The role of calcium in membrane condensation and spontaneous curvature variations in model lipidic systems

This journal is c the Owner Societies 2011 Phys. Chem. Chem. Phys., 2011, 13, 3115–3125 3115

The role of calcium in membrane condensation and spontaneous

curvature variations in model lipidic systemsw

Anan Yaghmur,*a Barbara Sartorib and Michael Rappolt*b

Received 30th June 2010, Accepted 30th September 2010

DOI: 10.1039/c0cp01036g

In this study, the dynamical behaviour of calcium-induced disordered to well-ordered structural

transitions has been investigated by time-resolved synchrotron small-angle X-ray scattering

(SAXS) in the milliseconds to seconds range. The in situ monitoring of the formed

non-equilibrium self-assembled structures was achieved by the successful combination of

synchrotron SAXS with stopped flow measurements. The effect of the rapid mixing of aqueous

dispersions of dioleoylphosphatidyalglycerol (DOPG)/monoolein (MO) with low concentrations

of Ca2+ ions is reported. Under static conditions and in the absence of Ca2+ ions, the evaluation

of SAXS data for DOPG/MO aqueous dispersions prepared with three different DOPG/MO

molar ratios indicates the formation of either a sponge-like L3 phase or uncorrelated bilayers.

Clearly, the lipid composition plays a vital role in modulating the structural behaviour of these

aqueous dispersions in the absence and also in the presence of Ca2+ ions. The rapid-mixing

experiments revealed that the fast and strong interactions of Ca2+ ions with the negatively

charged DOPG/MO membranes triggers the transformation from the L3 phase or the

uncorrelated bilayers to the well-ordered dehydrated La phase or to inverted type bicontinuous

cubic phases, V2, with either a symmetry of Pn3m or Im3m. Additionally, we recently reported

(A. Yaghmur, P. Laggner, B. Sartori and M. Rappolt, PLoS ONE, 2008, 3, e2072) that low

concentrations of Ca2+ ions trigger the formation of the inverted type hexagonal (H2) phase in

DOPG/MO aqueous dispersions with a molar DOPG/MO ratio of 30/70. These are also

temperature-sensitive structural transitions. Intriguingly, the strong association of Ca2+ ions with

the negatively charged DOPG/MO membranes leads to fast re-organization of the two lipids and

simultaneously induces fast tuning of the curvature.

1. Introduction

Self-assembly of biologically relevant lipids in aqueous media,

which induces a variety of well-defined nanostructures, is

attracting ever-growing interest in basic and applied research

due to its promising applications and biological relevance.

The self-assembled nano-objects are attractive due to their

potential applications in designing drug and food nanocarriers

with important and beneficial effects,2–6 and also due to their

remarkable resemblance to the highly ordered lipidic domains

observed in biological systems.7–9 In particular, there is great

interest in utilizing these self-assembled systems for the

solubilization and the controlled release of active materials,2,4,10

and in understanding how cells sense and respond to changes

in lipid composition and lipid–protein interactions.7–9 An

improved understanding of the biological role and the

potential utility of these self-assembled systems can be

achieved by studying both their nanoscale characteristics and

their static and dynamical structural transitions induced by

molecules with biological relevance such as peptides, proteins,

and metal ions.

In the literature, most studies are focused on characterizing

and mapping various binary and ternary phase behaviours of

biologically relevant lipids in aqueous media under static and

equilibrium conditions.11–18 Among them, phospholipids15–20

and monoglycerides13,14,21–23 are a unique class of lipids that

display complex phase behaviour in aqueous media. Examination

of the structural changes in these systems has been

performed under different experimental conditions of varying

temperature,13,14,21,24 pressure,25,26 lipid composition,27–29 and

in the presence of hydrophilic or hydrophobic guest

molecules.2,24,30,31 However, the number of studies that report

on the dynamical behavior of lipid-based self-assembled

systems including the formation of non-equilibrium structures

with short lifetimes is limited.1,32–38 The mechanisms of these

dynamical structural transformations could provide useful

information and important guidance with respect to self-assembly

pathways,32,37,38 the in situ formation of self-assembled

systems and their structural transitions,1,32,35,37 and the inter-

actions of proteins and other biomolecules with model

membranes having biological relevance. In this regard, the

combination of rapid mixing and small-angle X-ray (SAXS) or

aDepartment of Pharmaceutics and Analytical Chemistry,Faculty of Pharmaceutical Sciences, University of Copenhagen,Universitetsparken 2, DK-2100 Copenhagen, Denmark.E-mail: [email protected]; Fax: +45 35336030;Tel: +45 35 33 65 41

b Institute of Biophysics and Nanosystems Research (IBN),Austrian Academy of Sciences, Graz, Austria.E-mail: [email protected]; Fax: +45 35336030;Tel: +39 04 03 75 87 08

w This article was submitted as part of a special collection on scatteringmethods applied to soft matter, marking the 65th birthday of ProfessorOtto Glatter.

PAPER www.rsc.org/pccp | Physical Chemistry Chemical Physics

3116 Phys. Chem. Chem. Phys., 2011, 13, 3115–3125 This journal is c the Owner Societies 2011

small-angle neutron (SANS) scattering techniques has become

an important tool for investigating the dynamics of structural

transitions in self-assembled systems.1,32,37,39,40

The interaction mechanism of Ca2+ with biologically

relevant model lipids has attracted great interest in the literature

due to the important functional role of Ca2+ ions in living

cells.41–43 In this context, the remarkable influence of Ca2+

ions has been extensively observed in various phospholipid-

based vesicles.1,12,18,43–48 The calcium induced structural

transitions from fluid lamellar (La) to the inverted-type

hexagonal phase (H2) or the inverted-type bicontinuous cubic

(V2) phase are also frequently observed in different model

systems.1,12,18,46 The significance of this electrostatic lipid–Ca2+

interaction was particularly noticed after the addition of a low

concentration of the divalent cation to anionic phospholipid

systems.1,12,45,49,50 In a recent study,1 we investigated the

dynamics of Ca2+-induced self-assembled structural transitions

in dioleoyl–phosphatidylglycerol (DOPG)/monoolein (MO)

vesicles with a molar ratio of 30/70. The fast and unexpected

in situ structural transitions at 50 1C were monitored by

carrying out stopped-flow experiments combined with

synchrotron SAXS. We found that even at low Ca2+ concen-

trations, the strong binding of the divalent cation to the

negatively charged DOPG molecules enhances the negative

spontaneous curvature (H0 o 0) of the monolayers. It causes a

rapid collapse of the vesicles, and finally induces the trans-

formation within milliseconds from the La phase to the H2 phase.

At 20 1C, we observed that the La–H2 transition occurs via an

intermediate phase with a bilayer structure (possibly the Pn3m

phase). It was fascinating to observe that these dynamical

structural transitions are significantly different from those

previously reported12 under static and equilibrium conditions.

The study described here is the continuation of our recent

report on the combination of rapid mixing and time-resolved

synchrotron SAXS for the in situ investigations of structural

transitions of diluted DOPG/MO vesicles into well-ordered

nanostructures by the addition of low Ca2+ concentrations.

