Langmuir 1990,6, 1017-1023
1017
Monomolecular Film Behavior of Tetraether Lipids from a Thermoacidophilic Archaebacterium at the Air/Water Interface J. L. Dote,**+W. R. Barger,$ F. Behroozi,s E. L. Chang,*J S.-L. LO,^ C. E. Montague," and M. Nagumot Department of Biochemistry, Georgetown University, Washington, DC 20057, Naval Research Laboratory, Washington, DC 20375-5000, Department of Physics, University of Wisconsin-Parkside, Kenosha, Wisconsin 53141-2000, and Geo-Centers, Newton Upper Falls, Massachusetts 02164 Received August 22, 1989 The unusual cyclic bipolar structures of the lipids of thermoacidophilic archaebacteria suggest that they form monolayer membranes. These structures may enhance the ability of these bacteria to live in harsh environments. Monolayer films of conventional lipids have long provided simple models for biomembranes. We have studied the surface properties of monolayer films of two hydrolyzed lipids from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius, as well as several model compounds, at the air/water interface. The archaebacterial lipids studied, glycerol dialkyl nonitol tetraether (GDNT) and glycerol dialkyl glycerol tetraether (GDGT),which are mixtures of closely related molecules, do not form stable monolayer films at temperatures between 16 and 30 "C. The dependence of the surface pressure/area and surface potential/area isotherms on the temperature, ionic strength, and pH of the subphase is consistent with the lipids having only one polar head group at the lipid water interface. There is no evidence to support the existence of a substantial population of "n-shaped" molecules that have both polar head groups at the lipid/water boundary. The hydrocarbon chains dominate the lateral packing of the lipids. However, the sensitivity of GDNT films to subphase pH and ionic strength, compared to the insensitivity of GDGT films, indicates that the nonitol head groups of the GDNT lipids are at the lipid/water interface.
*
Sulfolobus are primarily a,w-bipolar amphiphilic molecules with two nonequivalent polar heads415 (see Figure 1). The head groups are linked in a macrocyclic tetraether structure by two c40 biphytanyl residues. These branched hydrocarbon chains are ether-linked a t both ends either to two glycerols (glycerol dialkyl glycerol tetraether, GDGT) or to one glycerol and one branched nonitol, CsHzoOe (glyceroldialkyl nonitol tetraether, GDNT). A variety of polar groups, mainly carbohydrates and phosphoinositols, are linked to the glycerol and nonitol groups (Figure l).3The c 4 0 hydrocarbon chains can also incorporate, at specific sites, up to four cyclopentane rings per chain5 The molecular structure of these lipids influences the structure and function of the membranes they form. The structures of ordinary membranes are based on a lipid bilayer stabilized by a balance of hydrophilic and hydrophobic interactions. The fluidity of the bilayer is achieved in part through alterations in the hydrocarbon chain length, unsaturation, or methyl branching.6 The introduction of methyl branches hinders close packing of the chains and thereby decreases the temperature of order/ disorder and chain-melting transition^.^^ Membrane permeability increases as the degree of unsaturation along the hydrocarbon chains increaseslO because the double
70.
(4) DeRosa, M.; Gambacorta, A.; Nicolaus, B.; Bu'lock, J. D. Phytochemistry 1980, 19, 821. ( 5 ) DeRosa, M.; Gambacorta, A.; Nicolaus, B.; Chappe, B.; Albrecht, P. Biochim. Biophys. Acta 1983, 753,249. (6) Melchior, D. L. Curr. Topics Membr. Tramp. 1982,17, 263. (7) Menger, F. M.; Wood,M. G., Jr.; Richardson, S.; Zhou, Q.;Elrington, A. R.; Sherrod, M. J. J. Am. Chem. SOC.1988,110,6797. (8) Menger, F. M.; Wood, M. G., Jr.; Zhou, Q. Z.; Hopkins, H. P.; Fumero, J. J. Am. Chem. SOC.1988,110,6804. (9) Silvius,J. R.; McElhaney, R. N. Chem. Phys. Lipids 1980,26,67.
