pubs.acs.org/Langmuir
© 2010 American Chemical Society
Water Replacement Hypothesis in Atomic Details: Effect of Trehalose
on the Structure of Single Dehydrated POPC Bilayers
E. A. Golovina,† A. Golovin,‡ F. A. Hoekstra,† and R. Faller*,§
†
Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands, ‡Faculty of
Bioengineering and Bioinformatics, Moscow State University, Moscow, Russia, and §Department of Chemical
Engineering & Materials Science, University of California, Davis, Davis, California 95616
Received March 3, 2010. Revised Manuscript Received June 4, 2010
We present molecular dynamics (MD) simulations to study the plausibility of the water replacement hypothesis
(WRH) from the viewpoint of structural chemistry. A total of 256 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine
(POPC) lipids were modeled for 400 ns at 11.7 or 5.4 waters/lipid. To obtain a single dehydrated bilayer relevant to the
WRH, simulations were performed in the NPxyhzT ensemble with hz >8 nm, allowing interactions between lipids in the
membrane plane and preventing interactions between neighboring membranes via periodic boundary conditions. This
setup resulted in a stable single bilayer in (or near) the gel state. Trehalose caused a concentration-dependent increase of
the area per lipid (APL) accompanied by fluidizing the bilayer core. This mechanism has been suggested by the WRH.
However, dehydrated bilayers in the presence of trehalose were not structurally identical to fully hydrated bilayers. The
headgroup vector was in a more parallel orientation in dehydrated bilayers with respect to the bilayer plane and maintained this orientation in the presence of trehalose in spite of APL increase. The total dipole potential changed sign in
dehydrated bilayers and remained slightly positive in the presence of trehalose. The model of a dehydrated bilayer
presented here allows the study of the mechanisms of membrane protection against desiccation by different compounds.
Introduction
It is well established that trehalose protects membranes in desiccation tolerant organisms.1,2 This “lesson from nature” is used to
protect the content of dry liposomes against leakage in the pharmaceutical industry.3,4 Trehalose can also ensure survival of human
blood cells during freeze-drying,5 which bears promise for blood
banking.
The water replacement hypothesis (WRH) describes the mechanism of membrane protection by trehalose.1,2 This mechanism is
based on replacement of water molecules by sugars in their interactions with polar groups of membrane lipids. These interactions
maintain spacing between lipids and prevent the increase of the
membrane main gel to fluid phase transition temperature Tm. As a
consequence, dry membranes remain in a fluid state at physiological temperatures and avoid a phase transition during rehydration. The transient coexistence of fluid and gel phases in a
membrane during rehydration causes leakage and is detrimental
for living organisms.
The WRH has considerable experimental support (see e.g.
refs 2 and 3 and references therein). However, all experimental
*To whom correspondence should be addressed.
(1) Crowe, J. H.; Crowe, L. M.; Chapman, D. Preservation of membranes in
anhydrobiotic organisms - The role of trehalose. Science 1984, 223 (4637), 701703.
(2) Crowe, J. H.; Hoekstra, F. A.; Crowe, L. M. Anhydrobiosis. Annu. Rev.
Physiol. 1992, 54, 579-599.
(3) Crowe, J. H.; Crowe, L. M.; Oliver, A. E.; Tsvetkova, N.; Wolkers, W.;
Tablin, F. The trehalose myth revisited: Introduction to a symposium on
stabilization of cells in the dry state. Cryobiology 2001, 43 (2), 89-105.
(4) Crowe, J. H.; Crowe, L. M.; Wolkers, W. F.; Oliver, A. E.; Ma, X.; Auh,
J.-H.; Tang, M.; Zhu, S.; Norris, J.; Tablin, F. Stabilization of Dry Mammalian
Cells: Lessons from Nature. Integr. Comp. Biol. 2005, 45, 810-820.
(5) Wolkers, W. F.; Walker, N. J.; Tablin, F.; Crowe, J. H. Human platelets
loaded with trehalose survive freeze-drying. Cryobiology 2001, 42, 79-87.
