High pressure effects on the structure and mesophase behaviour of supramolecular lipid aggregates and model membrane systems

R. Hayashi and C. Balny (Editors), High Pressure Bioscience and Biotechnology 9 1996 Elsevier Science B.V. All rights reserved.

21

High pressure effects on the structure and mesophase behaviour of supramolecular lipid aggregates and model membrane systems Roland Winter University of Dortmund, Department of Physical Chemistry, Otto-Hahn-Strafie 6, D-44227 Dortmund, Germany Abstract

Lipids, which provide valuable model systems for membranes, display a variety of polymorphic phases, depending on their molecular structure and environmental conditions. By use of X-ray and neutron di~action, infrared and fluorescence depolarization spectroscopy, calorimetry and volumetric measurements, the temperature and pressure dependent structure and phase behaviour of several lipid systems, differing in chain configuration and headgroup structure have been studied. Hydrostatic pressure has been used as a physical parameter, because high pressure is an important feature of certain natural membrane environments (e.g., marine biotopes), and because the high pressure phase behaviour of biomolecules is also of considerable biotechnological interest [1]. An understanding of the energetics of these lipid assembfies and of the various lipid phase transitions should help in assessing the role of such molecules in natural membranes. 1. INTRODUCTION Lipid bilayers, which constitute the basic structural component of biological membranes, exhibit a rich lyotropic and thermotropic phase behaviour [2]. Due to the large hydrophobic effect, most phospholipid bilayers associate in water already at extremely low concentrations (<10 12 M). Saturated phospholipids often exhibit two thermotropic lamellar phase transitions, a gel to gel (L~,/P~,) pretransition and a gel to liquid-crystalline (Pg,/Lc~) main transition at a higher temperature Tm. In the fluid-like L a phase, the hydrocarbon ~hains of the lipid bilayers are conformationally disordered, whereas in the gel phases, the chains are more extended and ordered. In addition to these thermotropic phase transitions, also pressure-induced phase transformations have been observed (see, e.g., [3-7]). Lamellar liquid-crystalline phases represent the fundamental structural element of cell membranes. However, it is assumed that the non-lamellar lipid structures, such as the inverse hexagonal (Hn) and bicontinuous cubic lipid phases, are also of biological relevance. They probably play an important functional role in some transient cell processes [8-11]. The bicontinuous cubic phases consist of a single lipid bilayer which partitions three-dimensional space into two congruent aqueous sub-volumes. The structures are based on periodic minimal surfaces. A pre-requisite for the formation of the HtI or inverse cubic phases is that the opposing monolayers wish to bend towards the water region. This desire arises because of differential lateral pressures which are present through the monolayer film. It increases, for instance, if the lateral chain pressure increases due to extensive cisJtrans-isomerisations at high temperatures. - First, we present data on the temperature and pressure dependent phase behaviour of single-component phospholipid bilayers. Second, we show results on lipid systems exhibiting also non-lamellar phases. Third, we discuss the effect of incorporated cholesterol on the structure and phase behaviour of phospholipid bilayers. In this overview we will mainly focus on the discussion of experimental results. Details of the experimental techniques are discussed elsewhere [ 12-15].

