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Bipolar tetraether lipid monolayers: correlation between interfacial properties and superficial structures as observed by electron microscopy
Chemistry and Physics of Lipids, 53 (1990) 341--346 Elsevier Scientific Publishers Ireland Ltd.
341
Bipolar tetraether lipid monolayers: correlation between interfacial properties and superficial structures as observed by electron microscopy A n n e t t e Gulik ~, Pierre T c h o r e l o f f ° and Jacques-Emile Proust c ~Centre de G~n~tique Mol~culaire, CNRS, avenue de la terrasse, 91190-G6r sur Yvette, ~Laboratoire de physicochimie des surfaces, UA 1218 CNRS, Facult~ de pharmacie, rue J-B Clement, 92296- Chatenay-Malabry and "UFR Sciences m~dicales et pharmaceutiques, Universit~ d'Angers, 16 bd Davier, 49000 Angers (France) (Received July 27th, 1989, revision received and accepted October 1lth, 1989) The purpose of the present study was to investigate by surface pressure measurements and electron microscopy, GDNT (glycerol dialkyl nonitol tetraether) lipid layers formed at an air-water interface. GDNT has an unusual chemical structure, it is a bipolar tetraether lipid derived from an extreme thermoacidophile archaebacterium. The results show that the molecules could adopt different orientations at the air-water interface, depending on the compression conditions; the upright orientation is metastable.
Keywords: monolayer; bipolar lipid; surface pressure; electron microscopy.
Introduction
One of the particular features of the archaebacteria is the chemical structure of their lipids which are sharply different from those of eukaryotes and eubacteria. Archaebacteria are comprised of three main classes: halophiles, methanogenes and thermoacidophiles. In all of them the hydrocarbon moiety of the lipids consists of isoprenoid chains (possibly containing some cyclopentanes) which are ether- (not ester-) linked to a glycerol (2,3-di-O-sn-glycerol, not 1,2-sn) [1--2]. In some extreme thermoacidophiles the lipids consist mainly of two C4o hydrocarbon chains ether-linked at both ends; this tetraether structure confers upon these lipids unique properties. Their chemical structures have been well established (3) and their physico-chemical properties have been investigated by several methods including X-ray diffraction, black-films study, fluorescence and surface pressure measurements. In the present work we report characteristics of films formed at the air-water interface of a hydrolytic fraction
from an extreme thermoacidophile archaebacteria, as assessed by measurements of pressuresurface isotherms and electron microscopy. Materials and methods
GDNT, whose chemical structure is shown in Fig. 1, is a hydrolytic fraction of the lipids extracted from a thermoacidophilic archaebacteria, Sulfolobus solfataricus. This lipid consists of a hydrophobic portion of two C40 residues chemically linked at each end to a hydrophilic group [3]. The surface pressures, I"1,were measured using a Pockels-Langmuir surface balance. In a typical experiment several/al of a chloroformic solution were spread with a microsyringe on the surface of a 2-1 tridistilled water bath kept at 22 °C. The pressure-area curves were automatically recorded, during compression, at a rate of 0.005 or 0.1 cm/sec the film area, A, varied from 392 cm 2 (18.6/21.0 cm) to 47 cm 2 (18.6/2.5 cm). The compressibility of the film is defined as (1/ A)(dA/dn/) m/mN. When collecting the lip-
0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
342 lnr mN/m
HO~-o-C4oH72.ao-O-]
I /
I
0
b
a
30
L-OC4oH72-80"O~_oH
I
I
I
20
OH OH OH OH OH OH 10
Fig, 1. Chemical structure of GDNT. --C4oH~2_80- represent the biphitanyl chains including 0 to 3 cyclopentanes (see Ref. 3),
0' Tt mN/rn
150 20~' 50
100 '~' 50
100
150 2
idic film a freshly cleaved mica piece (2 X 3 cm) was plunged into the water bath before lipid spreading. Subsequently the mica was withdrawn cautiously while keeping the desired pressure constant, during the first or second compression cycle. The mica was then mounted on the stage of a Balzers BAF 301 freeze-etch unit (used here only for shadowing). Unidirectional shadowing was performed at room temperature using platinumcarbon at an angle of 10 ° . The replicas were washed and then observed in a Philips 301 electron microscope. The step thickness was determined from the thickness measured on the images multiplied by tang 10 °.