(6) Lee, C. W. B.; Das Gupta, S. K.; Mattai, J.; Shipley, G. G.; Abdel-Mageed,
O. J.; Makriyannis, A.; Griffin, R. G. Characterization of the L-lambda phase in
trehalose-stabilized dry membranes by solid-state NMR and X-ray diffraction.
Biochemistry 1989, 28, 5000-5009.
11118 DOI: 10.1021/la100891x
data are indirect, and there is only limited structural data on lipids
that can support mechanisms described by WRH.6,7 Thus, alternative hypotheses which explain experimental data by other
mechanisms than interactions of disaccharides with lipid polar
groups have been proposed.8-11 They deny the role of sugar/lipid
interactions and consider sugar vitrification as the main mechanism of membrane protection by trehalose at low (<20%) water
content. Vitrification of the intermembrane layer causes the membranes to remain in the phase they were in at the time of vitrification. Therefore, if vitrification of the environment in the vicinity
of membranes occurs when membranes are in a fluid state, they
remain in this state in spite of dehydration.9 The preferential exclusion theory of Timasheff12 and the water entrapment hypothesis13
were developed for protein protection at dehydration stress. According to Timasheff, sugars are excluded from the vicinity of proteins,
thus preserving their hydration shell and maintaining the necessary level of hydration during osmotic stress. This theory is valid
for osmotic stress but does not relate to severe dehydration. Belton
and Gil13 extended the theory to dehydrated conditions and called
it water-entrapment hypothesis. According to this hypothesis, water,
(7) Lee, C. W. B.; Waugh, J. S.; Griffin, R. G. Solid-State NMR-Study of trehalose/
1,2-dipalmitoyl-sn-phosphatidylcholine interactions. Biochemistry 1986, 25 (13), 37373742.
(8) Koster, K. L.; Webb, M. S.; Bryant, G.; Lynch, D. V. Interactions between
soluble sugars and POPC (1-palmitoyl-2-oleoylphosphatidylcholine) during dehydration: vitrification of sugars alters the phase behavior of the phospholipid.
Biochim. Biophys. Acta 1994, 1193, 143-150.
(9) Wolfe, J.; Bryant, G. Freezing, drying, and/or vitrification of membranesolute-water systems. Cryobiology 1999, 39, 103-129.
(10) Koster, K. L.; Maddocks, K. J.; Bryant, G. Exclusion of maltodextrins
from phosphatidylcholine multilayers during dehydration: effects on membranephase behaviour. Eur. Biophys. J. 2003, 32, 96-105.
(11) Lenne, T.; Bryant, G.; Garvey, C. J.; Keiderling, U.; Koster, K. L. Location
of sugars in multilamellar membranes at low hydration. Physica B 2006, 385-386,
862-864.
(12) Arakawa, T.; Timasheff, S. N. Preferential interactions of proteins with
salts in concentrated solutions. Biochemistry 1982, 21 (25), 6545-6552.
(13) Belton, P. S.; Gil, A. M. IR and Raman spectroscopic studies of the interaction
of trehalose with hen egg white lysozyme. Biopolymers 1994, 34 (7), 957-961.
Published on Web 06/15/2010
Langmuir 2010, 26(13), 11118–11126
Golovina et al.
which was preferentially kept at the protein surface during
osmotic stress, remains near protein surface at dehydration due
to entrapment by sugar glasses.
In spite of different alternative hypotheses of membrane
protection at desiccation, WRH remains the most attractive due
to its simplicity. The first attempt to investigate the plausibility of
WRH using structural chemistry was based on molecular graphics
by applying docked structures improved by energy minimization.14,15
Reasonable binding geometries were found for trehalose with
a monolayer excised from the 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) crystal structure.
Nowadays, molecular dynamic simulations open new opportunities to study the effects of trehalose on dry bilayer structure in
atomic details. However, to date the interactions of trehalose with
lipid bilayers were studied mainly at high levels of hydration.16-22
This is mainly due to technical difficulties and long relaxation
times in simulations of dry bilayers.