22 2. RESULTS AND DISCUSSION

2.1 Lamellar phase transitions of single-component phospholipid bilayer dispersions Generally, the lamellar gel phases prevail at high pressure and low temperature. They give way to the lamellar liquid-crystalline L~ phase as pressure is lowered and temperature is raised. A common value for the La/gel transition slope of about 22 ~ has been observed for the saturated phosphatidylcholines DMPC, DPPC and DSPC [4-7,12-14,16] (see Fig. 1). The positive slope can be explained by the endothermic enthalpy change AHm and volume increase AVmat the gel to Lcx transition through the Clapeyron relation dTm/dp = TmA Vm/MJm. Similar transition slopes have been found for the mono-cis-unsaturated POPC, the phosphatidylserine DMPS, and for the phosphatidylethanolamine DPPE. Only those of cisunsaturated DOPC and DOPE have been found to be markedly smaller [4,17]. The transition slope does not significantly depend on the hydrocarbon chain length or the type of headgroup, they affect the transition temperature, mainly. The existence of cis double-bonds in the chain region drastically affects the transition slope, however. The introduction of cis double-bonds leads to the lowest transition temperatures and smallest transition slopes, presumably as the cis double-bonds impose a kink in the linearity of the acyl chains, thus creating si~ificant free volume in the bilayer, which reduces the ordering effect of high pressure. Further pressureinduced gel phases have been observed in single-component phospholipid dispersions, such as an interdigitated high pressure gel phase in DPPC and DSPC [4,12,16]. These studies clearly demonstrate that, by regulating the lipid composition of the cell membranes through changes in lipid chain length, degree of unsaturation and headgroup structure, biological organisms are already provided with a mechanism for efficiently modulating the physical state of their membranes in response to changes in the external environment, such as high hydrostatic pressure.

80L APPE/DPPc F/

/

6oF

DMPC

F / / ~

F 2 0 !/

/

80-

DEPC

/

-

-

60 H~

.,DOPE

O

40

o t...d

0

-20~ 0

t- 20

PC

ot

..... 1.......... n 1

2

3

p[kbar] Figure 1. T, p-phase diagram for the main transition of different phospholipid bilayer systems. The Lcx phase is observed at the low-pressure high-temperature comer of the phase diagram.

-200 - 4[)0" 860 "12'0018'002000 p [bar] Figure 2. T, p-phase diagram of DOPE in excess water (abbreviations are explained below).

23 2.2 Stable and metastable non-lamellar lipid phases For a series of lipid molecules, also non-lameUar phases occur as thermodynamically stable states or they can often be induced as long-lived metastable states. Here we discuss three examples, taken from different groups of amphiphilic molecules. Contrary to DOPC, the corresponding cis-unsaturated phospholipid with ethanolamine as headgroup (DOPE), in addition to the lamellar LI3/La transition exhibits a lamellar La to inverted hexagonal (Ha) transition. As pressure forces a closer packing of the chains, which results in a reduction of the number of gauche bonds within the chains, both transition temperatures increase with increasing pressure. Figure 2 displays the corresponding T, p-phase diagram for the two endothermic transitions. The initial transition slope of the Hii/Lot transition (dTh/dP = 40 ~ is almost three times steeper as the slope of the lamellar chain-melting transition. A similar steep slope for the HiI/L~-transition has also been observed for egg-PE by turbidity and volumetric measurements [4,20]. The Hii/La-transition is the most pressure-sensitive lipid phase transition found to date. In DOPE also two cubic phases of space group Pn3m and lm3m can be induced by subjecting the sample to an extensive temperature or pressure cycling process at conditions close to the transition region of the L~ and HII phase [4,18,19,21]. Fig. 3a displays diffraction patterns of a pressure-cycled DOPE dispersion. The Bragg reflections (10), (11), and (20) of the Hn phase, the (001) and (002) reflections of the La phase, and the Bragg peaks of the cubic structures of spacegroup lm3m and Pn3m are seen. It is also possible to induce metastable cubic structures in naturally derived lipid systems. Dispersions of egg-PE in excess water spontaneously form a lamellar L~, Lc~ and a Hn structure with increasing temperature, no equilibrium cubic phase is found. However, after a series of pressure-jumps passing the HIi/L~-transition we observe the formation of additional metastable cubic phases of space groups Im3m, Pn3m, and Ia3d (Fig. 3b). It has been shown, that in certain situations, the topology of bicontinuous cubic phases can result in a similar or even lower free

T - 62~

b) egg PE

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H,,(10)

P = Pn3m

T = 20~

p = 340 bar

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l-I(211)

_6 i,_

xS~~~_.~0

P(III)

I__1

1(200)

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,

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I

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~

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0.02

,

I

.