200 C
3C 2C 1C I
100
200
300
400
A (c m 21
Fig. 2. Pressure-area isotherm curves. Compression rate: 0.005 cm/s (o); 0.1 cm/s (a, b, c). Various amounts of a 6-mg/ml lipid chloroformic solution were spread on the water surface (392 cm2): 2 gl (a), 5 lal (o,b), 20 tal (c) corresponding respectively to =0.5 • 10'a, 1.3 • 10~ and 5.1 • 10~6molecules. Stars (*) indicate the points at which mica sheets were withdrawn for electron microscopy (see Fig. 4): *1 and *2 during the first compression cycle; *3 and *4 during the second cycle. Signs < and << refer to the first and second cycles.
Results
Surface-pressure isotherms The surface-pressure isotherms are shown in Fig. 2. They depend on the amount of lipids spread on the surface. During multiple cycles of compression-decompression some hysteresis was noted which suggests significant changes in the lipidic film. However, in all the cases a plateau was reached around 31 mN/m. During compression the increase in pressure from 0 to =18 m N / m was always very steep and corresponded to a compressibility of 6 • 10`3 m/mN (Fig. 3). A compression isotherm is shown in Fig. 2o: the experiment was carried out under quasi static conditions at a rate of 5 • 10-3 cm/sec. The increase of the pressure is steep, followed by a long plateau. The surface available per molecule was determined to be about 140 ,~2 at n = 0 and 120A 2 at the collapse pressure. These values
are consistent with molecules lying on the surface, possibly forming an arch with the two polar head groups at the air-water interface. This result is in good agreement with those already obtained [4]. Most of the following experiments were carried out at greater rate of compression with various amounts of lipids spread at the air-water interface. The first compression cycle was similar to that obtained in quasi-static conditions. Case a: with a short length of collapse plateau (corresponding to small quantities of lipid) the successive compression-decompression cycles were almost identical. Case b: with a longer collapse plateau (with increasing quantities of lipid), the second compression curve was quite different, the available area per molecule decreased to 100 A 2 or less (Fig. 2b), thus suggesting either a
343 rearrangement o f the lipid in the film or a superposition of several layers of lipids. The increase in pressure remained very sharp up to a certain value o f I'l (16--20 m N / m , depending on the amount of lipid), and then became smoother, finally reaching a plateau. The hypothesis of a loss of lipid into the bulk phase could be eliminated: if the surface was decompressed for about one hour the inital compression curve was restored. This means that the difference in the two compression cycles was caused not by a loss of lipid but by some remnant change (having occurred during the first collapse) such as superposition of lipids a n d / o r other orientations of the molecules at the interface requiring a smaller molecular area. Case c: when a larger amount of lipid was spread on the water surface so that the collapse pressure was reached without any compression, a large plateau was also observed around 31 m N / m. On further compression the pressure increased up to 35 m N / m or more (depending on the amount of lipid); on decompression the pressure fell to 0 and the second cycle gave results similar to case b but with a much more pronounced change in the increase of pressure around 16--20 m N / m (Fig. 2c). Here again when the surface was decompressed for one hour the surface pressure returned to the initial collapse pressure. This observation also excludes a loss of lipid into the bulk phase and suggests some change in the lipidic film. Since the pressure decreased to zero on decompression, islands of lipids separated with gaseous regions a n d / o r complete rearrangements of the molecules in the film occurred. The surface available per molecule decreased to 50/~ 2 or much less when I-I = 0 just when the pressure begins to increase during the second compression cycle. The compressibility was examined under different conditions. Under quasi-static conditions the compressibility was constant up to the collapse pressure (Fig. 3). This means that no rearrangements occurred under these conditions and that the surface available per molecule corresponds to the molecular surface. Alternatively, the compressibility remained at the same constant value during the first compression cycle
Cxl03 rn/mN 60
f
50
/
,40 30' 20
/
I0
1 0
!