The present structural analysis was performed on experiments

done on the effect of rapidly mixing Ca2+ with DOPG/

MO-based aqueous dispersions at three different DOPG/MO

molar ratios of 15/85 (sample A), 50/50 (sample B), and 70/30

(sample C), respectively. The obtained results are compared

with those previously reported on vesicles1 with a DOPG/MO

molar ratio of 30/70 (sample D). In absence of Ca2+ ions, how

the lipid composition can affect significantly the structure of

the DOPG/MO-based aqueous dispersions is also discussed in

this work. Fig. 1 schematically illustrates the applied

set-up and one representative example on the dynamics of

Ca2+-induced structural transitions.

2. Experimental details

Materials

The lipids: MO (1-monooleoyl-rac-glycerol, purity: 99%) was

purchased from Sigma Chemical Co. (St. Louis, Missouri,

USA), and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]

(sodium salt, purity: 99%) (DOPG) was obtained from Avanti

Polar Lipids (Alabaster, Alabama, USA). Chloroform

(CHCl3, purity: >99%) was supplied by Carl Roth GmbH

(Karlsruhe, Germany). Calcium chloride dihydrate of analytical

grade was supplied by Merck (Darmstadt, Germany). The

buffer used was PIPES (piperazine-N,N0-bis(2-ethanesulfonicacid) pH 7.0). For rapid mixing, the stock salt solution

containing 68 mM Ca2+ ions was prepared by dissolving the

salt in the PIPES buffer. All ingredients were used without

further purification.

Preparation of DOPG/MO-based vesicles

For the preparation of the vesicles, we followed a similar

procedure to that reported by Awad et al.12 for forming

multi-lamellar vesicles (MLVs). In the absence of Ca2+ ions,

the binary DOPG/MO mixtures with three different molar

ratios of 15/85, 50/50, and 70/30 were dispersed in the PIPES

buffer. To follow the kinetics of the phase transitions in the

rapid-mixing experiments, it was important to mix effectively

the salt solution with the vesicles and the sponge-like L3 phase.

This was achieved by carrying out the investigations with

diluted DOPG/MO vesicles containing a total lipid weight of

B7 wt%. Thus, the lipid concentration is different from that

used in ref. 12 (30 wt% total lipid content). Another important

difference is related to the preparation procedure of the

samples for SAXS investigations. In our study, we investigated

directly the formed vesicles and the sponge-like L3 phase

coexisting with excess water without applying any additional

protocols. In contrast, Awad et al.12 applied an additional step

of centrifuging the aqueous dispersions in order to remove

excess water, and hence used the so-called ‘pellets’ of lipids for

the SAXS measurements.

Time-resolved synchrotron X-ray scattering measurements

X-Ray scattering patterns were recorded at the Austrian

SAXS beamline51 (camera length 75 cm) at the synchrotron

light source ELETTRA (Trieste, Italy) using a 1D position

sensitive detector (Gabriel type), which covered the s-range

(s=2sin y/l, where l is the wavelength and 2y is the scatteringangle) of interest from about 1/300 to 1/15 A�1 at an X-ray

Fig. 1 Schematic illustration of the set-up combining synchrotron

SAXS with stopped-flow apparatus. In the stopped-flow apparatus,

one syringe contains a buffer with Ca2+ ions, whereas the other

contains DOPG/MO-based aqueous dispersion. The rapid mixing

was conducted within 10 milliseconds. The presented example shows

the in situ formation of the inverted type bicontinuous cubic (V2) phase

of the symmetry Pn3m.


energy of 8 keV. Silver behenate (CH3–(CH2)20–COOAg with

a d-spacing value of 58.38 A) was used as a standard to

calibrate the angular scale of the measured intensity.

Stopped-flow mixing experiments on the lipid dispersions were

performed with the commercial stopped-flow apparatus

SFM-400 (Bio-logic Company, Claix, France) in combination

with simultaneous time-resolved X-ray diffraction. The cell

consists of four reservoirs (each vertical syringe of 10 ml

volume is driven independently by a stepping-motor) and

three mixing chambers. The geometry of this system allowed

easy evacuation of air bubbles that can be form during filling.

For our study, two syringes were operated and a total shot

volume of 100 ml was chosen. When actuated by an electronic

trigger signal, both the DOPG/MO-based aqueous dispersion

and the solution of Ca2+ ions, respectively, were injected into

the mixing chamber and finally pressed into an X-ray quartz

capillary with a diameter of 1 mm (specified dead time 10

milliseconds), which was thermostated as well as the syringes

and the transfer lines of the stop-flow apparatus with a water

bath (�0.1 1C, Unistat CC, Huber, Offenburg, Germany). The

temperature was set to 20, 50, and 70 1C, respectively. Beforeeach time resolved experiment static exposures of the samples

were taken (typically exposed for 2 min). The time frame

protocol for the time-resolved experiments was as follows:

100 milliseconds exposures from frame 1–10, 2 seconds

exposures from frame 11–40, 10 seconds exposures from frame

41–53, and thereafter 20 seconds exposures were programmed.

X-Ray data-analysis

In the time resolved X-ray scattering experiments, all lattice

spacings of the La, the V2, and the H2 phases were deduced

from the reflections with the highest intensity. The SAXS

diffraction patterns were analyzed by standard procedures as

described in ref. 17. In short, all observed Bragg peaks were

fitted by Lorentzian distributions after detector efficiency

corrections and subtraction of the background scattering from

both the water and the sample cell. The fittings were carried

out with home-written procedures running under IDL 5.2

(Research Systems, Inc., USA).

3. Results and discussion

3.1 In situ monitoring of direct highly swollen L3 to V2

transition

As a control experiment, the structure of sample A

(an aqueous dispersion containing 15/85 mol/mol of DOPG/

MO) before performing the rapid-mixing experiment was

investigated. Fig. 2A shows that the structure of this aqueous

dispersion is significantly different than that investigated in

our recent study1 for an aqueous dispersion with a lower

DOPG content. In that study, we reported on the formation

of highly swollen large vesicles containing DOPG/MO at a

fixed molar ratio of 30/70 and having a d-spacing value of

140 A. The SAXS pattern of the vesicular dispersion mainly

consists of positionally uncorrelated membranes that do not

‘‘see’’ each other (Fig. 2 in ref. 1). In the present study,

lowering the DOPG/MO molar ratio to 15/85 induces a

significant change in the structure. As compared with those

of vesicular dispersions formed with a higher DOPG content

that will be discussed later, the basic distinguishing features of

the SAXS pattern presented in Fig. 2A are as following: (i) a

much broader diffuse maxima (peak) that is centered at sc, i.e.

having a higher value of full-width at half maximum (FWHM)

as compared to the first form factor maxima of the

DOPG/MO bilayers, and (ii) an appearance of the maxima

at lower s-value. Additionally, there is no indication for the

formation of any minima in the scattering pattern. Instead this

pattern closely resembles previous SAXS patterns obtained

from the L3 phase in different self-assembled systems52–56

indicating most likely that sample A forms a highly swollen

sponge-like structure. The proposed ‘‘sponge-like’’ L3 phase

can be considered as a precursor to the well-ordered V2 phase

and is composed of randomly oriented bilayer sheets.52,57 In

order to reveal the structure, we fitted the SAXS experimental

data of the highly swollen ‘‘sponge-like’’ L3 phase according to

previous theoretical works on the L3 phase.52,53,57 The scattering

function I(s) is calculated according to the following

equation:52,53,57

IðsÞ ¼ c

x�2 þ ðs� scÞ2ð1Þ

which describes the bilayer’s cell–cell correlations. The

correlation length is x, and the position of the maximum is

denoted as sc. The c term describes the intensity normalization.

