure to a 10% 2-propanol solution. The images reveal the gradual conversion of interdigitated domains to normal gel-phase membrane and surface defects resulting from the interaction between the tip and the sample: (a) scan 1, (b) scan 17, (c) scan 31, and (d) scan 47. The images were captured over a period of ∼45 min in the contact mode with a 0.58 N/m oxide-sharpened cantilever in 10 mM NaCl at room temperature. The images are 256 × 256 (5.08 Hz scan rate) and flattened (0 order).
are negligible, the relative lipid headgroup area can be determined by calculating the ratio of the interdigitated area in Figure 3a to the new normal membrane area in Figure 3b. Our experiments reveal this ratio to be 1.7 ( 0.2, which corresponds to a ∼30% expansion in the lipidlipid lattice spacing for an interdigitated membrane structure composed of two interpenetrating hexagonal lipid arrays. In gel-phase DPPC membranes, the area per lipid headgroup has been reported as 52 Å2.28 The factor of 1.7 discerned from the images in Figure 3 indicates that the area per DPPC headgroup in the interdigitated domains is 90 ( 10 Å2. Although it is clear that the predominant effect of the formation of the interdigitated phase is related to the decreased membrane thickness, the spacing of the (28) Marra, J.; Israelachvilli, J. Biochemistry 1985, 24, 4608-4618.
polar headgroups on the membrane surface is also important. This increased lateral spacing will affect the surface charge density of the membrane, the extent to which the membrane can bind charged species such as Ca2+ and Mg2+, and perhaps the permeability of the pores contained within the membrane.29 We are currently investigating the electrostatic environment of the interdigitated domains relative to that of the normal domains for DPPC and DPPG bilayers.30 Acknowledgment. The authors thank Professor Bradley Smith of the University of Notre Dame for his help and support of this project. LA010295+ (29) Rasper, D. M.; Merrrill, A. R. Biochemistry 1994, 33, 1298112989. (30) McClain, R. L.; Breen, J. J., manuscript in preparation.