/'/ 1=0
2O
I
30
lnr mN/m
Fig. 3. Compressibility curves, l: First compression; 2,3,4: second compression (with increasing quantities of lipid, from 30 to 120/ag).
up to the collapse pressure, thus indicating that the lipid remained in the same conformation. When the first collapse pressure plateau was long the second compression curve became different: the compressibility remained constant at the same value and from 16--20 m N / m it rapidly increased to an average value in the range of 55 • 10-3 m / r a N (Fig. 3). This increase in the compressibility may be explained by a change o f the lipidic film at the interface, the molecule being forced towards an upright position with variable tilt; this state would be metastable since after a sufficient time the first state could be restored.
Electron microscopy Lipid sampling was performed under different pressure conditions and with various amounts of lipid. Withdrawals of the mica are indicated by stars in Fig. 2. For a small quantity of lipids (corresponding to case a or b), no detectable step (which might correspond to a step between two lipid layers) could be observed at zero pressure: this is in agreement w i t h the presence of lipids in a gaseous state. At the collapse pressure at 31 m N / m
344 the images showed large areas with a well defined step of about 15 ,~ (Fig. 4, "1). With a larger quantity of lipid (corresponding to case c), at the collapse pressure (with only a very slight first compression if any), steps of about 15 /~, were observed (Fig. 4, *2). During the second compression steps of 35 /~ height were observed only at a pressure greater than 24
m N / m (Fig. 4, *4). At the end of the second decompression, at I] = 0, patches of 35 /~ height were still observed and often two small steps could be observed on their edge (Fig. 4,*3, see arrows). A thickness of 15/~ may correspond to molecules lying on the water interface in a horizontal position, possibly with molecules forming an
345 upside down U arch. A value o f 35 A is consistent with molecules standing in an upright position at the air-water interface. A change in the orientation of the molecules could be induced after a long collapse plateau; this state was very likely metastable and the initial state of molecules was partially restored upon decompression, as observed on the edges of the patches in Fig. 4 (*3, see arrows). Discussion and conclusion In a previous study o f monolayers o f ether lipids [4,5] it was suggested, on the basis of
pressure-area isotherms, that G D N T adopted a more or less horizontal position, with both polar groups at the air-water interface. Ellipsometric measurements gave an average thickness of the lipid layer of about 10 A . In the present study we were able to show a dynamic effect of compression on the behaviour of G D N T molecules and to visualize the lipidic film under different conditions. Both the pressure-area isotherm study and the electron microscopy observations are in agreement and lead to the same results: the molecules of G D N T are able to adopt different orientations at the air-water interface.
Fig. 4. Electron microscope images. Stars and numbers refer to the points at which the mica was withdrawn as indicated in Fig. 2. For *3, see arrows : two steps were observed.
346
Furthermore, this interpretation is confirmed by the chemical and physical features of GDNT. When the molecules are in a horizontal position, possibly in an arch-shape with the two polar groups in contact with water, both the molecular surface (=140 /~,2) as determined by surfacepressure isotherm and the thickness (=15 /~) as determined by electron microscopy give a good approximation of the molecular volume [6]. When the thickness, as determined by electron microscopy, increases to a value =35 /~, this is consistent with molecules standing in the upright position, possibly with some tilt to the normal of the water surface. In such a situation the surface per molecule would exceed 50 /~ 2 (depending on the angle of tilt), a value larger than commonly observed in lipids because of the unusual hydrocarbon composition [6]. Such a value is in the range of values found at the beginning of the increase of pressure during the second compression cycle (Fig. 2b,c). X-ray diffraction analysis of the GDNT-water mixture has already shown behavioural differences between the two polar groups of GDNT. Depending upon the conditions (temperature, water content) the smaller head group (unsubstituted glycerol) may or may not be at the polarapolar interface and may even tend to mix within the hydrocarbon matrix [6]. Thus, it is not surprising that the two polar groups of GDNT may also behave in different ways at the air-water interface, depending upon the compression conditions. Further, tetraether lipids with substituted glycerols at each extremity are found in an upright position at the air-water interface [5,7]. This result is consistent with the fact that when such a lipid is mixed with 'water it
forms a lamellar phase with each kind of polar group seggregated on opposite sides of the lamellae as long as the water content is low [8]. Thus, GDNT (and possibly other tetraethers with one substituted- and one unsubstituted-OH glycerol headgroup) might be quite exceptional examples of lipids able to adopt various conformations in films formed at the air-water interface.