The solid red line in Fig. 2A is the best fit to the experimental

data using eqn (1). The obtained fit results yield a cell–cell

peak position at sc B 0.018 A�1. For the vesicular dispersions

at a higher DOPG content, the observed diffuse peak position

has a higher s-value as will be discussed later. Taking into

consideration the similarities in local structures of the lamellar

and the sponge-like phases, we do not exclude as suggested

also previously56 the possible coexistence of the L3 phase with

a lamellar phase consisting mainly of fluid bilayers without

positional correlation. It should be pointed out that Awad

et al.12 reported that the DOPG/MO system with a similar

DOPG content but at a lower temperature (20 1C) which induces

the formation of Im3m cubic phase in the presence of calcium

ions. Here it must be mentioned that under their static

conditions, the SAXS experiments were performed on so called

‘pellets’ after removing excess water and therefore, the obtained

results are significantly different than those done under a realistic

excess of water conditions (DOPG/MO dispersions) as described

in our recent report.1 A detailed description of the importance of

the used experimental protocol prior to the SAXS investigations

is given in that published report.1

Fig. 2B shows the structural transitions observed in sample

A at 50 1C after rapid mixing with PIPES buffer (pH 7.4)

containing 68 mM Ca2+ ions. The rapid mixing investigation

was done at a volume ratio of the DOPG/MO-based dispersion

to the buffer of 50/50. The CaCl2 concentration after rapid

mixing was 34.0 mM. As presented in Table 1, the mixing was

done with a molar ratio of DOPG/Ca2+ at 0.4 (a low salt

concentration). The obtained SAXS patterns demonstrate the

drastic impact of Ca2+ ions on the curvature of the DOPG/MO

monolayers. The fast alteration in the structure of the

‘‘sponge-like’’ phase owing to the strong binding of the


divalent cation to the anionic DOPG molecules is one of the

remarkable findings in the present study and also in our recent

report on the fast La–H2 transition.1 The ease with which the

highly swollen L3 phase is transformed at least within the first

300 milliseconds after the rapid-mixing experiment to the V2

phase is clearly observed in the figure. It should be noted that

the first 10 frames had exposure times of 100 milliseconds

each. A representative example on the facilitated newly formed

V2 phase of the Pn3m symmetry with its 6 characteristic peaks

after an elapsed time of 290 seconds is presented in Fig. 2B.

Fig. 2C demonstrates in the presence of Ca2+ ions that the

initial unit cell parameter for the Pn3m phase, aPn3m, under

Fig. 2 The rapid calcium-triggered V2 phase formation at 50 1C. The investigated aqueous dispersion (sample A) is composed of the binary

DOPG/MO mixture at a molar ratio of 15/85. Panel (A) shows selected SAXS scattering patterns for three investigations: sample A in the absence

of Ca2+ ions (background subtracted scattering pattern of the proposed L3 phase), and two examples on the in situ formation of the V2 phases. In

panel (B), the contour plot clearly displays the characteristic reflections of the Pn3m phase (final Ca2+ concentration of 34.0 mM). Panel (C)

presents the time dependence of the unit-cell parameter of the rapidly formed Pn3m phase referring to the experiment given in panel (B). In panel

(D), the contour plot clearly displays the in situ formation of a biphasic system: coexistence of Im3m with Pn3m (final Ca2+ concentration of

6.8 mM). In both rapid-mixing experiments there is no indication for the formation of an intermediate phase. Panel (E) illustrates the indexing of

the cubic phases: the Pn3m (J) and the Im3m (%) phases. In panel (F), the plot shows the time dependence of the unit cell parameter of the

rapidly formed Pn3m and Im3m phases referring to the experiment given in panel (D). For further details refer to Table 1.


these non-equilibrium conditions is 93.3 A, and it rapidly

decreases over time in the first 100 seconds. Thereafter, the

results reveal only a slight decrease in the unit cell parameter.

After 300 seconds of the rapid-mixing experiment, the

obtained aPn3m, approaches a value of 89.5 A. In another

experiment, the rapid mixing was carried out at a volume ratio

of the highly swollen L3 phase to the buffer of 90/10 and the

CaCl2 concentration after the rapid-mixing experiment was

6.8 mM. Thus, the mixing was done with a molar ratio of

DOPG/Ca2+ of 3.3 (Table 1). Fig. 2D shows the Ca2+

induced structural transitions observed in sample A at this

lower Ca2+ concentration (at 50 1C). The dispersion forms a

biphasic system consisting of bicontinuous Im3m and Pn3m

cubic phases. At an elapsed time of 870 seconds, a representative

example for this system is shown in Fig. 2B. Fig. 2E illustrates

the indexing of both of these coexisting cubic phases in the

aqueous dispersion after 870 seconds of rapid mixing, and also

the indexing of the pure Pn3m phase, which is observed at a

higher Ca2+ concentration (34.0 mM) after an elapsed time of

290 seconds. The mixing time-dependent evolution of the

derived lattice parameters is illustrated in Fig. 2F for the

two newly formed Pn3m and Im3m bicontinuous cubic phases.

Under these non-equilibrium conditions, the unit cell parameters

of the two coexisting cubic phases in excess water reduce

significantly with the elapsed time. For instance, the lattice

parameter of the Im3m phase decreases 11.8 A (from 130.8 to

119.0 A) in the time interval between 181 and 1291 seconds,

whereas a decrease of 9.1 A is obtained for the Pn3m

phase in the same time interval. The values of the Bonnet

ratio (aIm3m/aPn3m) for the two phases lie in the range of

Table 1 The molar ratios of DOPG/[Ca2+] and (DOPG + MO)/[Ca2+] after rapidly mixing the DOPG/MO-based vesicles with PIPES buffer(pH 7.0) containing 68 mM Ca2+ ions

DOPG/MO Volume A (%) Volume B (%) Final Ca2+ conc./mM DOPG/[Ca2+] (DOPG + MO)/[Ca2+]

15/85 15 85 58 0.1 0.415/85 30 70 48 0.2 1.115/85 50 50 34 0.4 2.415/85 70 30 20 0.9 5.715/85 85 15 10 2.1 1415/85 90 10 6.8 3.3 2230/70a 10 90 61 0.1 0.230/70a 30 70 48 0.3 0.930/70a 50 50 34 0.6 2.130/70a 70 30 20 1.5 4.930/70a 90 10 6.8 5.5 18.430/70a 93 7 4.8 8.3 27.830/70a 94 6 4.1 9.9 32.830/70a 95 5 3.4 12.0 40.050/50 50 50 34 0.9 1.870/30 50 50 34 1.1 1.670/30 10 90 61 0.1 0.2

The investigated aqueous dispersions in the absence of Ca2+ ions were composed of DOPG and MO at constant molar ratios of 15/85 (sample A),

50/50 (sample B), 70/30 (sample C), and a30/70 (sample D) taken from ref. 1. All different rapid-mixing investigations with different volume ratios

of the DOPG/MO-based aqueous dispersion (volume A) to the buffer (volume B) are summarized. In addition, the final Ca2+ concentrations are

given.