Introduction Archaebacteria, a separate kingdom of microorganisms, thrive in extreme environments:l methanogens in anaerobic conditions, halophiles at high salt concentrations, and thermoacidophiles a t high temperatures and acidic pH (90 "C and pH 2 are not unusual). Their ability to live in these habitats may be due in part to the unusual character of the archaebacterial membrane lipids,2*3which have structural features that are not typically found in eubacteria or eukaryotes. The lipids consist primarily of two classes of compounds, isoprenoid hydrocarbons and alkylglycerol ether derived polar lipids. The ether functionality is more chemically and thermally resistant than the ester linkage ordinarily found in the glycolipids and phospholipids of other bacterial systems. The lipids also have an sn-2,3-glycerol stereoconfiguration whereas an sn-1,2 stereoconfiguration is found in other naturally occurring glycerophosphatides and diacylglycerols. The lipids of the thermoacidophile
* To whom correspondence should be addressed. + Georgetown University. Present address: Patent & Trademark Office, Arlington, VA. Naval Research Laboratory. 8 University of Wisconsin-Parkside. 1 Geo-Centers. Present address: NEN Products Dupont, Boston, MA 02118. 11 Geo-Centers. Present address: Pacific Scientific, Engineering Dept., Silver Spring, MD 20910. (1) Woese, C. R., Wolfe, R. S., Eds. The Bacteria; Academic Press: London, 1985; Vol. 8. (2) Langworthy, T. A. in The Bacteria; Woese, C . R., Wolfe, R. S., Eds.; Academic Press: London, 1985; Vol. 8, p 459. (3) DeRosa, M.; Gambacorta, A.; Gliozzi, A. Microbiol. Reu. 1986,50,
0743-7463/90/2406-lO17$02.50/0
0 1990 American Chemical Society
1018 Langmuir, Vol. 6, No. 5, 1990
Dote et al.
F+o-cn* 7 H C O
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p-D-Glucopyranosylsulphate
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Figure 1. Representative structures of the lipids of S.
acidocaldarius.3 The other molecular species differ in the degree of cyclization of the alkyl chains (0-4 cyclopentane rings/ chain). The hydrolyzed lipids GDGT and GDNT are shown as compounds 1 and 5, respectively. bonds prevent a tight association among the chains. In thermoacidophiles, the fluidity and permeability of the membrane may be modulated by changing the ratio of methyl branches to cyclopentane rings in the constituent lipids. The extent of cyclization in the biphytanyl moieties has been noted to increase when the thermoacidophile Sulfolobusll is grown at increasing temperatures. The introduction of rings is likely to reduce the thickness of the membrane and the rotational freedom of the chains, which would alter the viscosity, compressibility, and density in the membrane interior. Each ring incorporated in the chain shortens the effective carbon length of the chain by approximately 2.5 A. The dimensions and bipolar structure of the tetraether lipids may induce a different type of membrane self-organization,the “amphiphilic lipid monolayer”. This organization may account for thermal stability. Calorimetric studies of hydrated lipids extracted from the thermoacidophile Thermoplasma showed that these lipids do not undergo a thermotropic phase transition between 0 and 80 OC.12 (Thermoplasma lipid structures are similar to those of Sulfolobus GDGT (see Figure 1)where R1 is either a hydrogen or a carbohydrate moiety and R2 is either a carbohydrate moiety or a phosphoryl glycerol, respectively.) The monolayer structure may also provide a more effective barrier against the diffusion of hydrogen ions into the cell.3 Liposomal membranes composed of lipids extracted from Thermoplasma are less (10) Bittman, R.; Blau, L. Biochemistry 1972,11,4831. (11) DeRoea, M.;Esposito, E.; Gambacorta, A.; Nicolaus, B.; Bu’lock, J. D. Phytochemistry 1980, 19, 827. (12) Bllkher, D.; Guttermann, R.; Henkel, B.; Ring, K. Biochim. Biophys. Acta 1984, 778,74.