0.03 s[A -1]

~

I

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,

i

0.05

0

I

,

0.01

,

L

0.02 s[A -~3

~

i

0.03

,

0.04

Figure 3. SAXS patterns a) of DOPE/water, which has been pressure-cycled between the L~ and Hn phase, and b) of egg-PE/water, which has been pressure-cycled between 30 and 450 bar at 62 ~ (s = (2/2)sinO, 2 wavelength of radiation, 20 scattering angle).

24 energy than either the lamellar Lc~ or Hu phase, as the cubic phases have low curvature energies and do not suffer the extreme chain packing stress of the HII phase [ 10,21,22]. As a second example, the monoacylglycerides monoolein (MO, C18:1c9) and monoelaidin (ME, C18: l t9) are chosen, because they have received considerable interest due to their importance as intermediates in lipid digestion and their applications in food industry. For both systems, a temperature-pressure phase diagram has been determined [23], and drastic differences in phase behaviour are found for the two systems (see Fig. 4). In MO-water dispersions, the cubic phase Pn3m extends over a large phase field in the T, p-plane. At temperatures above 95 ~ the H~I phase is found. In the lower temperature region, a crystalline lamellar phase is induced at higher pressures. The phases found in ME-water include a lamellar crystalline L c phase, the L~ gel phase, the La phase, and two cubic phases belonging to the crystallographic space groups Im3m and Pn3m~ Obviously, even small changes in lipid chain configuration can lead to drastic changes in phase behaviour. The stability of the lamellar phases of ME at lower temperatures over the cubic and inverted hexagonal phase, as compared to MO, can be qualitatively explained by simple molecular packing arguments [23]. Compared to ME, the MO molecule is more wedge-shaped, thus leading to an increased tendency of the molecules to aggregate into structures with negative curvatures. I00

..I-

i00

~', ~

monoolein

80-

80

60

Pn3m

L) F--

0,U% I--

40

l

00

60~,i-I

,

Im3m

L

40 P ~ 20~ --''x

20

monoelaidin

Pn3m

L. LI3

t

500

I000 p [bar]

l(;

500 p [bar]

i000

Figure 4. T, p- phase diagram of monoolein (MO) and monoelaidin (ME) in excess water. According to the Gibbs phase rule, binary lipid mixtures may exhibit an even more complex phase behaviour with extensive phase coexistence regions. Here we focus on one class of system only: phospholipid/fatty acid mixtures. Fatty acids are known to affect important membrane properties, such as permeability and fusion. It is assumed that hydrogen bonded complexes are formed between the phospholipid and fatty acid molecules, which act as spacers, thus reducing the crowding of the relatively bulky phospholipid headgroups [24]. This change in the steric balance of the bilayer results in the non-bilayer phases being energetically favoured over the fluid lamellar phase, immediately that the chain-melting transition occurs. For the palmitic chain system DPPC/PA (1:2), the low temperature phase is a lamellar phase, but the high temperature fluid phase is an inverted hexagonal one. For the system DMPC/MA

25 (1:2), however, also an isotropic phase of cubic symmetry is observed at higher temperatures. From the combined results of high pressure DTA, X-ray and neutron diffraction experiments, a phase diagram has been constructed for these systems (Fig. 5). At the gel to fluid phase transition of DMPC/MA the change of monolayer curvature is probably that large that the Ha/cubic phases become more stable than the La-phase. The L~-phase is observed under nonequilibrium conditions, however. In the system DPPC/PA, with about 2.5 A longer chain length of the lipid molecules, the larger splay of the 'molten' hydrocarbon chains in the fluidlike state probably leads to such a large spontaneous (intrinsic) curvature that can only be adopted by the inverted hexagonal structure.

