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J . Phys. Chem. 1986, 90, 3198-3202
Long-Range Molecular Orientational Order in Monolayer Solid Domains of Phospholipid V. T. Moy, D. J. Keller, H. E. Gaub, and H. M. McConnell* Stauffer Laboratory f o r Physical Chemistry, Stanford University, Stanford, California 94305 (Received: January 13, 1986)
We report evidence for long-range molecular orientational order in monolayer solid phase domains of dipalmitoylphosphatidylcholine (DPPC) at the air-water interface. Monolayers of DPPC in the solid-fluid coexistence region of the pressure-area diagram were studied by using fluorescence excitaticn. A low concentration of a fluorescent lipid probe that dissolves in both the solid and fluid domains was included in the monolayer. When fluorescence is excited by a laser beam incident on a monolayer at glancing angles to the surface, the fluorescence emission depends on the orientation of the domains and the direction of the electric vector of illuminating light. The observed anisotropy of the fluorescence emission can be interpreted in terms of long-range molecular orientational order in the solid domains, an order that is doubtless associated with the tilt of the hydrocarbon chains. In monolayers composed of a single enantiomer of DPPC (R-DPPC), the solid domains have spiral, chiral shapes, and the molecular orientational order has a curvature that parallels the arms of these spirals.
Introduction Phospholipid monolayers at the air-water interface have been the topic of intense study in recent years. This interest stems in part from their two-dimensional properties, especially phase transitions of two-dimensional systems. Lipid monolayers are also relevant to models of biological membranes.' It has long been known from thermodynamic and spectroscopic data that when a lipid monolayer is compressed isothermally it undergoes a transition first from a low-density phase to a condensed "fluid" phase, and then, at higher surface pressures, to a still denser "solid" phase.* Recently, it has become possible to observe the formation of solid domains on compression of the fluid phase in phospholipid monolayers doped with trace amounts of a fluorescent p r ~ b e . ~In - ~these experiments the probe molecules are excluded from the solid regions of the monolayer with the result that the solid domains appear as dark patches in a bright fluid background. The solid domains are solid in the sense that lateral diffusion within these domains is low, characteristic of the gel or crystalline phases of phospholipid bilayer^.^ Even more recently, Weis and McConnell found that introducing cholesterol into monolayers of dipalmitoylphosphatidylcholine (DPPC) has a dramatic influence on the shape of the solid domains.6 In the presence of 1-2 mol % cholesterol, long chiral, spiral striplike domains form, unlike the relatively round, but still chiral shapes obtained in the absence of cholesterol. The widths of these striplike domains appear to be understandable in terms of equilibrium thermodynamics, in that they are remarkably uniform in magnitude and change reversibly with either concentration of cholesterol or surface pressure.' It has been pointed out previously that the chiral shapes of the solid-phase monomolecular domains imply some type of long-range molecular order.* Electron diffraction of solid monomolecular domains of phosphatidic acid (transferred to grids) shows evidence of positional ordering over long distances, but these crystals do not show the chiral shapes of interest to u s 9 Since the transfer (1) McConnell, H. M.; Watts, T.H.; Weis, R. M.: Brian, A . A . B B A Biomembr. Rev., submitted for publication. (2) Albrecht, 0.; Gruler, H.; Sackmann, E. J . Phys. (Paris) 1978, 39, 301-313. (3) Liische, M.; Sackmann, E.: Mohwald, H. Ber. Bunrenges. Phys. Ckem. 1983,87, 848-852. (4) Peters, R.: Beck, K. Proc. Narl. Acad. Sci. U.S.A. 1983. 80, 7. 1 83-7 1 87. .. . . ( 5 ) McConnell, H. M.; Tamm. L. K.; Weis, R. M. Proc. Nurl. Acad. Sci. U.S.A. 1984, 81, 3249-3253. ( 6 ) Weis, R. M.; McConnell, H. M. J . Phys. Chem. 1985,89,4453-4459. (7) McConnell, H. M.: Keller, D. J.: Gaub, H. E. J . Phvs. Chem. 1986, 90, 1717. (8) Weis, R. M.; McConnell, H. M. Nature (London) 1984, 310,47-49. (9) Fisher, A.; Losche, M.; Mohwald. H.: Sackmann. E. J . P k p . Letr. 1984, 45, L785-L791.
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of lipid monolayers from the air-water interface to solid substrates can change their properties, one must be cautious in relating the diffraction data to the present study. Here we report direct evidence for the existence of long-range molecular orientational order in the solid domains at the air-water interface. We have observed that when a DPPC monolayer is doped with a fluorescent probe that partitions partly into the solid and is illuminated at glancing angles some regions within the solid show greater fluorescence emission than others. From the results of our experiments we postulate that the chains of the lipid molecules are tilted with respect to the plane of the monolayer, and that this tilt order persists over distances of the order of at least 100 pm. The direction of this tilt order has a curvature in chiral, spiral solid domains. The curvature of the tilt order appears to parallel the curvature of the spiral arm of the chiral solid domains.
Materials and Methods (2R),3-Dipalmitoylglycero-1-phosphocholine (R-DPPC) and (2R),3-Dipalmitoylglycero- 1-phospho-N-(7-nitr0-2,1,3-benzoxadiazol-4-y1)ethanolamine(NBD-DPPE, head labeled) were obtained from Avanti Polar Lipids, Inc. (Birmingham, AL) and were used without further purification. Racemic DPPC from Sigma (St. Louis) was recrystallized in a 9: 1 hexane:ethanol mixture. Stock solutions of DPPC were diluted to a 1.5 pM concentration with the spreading solvent 9: 1 hexane:ethanol prior to use. Typically, the fluorescent probe concentration was 2 mol %. The fluorescent probe, NBD-DPPE (head labeled), is known to partition partially into the solid and thus permits observation of internal structures within the solid domaims Monolayers were formed over distilled and deionized Millipore filtered water at a room temperature of approximately 20 OC. A modified version of the fluorescence microscopy system described in Gaub et al. was used for our experiments.'O A Teflon Langmuir trough, mounted on a motorized stage, permitted observation of the entire monolayer. The monolayer compression rate was controlled by a motorized linear-actuator-driven barrier. In our experiments the relative compression rate was typically 1.8 X 10-3/s. At full expansion, the trough surface measured 25 mm X 80 mm. To minimize convection, the depth of the trough was made shallow and a thin piece of glass was positioned above the air-water interface. For total internal reflection (TIR) illumination, a glass trough was used. The illuminating laser beam passed through the side of the trough and reflected off the water side of the air-water interface. In all experiments, illumination of the monolayer was from the 488-nm line of a Spectra Physics argon laser or the mercury arc lamp of the microscope. Polarizers ( I O ) Gaub, H. E.: Moy, V . T.: McConnell, H . M. J . Phys. Ckem. 1986, 90. 1721
0 1986 American Chemical Society
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