Acknowledgements We are mostly indebted to Pr. M. DeRosa (Istituto di Biochimica delle Macromolecole, University of Naples, Italy) and to Pr. A. Gambacorta (Istituto di Chimica di Molecole di Interesse Biologico, CNR, Arco Felice, Italy) who provided us with the lipid sample prepared from a strain grown in their laboratory.
References 1 2
3 4 5 6 7 8
T.A. Langworthy and J.L. Pond (1986) System. Appl. Mirobiol. 7, 262--265. V. Luzzati, A. Gulik, M. DeRosa and A. Gambacorta (1987) in: Membrane Proteins : structures, functions assembly. Nobel Symposium, Chemica Scrypta 27B, pp. 211--219. M. DeRosa and A. Gambacorta (1986) System. Appl. Microbiol. 7,278--285. G. Gabrielli, A. Gliozzi, A. Sanguinetti and A. D'Agata (1989) Colloids. Surf. 35,261--273. R. Rolandi, H. Schindler, M. DeRosa and A. Gambacorta (1986) Eur. Biophys. J. 140, 19--27. A, Gulik, V. Luzzati, M. DeRosa and A. Gambacorta (1985) J. Mol. Biol. 182, 131--149. S. Strobl, L. Six and K. Heckmann (1985) Z. Naturforsch. 40c, 219--222. A. Gulik, V. Luzzati, M. DeRosa and A. Gambacorta (1988) J. Mol. Biol. 201,429--435.
341
Bipolar tetraether lipid monolayers: correlation between interfacial properties and superficial structures as observed by electron microscopy A n n e t t e Gulik ~, Pierre T c h o r e l o f f ° and Jacques-Emile Proust c ~Centre de G~n~tique Mol~culaire, CNRS, avenue de la terrasse, 91190-G6r sur Yvette, ~Laboratoire de physicochimie des surfaces, UA 1218 CNRS, Facult~ de pharmacie, rue J-B Clement, 92296- Chatenay-Malabry and "UFR Sciences m~dicales et pharmaceutiques, Universit~ d'Angers, 16 bd Davier, 49000 Angers (France) (Received July 27th, 1989, revision received and accepted October 1lth, 1989) The purpose of the present study was to investigate by surface pressure measurements and electron microscopy, GDNT (glycerol dialkyl nonitol tetraether) lipid layers formed at an air-water interface. GDNT has an unusual chemical structure, it is a bipolar tetraether lipid derived from an extreme thermoacidophile archaebacterium. The results show that the molecules could adopt different orientations at the air-water interface, depending on the compression conditions; the upright orientation is metastable.
Keywords: monolayer; bipolar lipid; surface pressure; electron microscopy.