100 f 90

Hr[

DMPC/MA /

80 70 c) o 60

100

/

50 4O

40

Lp

30201 0

I

500

~

Lp

30

MA - L~ ,

DPPC/PA

80 70 o o 60 I--50

l

HE

90

I

i000

p [bar]

~

,

t

1500

20 I

0

500

p [bar]

~

I

I000

Figure 5. T, p-phase diagrams of aqueous dispersions a) of DMPC/MA (1:2) - we encounter further metastable phases, such as a further cubic phase, upon cooling the sample, in particular at elevated pressures -, and b) ofDPPC/PA (1:2) in excess water. 2.3 The effect of cholesterol on the high pressure phase behaviour of phospholipids Not only the nature of pure phospholipid barotropic phase transitions, but also how they are affected by the incorporation of other species (ions, local anesthetics, steroids etc.) interacting with these membranes, has attracted considerable attention (see, e.g., [4] and references therein), recently. Here, we will focus on the effects of incorporating cholesterol into phospholipid bilayers, only. Cholesterol is an integral component of mammalian cell membranes with concentrations up to about 50 mol%. We investigated static and dynamic properties of unilameUar vesicles of the common phospholipid DPPC (Tin ~ 41.5 ~ containing different amounts of cholesterol. Incorporation of more than about 5 mol% cholesterol is sufficient to suppress the pretransition, and the main transition is abolished by 50 tool% of the sterol. We used the fluorescence depolarization technique for the study of the physical state of the membrane. It utilizes the fluorescence anisotropy elicited from the probe molecule TMA-DPH embedded in the lipid bilayers [15]. The steady-state fluorescence anisotropy rss has been analyzed in terms of a structural order parameter S = of the fluorophore, which reflects the average order parameter of the lipid bilayer at the position of the fluorophore (0 _ S _ 1). The rss data of TMA-DPH in DPPC/cholesterol mixtures as a function of temperature, pressure and sterol concentration are presented in Figs. 6. rss of

26 TMA-DPH in the La phase is significantly lower than that in the gel phase of DPPC, due to the greater static and/or dynamic molecular disorder present in the fluid-like phase of the bilayer; rss is about 0.30 in the gel phase and 0.17 in the fluid phase of pure DPPC, corresponding to a marked difference in the order parameter S of 0.83 and 0.45, respectively. Increase of pressure in the fluid phase leads to an about 20 % decrease of the population of gauche conformes per kbar [25]. The incorporation of cholesterol into the DPPC bilayer reduces the disorder in the liquid-crystalline state, as can be deduced from the observed larger steady-state anisotropy values. The rigid ring system of cholesterol significantly enforces the orientational ordering of the acyl chains in their fluid-like state. Contrary to the behaviour for T>Tm, the rss values slightly decrease for T
1

,.n 0.24 [._ 020L

~

-,-+\

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~

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***

t3 t3.~

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tool +

+

/

~

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9

20molZ

o

30 mol~ " .'

o ,0mo,

:

:

?o 32 34 36 as 40 42 44 4~ 48 so 52 s4 s~ s8 6o o.o o.~ o.2 0.3 o.4 o.s o.6 o.7 o.8 o.9 ~.o

T [~

p [kbar]

Figure 6. T- and p-dependence of the steady-state fluorescence anisotropy rss of TMA-DPH in DPPC/cholesterol unilamellar vesicles at different sterol concentrations. We found a center value for the fluorescence distribution of lifetimes rF of TMA-DPH of 5.8 ns for pure DPPC at 35 ~ in the gel phase, and of 3.0 ns at 55 ~ in the liquid-crystalline phase. The addition of cholesterol results in an increase in v~ in both lipid phases. It has been shown, that the excited-state lifetime is a function of the dielectric permittivity of the solvent cage [26]. As the fluorophore resides in the interfacial region of the membrane, it experiences quenching by probe-water interactions. Information on the hydration level at the location of the fluorophore position in the bilayer can thus be obtained by measurements of rF. In the La state, rF is significantly shorter, because greater static and/or dynamic molecular disorder is present in the fluid phase of the bilayer, which increases the probability of water penetration