Fig. 3 Rapid calcium-triggered V2 phase formation at 50 1C (A), and V2 plus H2 phase formation at 70 1C (B). The investigated aqueous

dispersion (sample A) was composed of the binary DOPG/MO mixture at a molar ratio of 15/85 and with a total lipid content of 7 wt%. In panel

(A), the lattice-spacing of the rapidly formed Pn3m phase is displayed as a function of the final Ca2+ concentration. The electroneutral point is

indicated by an arrow. Panel (B) shows a typical SAXS scattering pattern that was obtained 200 seconds after the rapid-mixing experiment at

70 1C. The final calcium concentration was 20 mM. At 70 1C, the scattering pattern indicates the formation of a biphasic sample consisting of the

Pn3m cubic phase coexisting with the H2 phase. The Bragg reflections of the cubic phase are marked with ‘‘D’’ and those of the inverse hexagonal

phase with ‘‘H2’’.


1.24–1.27 and therefore are consistent with the Bonnet relation,58

which ideally takes a value of 1.279. The findings are also in

good agreement with previously reported aIm3m/aPn3m ratios

for various lipid-based systems,22,27,36,59 which indicates that

the averaged Gaussian curvatures of the two coexisting cubic

bicontinuous phases are almost the same. In the present study,

this suggests also a fast re-arrangement of the lipids and water

molecules into the newly formed two cubic phases. Last, it is

worth noting the observation of a subtle intensity drop of the

diffraction peaks in Fig. 2D. The initial exponential lattice

spacing decrease of the cubic phases (as described also in

Fig. 2F) is accompanied with a constant but slow decrease in

the Bragg peak intensities, which reaches its minimum after

about 600 seconds. Thereafter, the Bragg peaks of the cubic

phases increase again in intensity, when the lattice changes are

only minor and after having reached a significant dehydration

level under the given experimental conditions. A possible

explanation could be that the rapid dehydration has caused

in this case some temporary lattice disorder. However, since

the lattice spacings change with time, the influence of the form

factor fluctuations is not totally excluded.

In Fig. 3, both the effect of the final salt concentration as

well as increasing the temperature to 70 1C on the final phase

formation are summarised. Here for comparison, all data refer

to 200 seconds after the rapid-mixing event. Obviously

increasing Ca2+ concentration reduces significantly the lattice

spacing, which implies a reduction in the spontaneous

curvature (Fig. 3A). Increasing temperature works in the same

direction as can be seen in panel B. Not only is the lattice

spacing of the Pn3m phase further reduced (from about 90 A

to 82 A), but also part of the sample A has converted into the

H2 phase, which is known to exhibit a greater negative

spontaneous curvature. However, while Ca2+ ions mainly

affect the polar interface of the membrane, the temperature

mainly influences the hydrophobic chain regions,1 i.e. increasing

significantly the hydrocarbon chain disorder while keeping the

polar interface area intact, therefore promoting the formation

of non-lamellar phases.

As illustrated above, the propensity for calcium-induced

DOPG/MO-based nanostructures is dependent to a great

extent upon the lipid composition. In the absence of Ca2+,

it is expected1 that the self-assembly of MO (a lipid favoring

the formation of non-lamellar phases) with DOPG (a rod-like

shaped lipid favoring the formation of La phase) in aqueous

media is controlling the mean membrane curvature, |H0|, of

the monolayers. For instance, one obtains a less negative

spontaneous curvature of the composite membrane by the

partial replacement of MO molecules by those of DOPG.1,12

These experimental observations can be rationalized by using

the critical packing parameter (CPP) concept.60 The usefulness

of this concept has been demonstrated in various studies on

lamellar to non-lamellar phase transitions in model membranes

consisting of mixtures of lipids with different lamellar and

non-lamellar propensities.1,6,22,26,27,46,53,61 The CPP parameter

is also known as the shape parameter of lipid molecules, S, and

is defined as the effective hydrophobic chain volume, v, divided

by the lipid chain length, lC, and its optimal headgroup area,

a0. It is worth mentioning that the CPP concept in various

published reports has mainly been applied to individual

lipid/water systems. Thus, the discussion of the molecular

geometry of the investigated lipids in the present study for

the binary DOPG/MO mixtures, which is similar to that

described on different binary lipid mixtures in previous

investigations in literature, is related of course to the effective

average molecular shape arising from the given molar ratios of

MO to DOPG. Further, it is important to stress that the

reason for any spontaneous monolayer curvature is always

given by the intrinsic curvatures of the constituents which built

up the membrane leaflets. Recent reports show up this

relationship between intrinsic and membrane curvatures, both

theoretically as well as in simulations.62,63 Returning to our

study, it should be noted that with increasing DOPG content,

the repulsive forces between the negatively charged DOPG

molecules in the DOPG/MO membrane induce an increase in

the a0 value which therefore favors the formation of nano-

structures with a higher tendency for flattened membranes1,12

(an unstressed fluid La phase has a zero mean spontaneous

curvature, |H0| = 0, S = 1).

It has been suggested based on different experimental

findings and theoretical considerations1,12,44,49,64–67 that the

strong binding of Ca2+ ions to the phosphate groups in

anionic phospholipids induces dehydration of the phospholipid

bilayer membrane (inducing a decrease in the effective a0 value

due to the screening of the negative charge of the lipid

molecules by Ca2+ ions) in an entropy-driven process, i.e.

the overall lipid–water interface area diminishes. This process

is also concomitant with a condensing effect (promoting the

tight packing of the fatty acyl chains in the membrane). In this

regard, it was demonstrated in a recent study on DOPG-based

vesicles that the lateral diffusion coefficient (DL) and the water

permeability of DOPG decreases with increasing Ca2+ ion

concentration due to the screening of the charged headgroups

of the investigated lipid and the induced tighter lipid packing.67

This strong binding impact of Ca2+ ions to the negatively

charged lipid molecules could even accelerate the membrane

fusion that involves lamellar to non-lamellar structural transitions

in a process that imposes a more negative spontaneous

curvature on the membrane. Lehrmann and Seelig48 proposed

the following two-step mechanism for this strong association:

(i) an increase in the local Ca2+ concentration at the lipid/

water interfacial area; and (ii) the formation of coordination

complexes. A recent study68 demonstrated that the complete

binding of Ca2+ ions is achieved when the electroneutrality of

the membrane is reached. This behavior is confirmed by the

fast formation of the Pn3m phase (Fig. 3A), which demonstrates

a strong Ca2+ concentration dependence of the lattice spacing

below the electroneutral point. In this regard, it should be

noted that the lattice spacing changes can be described well

with a double exponential decay function (solid line), whereas

a single exponential decay function does not fit well with the

data points. Also in our recent study,1 we suggested as

deduced from the rapid-mixing experiments combined with

SAXS on the sample D (a vesicular dispersion containing

DOPG/MO at a molar ratio of 30/70) that the membrane

re-organization in the newly formed H2 phase (Fig. 4) is

dominated by the membrane electroneutrality. The rapid-

mixing investigations were performed at different DOPG/Ca2+

ratios (Table 1). As shown in Fig. 4A, the obtained results


show a strong impact on the d10-spacing value of the calcium-

induced H2 phase at low Ca2+ concentrations (o10 mM

Ca2+, at DOPG/Ca2+ ratios higher than 2 as presented in

Fig. 4B). However, the membrane re-organization seems to be

less pronounced after reaching membrane electroneutrality

(>10 mM Ca2+, at DOPG/Ca2+ ratios less than 2 as

presented in Fig. 4B).

An interesting aspect for future investigations is to check the

reversibility of these non-equilibrium structural transitions. In

this context, a possible approach for investigating the reversible

non-equilibrium structural transitions from V2 or H2 phases to

vesicles or sponge-like L3 phase is to carry out these rapid-

mixing experiments in the presence of an effective chelating

agent such as EDTA (ethylenediaminetetraacetic acid), which

has a high tendency to bind Ca2+. Under static conditions,12

the performed experiments on the ‘pellets’ after 24 hours of

incubation the sample at 25 1C showed that EDTA induces a

transition from the cubic Pn3m phase to the cubic Im3m phase

but there was no indication for a reversible transition to the La

phase. It was suggested that the Im3m-La transition is slow

under the applied experimental conditions as a result of the

high activation energy. Another suggested explanation12 is

that the strong association of Ca2+ ions to the DOPG

molecules could lead to the segregation of the DOPG-rich

bilayers from the MO-rich membrane.

3.2 In situ formation of La or La/H2 phases

In this section, we report on the sensitivity of DOPG/MO

dispersions consisting of a higher concentration of DOPG to

the presence of Ca2+ ions at two different temperatures

(20 and 50 1C, respectively). In the control experiment, the

structure of sample B (an aqueous dispersion containing

DOPG/MO at a molar ratio of 50/50) was investigated before

performing the rapid-mixing experiment. Under static

conditions and in the absence of Ca2+ ions (Fig. 5A), the

obtained SAXS pattern was dominated by diffuse scattering.

A typical form-factor contribution with a maxima at

s B 0.020 A�1 arising from uncorrelated bilayers is observed.

In our recent report,1 we discussed in detail the structure of

similar vesicular aqueous dispersion based on DOPG/MO

before performing the rapid-mixing experiments. The determined

head to headgroup distance, dHH, in that dispersion was

36.6 A, i.e. the steric bilayer thickness was roughly B46 A

assuming the headgroup extension of DOPG to be about

8–10 A.69 Since the observed form factor of the dispersion

displayed in Fig. 5A is almost identical, it is plausible to

assume that the bilayer structure is alike. The only observed

difference in the present investigation as compared to the

previous study1 is the absence of the structure factor contribution

(no diffraction peaks). This means that the increased DOPG

content, which is equivalent to enhanced electrostatic bilayer

repulsions, suppresses even loose bilayer stacking and leads to

completely unbound membranes.

Sample B is rapidly mixed with the PIPES buffer at a

volume ratio of the vesicles to the buffer of 50/50. As presented

in Table 1, the CaCl2 concentration after the rapid-mixing

experiment and the calculated molar ratio of DOPG/Ca2+ are

34.0 mM and 0.9, respectively. At 20 1C, Fig. 5B shows a fast

and a direct disorder–order structural transition: a disordered

phase consisting of uncorrelated bilayers is transformed within

milliseconds to a highly ordered La phase with a single sharp

peak at s = 0.0212 A�1 (d-spacing = 47.2 A). A typical

example on this newly formed phase after 350 seconds of rapid

mixing is presented in Fig. 5A. Despite the appearance of only

the first order Bragg peak of the La phase, the identification of

this phase is supported by the fact that the presence of high

DOPG content1,12 favors the formation of the La phase by

pushing the |H0| value towards zero. It should also be noted

that the observed predominant first order Bragg peak suggests

that the La phase is only very poorly hydrated.70 Further

support arises from the fact that the steric bilayer thickness at

a DOPG/MO ratio of 30/70 is about 46 A, which is very close

to the measured d-spacing value of the induced La phase.

The same experiment was performed at 50 1C. The associationof Ca2+ ions to the DOPG molecules at a higher temperature

leads to significant alterations of the structure (Fig. 5C). There

is now evidence of the fast re-organization of the disordered

uncorrelated bilayers to form a dispersion consisting of two

coexisting structures: the La and the H2 phases (a typical

Fig. 4 Rapid calcium-triggered H2 phase formation at 50 1C. The investigated aqueous dispersion (sample D) was composed of the binary

DOPG/MO mixture at a molar ratio of 30/70 and with a total lipid content of 7 wt%. In panel (A), the d10-spacing of the rapidly formed H2 phase

is displayed as a function of the final Ca2+ concentration. Panel (B) shows the d-spacing in dependence of the DOPG/Ca2+ ratio. The

electroneutral regime is highlighted in light grey. This figure was reproduced from ref. 1.


example for the biphasic system after 510 seconds is illustrated

in Fig. 5A). The higher impact of Ca2+ ions on the DOPG/

MO vesicular dispersions at higher temperatures is discussed

also in our recent report.1 Similar to previous investigations on

lipidic systems containing monoglycerides,13,21,24,26,71–73 heating

up the samples leads to a decrease of the spontaneous mono-

layer curvature (H0 o 0), which thus causes a preferential

tendency to form non-lamellar phases. This is related to the

increase in the CPP value due to the significant and simultaneous

variations in both packing parameters: v increases due to the

less dense acyl chain packing, and a0 decreases due to the

headgroup’s dehydration. Hence, our study reveals that both

increasing temperature and mixing with Ca2+ ions have

similar effects of enhancing a more negative spontaneous

curvature (H0 o 0) of the monolayers.

In order to shed light on the kinetics of formation of both

the La and H2 phases through the association of Ca2+ ions

with the lipid bilayers, the time evolution of the normalized

intensity of the first order diffraction peak observed in both La

(Fig. 5B) and H2 phases (Fig. 5C) are displayed. As shown in

Fig. 5D, the results demonstrate that the formation of the La

phase is about 3 times faster. After 9.5 seconds B50% of the

lipid molecules are re-organized in an ordered La phase,

whereas it takes at least 35 seconds to form B50% of the

ordered H2 phase.