In molecular dynamics (MD) simulations periodic boundary
conditions (PBC) are usually used to avoid artifacts due to the
small size of the simulation box. This effectively results in simulations of a infinite stack of bilayers instead of a single bilayer.23
When lipids are fully hydrated (around 30 waters per lipid),
bilayers are separated by a sufficient water layer, which prevents
significant interactions between bilayers. However, when considerable water is removed from the interbilayer space to create
dehydrated bilayers, separation between bilayers is reduced and
bilayers might self-interact across periodic boundaries. Even if we
avoid self-interaction by simulating multiple layers, we cannot
compare to single dehydrated membranes as occurring in nature.
In a recent study of a dehydrated bilayer stack under periodic
boundary conditions we have shown small interpenetration of
headgroups of neighboring bilayers at 11.7 waters/lipid and
considerable overlap of bilayer interfaces at 5.4 waters per lipid.24
Interpenetration creates self-spacing of bilayers and prevents
(14) Chandrasekhar, I.; Gaber, B. P. Stabilization of the bio-membrane by small
molecules: interaction of trehalose with the phospholipids bilayer. J. Biomol. Struct.
Dyn. 1988, 5, 1163-1171.
(15) Rudolph, B. R.; Chandrasekhar, I.; Gaber, B. P.; Nagumo, M. Molecular
modelling of saccharide-lipid interactions. Chem. Phys. Lipids 1990, 53 (2-3),
243-261.
(16) Sum, A. K.; de Pablo, J. J. Molecular simulation study on the influence of
dimethylsulfoxide on the structure of phospholipid bilayers. Biophys. J. 2003, 85
(6), 3636-3645.
(17) Doxastakis, M.; Sum, A. K.; de Pablo, J. J. Modulating membrane properties:
The effect of trehalose and cholesterol on a phospholipid bilayer. J. Phys. Chem. B
2005, 109 (50), 24173-24181.
(18) Pereira, C. S.; Hunenberger, P. H. Effect of trehalose on a phospholipid
membrane under mechanical stress. Biophys. J. 2008, 95 (8), 3525-3534.
(19) Pereira, C. S.; Lins, R. D.; Chandrasekhar, I.; Carlos, L.; Freitas, G.;
H€unenberger, P. H. Interaction of the Disaccharide Trehalose with a Phospholipid
Bilayer: A Molecular Dynamics Study. Biophys. J. 2004, 86 (4), 2273-2285.
(20) Sum, A. K.; Faller, R.; de Pablo, J. J. Molecular simulation study of
phospholipid bilayers and insights of the interactions with disaccharides. Biophys.
J. 2003, 85 (5), 2830-2844.
(21) Skibinsky, A.; Venable, R. M.; Pastor, R. W. A Molecular Dynamics Study
of the Response of Lipid Bilayers and Monolayers to Trehalose. Biophys. J. 2005,
89 (6), 4111-4121.
(22) Villarreal, M. A.; Diaz, S. B.; Disalvo, E. A.; Montich, G. G. Molecular
dynamics simulation study of the interaction of trehalose with lipid membranes.
Langmuir 2004, 20 (18), 7844-7851.
(23) Tieleman, D. P.; Marrink, S. J.; Berendsen, H. J. C. A computer perspective
of membranes: Molecular dynamics studies of lipid bilayer systems. Biochim.
Biophys. Acta, Rev. Biomembr. 1997, 1331 (3), 235-270.
(24) Golovina, E. A.; Golovin, A. V.; Hoekstra, F. A.; Faller, R. Water
replacement hypothesis in atomic details - factors determining area per lipid in
dehydrated stack bilayers. Biophys. J. 2009, 97 (2), 490-499.
(25) Essmann, U.; Perera, L.; Berkowitz, M. L. The origin of the hydration
interaction of lipid bilayers from MD simulation of dipalmitoylphosphatidylcholine
membranes in gel and liquid crystalline phases. Langmuir 1995, 11, 4519-4531.