27 into the bilayer system Incorporation of the sterol reduces the probability of water penetration into the bilayer, thus leading to longer fluorescence lifetimes. Figure 7 presents some representative data for DPPC/cholesterol mixtures at T = 58 ~ as a function of pressure up to 1 kbar. Increasing pressure results in longer fluorescence lifetimes of TMA-DPH in DPPC vesicles in their fluid-like state, rv increases slightly from 2.5 ns at 1 bar to 3.5 ns at 700 bar. At the pressure-induced fluid to gel phase transition, which takes place around 750 bar at that temperature, VF increases up to about 5.7 ns. Further increase of pressure leads to a slight increase of rF only. Addition of for example 30 mol% cholesterol causes a 2.5-fold increase of VF at atmospheric pressure. Hydrostatic pressure increase has only a small effect on fluorescence lifetime in this mixture. Above about 400 bar, r~ reaches a plateau value around 7 ns, which is slightly higher than that in the gel phase of pure DPPC.

9~7'

I'

1'

I'

I'

I'

1'

I'

I'

I'

1

8 m ..... -0- ..... E3 ..... [] ...... El- ..... :~ ..... .~._ - - e.

. . . .

~.._._.l~e.....

~ c~,5

100m~ %

- I ..... _~ ............ 20 rnol% ....~ . . . . . . - ~

~f-" ,--7 6 1

~ ~

~ . . . . ~ .......

......... ~

........ ~

~ .../~.~

....

<> ....

J. ----r . . . . ...... [] ......

30

mol

%

40 tool% 50 tool %

b_ ~

4

.....

3 ~---.- X L 2

0

o.

'

I'

0.~

1

~

X ------'X~

J,

0.2

l , ' ,

0.3

I,

i , , ,

0.4 o.s 06 p [kbar]

0.7

I,

o.s

!

0.9

,

1

1.0

Figure 7. Center value rF of the fluorescence lifetime distribution of TMA-DPH in DPPC/cholesterol unilamellar vesicles as a function of pressure (T-- 58 ~ These results clearly indicate that the incorporation of cholesterol into the DPPC bilayer leads to a significant increase in hydrophibicity of the membrane. An increase in pressure up to the 1 kbar range is much less effective in suppressing water permeability than cholesterol embedded in fluid DPPC bilayers at concentration levels higher than about 10 mol% sterol. Abbreviations MA: myristic acid, PAL: palmitic acid, MO: monoolein, ME: monoelaidin, DMPC: 1,2dimyristoyl-sn-glycero-3-phosphatidylcholine (di-C 14:0), DPPC: 1,2-dipalmitoyl-sn-glycero3-phosphatidylcholine (di-C16:0), DSPC: 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (diC18:0), DOPC: 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (di-C18:l,cis), DOPE: 1,2dioleoyl-sn-glycero-3-phosphatidylethanolamine (di-C 18:1,cis), POPC: 1-palmitoyl-2-oleoylsn-glycero-3-phosphatidylcholine, (C16:0, C18:1,cis), DEPC: 1,2-dielaidoyl-sn-glycero-3phosphatidylcholine (di-C18:1,trans); DPPE: 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (di-C16:0), DMPS: 1,2-dimyristoyl-sn-glycero-3-phosphatidylserin (di-C14:0), egg-PE: egg-yolk phosphatidylethanolamine, TMA-DPH: 1-(4-trimethylammonium-phenyl)-6phenyl-l,3,5-hexatriene, SAXS small-angle X-ray scattering, DTA differential thermal

analysis.

