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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: roland.winter@tu-dortmund.de
† 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
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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,
This journal is ª The Royal Society of Chemistry 2009
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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.
This journal is ª The Royal Society of Chemistry 2009
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
This journal is ª The Royal Society of Chemistry 2009
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.
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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
This journal is ª The Royal Society of Chemistry 2009
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
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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,
This journal is ª The Royal Society of Chemistry 2009
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
This journal is ª The Royal Society of Chemistry 2009
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.
This journal is ª The Royal Society of Chemistry 2009
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).
This journal is ª The Royal Society of Chemistry 2009
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
This journal is ª The Royal Society of Chemistry 2009
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
phosphatidylethanolaminejournal is ª The Royal Society of Chemistry 2009
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PS
This journal is ª T
phosphatidylserine
FA
fatty acidLA
lauric acidMA
myristic acidPA
palmitic acidSA
stearic acidMO
monooleinME
monoelaidinDMPC
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-phosphatidylcholine (di-C16:0)
DPPE
1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (di-C16:0)
DSPC
1,2-distearoyl-sn-glycero-3-phosphatidylcholine (di-C18:0)
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 phosphatidylcholineegg-PE
egg-yolk phosphatidylethanolamineSDS
sodium dodecylsulfateAOT
bis[2-ethylhexyl]sulfosuccinateTDMAO
tetradecyldimethylaminoxideCiEj
n-alkyl polyoxyethylene etherTTAB
tetradecyl-trimethylammonium bromideGD
gramicidin DHHP
high hydrostatic pressureChol
cholesterolSM
sphingomyelinDSC
differential scanning calorimetrySAXS (WAXS)
small(wide)-angle X-ray scatteringSANS
small-angle neutron scatteringFT-IR
Fourier-transform infraredNMR
nuclear magnetic resonanceGUV
giant unilamellar vesicleTTC
tetracaineAFM
atomic force microscopy.Acknowledgements
Financial support from the Deutsche Forschungsgemeinschaft
(DFG) is gratefully acknowledged.
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