Two additional examples on the sensitivity of DOPG/MO

aqueous dispersions with an even higher DOPG content are

illustrated in Fig. 6. At 20 1C, the control static experiment of

sample C (vesicular dispersion containing DOPG/MO at a

molar ratio of 70/30) in the absence of Ca2+ ions indicates the

formation of uncorrelated bilayers. It should be noted that the

observed SAXS patterns are very much the same in both

samples B (Fig. 5A) and C (Fig. 6A) displaying the typical

maxima and minima characteristic for bilayer form factors.

These patterns are different from those observed for the

proposed L3 phase as discussed in detail in the first section.

Fig. 5 Rapid calcium-triggered formation of La or La/H2 phases at two temperatures 20 and 50 1C, respectively (final Ca2+ concentration of

34.0 mM). Panel (A) shows selected SAXS scattering patterns for three investigations: sample B in the absence of Ca2+ ions, and two examples on

the in situ formation of La (at 20 1C) and La/H2 phases (at 50 1C). Both time-resolved experiments are given in panels (B) and (C). No indication for

an intermediate phase formation is spotted. Panel (D) presents the time dependence of the normalized intensity of the first order reflection peaks of

the La (J) and the H2 ( ) phases referring to the experiments given in panel (B) and (C), respectively. The red and the blue lines are used only to

guide the eye. For experimental details refer to Table 1.


The rapid-mixing experiments of sample C with the PIPES

buffer was performed at two different volume ratios of the

vesicles to the buffer of 50/50 and 10/90, respectively. As

presented in Table 1, the two final CaCl2 concentrations after

the rapid-mixing experiment are 34 and 61 mM, respectively;

whereas the calculated two molar ratios of DOPG/Ca2+ are

1.1 and 0.1, respectively. In both experiments done at 20 1C,there is an indication of a fast disordered-ordered structural

transition (Fig. 6) similar to that obtained for sample B. At the

DOPG/Ca2+ molar ratio of 1.1, a highly ordered La phase is

rapidly formed (Fig. 6A and B). However at the relatively very

low DOPG/Ca2+ ratio of 0.1 (Fig. 6A and C), we note that the

salt concentration is not sufficient to induce a complete and

full transition to the highly ordered La phase. The results

suggest the formation of a biphasic dispersion consisting of

an ordered La phase that coexists with a great amount of

uncorrelated bilayers.

3.3 Mechanism of the sponge (L3) to the bicontinuous cubic

(V2) phase transitions

In this work, we propose a structural mechanism for how

Ca2+ ions induce the re-crystallization of the molten

disordered L3 phase, which is different from the V2 phases

due to the lack of long-range order, to form the two well-

ordered V2 phases of the symmetries Pn3m and Im3m. Clearly,

our results reveal that the L3–V2 transition depends on

the Ca2+ concentration. However, we shall first discuss the

formation mechanism of the DOPG/MO L3 phase in the

absence of Ca2+ ions. In the literature, the enlargement of

water channels in the MO-based cubic Pn3m phase, which is

important for different applications, is accomplished by

different routes including (i) the addition of charged phos-

pholipid similar to our present study or highly hydrophilic

neutral lipids,27,74,75 (ii) the addition of different water-

miscible solvents,75–78 and (iii) the addition of hydrophilic

polymers77 or copolymers.27,79 Here, we investigated the

impact of the partial replacement of the neutral lipid MO by

the negatively charged phospholipid DOPG. As mentioned

above, the inclusion of the DOPG molecules in the polar/

apolar interfacial film is associated with an increase in the

electrostatic repulsions and therefore it induces a significant

increase in solubilized water in the self-assembled system. The

significant increase in the hydrophilic channels of the fully

hydrated Pn3m phase with increasing DOPG content leads at

a certain DOPG/MO ratio to the formation of the swollen and

disordered sponge L3 phase with bicontinuous nature similar

to the ordered Pn3m phase. It was suggested in different

studies that the mechanism of the V2–L3 transition is due to

the enhanced up-take of water, which causes an increase in

the diameter of the hydrophilic channels of up to three

times.75,80,81 These highly swollen soft sponge phases are easily

handled and are also interesting for different technological

applications77–78,81 including the crystallization of membrane

proteins.78,81 In a recent study,75 it was reported that the

partial replacement of MO and phytantriol by the similar

negatively charged phospholipid distearoylphosphatidyl-

glycerol (DSPG), which has the same hydrophilic headgroup

but with a stronger hydrophobic moiety, induces the structural

transition from Pn3m to Im3m phase in the MO system and

the formation of a highly swollen Pn3m phase in the phytantriol

mixture (the lattice parameter of the Pn3m phase increases

from 68 to 141 A after the addition of DSPG). The formation

of the L3 phase in the present work suggests that the addition

of DOPG increases significantly the bending flexibility of the

Fig. 6 Rapid calcium-triggered formation of the La phase at 20 1C.Panel (A) shows selected SAXS scattering patterns for three investigations:

sample C in the absence of Ca2+ ions, and two examples on the in situ

formation of two highly dehydrated La phases. In panel (B), the contour

plot clearly displays the characteristic single reflection of the La phase.

Panel (C) shows the contour plot that clearly displays the in situ formation

of the biphasic system: coexistence of the dehydrated La phase with

uncorrelated vesicles at a very low ratio of DOPG/Ca2+ (Table 1).


MO bilayers, which is an important parameter for the formation

of the highly dynamical disordered sponge phase. With a

further increase in DOPG content, the interfacial film becomes

more flattened and leads to the formation of the planar La

phase at relatively high DOPG content. This means that the

degree of stability of the La and the L3 phases can be easily

changed by varying the lipid composition due to the significant

influence on both the bending flexibility and the monolayer

spontaneous curvature. Note though, that the interplay of the

different contributions to the free bending energy of the

membrane is complex, since the electrostatic interactions are

involved in the presence of DOPG and/or Ca2+ ions. An

interesting attempt to extend the classical Helfrich formalism

was proposed earlier by separating the bending free energy

into an electrostatic and a non-electrostatic contribution.82

Following the same train of thought leads us to suggest that at

high DOPG content and very low Ca2+ concentrations, the

self-assembled structures (L3 and La phases) are dominated by

the electrostatic contribution, whereas at lower DOPG content

and high Ca2+ concentrations our results indicate that the

non-electrostatic contribution dominates, leading to stronger

negatively spontaneous curvatures, and therefore stable V2

phases are formed.

4. Conclusions

The dynamics of the electrostatic interactions of Ca2+ ions

with a series of aqueous dispersions containing different

DOPG/MO ratios was investigated by the combination of

synchrotron SAXS with stopped-flow measurements. The

in situ investigations of the calcium triggered phase transitions

reveal a very fast re-organization of lipid molecules in the

aqueous media to form a variety of nanostructures. Fig. 7

shows a schematic illustration of the various intriguing and

fast structural transitions observed in the present study, which

also includes the fast La–H2 transitions that were monitored in

our recent report.1 One of the most striking features in these

investigations is the occurrence of disordered-ordered transitions

within the milliseconds to seconds range. While the binding

steps of Ca2+ ions to the negatively charged DOPG/MO

membrane are not detectable in this time window, the rapid

condensation of bilayers due to screened electrostatic repulsion

forces and the subsequent formation of one-, two- and three-

dimensional nanostructures are well documented.