permeable to low molecular weight hydrophilic solutes than membranes from bilayer-forming 1e~ithins.l~ There are several lines of evidence that the membranes of the thermoacidophiles Thermoplasma and Sulfolobus may occur as lipid monolayers. First, there is no preferential fracture plane at the middle of the lipid layer in freezefracture electron micrographs of T h e r m ~ p l a s m a and l~ Sulfolobus cells.15 Second, glycosidase digestion of the membrane lipids of intact cells of Sulfolobus showed that 82% of the total lipids have a polar head exposed outside the membrane while 92% of the total lipids have glycosidic linkages on a t least one of the polar head groups.16 Finally, results of current-voltage and capacitance experiments on black lipid membranes of the hydrolyzed lipids GDNT of Sulfolobus solfataricus (previously known as Caldariella acidophila) suggested monolayer organization.’7-’9 Understanding how these lipids form membrane systems that withstand harsh environments is of great interest in lipid research. In this work, we examine the surface properties of the hydrolyzed tetraether lipids of the archaebacterium S. acidocaldarius at the airlwater interface. There are several reasons to study the hydrolyzed lipids, which are not major components of archaebacterial membranes. As a practical matter, the native polar lipids have proven to be difficult to isolate and purify in quantities adequate for physical characterization. Hydrolysis of the head groups reduces the number of lipid species present and thus increases the amount of usable material. The simpler constitution of the hydrolyzed head groups allows the role of the branched cyclic hydrocarbon chains to be assessed. Finally, there is a body of work on hydrolyzed lipids extracted from archaebacterial membranes. The lipids used in this study are extracted and purified by procedures developed in our laboratory, which differ from those previously p ~ b l i s h e d . ~The ,~,~~ properties of the hydrolyzed lipids are examined by film balance experiments, which provide direct information on packing properties and limiting molecular areas of membrane lipids in planar layers. The compressibility of the film and the area per molecule at a given pressure reflect molecular interactions and packing. Surface potential measurements yield information on molecular dipole orientation. The effects of methyl branches and cyclopentane rings on chain packing are examined by comparing the properties of monomolecular films of analogous straight-chain and methyl-branched alcohols and lecithins to those of the tetraether lipids. The effects of chain substituents have been studied in other lipid systems as ~ e l l . ~ Rolandi l - ~ ~ et al. have presented film balance studies on several lipids extracted from the archaebacterium (13) Bauer, S.;Heckmann, L.; Six, L.; Strobl, Chr.; Blocher, D.; Henkel, B.; Garde, Th.; Ring, K.Desalination 1983,46, 369. (14) Langworthy, T. A. in Biochemistry of Thermophily; Friedman, S. M., Ed.; Academic Press: New York, 1978; p 11. (15) Weiss, R. L. J. Bacteriol. 1974, 118,275. (16) DeRosa, M.; Gambacorta, A.; Nicolaus, B. J. Membr. Sci. 1983, 16,287. (17) Gliozzi, A.; Paoli, G.; Rolandi, R.; DeRosa, M.; Gambacorta, A. J.Bioelectrochem. Bioenerp. 1982. 9.591. (18) Gliozzi, A.; Rolandi; R.; DeRosa, M.; Gambacorta, A. Biophys. J . 1982, 37,563. (19) Gliozzi, A.; Rolandi, R.; DeRosa, M.; Gambacorta, A. J. Membr. Bid. 1983,75,45. (20) Rolandi, R.;Schindler, H.; DeRosa, M.; Gambacorta, A. Eur. Biophys. J . 1986,14, 19. (21) Suzuki, A.; Cadenhead, D. A. Chem. Phys. Lipids 1985, 37,69. (22) Rice, D. K.;Cadenhead, D. A.; Lewis, R. N. A. H.; McElhaney, R. N.Biochemistry 1987,26,3205. (23) Balthasar, D. M.; Cadenhead, D. A.; Lewis, R. N. A. H.; McElhaney, R. N.Langmuir 1988,4, 180. (24) Asgharian, B.; Rice, D. K.; Cadenhead, D. A.; Lewis, R. N. A. H.; McElhaney, R. N.Langmuir 1989,5, 30.
