b901690b

REVIEW www.rsc.org/softmatter | Soft Matter

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Effect of pressure on membranes†

Roland Winter* and Christoph Jeworrek

Received 26th January 2009, Accepted 27th March 2009

First published as an Advance Article on the web 7th May 2009

DOI: 10.1039/b901690b

Besides temperature, hydrostatic pressure has been used as a physical-chemical parameter for studying

the energetics and phase behavior of membrane systems. First we review some theoretical aspects of

lipid self-assembly. Then, the temperature and pressure dependent structure and phase behavior of lipid

bilayers, differing in chain configuration, headgroup structure and composition as revealed by using

thermodynamic, spectroscopic and scattering experiments is discussed. We also report on the lateral

organization of phase-separated lipid membranes and model raft mixtures as well as the influence of

peptide and protein incorporation on membrane structure and dynamics upon pressurization. Also the

effect of other additives, such as ions, cholesterol, and anaesthetics is discussed. Furthermore, we

introduce pressure as a kinetic variable. Applying the pressure-jump relaxation technique in

combination with time-resolved synchrotron X-ray diffraction, the kinetics of various lipid phase

transformations was investigated. Finally, also new data on pressure effects on membrane mimetics,

such as surfactants and microemulsions, are presented.

Introduction

The interest in using—next to temperature and the chemical

potentials of the species involved—pressure as a thermodynamic

and kinetic variable has been largely growing in physical-chem-

ical and biophysical studies of biological systems in recent

years.1–7 To describe the energy landscape and the set of

parameters necessary to provide an understanding of the phase

behavior of biomolecular systems, one needs to scan the

TU Dortmund University, Physical Chemistry I - Biophysical Chemistry,Otto-Hahn Str. 6, D-44227 Dortmund, Germany. E-mail: [email protected]

† This paper is part of a Soft Matter themed issue on MembraneBiophysics. Guest editor: Thomas Heimburg.

Roland Winter graduated in

Chemistry at the University of

Karlsruhe and received his PhD

in Physical Chemistry in 1982.

He then joined Professor Hen-

sel’s group at the University of

Marburg as a postdoctoral

fellow working on liquid matter

under extreme conditions. After

completing postdoctoral training

in Prof. Jonas’ laboratory,

University of Illinois, he was

appointed Professor at the

University of Bochum. Since

1993 he has held a Chair of

Physical Chemistry (Biophysical Chemistry) at TU Dortmund

University. His research interests comprise the study of the struc-

ture, dynamics and phase behavior of model biomembranes and

proteins, and pressure effects in molecular biophysics.

This journal is ª The Royal Society of Chemistry 2009

appropriate parameter space experimentally.1–8 To this end, also

pressure dependent studies have proven to be a valuable tool.

High hydrostatic pressure (HHP) acts predominantly on the

spatial (secondary, tertiary, quaternary and supramolecular)

structures of biomacromolecules. Besides the general physical-

chemical interest in using high pressure as a tool for under-

standing the structure, energetics, phase behavior and dynamics

of biomolecules, HHP is also of biotechnological and physio-

logical interest e.g., for understanding the physiology of deep-sea

organisms living in cold and high-pressure habitats, which exist

at pressures up to about 1 kbar (0.1 MPa ¼ 1 bar, 1 GPa ¼ 10

kbar).7–12 High hydrostatic pressure harbors also the potential to

inactivate microorganisms, viruses and enzymes while the effect

on the flavor and nutrient content of food is low compared to

Christoph Jeworrek studied

Chemistry and Molecular

Materials at the Gerhard

Mercator University Duisburg

where he received his Bachelor

degree in 2005. In 2007 he

finished his Master thesis at the

Department of Chemistry of TU

Dortmund University.

Currently, he is working on his

PhD thesis in Prof. Winter’s

group. His research interests are

proteins under high hydrostatic

pressure, the structure and

dynamics of model bio-

membranes as well as the interaction of lipid membranes with

steroids and peptides by using different scattering techniques such

as X-ray-, synchrotron- and neutron-small angle scattering and

reflectivity.

Soft Matter, 2009, 5, 3157–3173 | 3157

http://dx.doi.org/10.1039/b901690b

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usual thermal treatments. Hence, high pressure food processing

has been introduced in several countries, now.13

Pressure stress affects all levels of cellular physiology including

metabolism, membrane physiology, transport, transcription and

translation.12 Interestingly, the biological membrane seems to be

one of the most pressure sensitive cellular components. In this

review, we discuss results of studies of the effects of pressure on

the structure and phase behavior of lyotropic lipid mesophases,

model and natural membrane systems as well as pressure effects

on the interaction of peptides and drugs with membranes. At

a more empirical level, there exists also a quasi-pharmacological

aspect of pressure in which it is used to perturb membrane–drug

interactions. High pressure biophysical studies generally call for

unique methods, which have largely been developed in recent

years. The principle designs can be found elsewhere.6,7,14–19 In this

review, we focus on basic concepts and results, only.

Fig. 2 Schematic drawing of various lamellar and nonlamellar lyotropic

lipid mesophases adopted by membrane lipids (Lc, lamellar crystalline;

Lb0, Pb0, lamellar gel; La, lamellar liquid-crystalline (fluid-like); QIIG

(space group Ia3d, number 230), QIIP (space group Im3m, number 229),

QIID (space group Pn3m, number 224), inverse bicontinuous cubics of

different space group - the cubic phases are represented by the G, D, and

P minimal surfaces, which locate the midplanes of fluid lipid bilayers; HII,

inverse hexagonal). Numerous factors determine the particular meso-

phase structure, e.g., the type of lipids, lipid chain length and degree of

Lipid self-assembly

The amphiphilic properties of lipid or surfactant molecules lead

to self-aggregation in water solution. The main driving force

behind this self-assembly is the hydrophobic effect, which on its

own would lead to a macroscopic phase separation (one polar

and one non-polar phase), but which is prevented owing to the

requirement that the polar headgroups like to be in contact with

water. Instead, packing restrictions of the lipid molecules in the

aggregate structures have to be fulfilled.20–23 A useful concept for

a qualitative understanding of the phase behavior of amphiphilic

systems is based on a consideration of the shape of the lipid

molecules (Fig. 1a). The self-assembly of lipid molecules can be

rationalized by a dimensionless packing parameter, P, defined by

n(al)�1, where n is the molecular volume, l is the molecular length,

and a is the molecular area at the hydrocarbon–water interface.

When the packing parameter P z 1 (cylindrical-like molecules),

these are optimal conditions for the formation of a bilayer

structure. For P > 1, the molecules are wedge-shaped and the

lipid monolayer prefers to curve towards the water region, and,

for example, an inverse bicontinuous cubic (QII) or hexagonal

(HII) phase may form (e.g., for phosphatidylamines at high

temperatures) (Fig. 1, 2).20–23 It is generally assumed that the

nonlamellar lipid structures, such as the HII and QII lipid phases,

Fig. 1 Possible spontaneous curvatures of a lipid monolayer arising

from differences in the distribution of lateral forces within the headgroup

and acyl-chain regions and the corresponding packing parameter P of

the molecular building blocks (P-values: 0–1/3: spheres, 1/3–1/2: cylinder,

1/2–1: lamellar, >1: inverse phases).

unsaturation, headgroup area and charge, solvent properties, pH,

temperature and pressure.

3158 | Soft Matter, 2009, 5, 3157–3173

are also of biological relevance. Fundamental cell processes, such

as endo- and exocytosis, vesicular protein trafficking and fat

digestion, involve a rearrangement of membranes where locally

nonlamellar lipid structures are involved. Furthermore, the cubic

phases can be used as controlled-release drug carriers and crys-

tallization media for membrane proteins.24,25

Lipid mesophases and model biomembrane systems

Lamellar lipid bilayer phases

Lyotropic lipid mesophases are organized soft matter systems

formed by amphiphilic molecules, mostly phospholipids, in the

presence of water. They exhibit a rich structural polymorphism,



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depending on their molecular structure, hydration level, pH,

ionic strength, temperature and pressure.14,15,20–23,26–42 The basic

structural element of biological membranes consists of a lamellar

phospholipid bilayer matrix (Fig. 2a). Even though most lipids

possess two acyl-chains and one hyphrophilic headgroup, the

composition of the chains and the headgroup can vary signifi-

cantly in cellular membranes. Also, the lipid composition is very

different in different cell types of the same organism, or even in

different organelles of the same cell. Not only is the entire cell

membrane very complex, containing a large variety of different

lipid molecules and a large body (ca. 50%) of proteins performing

versatile biochemical functions, but also the simplest lipid bilayer

consisting of only one or two kinds of lipid molecules exhibits

already a very complex phase behavior as discussed in the

following. Lipid bilayers display various phase transitions

including a chain melting transition. One good way to measure

such transitions is the calorimetric determination of the heat

capacity, Cp.20,21 In excess water, saturated phospholipids often

exhibit two thermotropic lamellar phase transitions, a gel-to-gel

(Lb0-Pb0) pretransition and a gel-to-liquid-crystalline (Pb0-La)