In general, the obtained results expand our knowledge

regarding both the strong impact of Ca2+ ions on the negatively

charged membranes as shown in this study in the DOPG/MO-

based vesicular and sponge-like systems, especially regarding

the very fast re-ordering of the lipids in the self-assembled

structures. Clearly, the lipid composition and the applied

experimental conditions including the temperature and the

investigated Ca2+ ion concentration are important factors in

the modulation of the calcium-induced structures.

5. Dedications

From Anan Yaghmur: it is a privilege and great honor to

dedicate this article (in collaboration with M.R. and B.S.) with

respect and friendship to Prof. Dr Otto Glatter on the occasion

of his 65th birthday and in recognition of his pioneering, and

outstanding contributions to the research of soft matter,

the studies of nanostructured aqueous dispersions, and the

development of various scattering analysis methods.

References

1 A. Yaghmur, P. Laggner, B. Sartori and M. Rappolt, PLoS One,2008, 3, e2072.

2 A. Yaghmur and O. Glatter, Adv. Colloid Interface Sci., 2009,147–148, 333–342.

3 R. Mezzenga, P. Schurtenberger, A. Burbidge and M. Michel, Nat.Mater., 2005, 4, 729–740.

4 C. J. H. Porter, N. L. Trevaskis and W. N. Charman, Nat. Rev.Drug Discovery, 2007, 6, 231–248.

5 A. Yaghmur and M. Rappolt, in Colloids in Drug Delivery,ed. M. Fanun, Taylor & Francis Group LLC Press, Karnataka,2010, pp. 339–355.

6 A. Yaghmur, L. Paasonen, M. Yliperttula, A. Urtti andM. Rappolt, J. Phys. Chem. Lett., 2010, 1, 962–966.

Fig. 7 Schematic illustration of the lipid composition dependence of the rapid calcium-triggered nanostructures in DOPG/MO-based aqueous

dispersions. Four different examples for fast disordered-ordered structural transitions are given. The in situ formation of the well-ordered

nanostructures: the two V2 phases of the symmetries Pn3m and Im3m, the H2 phase, and the La phase. The dynamical behaviour of these

calcium-induced nanostructures have been followed by millisecond time-resolved synchrotron SAXS.


7 H. T. McMahon and J. L. Gallop, Nature, 2005, 438, 590–596.8 Z. A. Almsherqi, S. D. Kohlwein and Y. Deng, J. Cell Biol., 2006,173, 839–844.

9 K. Schara, V. Jansa, V. Sustar, D. Dolinar, J. I. Pavlic, M. Lokar,V. Kralj-Iglic, P. Veranic and A. Iglic, Cell. Mol. Biol. Lett., 2009,14, 636–656.

10 D. Paolino, D. Cosco, F. Cilurzo and M. Fresta, Curr. Bioact.Compd., 2007, 3, 262–277.

11 S. Hyde, Z. Blum, T. Landh, S. Lidin, B. W. Ninham,S. Andersson and K. Larsson, in The Language of Shape: TheRole of Curvature in Condensed Matter: Physics, Chemistry,Biology, Elsevier, Amsterdam, 1997.

12 T. S. Awad, Y. Okamoto, S. M. Masum and M. Yamazaki,Langmuir, 2005, 21, 11556–11561.

13 L. de Campo, A. Yaghmur, L. Sagalowicz, M. E. Leser, H. Watzkeand O. Glatter, Langmuir, 2004, 20, 5254–5261.

14 Y. D. Dong, I. Larson, T. Hanley and B. J. Boyd, Langmuir, 2006,22, 9512–9518.

15 V. Cherezov, D. P. Siegel, W. Shaw, S. W. Burgess andM. Caffrey,J. Membr. Biol., 2003, 195, 165–182.

16 D. P. Siegel and R. M. Epand, Biophys. J., 1997, 73, 3089–3111.17 M. Rappolt, in Planar Lipid Bilayers and Liposomes, ed. A.

Leitmannova, Elsevier, Amsterdam, 2006, pp. 253–283.18 R. N. Lewis and R. N. McElhaney, Biochim. Biophys. Acta,

Biomembr., 2009, 1788, 2069–2079.19 M. Rappolt, A. Hickel, F. Bringezu and K. Lohner, Biophys. J.,

2003, 84, 3111–3122.20 J. M. Seddon, J. Robins, T. Gulik-Krzywicki and H. Delacroix,

Phys. Chem. Chem. Phys., 2000, 2, 4485–4493.21 H. Qiu and M. Caffrey, Biomaterials, 2000, 21, 223–234.22 A. Yaghmur, P. Laggner, M. Almgren and M. Rappolt, PLoS

One, 2008, 3, e3747.23 B. J. Boyd, Y. D. Dong and T. Rades, J. Liposome Res., 2009, 19,

12–28.24 A. Yaghmur, L. de Campo, L. Sagalowicz, M. E. Leser and

O. Glatter, Langmuir, 2005, 21, 569–577.25 R. Winter, Curr. Opin. Colloid Interface Sci., 2001, 6, 303–312.26 A. Yaghmur, M. Kriechbaum, H. Amenitsch, M. Steinhart,

P. Laggner and M. Rappolt, Langmuir, 2010, 26, 1177–1185.27 A. Yaghmur, L. de Campo, L. Sagalowicz, M. E. Leser and

O. Glatter, Langmuir, 2006, 22, 9919–9927.28 M. Johnsson, J. Barauskas and F. Tiberg, J. Am. Chem. Soc., 2005,

127, 1076–1077.29 M. Johnsson, Y. Lam, J. Barauskas and F. Tiberg, Langmuir,

2005, 21, 5159–5165.30 A. Yaghmur, L. de Campo, S. Salentinig, L. Sagalowicz,

M. E. Leser and O. Glatter, Langmuir, 2006, 22, 517–521.31 I. Amar-Yuli, D. Libster, A. Aserin and N. Garti, Curr. Opin.

Colloid Interface Sci., 2009, 14, 21–32.32 T. Narayanan, Curr. Opin. Colloid Interface Sci., 2009, 14, 409–415.33 M. Rappolt, G. M. Di Gregorio, M. Almgren, H. Amenitsch,

G. Pabst, P. Laggner and P. Mariani, Europhys. Lett., 2006, 75,267–273.

34 X. Mulet, X. Gong, L. J. Waddington and C. J. Drummond, ACSNano, 2009, 3, 2789–2797.

35 T. M. Weiss, T. Narayanan, C. Wolf, M. Gradzielski, P. Panine,S. Finet and W. I. Helsby, Phys. Rev. Lett., 2005, 94, 038303.

36 Y. D. Dong, A. J. Tilley, I. Larson, M. J. Lawrence, H. Amenitsch,M. Rappolt, T. Hanley and B. J. Boyd, Langmuir, 2010, 26,9000–9010.