Monomolecular Film Behavior of Tetraether Lipids
S. solfataricus.20 They found that the hydrolyzed lipids GDGT and GDNT formed unstable films a t the air/ water interface, while the other lipids with complex polar head groups (R1 or Rz # H, cf. Figure 1) formed stable compressible films. Surface films of the bipolar main tetraether phospholipid MPL (MPL is analogous to GDGT, but R1 = p-D-glucoside and R2 = phosphoryl glycerol, cf. Figure 1)of Thermoplasma acidophilum26 were also found to be highly stable. We have reexamined and extended the work of Rolandi et a1.20 on the hydrolyzed tetraethers. We find that the lipids do not form mechanically stable monomolecular films. The films do not maintain any substantial surface pressure: when compression halts the pressure decreases. The instability observed differs from that reported by Rolandi et aLZo Our data show that the hydrolyzed tetraethers GDNT and GDGT pack similarly to each other at high surface pressure; i.e., the head groups do not significantly determine the packing. Furthermore, the simplest consistent model of the lipid monolayer is one in which the lipid molecules are oriented in an upright position with respect to the air/ water interface with only one head group attached to the aqueous subphase. It is not necessary to invoke a substantial population of the molecules configured with both polar head groups attached to the subphase in an n-shaped configuration, as others have proposed,2O to explain the data. Experimental Section The dialkylglycerol tetraether lipids used in this study were extracted from the thermoacidophilic archaebacteria S. acidocaldarius (American Tissue Culture Collection, Strain 33909). The procedures for cell growth, lipid isolation, and purification of the hydrolyzed lipids GDNT and GDGT are described in detail elsewhere.26~27 The lipid isolation and purification procedures were developed in our laboratory and differ from those used by Rolandi et a1.20 The lipids were Soxhlet-extracted from dried cells with a 5% trichloroacetic acid solution in (1:l) chloroform/methanol and then hydrolyzed with methanolic HC1. The hydrolyzed lipids were fractionated by silicic acid column chromatography. The GDGT lipids were purified as benzoate derivatives by thin-layer chromatography (TLC), hydrolyzed, and finally precipitated in acetone. The GDNT lipids were purified directly by TLC without derivatization and then precipitated in acetone. Nearly colorless products were obtained from these preparations. In addition, both lipids were tested for purity by TLC. Both GDGT (lot 127) and GDNT (lots 116 and 125) are mixed lipid systems composed of molecules with varying numbers of cyclopentane rings incorporated in the hydrocarbon backbones. It has been shown by chemical ionization mass spectrometry that the principal degrees of cyclization for GDGT (lot 127)27 are two and four rings per molecule and for GDNT (lot 056)26are one, three, and five rings per molecule. The mass spectrometry was performed on a different lot of GDNT than those used in this study. Phytanol was synthesized by reducing phytol (Aldrich Chemical Co., purity 97%) with Pd over carbon. Phytanol was then purified by HPLC on a Waters p Bondapak CIS column with a methanol/water (1O:l) solvent system. The synthetic L-W diphytanoylphosphatidylcholine(lot PPC-54) and L-a-dipalmitoylphosphatidylcholine (lot (2160-115) were purchased from Avanti polar lipids (purity 99%). The hexadecanol was obtained from Applied Science Laboratories,Inc. (lot 1171, purity 99.9+%). Monolayers of the hydrolyzed lipids and synthetic lecithins were spread by using 0.1-0.2 mL of 0.3-0.6 mg/mL lipid dissolved in analytical reagent grade chloroform onto subphases of triply distilled water (pH 5.5, with the last two distillations (25) Strobl, Chr.; Six, L.; Heckmann,K.; Henkel, B.; Ring,K. Z. Naturforsch. 1985,40c, 219. (26) Lo, S.-L.; Montague, C. E.; Chang, E. L. J.Lipid Res. 1989, 30, 944. (27) Lo, S.-L.; Chang, E. L., manuscript in preparation.