main (chain melting) transition at a higher temperature, Tm

(Fig. 2). The prime index ‘‘ 0 ’’ indicates that the lipids are tilted

with respect to the membrane normal. Phosphatidylcholines

display a tilt angle of about 30�, while phosphatidylethanol-

amines do not display such a tilt. The pretransition is linked to

the formation of linear defects of disordered chains,21 which is

probably a consequence of the coupling between geometry

changes and chain melting. The ripples in the Pb0 phase display

a periodicity of typically 15–30 nm. In the fluid-like La phase, the

acyl-chains of the lipid bilayers are conformationally disordered

(‘‘melted’’), whereas in the gel phases, the chains are more

extended and ordered. The lipids in the Lb0 phase are arranged on

a two-dimensional triangular lattice in the membrane phase. This

phase is also called solid-ordered (so) phase. Besides neutral or

zwitterionic lipids, also negatively charged lipids are present in

the cell membranes. The melting temperature of negatively

charged lipid membranes generally increases when neutralizing

the charges by proteins or divalent ions. In addition to these

thermotropic phase transitions, a variety of pressure-induced

phase transformations have been observed.26–28,30–33

Because the average end-to-end distance of disordered

hydrocarbon chains in the La-phase is smaller than that of

ordered (all-trans) chains, the bilayer becomes thinner during

melting at the Pb0/La-transition, even though the partial lipid

volume increases. This is demonstrated in Fig. 3a which shows

Fig. 3 Effect of (a) temperature and (b) pressure (at T ¼ 30 �C) on the

partial lipid volume VL of DMPC bilayers as obtained from densimetric

measurements. The gel-to-gel (Lb0 to Pb0,) and gel-to-fluid (La) lamellar

phase transitions appear at temperatures Tp and Tm, respectively.


the temperature dependence of the specific partial lipid volume

VL of DMPC (a C14 double-chain phospholipid, see list of

abbreviations) in water.37 The change of VL near 14 �C corre-

sponds to a small volume change in course of the Lb0-to-Pb0

transition. The main transition at Tm ¼ 23.9 �C for this phos-

pholipid is accompanied by a well pronounced 3% change in

volume, which is mainly due to changes of the chain cross-

sectional area, because the chain disorder increases drastically at

the transition. The compression of the bilayer as a whole is

anisotropic, lateral shrinking being accompanied by an increase

in thickness due to a straightening of the acyl-chains. Fig. 3b

exhibits the pressure dependence of VL at a temperature above

Tm, e.g. 30 �C. Increasing pressure triggers the phase trans-

formation from the La to the gel phase, as can be seen from the

rather abrupt decrease of the lipid volume at 270 bar.

The volume change DVm at the main transition decreases

slightly with increasing temperature and pressure along the main

transition line. Other thermodynamic parameters have been

determined as well. The coefficient of isothermal compressibility

of the Pb0 gel phase is substantially lower than that of the liquid-

crystalline phase (typically, kT(Pb0) z 5�10�5 bar�1 and kT(La) z13�10�5 bar�1).37 Whereas the lateral compressibility of the lipid

chains is rather high, a slight lateral compression is observed for

the polar headgroups, only. Hence, this will have little effect on

the nature of the electrical properties in the interfacial region, but

pressure would generally be expected to favor ionization of polar

groups and electrostriction of the surrounding water.21

Biological lipid membranes can also melt. Typically, such

melting transitions are found about 10 �C below body or growth

temperatures.21 It seems that biological membranes adapt their

lipid compositions such that the temperature distance to the

melting transition is maintained. The same may hold true for

adaptation to high pressure conditions. Hence it is likely that

such behavior serves a purpose in the biological cell. Close to the

melting transition, in lipid bilayers the fluctuations in enthalpy,

volume and area are high. High enthalpy fluctuations lead to

high heat capacity, high volume fluctuations lead to a high

volume compressibility, and high area fluctuations lead to a high

area compressibility. In turn, area fluctuations lead to fluctua-

tions in curvature and bending elasticity.21 These properties may

be required for optimal physiological function.

A common slope of �22 �C/kbar has been observed for the

gel–fluid phase boundary of saturated phosphatidylcholines as

shown in Fig. 4.26–28 Assuming the validity of the Clapeyron

relation describing first-order phase transitions for this

quasi-one-component lipid system, dTm/dp ¼ TmDVm(Tm, p)/

DHm(Tm, p), the positive slope can be explained by an

endothermic enthalpy change, DHm, and a partial molar volume

increase, DVm, for the gel-to-fluid transition, which have indeed

been determined in direct thermodynamic measurements.32,33,37

The transition enthalpy at atmospheric pressure is about

36 kJ/mol, for DPPC at ambient pressure and decreases slightly

with pressure (dDHm/dp)¼�3.4 kJ mol�1 kbar�1).39 As dDHm/dp

¼ �Tm(dDVm/dT)p + DCp,m(dTm/dp), the drop of enthalpy

change with pressure evidences a significant difference in the

coefficients of thermal expansion of the two phases. Similarly,

DVm decreases linearly with increasing pressure (from 22.9 cm3

mol�1 at 1 bar to �13 cm3 mol�1 at 2 kbar, i.e., dDVm/dp) ¼�4.93 cm3 mol�1 kbar�1).39 According to dDVm/dp¼ (dDVm/dp)T

Soft Matter, 2009, 5, 3157–3173 | 3159


Fig. 4 T,p-phase diagram for the main (chain-melting) transition of

different phospholipid bilayer systems. The fluid-like (liquid–crystalline)

La-phase is observed in the low-pressure, high-temperature region of the

phase diagram; the ordered gel phase regions appear at low temperatures

and high pressures, respectively. The gel-to-fluid transition lines of the

phosphatidylcholines are drawn as a full line, those of the phospholipids

with different headgroups as dashed lines. The lengths and degree of

unsaturation of the acyl-chains of the various phospholipids are denoted

on the right-hand side of the figure.

Fig. 5 (a) T,p-phase diagram of DPPC bilayers in excess water. Besides

the Gel 1 (Pb0), Gel 2 (Lb0) and Gel 3 phase, an additional crystalline gel

phase (Lc) can be induced in the low-temperature regime after prolonged

cooling which is not shown here. (b) Phase diagram of DPPC-gramicidin

D (GD) (5 mol%) in excess water as obtained from diffraction and

spectroscopic data. The inset shows a schematic view of the helical dimer

(HD) and double helix (DH) conformation of GD.

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+ (dDVm/dT)p$(dTm/dp), this is due to the significant difference

in the lipid compressibility coefficients in the fluid and gel phase,

respectively. The transition half-width (DTm,1/2), which can be

estimated as the ratio of the calorimetric peak area DHm,cal to its

amplitude Cp,max, can be determined form the van’t Hoff

enthalpy change by using DHm,vH ¼ 4RTm2Cp,max/DHm,cal ¼

4RTm2/DTm,1/2). The transition half-width does not change with

pressure, and the average number of lipid molecules (N ¼DHm,cal/DHm,vH) comprising the cooperative unit N of the

transition grows slightly with the increase of pressure and

temperature.39

Similar transition slopes have been determined for the mono-

cis-unsaturated lipid POPC, the phosphatidylserine DMPS, and

the phosphatidylethanolamine DPPE. Only the slopes of the

di-cis-unsaturated lipids DOPC and DOPE have been found to

be markedly smaller. The two cis-double bonds of DOPC and

DOPE lead to very low transition temperatures and slopes, as

they impose kinks in the linear conformations of the lipid acyl-

chains, thus creating significant free volume fluctuations in the

bilayer so that the ordering effect of high pressure is reduced.

Hence, in order to remain in a physiological relevant, fluid-like

state at high pressures, more of such cis-unsaturated lipids are

incorporated into cellular membranes of deep sea organisms,

another example of homeoviscous adaptation.42–44 While

increasing pressure is accompanied by an increase of the

concentration of unsaturated chains, the abundance of saturated

chains is lowered. For example, the ratio of unsaturated to

saturated fatty acids of the barophilic deep sea bacterium

CNPT3 is linearly dependent on the hydrostatic pressure at

3160 | Soft Matter, 2009, 5, 3157–3173

which they were cultivated.45 The unsaturated to saturated ratio

increases from 1.9 at ambient pressure up to about 3 at 690 bar

at 2 �C.