37 M. Gradzielski, Curr. Opin. Colloid Interface Sci., 2004, 9, 256–263.38 G. V. Jensen, R. Lund, T. Narayanan, J. Gummel and

J. S. Pedersen, presented in part at the Seventh Nordic Workshopon Scattering from Soft Matter, Helsinki, 2010.

39 P. Panine, S. Finet, T. M. Weiss and T. Narayanan, Adv. ColloidInterface Sci., 2006, 127, 9–18.

40 C. Moitzi, S. Guillot, G. Fritz, S. Salentinig and O. Glatter, Adv.Mater., 2007, 19, 1352–1358.

41 J. A. Szule, S. E. Jarvis, J. E. Hibbert, J. D. Spafford, J. E.A. Braun, G. W. Zamponni, G. M. Wessel and J. R. Coorssen,J. Biol. Chem., 2003, 278, 24251–24254.

42 T. Yeung, G. E. Gilbert, J. Shi, J. Silvius, A. Kapus andS. Grinstein, Science, 2008, 319, 210–213.

43 A. Ortiz, J. A. Killian, A. J. Verkleij and J. Wilschut, Biophys. J.,1999, 77, 2003–2014.

44 C. G. Sinn, M. Antonietti and R. Dimova, Colloids Surf., A, 2006,282–283, 410–419.

45 P. Garidel and A. Blume, Chem. Phys. Lipids, 2005, 138, 50–59.46 M. Pisani, V. Fino, P. Bruni, E. D. Cola and O. Francescangeli,

J. Phys. Chem. B, 2008, 112, 5276–5278.47 D. Uhrikova, J. Teixeira, A. Lengyel, L. Almasy and P. Balgavy,

Spectrosc.-Int. J., 2007, 21, 43–52.48 R. Lehrmann and J. Seelig, Biochim. Biophys. Acta, Biomembr.,

1994, 1189, 89–95.49 P. Garidel and A. Blume, Langmuir, 2000, 16, 1662–1667.50 S. D. Shoemaker and T. K. Vanderlick, J. Colloid Interface Sci.,

2003, 266, 314–321.51 H. Amenitsch, M. Rappolt, M. Kriechbaum, H. Mio, P. Laggner

and S. Bernstorff, J. Synchrotron Radiat., 1998, 5, 506–508.52 N. Lei, C. R. Safinya, D. Roux and K. S. Liang, Phys. Rev. E: Stat.

Phys., Plasmas, Fluids, Relat. Interdiscip. Top., 1997, 56, 608–613.53 B. Angelov, A. Angelova, U. Vainio, V. M. Garamus, S. Lesieur,

R. Willumeit and P. Couvreur, Langmuir, 2009, 25, 3734–3742.54 D. Roux, C. Coulon and M. E. Cates, J. Phys. Chem., 1992, 96,

4174–4187.55 D. Kitamoto, T. Morita, T. Fukuoka, M. Konishi and T. Imura,

Curr. Opin. Colloid Interface Sci., 2009, 14, 315–328.56 A. Zapf, R. Beck, G. Platz and H. Hoffmann, Adv. Colloid

Interface Sci., 2003, 100–102, 349–380.57 D. Roux, C. Coulon and M. E. Cates, J. Phys. Chem., 1992, 96,

4174–4187.58 S. T. Hyde, Curr. Opin. Solid State Mater. Sci., 1996, 1, 653–662.59 K. Larsson and F. Tiberg, Curr. Opin. Colloid Interface Sci., 2005,

9, 365–369.60 J. N. Isrealachvili, in Intermolecular and Surface Forces, Academic

Press, London, 2nd edn, 1991.61 D. P. Siegel, J. Banschbach, D. Alford, H. Ellens, L. J. Lis,

P. J. Quinn, P. L. Yeagle and J. Bentz, Biochemistry, 1989, 28,3703–3709.

62 T. Mares, M. Daniel, S. Perutkova, A. Perne, G. Dolinar, A. Iglic,M. Rappolt and V. Kralj-Iglic, J. Phys. Chem. B, 2008, 112,16575–16584.

63 S. Perutkova, M. Daniel, G. Dolinar, M. Rappolt, V. Kralj-Iglicand A. Iglic, in Advances in Planar Lipid Bilayers and Liposomes,ed. A. Leitmannova Liu and H. T. Tien, Academic Press, Burlington,2009, vol. 9, pp. 237–278.

64 P. Garidel, A. Blume and W. Hubner, Biochim. Biophys. Acta,Biomembr., 2000, 1466, 245–259.

65 U. R. Pedersen, C. Leidy, P. Westh and G. H. Peters, Biochim.Biophys. Acta, Biomembr., 2006, 1758, 573–582.

66 P. Garidel, G. Forster, W. Richter, B. H. Kunst, G. Rapp andA. Blume, Phys. Chem. Chem. Phys., 2000, 2, 4537–4544.

67 A. Filippov, G. Oradd and G. Lindblom, Chem. Phys. Lipids,2009, 159, 81–87.

68 M. Arseneault and M. Lafleur, Chem. Phys. Lipids, 2006, 142, 84–93.69 G. Pabst, R. Koschuch, B. Pozo-Navas, M. Rappolt, K. Lohner

and P. Laggner, J. Appl. Crystallogr., 2003, 36, 1378–1388.70 M. Rappolt, J. Appl. Phys., 2010, 107, 084701.71 A. Yaghmur, P. Laggner, S. Zhang and M. Rappolt, PLoS One,

2007, 2, e479.72 C. Czeslik, R. Winter, G. Rapp and K. Bartels, Biophys. J., 1995,

68, 1423–1429.73 J. Barauskas and T. Landh, Langmuir, 2003, 19, 9562–9565.74 J. Engblom, Y. Miezis, T. Nylander and V. Razumas, Prog.

Colloid Polym. Sci., 2001, 116, 9–15.75 P. Wadsten-Hindrichsen, J. Bender, J. Unga and S. Engstrom,

J. Colloid Interface Sci., 2007, 315, 701–713.76 A. Imberg, H. Evertsson, P. Stilbs, M. Kriechbaum and

S. Engstrom, J. Phys. Chem. B, 2003, 107, 2311–2318.77 K. Alfons and S. Engstrom, J. Pharm. Sci., 1998, 87, 1527–1530.78 V. Cherezov, J. Clogston, M. Z. Papiz and M. Caffrey, J. Mol.

Biol., 2006, 357, 1605–1618.79 T. Landh, J. Phys. Chem., 1994, 98, 8453–8467.80 A. Ridell, K. Ekelund, H. Evertsson and S. Engstrom, Colloids

Surf., A, 2003, 228, 17–24.81 A. B. Wohri, L. C. Johansson, P. Wadsten-Hindrichsen,

W. Y. Wahlgren, G. Fischer, R. Horsefield, G. Katona,M. Nyblom, F. Oberg, G. Young, R. J. Cogdell, N. J. Fraser,S. Engstrom and R. Neutze, Structure, 2008, 16, 1003–1009.

82 I. Carlsson and H. Wennerstrom, Langmuir, 1999, 15, 1966–1972.

Documents

The role of calcium in membrane condensation and spontaneous curvature variations in model lipidic systems