28 Acknowledgements We thank the Laboratory of Fluorescence Dynamics at the University of Illinois at Urbana/Champaign (U.S.A.) for the opportunity to carry out the time-resolved fluorescence measurements. The Synchrotron-X-ray diffraction experiments have been carried out at the EMBL outstation at DESY in Hamburg. Financial support from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen lndustrie is gratefully acknowledged.

6. REFERENCES 1 C. Balny, 1L Hayashi, K. Heremans, and P. Masson (eds.), High Pressure and Biotechnology, Colloque Inseram, Vol. 224, John Libbey Eurotext, 1992. 2 G. Cevc and D. Marsh, Phospholipid Bilayers, John Wiley and Sons, New York, 1987. 3 1L Winter and J. Jonas (eds.), High Pressure Chemistry, Biochemistry and Materials Science, Kluwer Academic Publishers, Dordrecht, Netherlands, 1993. 4 IL Winter, A. Landwehr, Th. Brauns, J. Erbes, C. Czeslik, and O. Reis, Proceedings 23rd Steenbock Symposium on High Pressure Effects in Molecular Biophysics and Enzymology, Madison, 1994. 5 P.-L. G. Chong and G. Weber, Biochemistry 22 (1983) 5544. 6 P.T.T. Wong, D.J. Siminovitch, and H.H. Mantsch, Biochim. Biophys. Acta 47 (1988) 139. 7 D.A. Driscoll, J. Jonas, and A. Jonas, Chem. Phys. Lipids 58 (1991) 97. 8 P. Mariani, V. Luzzati, and H. Delacroix, J. Mol. Biol. 204 (1988) 165. 9 G. Lindblom and L. Rilfors, Biochim. Biophys. Acta 988 (1989) 221. 10 J.M. Seddon, Biochim Biophys. Acta 1031 (1990) 1. 11 M.W. Tate, E.F. Eikenberry, D.C. Turner, E. Shyamsunder, and S.M. Gnmer, Chem~ Phys. Lipids 57 (1991) 147. 12 R. Winter and W.-C. Pilgrim, Ber. Bunsenges. Phys. Chem. 93 (1989) 708. 13 M. Bfttner, D. Cell, U. Jacobs, and R. Winter, Z. Phys. Chem. 184 (1994) 205. 14 A. Landwehr and R. Winter, Ber. Bunsenges. Phys. Chem 98 (1994) 214. 15 C. Bemsdorff, R. Winter, T.L. Hazlett, and E. Gratton, Bet. Btmsenges. Phys. Chenl 99 (1995) 1479. 16 L.F. Braganza and D.L. Worcester, Biochemistry 25 (1986) 2591. 17 IL Winter and P. Thiyagarajan, Progr. Colloid. Polym. Sci. 81 (1990) 216. 18 J. Erbes, C. Czeslik, W. Hahn, M. Rappolt, G. Rapp, and R. Winter, Ber. Btmsenges. Phys. Chem. 98 (1994) 1287. 19 P.T.C. So, S.M. Gnmer, and E.S. Shyamsunder, Phys. Rev. Lett. 70 (1993) 3455. 20 E.L. Chang and P. Yager, Mol. Cryst. Liq. Cryst. 98 (1983) 125. 21 E. Shyamsunder, S.M. Grtmer, M.W. Tate, D.C. Turner, P.T.C. So, and C.P.S. Tilcock, Biochemistry 27 (1988) 2332. 22 J.M. Seddon and 1LH. Templer, Phil. Trans. R. Soc. Lond. A 344 (1993) 377. 23 C. Czeslik, R. Winter, G. Rapp, and K. Bartels, Biophys. J. 68 (1995) 1423. 24 J.M. Seddon, J.L. Hogan, N.A. Warrender, and E. Pebay-Peyroula, Progr. Colloid Polym. Sci. 81 (1990) 189. 25 O. Reis, 1L Winter, and T.W. Zerda, Biochim. Biophys. Acta, in press. 26 E. Gratton and T. Parasassi, J. of Fluorescence 5 (1995) 51.