As seen in Fig. 4, pressure generally increases the order of

membranes, thus mimicking the effect of cooling. But we note

that applying high pressure can lead to the formation of addi-

tional ordered phases, which are not observed under ambient

pressure conditions, such as a partially interdigitated high pres-

sure gel phase, Lbi, found for phospholipid bilayers with acyl-

chain lengths $C16.31,33 To illustrate this phase variety, the

results of a detailed SAXS/SANS and FT-IR spectroscopy study

of the p,T-phase diagram of DPPC in excess water are shown in

Fig. 5a. At much higher pressures as shown here, even further

ordered gel phases appear, differing in the tilt angle of the acyl-

chains and the level of hydration in the headgroup area. Even at

pressures where the bulk water freezes, the lamellar structure of

the membrane is preserved.30

So far, the response to pressurization has been interpreted in

terms of conformational changes induced in the amphiphilic

molecule, which in fact are very pressure sensitive. In addition,

pressurization might affect the structural properties of the

surrounding solvent (water) as well, which might indirectly

induce structural and dynamical changes of the biomolecule.

Generally, in the pressure range considered here, the changes in

water structure are rather small.46–50 Whereas the first shell

neighbors are hardly affected by pressure, pressure-induced

changes essentially occur in the second shell of water molecules

upon pressurization. Pressure increase causes a gradual collapse

of the second-shell water molecules leading to an increase of the

population of water molecules in the interstitial positions. The

local hydrogen-bonded molecular network remains almost intact

even at high pressures such a 10 kbar. For example, in a neutron

diffraction study of the structure of water confined in cubic lipid–

water mesophases no evidence to suggest that the nature of the

interbilayer water is substantially altered has been found in the

pressure range up to about 1 kbar.51

Cholesterol effect

Cholesterol constitutes up to�50% of the lipid content of animal

cell membranes. Due to both its amphiphilic character and size



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(intermediate between that of the shortest and longest acyl-

chains, C14 and C24, 19.1 A hydrophobic length), it is inserted

into phospholipid membranes. Cholesterol thickens liquid-crys-

talline bilayers and increases the packing density of the lipid acyl-

chains in a way that has been referred to as ‘‘condensing effect’’.

Increasing cholesterol incorporation leads to a drastic reduction

of the main transition enthalpy, DHm, until at cholesterol

contents higher than�30 mol% the main transition vanishes. For

phospholipid–cholesterol lipid mixtures, a rather complex phase

behavior has been found.20–22,52–54 Measurements of the acyl-

chain orientational order of the lipid bilayer system by measuring

the 2H-NMR spectra or the steady-state fluorescence anisotropy,

rss, of the fluorophore TMA-DPH clearly demonstrate the ability

of cholesterol and other plant or bacterial sterols to efficiently

regulate the structure, motional freedom and hydrophobicity of

biomembranes.15,34,35

The pressure dependencies of the order parameter, S, of the

fluorophore TMA-DPH in DPPC and DPPC/cholesterol

mixtures as obtained from fluorescence anisotropy measure-

ments are shown in Fig. 6. The S-value of pure DPPC at

Fig. 6 Pressure dependence of the order parameter as determined from

steady-state fluorescence anisotropy measurements of TMA-DPH in

DPPC unilamellar vesicles at different cholesterol concentrations

(at T ¼ 50 �C).

Fig. 7 T,p-phase diagram of equimolar DMPC/DPPC (di-C14/di-C16) an


T ¼ 50 �C increases slightly up to about 400 bar, where the

pressure-induced liquid-crystalline to gel phase transition takes

place. Since S essentially reflects the mean order parameter of the

lipid acyl-chains, these results indicate that increased pressures

cause the chain region to be ordered in a manner similar to that

which occurs upon decreasing the temperature. Addition of

increasing amounts of cholesterol leads to a drastic increase of

S-values in the lower pressure region, whereas the corresponding

data at higher pressures in the gel-like state of DPPC are slightly

reduced. For concentrations above �30 mol% cholesterol, the

main phase transition can hardly be detected any more. At

a concentration of �50 mol% cholesterol, the order parameter

values are found to be almost independent of pressure. Incor-

poration of the sterol also significantly changes the expansion

coefficient and isothermal compressibility of the membrane,52

and it significantly increases the hydrophobicity and hence

decreases the water permeability of the bilayer.34,35 Notably, 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 high concentration levels. These data and

further FT-IR spectroscopic pressure studies38 clearly demon-

strate the ability of sterols to efficiently regulate the structure,

motional freedom and hydrophobicity of lipid membranes, so

that they can withstand even drastic changes in environmental

conditions, such as in external pressure and temperature.

Lipid mixtures and model raft membranes

To increase the level of complexity, T,p-phase diagrams of binary

mixtures of saturated phospholipids have been determined as

well.26–28,55 They are typically characterized by lamellar gel pha-

ses at low temperatures, a lamellar fluid phase at high tempera-

tures, and an intermediate fluid–gel coexistence region (Fig. 7).

The narrow fluid–gel coexistence region in the DMPC(di-C14)-

DPPC(di-C16) system indicates a nearly ideal mixing behavior of

the two components (isomorphous system). In comparison, the

coexistence region in the DMPC(di-C14)-DSPC(di-C18) system is

broader and reveals pronounced deviations from ideality. As

seen in Fig. 7, with increasing pressure the gel–fluid coexistence

region of the binary lipid systems is shifted toward higher

temperatures. A shift of about 22 �C/kbar is observed, similar to

d DMPC/DSPC (di-C14/di-C18) multilamellar vesicles in excess water.

Soft Matter, 2009, 5, 3157–3173 | 3161


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the slope of the gel–fluid transition line of the pure lipid

components.32,26–28

Studies were also carried out on the phase behavior of

cholesterol-containing ternary lipid mixtures, generally contain-

ing an unsaturated lipid like a phosphatidylcholine and a satu-

rated lipid like sphingomyelin (SM) or DPPC. Such lipid systems

are supposed to mimic distinct liquid-ordered lipid regions,

called ‘‘rafts’’,56 which seem to be also present in cell membranes

and are thought to be important for cellular functions such as

signal transduction and the sorting and transport of lipids and

proteins.56–59 Lipid domain formation can be influenced by

temperature, pH, calcium ions, protein adsorption, and may be

expected to change upon pressurization as well. Recently, we

determined the liquid-disordered/liquid-ordered (ld/lo) phase

coexistence region of canonical model raft mixtures such as

POPC/SM/Chol (1 : 1 : 1), which extends over a rather wide

temperature range. An overall fluid phase without domains is

reached at rather high temperatures (above �50 �C), only.60

Upon pressurization at ambient temperatures (20–40 �C), an

overall (liquid- and solid-) ordered state is reached at pressures of

about 1–2 kbar. A similar behavior has been observed for the

model raft mixture DOPC/DPPC/Chol (1 : 2 : 1) (Fig. 8).61

Interestingly, in this pressure range of �2 kbar, ceasing of

membrane protein function in natural membrane environments

has been observed for a variety of systems,62–68 which might be

related with the membrane matrix reaching a physiologically

unacceptable overall ordered state at these pressures. Moreover,

many bacterial organisms have been shown to completely loose

activity at these pressures.

The results discussed in this chapter demonstrate that organ-

isms are able to modulate the physical state of their membranes

in response to changes in the external environment by regulating

the fractions of the various lipids in a cell membrane differing in

chain length, chain unsaturation or headgroup structure

(‘‘homeoviscous adaption’’). Moreover, nature has further means

to regulate membrane fluidity, such as by changing the

membrane concentration of its sterols and by a lateral redistri-

bution of their various lipid components and domains. In fact,

several studies have demonstrated that membranes are signifi-

cantly more fluid in barophilic and/or psychrophilic species,

Fig. 8 p,T-phase diagram of the ternary lipid mixture DOPC/DPPC/

Chol (1 : 2 : 1) in excess water as obtained from FT-IR spectroscopy (C)

and SAXS (O) data. The ld + lo two-phase coexistence region is marked

in grey and depicted schematically in the adjacent drawing.

3162 | Soft Matter, 2009, 5, 3157–3173

which is principally a consequence of an increase in the unsatu-

rated to saturated lipid ratio.

Morphological transitions of lipid vesicles

Theoretical studies have shown that lipid vesicles may also

undergo morphological changes upon exposure to environ-

mental changes, such as temperature or osmotic stress.69 A large

variety of vesicle shapes which minimize the energy for certain

physical parameters, such as the enclosed volume and the area of

the vesicle, may be adopted, which are then organized in phase

diagrams, in which trajectories predict how the shape transforms

as, e.g., the temperature is varied. For example, an increase in

temperature transforms a quasi-spherical vesicle via thermal

expansion of the bilayer to a prolate shape and then to a pear.69

Finally, a small bud may be expelled from the vesicle. Here, the

relevant quantity for a change in shape is the deviation of the

equilibrium area-difference in the two monolayers, DA0, leading,

next to the bending energy as quantified by Helfrich,70 to an

additional, so-called area-difference elasticity free energy

contribution being proportional to (k/A$D2)$(DA� DA0)2, where

DA is the actual area difference, and D is the distance between the

neutral surfaces of the two monolayers, i.e., roughly half the

bilayer thickness.69 DA0 is expected to sensitively depend on

several factors including temperature, and is certainly also

expected to change upon pressurization. Since the thermal

expansivity of a lipid bilayer is large compared to that of water,

the vesicle area changes more rapidly with temperature than the

vesicle volume, and hence the volume-to-area ratio increases with

increasing temperature (the volume-to-area ratio of spherical

vesicles is given by V/A ¼ R/3). If the membrane is largely water

impermeable—which is essentially the case outside the melting

transition region—the vesicle cannot rapidly assume spherical

sheets any more. If the outer monolayer expands more than the

inner one, the additional area accumulated in this outer layer

will cause budding since the formation of buds increases the

area difference. Likewise, a stronger increase of the area of the

inner monolayer may induce a transition to discotyes and

stomatocytes.