Introduction
One of the particular features of the archaebacteria is the chemical structure of their lipids which are sharply different from those of eukaryotes and eubacteria. Archaebacteria are comprised of three main classes: halophiles, methanogenes and thermoacidophiles. In all of them the hydrocarbon moiety of the lipids consists of isoprenoid chains (possibly containing some cyclopentanes) which are ether- (not ester-) linked to a glycerol (2,3-di-O-sn-glycerol, not 1,2-sn) [1--2]. In some extreme thermoacidophiles the lipids consist mainly of two C4o hydrocarbon chains ether-linked at both ends; this tetraether structure confers upon these lipids unique properties. Their chemical structures have been well established (3) and their physico-chemical properties have been investigated by several methods including X-ray diffraction, black-films study, fluorescence and surface pressure measurements. In the present work we report characteristics of films formed at the air-water interface of a hydrolytic fraction
from an extreme thermoacidophile archaebacteria, as assessed by measurements of pressuresurface isotherms and electron microscopy. Materials and methods
GDNT, whose chemical structure is shown in Fig. 1, is a hydrolytic fraction of the lipids extracted from a thermoacidophilic archaebacteria, Sulfolobus solfataricus. This lipid consists of a hydrophobic portion of two C40 residues chemically linked at each end to a hydrophilic group [3]. The surface pressures, I"1,were measured using a Pockels-Langmuir surface balance. In a typical experiment several/al of a chloroformic solution were spread with a microsyringe on the surface of a 2-1 tridistilled water bath kept at 22 °C. The pressure-area curves were automatically recorded, during compression, at a rate of 0.005 or 0.1 cm/sec the film area, A, varied from 392 cm 2 (18.6/21.0 cm) to 47 cm 2 (18.6/2.5 cm). The compressibility of the film is defined as (1/ A)(dA/dn/) m/mN. When collecting the lip-
0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
342 lnr mN/m
HO~-o-C4oH72.ao-O-]
I /
I
0
b
a
30
L-OC4oH72-80"O~_oH
I
I
I
20
OH OH OH OH OH OH 10
Fig, 1. Chemical structure of GDNT. --C4oH~2_80- represent the biphitanyl chains including 0 to 3 cyclopentanes (see Ref. 3),
0' Tt mN/rn
150 20~' 50
100 '~' 50
100
150 2
idic film a freshly cleaved mica piece (2 X 3 cm) was plunged into the water bath before lipid spreading. Subsequently the mica was withdrawn cautiously while keeping the desired pressure constant, during the first or second compression cycle. The mica was then mounted on the stage of a Balzers BAF 301 freeze-etch unit (used here only for shadowing). Unidirectional shadowing was performed at room temperature using platinumcarbon at an angle of 10 ° . The replicas were washed and then observed in a Philips 301 electron microscope. The step thickness was determined from the thickness measured on the images multiplied by tang 10 °.
200 C
3C 2C 1C I
100
200
300
400
A (c m 21
Fig. 2. Pressure-area isotherm curves. Compression rate: 0.005 cm/s (o); 0.1 cm/s (a, b, c). Various amounts of a 6-mg/ml lipid chloroformic solution were spread on the water surface (392 cm2): 2 gl (a), 5 lal (o,b), 20 tal (c) corresponding respectively to =0.5 • 10'a, 1.3 • 10~ and 5.1 • 10~6molecules. Stars (*) indicate the points at which mica sheets were withdrawn for electron microscopy (see Fig. 4): *1 and *2 during the first compression cycle; *3 and *4 during the second cycle. Signs < and << refer to the first and second cycles.