Langmuir, Vol. 6, No. 5, 1990 1019 from an all-quartz still). The alcohols were spread from solutions of analytical reagent grade n-hexane. The hydrolyzed lipid solutions were also deposited onto subphases of aqueous solutions of 0.1 F NaCl (Mallinckrodt, analytical reagent grade) in triply distilled water (pH 5.5) and onto subphases of pH 7.4 buffered solutions that consisted of 0.1 F NaCl and 2 mM Hepes buffer (Sigma Chemical Co., lot 66F-623) prepared in triply distilled water at room temperature, 22-25 "C. The pH was adjusted by titration with an aqueous solution of 0.1 F NaOH (Mallinckrodt, analytical reagent grade). Monolayer films were prepared at the air/water interface on one of two Langmuir trough systems designed and constructed in our laboratory.28 Both troughs were paraffin-coated with one having dimensions of 14 cm X 82.5 cm (system 1)and the other having dimensions of 12 cm X 72.5 cm (system 2). Both had a depth of 3 mm. The surface pressure in both systems was measured by a 1.7-cm-widePt foil Wilhelmy plate suspended from a Gould UC-2 strain gauge interfaced to an Apple 11-e computer, which also controlled the drive motor for the compression bar. The rate of compression was approximately 6.7 X 1015 A2/s for system 1 and 4.0 X 1016 A2/s for system 2. System 1 was used for temperature-controlled experiments. The trough of system 1was thermostated by a circulating water system connected to a refrigerated Fisher-Scientific Model 90 water bath. The temperature at the airfwater interface was monitored with a Yellow Springs instrument Model 46 Tele-thermometer connected to a surface thermocouple probe that was accurate to f0.15 "C. The temperature at the air/water interface was stable to f1.0 "C. Surface potential measurements were conducted on system 2. Since the trough of system 2 was not thermoregulated, the experiments were performed at room temperature, 22-25 "C. The surface potential was measured with an air ionization electrode, a pinpoint 1-pCi z4lAm source centered on a 2.1-cm2 nickel surface, which was suspended 1 cm above the airfwater interface. A AgfAgCl reference electrode was submerged in the subphase. The potential change was read by a Keithley 617 programmable electrometer connected to an Apple 11-ecomputer through an IEEE-488 interface. The potential change and surface pressure were recorded simultaneously as a function of the trough's surface area at intervals of 8.5 s. All data reported are the averages of at least three different experiments. Force-area compression-expansion experiments were performed on monolayers of the GDNT lipid system. The monolayers of the GDNT (lot 116) were spread from solutions of chloroform onto a subphase of laboratory deionized water that was further purified by passing it through a Sybron NanoPure I1 ion exchanger and then through a Millipore filter. The water had a resistivity of 18 Ma-cm. A Teflon trough fitted with a Langmuir balancem.30 was used for these measurements. The films were compressed at a constant rate at speeds that ranged from 2.25 X 1014 to 1.78 X 10'5 Az/s.
Results and Discussion The films of both hydrolyzed lipids (GDNT and GDGT) could not maintain any substantial surface pressure; i.e., the pressure dropped rapidly when compression was halted under all conditions studied. A series of compressiondecompression isotherms for the hydrolyzed lipids GDNT on aqueous subphases was obtained with a barrier speed of 2.25 X 1014A2/s. The film was first compressed to 25 mN/m and then decompressed to zero surface pressure and recompressed. In each subsequent cycle, the area per molecule at zero surface pressure, determined by linear extrapolation of the pressurelarea isotherm to zero pressure, was smaller than the previous one. After four cycles, the area at zero pressure was 50% less than that of the first cycle. The isotherms were similar in shape. (28) Wohltjen, H.; Barger, W. R.; Snow,A. W.; Jarvis, N. L. IEEE Trans. Electron Deuices 1985,32, 1170. (29) Abraham, B. M.; Miyano, K.; Buzard, K.; Ketterson, J. B. Rev. Sci. Instrum. 1980,51,1083. (30) Abraham, B. M.; Behroozi, F. J.Colloid Interface Sci. 1989,127, 346.
1020 Langmuir, Vol. 6, No. 5, 1990
Dote et al. Table I. Ao,Azo, K , and A V for Lipid Monolayers (See Figure 2) and the Lipid Mixtures GDGT and GDNT at the Air/Water Interface' Ao, Az/ Azo, AZ/ K X IOs, AV,,,
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molecule m /mN mV 18.8 f 0.4 7.70 f 0.78 386 f 8 50.1 f 1.0 36.2 f 1.6 16.5 f 1.3 142 f 2 89.4 f 2.0 44.8 f 1.5 29.0 f 2.0 534 f 9 104.2 f 2.8 76.3 f 1.9 15.6 f 1.2 419 f 3 154.1f 5.9 130.2 f 3.8 8.74 f 1.11 264 f 4 154.4 f 3.0 128.8 f 1.6 8.48 f 0.73 252 f 2 a The data reported are the averaged values for at least three separate experiments. The values for A0 and K are determined from eq 1. The area per molecule at a surface pressure of 20 mN/m, Azo, and AV are directly measured experimental values.
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