Shape transformations are also predicted to arise in vesicles

consisting of bilayers with different components due to different

mechanisms.71–74 In phase-segregated membranes, the free energy

of the membrane consists essentially of the curvature energy of

the domains and the line energy of the one-dimensional domain

interfaces. As far as the edge energy is concerned only, a flat

circular disk does not represent the state of lowest energy since

the length of the edge can be further reduced if the domains form

a bud. But on the other hand, the curvature energy increases

concomitantly. Hence, in addition to the bending energy term

Ebi, there is a contribution from the line tension of the boundary

of each domain i, Eis ¼ li

ð

Ai

dA. The tendency towards budding is

proportional to Llk �1, where L is the radius of the domain with

area A in the limit of flatness, l is the line tension of the domain

interface, and k is the bending rigidity of the membrane. Hence,

the tendency towards budding depends on domain size, line

tension, and membrane stiffness. It has been predicted that above

a limiting boundary length L0 z 8k/l z 80 nm, an initially flat



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domain would transform into a complete spherical bud with

vanishing neck radius, provided that sufficient membrane area is

available.71 Certainly, pressure is also expected to markedly

influence these parameters as well, but this has not been explored

theoretically so far.

Recently, we used a technique that allows us to visualize

morphological changes of the membrane of giant more-compo-

nent unilamellar vesicles (GUVs) upon pressure perturbation.75

Under these conditions, the bending rigidity and the line tension,

which control the domain structure of heterogeneous vesicles,

and the differential area of the opposing monolayers in the lipid

vesicle are influenced by pressure-induced changes in lipid

molecular packing and shape. It has been shown by two-photon

excitation fluorescence microscopy on these multidomain GUVs

that budding and fission of daughter vesicles may occur, and this

at surprisingly low pressures of 200–300 bar, already. Theses

budding processes are not directly related to phase transitions to

an overall ordered conformational state of the lipid membrane,

which occur at much higher pressures (see for example Fig. 8). A

similar effect has been observed for egg yolk phospholipid giant

unilamellar vesicles.76 The topological changes of the lipid vesi-

cles are irreversible and exhibit a different behavior depending if

the pressure is increased or decreased. Hence, this scenario

provides a further mechanism that may be responsible for

disruption of natural membranes upon compression.

Fig. 9 Effect of the pressure of various gases on the melting transition of

DPPC vesicles. For comparison, the gel-to-fluid transition for pressuri-

zation under hydrostatic conditions is depicted as well.

Amphiphilic drugs and ions

The lipid conformation, membrane structure and phase behavior

is also influenced by the ionic strength of the surrounding solvent

(in particular for lipids with charged headgroups) and the

incorporation of amphiphilic molecules, such as anaesthetics and

drugs.7,21,26,77–83 The group of substances that cause general

anaesthetic effects comprises a wide range of chemically and

structurally dissimilar molecules (e.g., alcohols, ethers, noble

gases, etc.). It has been demonstrated that general anaesthetics,

such as halothane (CF3CHBrCl) and enflurane

(CHF2OCF2CHFCl), decrease the gel-to-liquid crystalline phase

transition temperature of phospholipid bilayers and that this

effect can be reversed by application of moderate pressures to the

system. For example, in 1950 Johnson and Flagler reported that

tadpoles anaesthetized in 3–6 vol% EtOH solution awake upon

application of 140–350 bar of hydrostatic pressure.84 The

unusual anaesthetic effects of chemically inert gases, such as

xenon, nitrous oxide, and nitrogen narcosis under hyperbaric

conditions, have long provided an astonishing phenomenon for

neuroscience as well.

The question of specific sites at which various anaesthetics act

is still a matter of considerable debate. One of the earliest

quantitative theories of anaesthesia is attributed to Meyer and

Overton based on their discovery that the potencies of various

anaesthetic species are generally proportional to the solubility in

fatty acids (calibrated in olive oil at ambient pressure for gases at

that time, in octane or lipid bilayers later on).85 Those findings

led to the theory that anaesthetics dissolve in the lipid fraction of

the cell membrane, thus altering—by changes in membrane

fluidity, volume expansion, and lateral structure (which might be

reversed upon pressurization)—the physiological properties. It is

now thought that ion channels and neurotransmitter receptor


sites formed within the neuronal cell membranes constitute

essentially the primary sites of anaesthetic action.77 But it is also

likely that, depending on the concentration and pressure, both

effects play a decisive role.

Fig. 9 shows the effect of various gases on the gel-to-fluid

transition of DPPC bilayers as a function of pressure as revealed

by high pressure calorimetric, light scattering and fluorescence

anisotropy measurements.86–88 For comparison, the effect of

hydrostatic pressure is shown as well. The least lipid soluble of

the inert gases, He, fails to narcotise animals, it merely acts as

a pressure-transmitting fluid and exhibits a similar Clapeyron

slope of the main transition as hydrostatic pressure (dTm/dp z0.022 �C/bar).86,87,33 With increasing solubility in the fluid phase,

a reduction on Tm should be expected (similar to ‘‘freezing-point

depression’’). Nitrogen shows such a reduction in transition slope

(dTm/dp z 0.006 �C/bar). The anaesthetic gas nitrous oxide

(N2O) lowers the Tm drastically with increasing gas pressure (at

a rate of�0.5� 0.1 �C/bar), indicating an increasing solubility in

the lipid phase. Also Ar has been shown to exhibit slightly

negative slopes.86 Inert gases, such as N2O, may exert narcotic

effects apparently by a mechanism similar to that of the more

potent inhalational general anaesthetics.

Also the influence of local anaesthetics, such as tetracaine

(TTC), on the thermodynamic properties and the temperature

and pressure dependent phase behavior of phospholipids has

been studied.78–82 From volumetric measurements it has been

found that the main transition at ambient pressure shifts to

a lower temperature and the isothermal compressibility increases

in both lipid phases by addition of TTC, and the addition of the

TTC shifts the pressure-induced liquid-crystalline to gel phase

transition towards somewhat higher pressures. kT is drastically

reduced at the main transition point, enhanced, however, in the

direct neighborhood of the transition. Hence, the binding of the

anaesthetic in membranes seems to strongly couple to the

thermal density and concentration fluctuations of the lipid

system near its gel to liquid-crystalline phase transition, thus

leading to a strong enhancement of the volume fluctuations in the

Soft Matter, 2009, 5, 3157–3173 | 3163


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neighborhood of Tm. These findings might also be of biochemical

relevance, as in lipid bilayer membranes, strong density or

concentration fluctuations may be related to the transmembrane

permeability of ions and small molecules.21 Also the biochemical

action of local anaesthetics is still controversial as to whether or

not the action is lipid mediated. One thing which is clear,

however, is that they do strongly perturb the lipid bilayer system

and change their thermomechanical properties.

It is well known that the effect of inorganic ions on the melting

transition temperature of phospholipids may depend on the

nature of the ions and the charge of the lipid membrane.20,21 The

effect is especially pronounced when Ca2+ ions are adsorbed on

negatively charged membranes, because of the formation of more

or less stable complexes between the divalent ion and the phos-

phate group. To assess the effect of salts on the thermodynamic

parameters of the gel-to-fluid transition of zwitterionic and

anionic phospholipid bilayers, experiments were performed in

the presence of various salts, such as NaCl and CaCl2. The

addition of 0.1 M NaCl to the zwitterionic DPPC lipid dispersion

does not virtually change the transition temperature of DPPC. 20

mM CaCl2 raises the transition temperature by about 1 �C,

however. The dTm/dp transition slope of the main transition is

hardly affected by the addition of these salts, but the rates of the

enthalpy and volume changes decrease slightly upon addition of

the salts.39 Calorimetric measurements on DMPC/Ca2+ disper-

sions revealed that increasing Ca2+ concentration leads to an

increase in main transition temperatures with little change in

transition enthalpy, and also to an increase of the Lb0-Pb0 gel to

gel transition temperature, until both transitions merge at high

salt concentrations. The main transition is shifted towards

smaller pressures with increasing temperature in comparison to

that of pure DMPC dispersions. Otherwise, the transition slope

dTm/dp is parallel to that of pure DMPC, and the volume change

DVm at the main transition is of similar magnitude. A similar

behavior has been observed for the negatively charged lipid

DMPS with addition of Ca2+.26

Effects of peptide incorporation

Membrane proteins can constitute about 30% of the entire

protein content of a cell and so rest to a varying extent in the lipid

environment where they act as anchors, enzymes or transporters.