(26) Feller, S. E.; Yin, D.; Pastor, R. W.; MacKerell, A. D., Jr. Molecular
dynamics simulation of unsaturated lipid bilayers at low hydration: parameterization and comparison with diffraction studies. Biophys. J. 1997, 73 (5), 22692279.
Langmuir 2010, 26(13), 11118–11126
Article
gel-phase formation.24 Interpenetration of bilayers in stack models
has been seen earlier in MD of dry bilayers.25-27 We conjectured
that interpenetration becomes possible due to reorientation of
headgroup PN vectors from facing outward to inward which
changes electrostatic potentials.24 Conditions for interpenetration
can only be created by approaching of two bilayers. Thus, MD on
dehydrated bilayer stacks is relevant to X-ray studies of bilayer
structure. Bilayer stacks at hydration levels below 14.5 waters/
lipid are used to obtain trusted diffraction intensities.28 However,
the disappearance of water layer between bilayers and overlap of
headgroups at low water contents cause changes, which do not
allow calculation of bilayer area per lipid and other structural
parameters.28 Diffraction patterns of dehydrated lipd/trehalose
mixture show a large number of reflections in both low- and wideangle regions.6 The low-angle diffraction spacing index is indicative of a lamellar phase with bilayer periodicity. The complexity of
the wide-angle diffraction pattern due the presence of trehalose
complicates the analysis of bilayer structure.6 MD simulations on
dehydrated stacks with and without trehalose complete the picture of interactions between dry bilayers and trehalose, obtained
by X-ray scattering and solid-state NMR.6,7,29
However, the fundamental idea of the WRH is formulated for a
single bilayer, and interactions with adjacent bilayers are not
considered a priori. This hypothesis relates to the situation in vivo,
where cell membranes do not form stacks at dehydration but are
embedded in a glassy matrix formed by the dry cytoplasm or dried
extracellular media. Therefore, the plausibility of the WRH from
the point of structural chemistry needs to be investigated by MD
simulations of a single dry bilayer. Such conditions of single
bilayers obviously occur in dry cells.
The only attempt to separate dry bilayers in an MD simulation
and study the effect of sugars on them was carried out by Leekumjorn
and Sum.30 The separation of neighboring 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) bilayers was provided by
argon molecules introduced between bilayers. Their results show
that unilamellar bilayers become unstable and disintegrate to nonbilayer structures at 10 waters/lipid in the absence of sugars and
maintain their stability with sugars. This breakdown was attributed to changing hydrophobic/hydrophilic interactions between
water and lipids. However, such a model is not directly experimentally relevant. According to the molecular shape concept of
lipid polymorphism, DPPC molecules organize themselves into
bilayers in both the hydrated and dehydrated state.31,32 Nonetheless, the attempt to simulate a single dry bilayer is a valid
approach to study the mechanisms of membrane protection
against desiccation and needs further development.
Methods
Details of the Simulation. All MD simulations were carried
out using the GROMACS molecular dynamics package; primarily
(27) Mashl, R. J.; Scott, H. L.; Subramaniam, S.; Jakobsson, E. Molecular
Simulation of Dioleoylphosphatidylcholine Lipid Bilayers at Differing Levels of
Hydration. Biophys. J. 2001, 81 (6), 3005-3015.
(28) Tristram-Nagle, S.; Nagle, J. F. Lipid bilayers: thermodynamics, structure,
fluctuations and interactions. Chem. Phys. Lipids 2004, 127, 3-14.
(29) Quinn, P. J.; Koynova, R. D.; Lis, L. J.; Tenchov, B. G. Lamellar gellamellar liquid crystal phase transition of dipalmitoylphospatidylcholine multilayers freez-dried from aqueous trehalose solutions. A real-time X-ray difraction
study. Biochim. Biophys. Acta 1988, 942, 315-323.