Results
Surface-pressure isotherms The surface-pressure isotherms are shown in Fig. 2. They depend on the amount of lipids spread on the surface. During multiple cycles of compression-decompression some hysteresis was noted which suggests significant changes in the lipidic film. However, in all the cases a plateau was reached around 31 mN/m. During compression the increase in pressure from 0 to =18 m N / m was always very steep and corresponded to a compressibility of 6 • 10`3 m/mN (Fig. 3). A compression isotherm is shown in Fig. 2o: the experiment was carried out under quasi static conditions at a rate of 5 • 10-3 cm/sec. The increase of the pressure is steep, followed by a long plateau. The surface available per molecule was determined to be about 140 ,~2 at n = 0 and 120A 2 at the collapse pressure. These values
are consistent with molecules lying on the surface, possibly forming an arch with the two polar head groups at the air-water interface. This result is in good agreement with those already obtained [4]. Most of the following experiments were carried out at greater rate of compression with various amounts of lipids spread at the air-water interface. The first compression cycle was similar to that obtained in quasi-static conditions. Case a: with a short length of collapse plateau (corresponding to small quantities of lipid) the successive compression-decompression cycles were almost identical. Case b: with a longer collapse plateau (with increasing quantities of lipid), the second compression curve was quite different, the available area per molecule decreased to 100 A 2 or less (Fig. 2b), thus suggesting either a
343 rearrangement o f the lipid in the film or a superposition of several layers of lipids. The increase in pressure remained very sharp up to a certain value o f I'l (16--20 m N / m , depending on the amount of lipid), and then became smoother, finally reaching a plateau. The hypothesis of a loss of lipid into the bulk phase could be eliminated: if the surface was decompressed for about one hour the inital compression curve was restored. This means that the difference in the two compression cycles was caused not by a loss of lipid but by some remnant change (having occurred during the first collapse) such as superposition of lipids a n d / o r other orientations of the molecules at the interface requiring a smaller molecular area. Case c: when a larger amount of lipid was spread on the water surface so that the collapse pressure was reached without any compression, a large plateau was also observed around 31 m N / m. On further compression the pressure increased up to 35 m N / m or more (depending on the amount of lipid); on decompression the pressure fell to 0 and the second cycle gave results similar to case b but with a much more pronounced change in the increase of pressure around 16--20 m N / m (Fig. 2c). Here again when the surface was decompressed for one hour the surface pressure returned to the initial collapse pressure. This observation also excludes a loss of lipid into the bulk phase and suggests some change in the lipidic film. Since the pressure decreased to zero on decompression, islands of lipids separated with gaseous regions a n d / o r complete rearrangements of the molecules in the film occurred. The surface available per molecule decreased to 50/~ 2 or much less when I-I = 0 just when the pressure begins to increase during the second compression cycle. The compressibility was examined under different conditions. Under quasi-static conditions the compressibility was constant up to the collapse pressure (Fig. 3). This means that no rearrangements occurred under these conditions and that the surface available per molecule corresponds to the molecular surface. Alternatively, the compressibility remained at the same constant value during the first compression cycle
Cxl03 rn/mN 60
f
50
/
,40 30' 20
/
I0
1 0
!
/'/ 1=0
2O
I
30
lnr mN/m
Fig. 3. Compressibility curves, l: First compression; 2,3,4: second compression (with increasing quantities of lipid, from 30 to 120/ag).
up to the collapse pressure, thus indicating that the lipid remained in the same conformation. When the first collapse pressure plateau was long the second compression curve became different: the compressibility remained constant at the same value and from 16--20 m N / m it rapidly increased to an average value in the range of 55 • 10-3 m / r a N (Fig. 3). This increase in the compressibility may be explained by a change o f the lipidic film at the interface, the molecule being forced towards an upright position with variable tilt; this state would be metastable since after a sufficient time the first state could be restored.
Electron microscopy Lipid sampling was performed under different pressure conditions and with various amounts of lipid. Withdrawals of the mica are indicated by stars in Fig. 2. For a small quantity of lipids (corresponding to case a or b), no detectable step (which might correspond to a step between two lipid layers) could be observed at zero pressure: this is in agreement w i t h the presence of lipids in a gaseous state. At the collapse pressure at 31 m N / m
344 the images showed large areas with a well defined step of about 15 ,~ (Fig. 4, "1). With a larger quantity of lipid (corresponding to case c), at the collapse pressure (with only a very slight first compression if any), steps of about 15 /~, were observed (Fig. 4, *2). During the second compression steps of 35 /~ height were observed only at a pressure greater than 24
m N / m (Fig. 4, *4). At the end of the second decompression, at I] = 0, patches of 35 /~ height were still observed and often two small steps could be observed on their edge (Fig. 4,*3, see arrows). A thickness of 15/~ may correspond to molecules lying on the water interface in a horizontal position, possibly with molecules forming an
345 upside down U arch. A value o f 35 A is consistent with molecules standing in an upright position at the air-water interface. A change in the orientation of the molecules could be induced after a long collapse plateau; this state was very likely metastable and the initial state of molecules was partially restored upon decompression, as observed on the edges of the patches in Fig. 4 (*3, see arrows). Discussion and conclusion In a previous study o f monolayers o f ether lipids [4,5] it was suggested, on the basis of
pressure-area isotherms, that G D N T adopted a more or less horizontal position, with both polar groups at the air-water interface. Ellipsometric measurements gave an average thickness of the lipid layer of about 10 A . In the present study we were able to show a dynamic effect of compression on the behaviour of G D N T molecules and to visualize the lipidic film under different conditions. Both the pressure-area isotherm study and the electron microscopy observations are in agreement and lead to the same results: the molecules of G D N T are able to adopt different orientations at the air-water interface.