Membrane lipids and proteins influence each other directly as

a result of their biochemical nature and in reaction to environ-

mental changes. Pressure studies are still very scarce. Here, we

discuss the effect of the incorporation of the model channel

peptide gramicidin D (GD) on the structure and phase behavior

of phospholipid bilayers.89–91 Gramicidin is polymorphic, being

able to adopt a range of structures with different topologies.

Common forms are the dimeric single-stranded right-handed

b6.3-helix with a length of 24 A, and the antiparallel double-

stranded b5.6-helix, being approximately 32 A long. For

comparison, the hydrophobic fluid bilayer thickness is about

30 A for DPPC bilayers, and the hydrophobic thickness of the gel

phases is larger by 4–5 A. Depending on the gramicidin

concentration, significant changes of the lipid bilayer structure

and phase behavior were observed. These include disappearance

of certain gel phases formed by the pure DPPC system, and the

formation of broad two-phase coexistence regions at higher

3164 | Soft Matter, 2009, 5, 3157–3173

gramicidin concentrations (Fig. 5b) and vice versa, also the

peptide conformation is influenced by the lipid environment.

Depending on the phase state and lipid acyl-chain length,

gramicidin adopts at least two different types of quaternary

structures in the bilayer environment, a double helical pore (DH)

and a helical dimer channel (HD) (see Fig. 5). When the bilayer

thickness changes at the gel-to-fluid main phase transition of

DPPC, the conformational equilibrium of the peptide also

changes. In gel-like DPPC bilayers, the equilibrium of the

gramicidin species in the lipid bilayer is shifted in favor of the

longer double helical configuration.89 Hence, not only the lipid

bilayer structure and T,p-dependent phase behavior drastically

depends on the polypeptide concentration, but also the peptide

conformation (and hence function) can be significantly influ-

enced by the lipid environment. No pressure-induced unfolding

of the polypeptide is observed up to 10 kbar. For large integral

and peripheral proteins, however, pressure-induced changes in

the physical state of the membrane may lead to a weakening of

protein–lipid interactions as well as to protein dissociation.

Dynamical properties

Little is known about pressure effects on the dynamical proper-

ties of lipid bilayers at elevated pressures.7,15,18 Of particular

interest is the effect of pressure on lateral diffusion, which is

related to biological functions such as transport and membrane-

associated signalling processes. Pressure effects on the lateral self

diffusion coefficient, Dlat, of DPPC and POPC vesicles have been

studied by Jonas et al.18 The lateral diffusion coefficient of DPPC

in the liquid-crystalline phase decreases by about 30% from 1 to

300 bar at 50 �C. A further 70% decrease in the Dlat-value occurs

at the pressure-induced La to gel phase transition. Compared to

the lateral diffusion, the rotational dynamics of lipids and small

amphiphilic molecules in membranes seems to be less influenced

by high pressure.34,35 Notably, phospholipid flip-flop and lipid

transfer between membranes is also slowed down by high pres-

sure.92,93

Nonlamellar lipid phases

For a series of lipid molecules, nonlamellar lyotropic phases are

observed as thermodynamically stable phases or as long-lived

metastable phases after special sample treatment, including

pressurization23–28,94–96 (Fig. 2). Lipids, which can adopt

a hexagonal phase, are present at substantial levels in biological

membranes, usually with at least 30 mol% of the total lipid

content. Fundamental cell processes, such as endo- and exocy-

tosis, fat digestion, membrane budding and fusion, involve

a rearrangement of biological membranes where such non-

lamellar highly curved lipid structures are probably involved, but

probably also static cubic structures (cubic membranes) occur in

biological cells.24 The cubic lipid phases are mostly bicontinuous

unilamellar lipid bilayer phases with periodic three-dimensional

order (Fig. 2b).

In recent years, the temperature and pressure dependent

structure and phase behavior of a series of phospholipid systems,

such as phospholipid/fatty acid mixtures (e.g., DLPC/LA,

DMPC/MA, DPPC/PA) and monoaclyglycerides (MO, ME),

exhibiting nonlamellar phases have been studied.23–27,94–96 Here,



Fig. 10 T,p-phase diagram of DOPE in excess water.

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we just discuss one example. Contrary to DOPC which shows

a lamellar Lb-to-La transition, only (Fig. 4) the corresponding

lipid DOPE with ethanolamine as (smaller) headgroup exhibits

an additional phase transition from the lamellar La to the non-

lamellar, inverse hexagonal HII phase at high temperatures

(Fig. 10). As pressure forces a closer packing of the lipid chains,

which results in a decreased number of gauche bonds and kinks in

the chains, both transition temperatures, of the Lb-La and the

La-HII transition, increase with increasing pressure. The La-HII

transition observed in DOPE/water and also in egg-PE/water

(egg-PE is a natural mixture of different phosphatidylethanol-

amines) is the most pressure-sensitive lyotropic lipid phase

transition found to date (dT/dp z 40 �C/kbar). The reason why

this transition has such a strong pressure dependence is the

strong pressure dependence of the chain length and volume of its

cis-unsaturated chains. Generally, at sufficiently high pressures,

hexagonal and cubic lipid mesophases give way to lamellar

structures as they exhibit smaller partial lipid volumes. Inter-

estingly, in these systems inverse cubic phases QIID and QII

P can

be induced in the region of the La-HII transition by subjecting the

sample to extensive temperature or pressure cycles across the

phase transition.26–28 It has been shown that for conditions,

which favor a spontaneous curvature of a lipid monolayer which

is not too high, the topology of an inverse bicontinuous cubic

phase (Fig. 2b) can have a similar or even lower free energy than

the lamellar or inverse hexagonal phase, as the cubic phases are

characterized by a low curvature free energy and do not suffer

the extreme chain packing stress predominant in the HII-phase.

Kinetic studies

Phase transitions between lipid mesophases are associated with

deformations of the interfaces which, very often, imply also their

fragmentation and fusion. Depending on the topology of the

structures involved, transition phenomena of different

complexity are observed. We have used the pressure-jump tech-

nique in conjunction with synchrotron X-ray diffraction to study

the time course of lipid phase transitions and to search for

possible transient intermediate structures, with a view to

unravelling the underlying transition mechanisms.26,27,97–103 The

pressure-jump technique offers several advantages over the

temperature-jump approach: (1) pressure propagates rapidly so

that sample inhomogeneity is a minor problem. (2) Pressure-

jumps can be performed bidirectional, i.e., with increasing or

decreasing pressure. (3) In the case of fully reversible structural

changes of the sample, pressure-jumps can be repeated with


identical amplitudes to allow for an averaging of the diffraction

data over several jumps and hence an improvement of the

counting statistics.

Here, we focus on an interesting phase transition where

membrane fusion occurs, a fluid lamellar to inverse bicontinuous

cubic phase transition. Models for the process of membrane fusion

between apposed lipid bilayers have been proposed by a number of

groups.104,105 All rely on the formation of transient lipid contacts

known as stalks, which subsequently break through to form the

beginnings of the tubular connections (fusion pores) that are the

fundamental connecting element in the inverse bicontinuous cubic

phases, which consist of ordered arrays of such connections. As an

example of a lamellar to cubic lipid phase transformation, we

present data on the monoolein–water system. A pressure-jump

from 1500 to 1 bar was used to induce the La / Ia3d transition at

20 wt% H2O and 35 �C. The time-dependent SAXS patterns after

the jump are shown in Fig. 11a. The first lamellar reflection (001) of

the La phase and, at longer times, the reflections (O6, O8, O14,

O16,.) of the developing Ia3d phase are clearly visible. The cor-

responding intensities and lattice constants as a function of time are

plotted in Fig. 11 as well. The system starts in the lamellar phase

with an initial lattice parameter of a¼ 44 A. The intensity of the La

phase decays rapidly while the cubic phase Ia3d is formed

concomitantly. The first peak of Ia3d appears after 1.5 s, and the

intensity of the diffraction peaks of the La phase vanishes at�2.5 s.