(30) Leekumjorn, S.; Sum, A. K. Molecular dynamics study on the stabilization
of dehydrated lipid bilayers with glucose and trehalose. J. Phys. Chem. B 2008,
112, 10732-10740.
(31) Cullis, P. R.; de Kruijff, B. Lipid polymorphism and the functional roles of
lipids in biological membranes. Biochim. Biophys. Acta 1979, 559, 399-420.
(32) Epand, R. M. Membrane lipid polymorphism. Relationship to bilayer
properties and protein function. In Methods in Membrane Lipids; Dopico, A. M.,
Ed.; Humana Press: New York, 2007; pp 15-26.
DOI: 10.1021/la100891x
11119
Article
short simulations were done with version 3.3.3,33 and long
trajectories were produced with version 4.0.34 The authors have
done previously extensive test simulations to ensure that different
versions of the Gromacs code behave equivalently. A united atom
description for the lipids was used. Parameters for bonded and
nonbonded interactions were taken from a study of DPPC bilayers,35 electronically available at http://moose.bio.ucalgary.ca/
files/lipid.itp. Partial charges were obtained from Tieleman and
Berendsen36 which are based on calculations by Chiu et al.37 and
can be found at http://moose.bio.ucalgary.ca/files/popc.itp. The
SPC water model38 was used. All runs were carried out with orthorhombic periodic boundary conditions in all three dimensions. To
increase the time of simulation to 400 ns and the size of the
simulation box to 256 lipids, we had to increase a time step to 4 fs.
Application of the fast constraint algorithm LINCS39 allows such
a time step. We are aware that this time step is on the larger end of
acceptable time steps. But it was shown that using LINCS time
steps of 5 and 2 fs provides similar accuracy in bilayer simulations.40 The water geometry (as a rigid molecule) was maintained
with the SETTLE algorithm.41 In some cases LINCS cannot
handle the highly connected constraints that arise from constraining both bonds and angles. To solve this issue, Feenstra et al.42
introduced dummy atoms and increased the hydrogen mass. For
our simulations this was not necessary. For Lennard-Jones interactions we used a plain cutoff (without shift function) of 1.2 nm.
Electrostatic interactions within 1.0 nm were calculated each time
step, while interactions beyond this range every 10 time steps.
Long-range electrostatics was handled by means of the particlemesh Ewald technique.43 Neighbor searching used a twin-range
approach with the cutoff of 1 nm. POPC lipids, trehalose, and
water molecules were separately coupled to a heat bath at T=310 K,
using the Berendsen algorithm44 with a coupling constant of
0.1 ps. The box size was maintained constant in the z direction.
Therefore, the simulations were carried out in the NhzpxyT ensemble. Pressure was separately coupled to 1 atm for x and y
directions with coupling constant of 1 ps using the Berendsen
weak coupling algorithm.44 The starting point for all simulations
of dry and hydrated bilayers was the self-assembled POPC bilayer
from our earlier work24 with 32 lipids per leaflet and excess water.
According to area per lipid value, this bilayer configuration was in
a fluid state. For the systems with 11.7 and 5.4 waters per lipid,
(33) Van Der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.;
Berendsen, H. J. C. GROMACS: Fast, flexible, and free. J. Comput. Chem. 2005,
26 (16), 1701-1718.
(34) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4:
Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theory Comput. 2008, 4 (3), 435-447.
(35) Berger, O.; Edholm, O.; Jahnig, F. Molecular dynamics simulations of a
fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature. Biophys. J. 1997, 72 (5), 2002-2013.
(36) Tieleman, D. P.; Berendsen, H. J. C. Molecular dynamics simulations of
fully hydrated dipalmitoylphosphatidylcholine bilayer with different macroscopic
boundary conditions and parameters. J. Chem. Phys. 1996, 105 (11), 4871-4880.
(37) Chiu, S. W.; Clark, M.; Balaji, V.; Subramaniam, S.; Scott, H. L.; Jakobsson,
E. Incorporation of Surface Tension into Molecular Dynamics Simulation of an
Interface: A Fluid Phase Lipid Bilayer Membrane. Biophys. J. 1995, 69, 1230-1245.