Fig. 4. Electron microscope images. Stars and numbers refer to the points at which the mica was withdrawn as indicated in Fig. 2. For *3, see arrows : two steps were observed.
346
Furthermore, this interpretation is confirmed by the chemical and physical features of GDNT. When the molecules are in a horizontal position, possibly in an arch-shape with the two polar groups in contact with water, both the molecular surface (=140 /~,2) as determined by surfacepressure isotherm and the thickness (=15 /~) as determined by electron microscopy give a good approximation of the molecular volume [6]. When the thickness, as determined by electron microscopy, increases to a value =35 /~, this is consistent with molecules standing in the upright position, possibly with some tilt to the normal of the water surface. In such a situation the surface per molecule would exceed 50 /~ 2 (depending on the angle of tilt), a value larger than commonly observed in lipids because of the unusual hydrocarbon composition [6]. Such a value is in the range of values found at the beginning of the increase of pressure during the second compression cycle (Fig. 2b,c). X-ray diffraction analysis of the GDNT-water mixture has already shown behavioural differences between the two polar groups of GDNT. Depending upon the conditions (temperature, water content) the smaller head group (unsubstituted glycerol) may or may not be at the polarapolar interface and may even tend to mix within the hydrocarbon matrix [6]. Thus, it is not surprising that the two polar groups of GDNT may also behave in different ways at the air-water interface, depending upon the compression conditions. Further, tetraether lipids with substituted glycerols at each extremity are found in an upright position at the air-water interface [5,7]. This result is consistent with the fact that when such a lipid is mixed with 'water it
forms a lamellar phase with each kind of polar group seggregated on opposite sides of the lamellae as long as the water content is low [8]. Thus, GDNT (and possibly other tetraethers with one substituted- and one unsubstituted-OH glycerol headgroup) might be quite exceptional examples of lipids able to adopt various conformations in films formed at the air-water interface.
Acknowledgements We are mostly indebted to Pr. M. DeRosa (Istituto di Biochimica delle Macromolecole, University of Naples, Italy) and to Pr. A. Gambacorta (Istituto di Chimica di Molecole di Interesse Biologico, CNR, Arco Felice, Italy) who provided us with the lipid sample prepared from a strain grown in their laboratory.
References 1 2
3 4 5 6 7 8
T.A. Langworthy and J.L. Pond (1986) System. Appl. Mirobiol. 7, 262--265. V. Luzzati, A. Gulik, M. DeRosa and A. Gambacorta (1987) in: Membrane Proteins : structures, functions assembly. Nobel Symposium, Chemica Scrypta 27B, pp. 211--219. M. DeRosa and A. Gambacorta (1986) System. Appl. Microbiol. 7,278--285. G. Gabrielli, A. Gliozzi, A. Sanguinetti and A. D'Agata (1989) Colloids. Surf. 35,261--273. R. Rolandi, H. Schindler, M. DeRosa and A. Gambacorta (1986) Eur. Biophys. J. 140, 19--27. A, Gulik, V. Luzzati, M. DeRosa and A. Gambacorta (1985) J. Mol. Biol. 182, 131--149. S. Strobl, L. Six and K. Heckmann (1985) Z. Naturforsch. 40c, 219--222. A. Gulik, V. Luzzati, M. DeRosa and A. Gambacorta (1988) J. Mol. Biol. 201,429--435.