During the phase transition, the lamellar lattice constant decreases

from 43.4 to 42.3 A, while that of the cubic phase decreases from an

initial high value of 114.2 A (more swollen state) to an equilibrium

value of 109.1 A. Hence, within the accuracy and time-resolution of

the measurement, a two-state transition can be assumed. Following

the step change in thermodynamic conditions by the pressure-

jump, the lamellar phase shrinks to a specific threshold inter-

lamellar spacing. This shrinkage of the lamellar lattice is probably

occurring at the same time that stalks are formed between bilayers

(Fig. 12b). In natural membranes, it is very likely that membrane

fusion-mediating proteins have to act by reducing the energy

necessary to create these fusion intermediates. A similar scenario

has been found for other lipid systems undergoing lamellar to cubic

phase transitions.102

Generally, as has also been found in studies of lipid mesophase

transitions of other systems, the relaxation behavior and the

kinetics of pressure-induced lipid phase transformations depend

drastically on the topology of the lipid mesophase, and also on

the temperature and the driving force, i.e., the applied pressure

jump amplitude Dp.26,27,103,106–108 Often multicomponent kinetic

behavior is observed, with short relaxation times (probably on

the nanosecond to microsecond time-scale) referring to the

relaxation of the lipid acyl-chain conformation in response to the

pressure change. Longer relaxation times as measured here are

due to the kinetic trapping of the system. In most cases the rate of

the transition is limited by the transport and redistribution of

water into and in the new lipid phase, and the obstruction factor

of the different structures, especially in cases where nonlamellar

(hexagonal and cubic) phases are involved. In addition, nucle-

ation phenomena and domain size growth of the structures

evolving play a role. Slow relaxation processes may correspond

to fluctuations in domain size, i.e. domain growth. Maximum

values of up to �1 min have been observed at the melting tran-

sition of pure phospholipids.21

Soft Matter, 2009, 5, 3157–3173 | 3165


Fig. 12 Activity k (in arbitrary units) of Na+, K+-ATPase—as measured

using an enzymatic assay—at selected pressures and T ¼ 37 �C. The free

energy of hydrolysis of one ATP molecule is converted to up-hill trans-

port by actively transporting 3 Na+ out of and 2 K+ into the cell.

Fig. 11 Top: time-resolved SAXS data of monoolein (MO) in 20 wt% H2O at 35 �C. At time zero, a 5 ms pressure-jump from 1500 to 1 bar induces

a phase transition from the lamellar La to the Ia3d cubic phase (a schematic view of the two phases is added). Indexing of the Bragg reflections is

indicated. Bottom: the corresponding intensities and lattice constants of the corresponding phases as a function of time.

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The pressure-jump relaxation technique may also be applied to

more complex biochemical processes. For example, it has been

applied to study the pressure dependence of the photocycle

3166 | Soft Matter, 2009, 5, 3157–3173

kinetics of bacteriorhodopsin (bR) from Halobacterium salina-

rium.109 Certainly, time-resolved studies of lyotropic lipid phase

transitions are still at an early stage, but clearly will be invaluable

for helping to clarify transition pathways and mechanisms.

Biological and reconstituted membranes

The cytoplasmic membrane is a complex heterogeneous aggre-

gate structure, largely held together by the hydrophobic effect,

that can be disturbed by rather low pressures in its structure and

function. The integrity and functionality of natural membranes

are vital for the cell, e.g., for energy generation, transport, sig-

nalling processes, and maintenance of osmotic pressure and

intracellular pH. Although some ion transporters are unaffected

or even activated upon mild compression, certain other channels

and pumps are inactivated at moderate pressures. It has generally

been observed, however, that at sufficiently high pressures of

several kbar, membrane protein function ceases, and integral and

peripheral proteins may even become detached from the

membrane when its bilayer is sufficiently ordered by pressure,

and depolymerization of cytoskeletal proteins may be involved as

well.110



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In a detailed study, the influence of hydrostatic pressure on the

activity of Na+, K+-ATPase enriched in the plasma membrane

from rabbit kidney outer medulla was studied using a kinetic

assay that couples ATP hydrolysis to NADH oxidation.64 The

data shown in Fig. 12 reveal that the activity, k, of Na+, K+-

ATPase is inhibited by pressures below 2 kbar. The plot of lnk vs.

p revealed an apparent activation volume of the pressure-induced

inhibition reaction which amounts to DVs ¼ 47 mL mol�1. At

higher pressures, exceeding 2 kbar, the enzyme is inactivated

irreversibly, in agreement with literature data.62,65 Kato et al.63

suggested that the activity of the enzyme shows at last three step

changes induced by pressure: at pressures below and around

1 kbar, a decrease in the fluidity of the lipid bilayer and

a reversible conformational change in the transmembrane

protein is induced, leading to functional disorder of the

membrane associated ATPase activity. Pressures of 1–2 kbar

cause a reversible phase transition and the dissociation or

conformational changes in the protein subunits, and pressures

higher that 2200 bar irreversibly destroy the membrane structure

due to protein unfolding and interface separation. In fact, pressure

dissociation of water-soluble oligomeric proteins in this pressure

range is well documented as well.1,3,7 To be able to explore the effect

of the lipid matrix on the enzyme activity, the Na+, K+-ATPase was

also reconstituted into various lipid bilayer systems of different

chain length, configuration, phase state and heterogeneity

including model raft mixtures. Interestingly, in the low-pressure

region, around 0.1 kbar, a significant increase of the activity was

observed for the enzyme reconstituted into DMPC and DOPC

bilayers. We found that the enzyme activity decreases upon further

compression, reaching zero activity around 2 kbar for all recon-

stituted systems measured, similar to the natural system.

A similar behavior has been found for the chloroplast ATP-

synthase. This enzyme is a H+/ATP-driven rotary motor in which

a hydrophobic multi-subunit assemblage rotates within a hydro-

philic stator, and subunit interactions dictates alternate-site

catalysis. Souza et al.111 used hydrostatic pressure to induce

conformational changes and/or subunit dissociation, and the

resulting changes in the ATPase activity and oligomer structure

were evaluated. Under moderate hydrostatic pressures (up to

0.8 kbar), the ATPase activity increased by 1.5-fold, which did

not seem to be related to an increase in the affinity for ATP, but

rather seemed to correlate with an enhanced turnover induced by

pressure. The activation volume determined for the ATPase

reaction was found to be�23.7 mL mol�1. Higher pressures of up

to 2 kbar lead to dissociation of the enzyme. At these high

pressures, dissociation seemed to impair the contacts needed for

rotational catalysis.

The effect of pressure was also determined for the HorA

activity of L. plantarum,66 an ATP-dependent multi-drug-resis-

tance transporter of the ABC family. Changes were determined

in the membrane composition of L. plantarum induced by

different growth temperatures and their effect on the pressure

inactivation, and a temperature-pressure phase diagram was

constructed for the L. plantarum membranes that could be

correlated with the respective kinetics of high pressure inactiva-

tion. Upon pressure-induced transitions to rigid (e.g., gel-like)

membrane structures, at pressures around 0.5–1.5 kbar for

temperatures between 20 and 37 �C, fast inactivation of HorA

was observed.


The effect of pressure and the influence of the lipid matrix on

lipid-protein interactions was also studied for the multidrug

resistance protein LmrA, which was expressed in L. lactis and

functionally reconstituted in different model membrane systems.67

The membrane systems were composed of DMPC, DOPC,

DMPC + 10 mol% Chol, and the model raft mixture DOPC :

DPPC : Chol (1 : 2 : 1). Teichert showed that a sharp pressure-

induced fluid-to-gel phase transition without the possibility for

lipid sorting, such as in DMPC bilayers, has a drastic inhibitory

effect on the LmrA activity. As inferred from the experiments

performed so far, inactivation of membrane protein function upon

entering a rigid gel-like (solid-ordered) membranous state seems to

be a rather common phenomenon.112 Otherwise, an overall fluid-

like membrane phase over the whole pressure-range covered, with

suitable hydrophobic matching, such as for DOPC, prevents the

membrane protein from total high pressure inactivation even up to

2 kbar. Also the systems exhibiting thicker membranes with higher

lipid order parameters, such as DMPC/10 mol% Chol and the

model raft mixture, show remarkable pressure stabilities. The

results also revealed that an efficient packing with optimal lipid

adjustment to prevent (also pressure-induced) hydrophobic

mismatch might be a particular prerequisite for the homodimer

formation and hence function of LmrA.

Recently, pressure has also been found to be of interest for

understanding perturbations of signalling events, such as phos-

pholipase activation by G-proteins or ToxR/ToxS interac-

tion.113,68 Scarlata revealed perturbations of phospholipase

PLb-Gbg association caused by the Ga(GDP) subunit in the

membranous context that where not observable by atmospheric

association measurements.113 PLCb membrane binding was stable

throughout the 1–2000 bar range, Gbg only at high concentra-

tions, whereas Ga(GDP) dissociated from membranes above

1 kbar. Recently, we also studied HHP induced dimer dissociation

of membrane proteins in vivo with Vibrio cholerae ToxR variants

in E. coli reporter strains carrying ctx:lacZ fusions. Dimerization

ceased at 0.2 to 0.5 kbar, depending on the nature of the trans-

membrane segment rather than on changes in a pressure-induced

lipid bilayer environment.68 Pressure experiments on ion channels

at high pressure are still scarce as well. They revealed that high

pressures below 1 kbar affect the kinetics of gating (generally,

a retardation is observed due to a positive activation volume,

possibly owing to a conformational change following a change in

dipole moment) but not the conductance of the channel.92

The study of high-pressure effects on membrane proteins and

in particular on ion channels114 and signalling processes is still in

its infancy, but there are sufficient published experiments now to

encourage further work in this growing area. What is already

clear is that the membrane0s physical-chemical effects markedly

influence the lipid–protein interaction, activity and pressure

stability of the embedded membrane protein. It also seems to be

clear that the specific nature of the membrane protein (e.g.,

oligomeric assembly, a required dimerization reaction upon

signalling, etc.) plays an important role in its pressure-dependent

stability and activity.

Membrane mimetics—surfactants and microemulsions

Also the phase preference of surfactants is essentially related to

the overall shape of the molecules under the experimental

Soft Matter, 2009, 5, 3157–3173 | 3167


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conditions used. The molecular shape is in turn dependent on the

relative sizes of the polar and nonpolar regions of the amphiphilic

molecules, in particular on the relative cross-sectional areas

occupied by the polar headgroup and nonpolar hydrocarbon

chains, and these strongly depend on temperature, pressure and

the ionic strength. If the effective cross-sectional area occupied by

the polar headgroup significantly exceeds that occupied by the

nonpolar region of the amphiphile (Fig. 1), then these wedge-

shaped molecules aggregate in water to form spherical or elliptical

micelles. So far, only few studies have been carried out on the

effect of pressure on surfactant phases, though pressure studies on

these systems are of interest not only from a physical-chemical

point of view, but they are also of biochemical interest, for

example, regarding the modulation of enzyme activity in micelles

by pressure or protein extraction by surfactants.7

A few studies have been performed on the effect of pressure on

the critical micelle concentration (cmc) of ionic and nonionic

surfactants (e.g., SDS) in aqueous solutions.115–119 It has been

shown that the cmc vs. pressure curves have a maximum in the

pressure range 0.001–4 kbar. The initial compression causes the

dissociation of micelles whereas successive compression above

a certain pressure causes aggregation of monomers to micelles

again. These data imply that the partial molar volume change

upon micellation, DVmic, is positive in the lower pressure region

and negative for the higher pressures. The similar characteristic

pressure dependence of the cmc for both, ionic and nonionic

surfactants, as well as the existence of a marked alkyl chain

length dependence for ionic surfactants suggest that the effect of

pressure is largely independent of the hydrophilic headgroup of

the surfactant.

Compression of concentrated micellar systems and micro-

emulsions (e.g. sodium decanoates, TDMAO, AOT-water-

decane microemulsions, etc.) can lead to changes in aggregation

number and will finally induce aggregate structures with smaller

partial molar volumes of their amphiphiles, such as coagel or

ordered lamellar states.120–127

Fig. 13 (a) SANS patterns of a 10 wt% SDS solution in D2O as a function of

SDS solution in D2O as a function of pressure from 1 to 2000 bar (T ¼ 20 �

a 50 wt% SDS solution in D2O as a function of pressure from 50 to 2000 ba

3168 | Soft Matter, 2009, 5, 3157–3173

We have recently looked into pressure effects on an important

surfactant, sodium dodecyl sulfate ([C12H25OSO3�]Na+, SDS,

dispersed in water.127 Above �20–25 �C and up to a concentra-

tion of about 35 wt%, SDS forms spherical micelles of a diameter

of about 36 A. At higher concentrations, elongated micellar

structures and other lyotropic liquid-crystalline mesophases are

found, such as a hexagonal (HI), and lamellar La phase as well as

a series of further 2- and 3-dimensional phases including an

isotropic cubic phase.127 The mesophases of SDS in water form

above room temperature only because hydrated crystals of SDS

coexist with water in the low temperature region. We performed

SAXS and SANS measurements on 5–75 wt% SDS solutions at

selected temperatures up to pressures of about 2 kbar. Fig. 13a

depicts SANS patterns of a 10 wt% SDS solution at selected

pressures for T ¼ 30 �C. A broad peak around a momentum

transfer Q ¼ 0.086 A�1 appears, which represents the inter-

micellar correlation peak of the SDS micelles. The peak position

corresponds to a mean intermolecular distance dinter of the

micelles of about 90 A and, up to a pressure of 2 kbar, no

structural changes occur. Fig. 13b displays the corresponding

SANS data for a 30 wt% SDS solution at 20 �C. With increasing

pressure, already above �100 bar, the correlation peak shifts

markedly towards smaller Q-values, i.e. larger distances, which

might be explained by the continuous formation of elongated

micellar particles upon pressurization. Above about 1 kbar, no

small-angle peak is detected any more and the diffuse small-angle

scattering below 0.02 A�1 drastically increases—a broad phase

transition to a new, possibly crystalline, mesophase with smaller

partial molar volume, occurs. An increase of temperature shifts

this transition to higher pressures. For example, at 30 �C, the

onset of the shift of the correlations peak sets in around 1000 bar,

with a concomitant peak appearing at 0.2 A�1 (data not shown),

which indicates formation of coexisting SDS crystals. In Fig. 13c,

the pressure-effect on the hexagonal phase of SDS in a 50 wt%

solution at 30 �C is shown. At Q¼ 0.14 A�1, the (10) reflection of

the HI phase is seen, which corresponds to a lattice constant of

pressure from 1 to 2000 bar (T ¼ 30 �C). (b) SANS patterns of a 30 wt%

C). Only the intermediate Q-range is shown here. (c) SANS patterns of

r (T ¼ 30 �C).



Fig. 14 (a) Small-angle X-ray scattering patterns of the lipid mixture

DMPC/DHPC (3.2 : 1, 15 %wt lipid in water) between 6 and 62 �C at

ambient pressure (1 bar). In the bicellar phase, the scattering intensity

I(Q) is flat at low Q, which can be fitted with a model of disc-shaped

objects. In the intermediate temperature range, a nematic phase is found.

The broad peak observed corresponds to the average interparticle sepa-

ration, At small Q-values, I(Q) � Q�1, indicating the presence of elon-

gated, wormlike structures. The nematic phase gives way to

a multilamellar phase with equally spaced Bragg reflections. The corre-

sponding d-spacings are observed at Q¼ 2ph/d, where h is an integer and

d is the lamellar periodicity. (b) Small-angle X-ray scattering patterns of

the pressure dependent measurement on the lipid system DMPC/DHPC

(3.2 : 1) at 61.9 �C.

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a ¼ 51.5 A. Above about 1 kbar, a phase transition occurs,

possibly to a micellar-crystalline phase coexistence region again, as

indicated by an additional peak appearing around 0.2 A�1, which

corresponds to a d-spacing of the crystalline phase of 31.4 A.

Hence, we note that an increase of pressure of a few hundred to

thousand bars in surfactant systems leads to drastic effects on

their structure and phase behavior. In this micellar system, the

low volume high pressure phase is a crystalline phase. The

micellar system tetradecyl-trimethylammonium bromide (TTAB,

C14H29N(CH3)3Br)) exhibits a similar behavior.125 Upon pres-

surization, the system undergoes a phase transition from a liquid

micellar to crystalline solid phase (hydrated crystals) with an

intermediate two-phase coexistence region.

Notably, enzymes entrapped in reverse micelles were found to

have a different, compared to aqueous solution, response to pres-

sure application. It has been demonstrated that the application of

hydrostatic pressure to enzymes placed in surfactant nano-

containers, can bring additional advantages for both increasing the

enzyme stability and modulating the enzyme activity. For some

proteins, such as liver alcohol dehydrogenase (LADH), a drastic

increase in the enzyme catalytic activity has been observed upon

increasing the pressure in such a system.7 The lipid confinement

may stabilize the transition state and help facilitate substrate

desorption from the enzyme active site that is the limiting step of

the catalytic process. Ternary amphiphilic systems have also been

used as mimetic for cellular conditions—including macromolec-

ular crowding and the presence of lipid interfaces—for studying

pressure induced unfolding reactions of proteins.128

In high pressure SANS and FT-IR studies, the effect of micellar

confinement of bis(2-ethylhexyl)-sodium sulfosuccinate (AOT)-

octane-water was studied on the pressure-induced unfolding

behavior of enzymes such as a-chymotrypsin.101,129 In the

confinement, the protein is located in the water pools and does not

adsorb to AOT, at ambient pressures. The unfolding pressure of

7.5 kbar at ambient temperature is not altered, the confinement

fosters more complete, random coil-like unfolding and subse-

quent aggregation after pressure release, however, which might be

due to the combined effect of confinement and the possibility to

accommodate exposed hydrophobic residues in the apolar part of

the surrounding matrix. But upon pressurization, pressure-

induced lipid phase transitions, such as those from the micellar

(L2) to a lamellar phase (L2-to-La transition) occurs. For

confinement studies, bicontinuous cubic phases have also been

explored. The effects of protein entrapment on the structure and

phase behavior of cubic mesostructures, such as the cubic Ia3d

phase of monoolein (MO) have been examined by synchrotron

small-angle X-ray diffraction and FT-IR spectroscopy.128 The

stability of the protein depends on the relative size of the protein

and lipid confinement, respectively. While the secondary struc-

ture of cyt c remains unaffected in the confining lipid environ-

ment, the structure of the larger a-chymotrypsin gets destabilized

slightly, and the protein tends to aggregate even at relatively low

concentrations. But also the lipid matrix is prone to undergo

structural changes. Above a critical protein concentration, even

new micellar cubic phases may be formed. Hence, protein stabi-

lisation or enzymatic reactions in reversed micellar systems is only

feasible in a restricted pressure-temperature range.

Whereas the effect of pressure on one-component phospho-

lipid systems is rather straightforward to predict now, the effects


of pressure on mixtures of long-chain phospholipids and short-

chain detergents are more difficult to foresee. Such mixtures are

able to form disk-like micelles (bicelles), which have also emerged

as important substrates for high-resolution NMR studies of

biomolecules. Depending on the mixing ratio of the constituents,

the total lipid concentration as well as temperature, also addi-

tional liquid-crystalline phases, such as a nematic and lamellar

phase, have been observed in these systems.130,131 Here we report

on the first measurements of the effects of pressure on the system

DMPC/DHPC. This system, which forms magnetically alignable

bicellar structures, has also been explored for high pressure

NMR studies of proteins.132 The preference for the use of bicelles

over micelles in NMR studies of membrane-peptide interactions

is that the bicelle interior consists of a true bilayer arrangement,

necessary for the proper conformation and activity of

a membrane protein. The temperature dependent phase behavior

of the binary lipid system DMPC/DHPC is well established.130 At

low temperatures, the system forms bicelles. Upon increasing the

Soft Matter, 2009, 5, 3157–3173 | 3169


Fig. 15 Tentative p,T-phase diagram of the binary lipid mixture DMPC/

DHPC (3.2 : 1, 15 %wt lipid in water).

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temperature, a transition to a nematic phase is observed which

gives way to formation of multilamellar vesicles with porelike

defects in the bilayers above about 50 �C.130,131 In Fig. 14a, we

show the effect of temperature on the SAXS pattern of a DMPC/

DHPC (3.2 : 1; 15 wt% lipid in water) mixture at ambient pres-

sure. At the initial temperature of 6 �C, the system is in the

bicellar phase. The thickness of the bilayer in the bicelles is

approximately 4 nm and the diameter of the disks is � 60 nm.130

Between 15 and 25 �C, a continuous transition to the nematic

phase takes place. This phase consists of elongated flexible lipid

aggregates, wormlike micelles. At �49 �C, a Bragg peak can be

observed in the scattering pattern, corresponding to the formation

of stacked lamellar bilayers in the system with a lamellar d-spacing

of 7.1 nm, which shifts to slightly smaller values with increasing

temperature. The d-spacings comprise the sum of the lipid bilayer

thickness and the interlamellar water layer around the lipid

headgroups (d ¼ dlipid + dwater). The nematic-lamellar phase

coexistence region extends at least up to 57.2 �C. The SAXS data

of the pressure-dependent measurements at T ¼ 61.9 �C are

depicted in Fig. 14b. The d-spacing of the initial single lamellar

phase increases slightly with increasing pressure, from 6.9 nm at

ambient pressure to a final value of 7.1 nm at 1 kbar. At this

pressure, the lamellar phase disappears and a nematic phase is

induced, which is stable up to 2.2 kbar. At higher pressures,

between 2.2 and 2.6 kbar, a two-phase region is observed, followed

by the pure bicellar phase at pressures above 2.6 kbar. Fig. 15

displays the corresponding p,T-phase diagram of the system.

Osmotic vs. hydrostatic pressure effects

In the 1980s, Parsegian et al. introduced a new approach,

osmotic stress (pressure), to investigate the role of water mole-

cules in biological processes.133 In this approach, osmolytes were

used to decrease the activity of water, which can be varied by the

addition of osmolytes (mainly polyols such as polyethylene-

glycol). The generated osmotic stress affects any water molecules

that are implicated in the conformation or activity of the

biomolecule (e.g., lipid headgroup or protein). By measuring

characteristic system parameters as a function of osmotic pres-

sure, it is possible to determine the number of water molecules

associated to the biomolecule that play a key role in the process.

Numerous examples have demonstrated the validity of this

methodology, as in ligand binding reactions to proteins,

3170 | Soft Matter, 2009, 5, 3157–3173

DNA-protein interactions, protein–protein complex formation

and lipid bilayer interaction and fusion.133–140

For example, the osmotic stress method has been applied to

determine interbilayer forces in multilamellar lipid systems, such

as the repulsive hydration pressure, which is essentially expo-

nential with decay distances of several tenth of a nm. As

a consequence of the strong mutual repulsion when the lipid

membranes are approaching each other, the bilayers thicken, the

molecular area decreases, the dipoles of the headgroups become

more perpendicular to the plane to the bilayer, and the main

transition temperature Tm increases. Dehydration of the hydro-

philic membranes has also been shown to induce phase separa-

tion and lamellar-to-hexagonal-II phase transitions. Also, partial

dehydration of phospholipid membranes is a prerequisite to the

fusion of membranes.139

But clearly, hydrostatic pressure—as discussed in this paper—

and osmotic stress (pressure) perturbation are not probing the

same process. The volume change measured with hydrostatic

pressure is a complex parameter, taking into account the volume

change resulting from a conformational change of the biomole-

cule (including void volume contributions) subjected to pressure

and a hydration contribution taking into account the groups

exposed to the solvent. The volume change measured with

osmotic pressure would rather reflect the biomolecule-bound

water molecules displaced when the activity of the water is

decreased. The number of these osmotically active water mole-

cules can be determined taking into account the partial molar

volume of the water. Hence, the volume changes measured by

osmotic pressure and hydrostatic pressure are generally different.

Hydrostatic pressure probes differences in density, whereas

osmotic pressure probes a difference in the number of water

molecules associated with different conformational states of the

biomolecule. Increasing osmotic stress drives a system from the

more hydrated state to a less hydrated one according to the

number of solute-inaccessible waters that connect the two states.

In fact, both pressures can induce opposite or antagonistic

effects. For example, hydrostatic pressure generally leads to an

increase of hydration upon pressure-induced unfolding of

proteins, osmotic stress to desolvation.

Concluding remarks

We conclude that pressure work on lipid and surfactant systems

can yield a wealth of enlightening new information on their

structure, energetics and phase behavior and on the transition

kinetics between mesophases, and might promise fulfilment of

the challenge set forth by W. Kauzmann: ‘‘Until more searching

is done in the darkness of high-pressure studies, our under-

standing of the hydrophobic effect must be considered incom-

plete’’.141 It is clear that the application of the pressure variable in

this research area has only just started and many interesting

results are expected in the near future. These pressure studies are

also important for the development of new technological and

pharmaceutical applications, such as high pressure food pro-

cessing, tissue engineering and micellar baroenzymology.7,13,142

Abbreviations

PC

This

phosphatidylcholine

PE
phosphatidylethanolamine
journal is ª The Royal Society of Chemistry 2009


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PS

This journal is ª T

phosphatidylserine

FA
fatty acid
LA
lauric acid
MA
myristic acid
PA
palmitic acid
SA
stearic acid
MO
monoolein
ME
monoelaidin
DMPC
1,2-dimyristoyl-sn-glycero-3-
phosphatidylcholine (di-C14:0)

DMPS
1,2-dimyristoyl-sn-glycero-3-
phosphatidylserin (di-C14:0)

DPPC
1,2-dipalmitoyl-sn-glycero-3-

DPPE
1,2-dipalmitoyl-sn-glycero-3-
phosphatidylethanolamine (di-C16:0)

DSPC
1,2-distearoyl-sn-glycero-3-

DOPC
1,2-dioleoyl-sn-glycero-3-phosphatidylcholine
(di-C18:1,cis)

DOPE
1,2-dioleoyl-sn-glycero-3-
phosphatidylethanolamine (di-C18:1,cis)

DEPC
1,2-dielaidoyl-sn-glycero-3-
phosphatidylcholine (di-C18:1,trans)

POPC
1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphatidylcholine (C16:0,C18:1,cis)

DHPC
dihexanoyl phosphatidylcholine
egg-PE
egg-yolk phosphatidylethanolamine
SDS
sodium dodecylsulfate
AOT
bis[2-ethylhexyl]sulfosuccinate
TDMAO
tetradecyldimethylaminoxide
CiEj
n-alkyl polyoxyethylene ether
TTAB
tetradecyl-trimethylammonium bromide
GD
gramicidin D
HHP
high hydrostatic pressure
Chol
cholesterol
SM
sphingomyelin
DSC
differential scanning calorimetry
SAXS (WAXS)
small(wide)-angle X-ray scattering
SANS
small-angle neutron scattering
FT-IR
Fourier-transform infrared
NMR
nuclear magnetic resonance
GUV
giant unilamellar vesicle
TTC
tetracaine
AFM
atomic force microscopy.
Acknowledgements

Financial support from the Deutsche Forschungsgemeinschaft

(DFG) is gratefully acknowledged.

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