(38) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; Hermans, J.
Interaction models for water in relation to protein hydration. In Intermolecular
Forces; Pullman, B., Ed.; Reidel: Dordrecht, 1981; pp 331-342.
(39) Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. LINCS: A
Linear Constraint Solver for Molecular Simulations. J. Comput. Chem. 1997, 18
(12), 1463-1472.
(40) Anezo, C.; de Vries, A. H.; Holtje, H.; Tieleman, D. P.; Marrink, S. J.
Methodological issues in lipid bilayer simulations. J. Phys. Chem. B 2003, 107 (35),
9424-9433.
(41) Miyamoto, S.; Kollman, P. A. Settle: An analytical version of the SHAKE
and RATTLE algorithm for rigid water models. J. Comput. Chem. 1992, 13 (8),
952-962.
(42) Feenstra, K. A.; Hess, B.; Berendsen, H. J. C. Improving efficiency of large
time-scale molecular dynamics simulations of hydrogen-rich systems. J. Comput.
Chem. 1999, 20 (8), 786-798.
(43) Essman, U.; Perela, L.; Berkowitz, M. L.; Darden, H. L. T.; Pedersen, L. G.
A smooth particle mesh Ewald method. J. Chem. Phys. 1995, 103 (19), 8577-8592.
(44) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.;
Haak, J. R. Molecular dynamics with coupling to an external heat bath. J. Chem.
Phys. 1984, 81 (8), 3684-3690.
11120 DOI: 10.1021/la100891x
Golovina et al.
Table 1. Vacuum Model Composition and Nomenclature
Used in This Work
model
name
lipids/
box
water/
lipid
trehalose/
box
trehalose/
lipid
box height
[nm]
h28-00
v11-00
v11-03
v11-05
v11-10
v11-14
v5-00
v5-03
v5-05
v5-10
v5-14
256
256
256
256
256
256
256
256
256
256
256
28.5
11.7
11.7
11.7
11.7
11.7
5.4
5.4
5.4
5.4
5.4
0
0
80
128
256
320
0
80
128
256
320
0
0
0.31
0.5
1
1.41
0
0.31
0.5
1
1.41
6.23 ( 0.056
8.7
12
13
15
17
8.7
12
13
15
17
Figure 1. Time evolution of area per lipid at intermediate (A; 11.7
waters/lipid) and low (B; 5.4 waters/lipid) water contents and
different trehalose concentrations (Table 1). For comparison, the
data for the fully hydrated bilayer (h28-00; 28.5 waters/lipid) are
also plotted. Gray lines represent exponential fits. Insets: averaged
over the last 50 ns area per lipid in dehydrated bilayers at different
trehalose:lipid ratio. Standard deviation (SD) bars are visible if
their sizes exceed the sizes of the symbols. Red dashed line: APL for
the fully hydrated bilayer h28-00.
water was removed from the midplane of the interlamellar region
between bilayers. To create a bilayer with trehalose, different
numbers of sugars were placed randomly in the empty space after
water removal. Initial constraints on the lipids resulted in preferential location of trehalose and water along the lipid interface
before any changes in area per lipid take place. We used a void
space to keep the bilayers and their associated water separate from
their periodic images. The vapor pressure of water is negligible
such that water does not boil and the void space remains a vacuum.
To keep the same thickness of this vacuum layer (4 nm) between
bilayers through PBC, the box height was set separately for each
sugar content (8.7-17 nm). Equilibration for 20 ns under NhzpxyT
conditions was performed. To avoid artifacts due to the small
number of lipids in the box, we multiplied the system 4 times in the
xy plane. After multiplication we run each system for 400 ns with
256 POPC lipids and a corresponding number of water and
trehalose molecules. The exact compositions and our nomenclature for all systems are presented in Table 1.
Analysis Techniques. All analysis was averaged over the last
50 ns (350-400 ns) in the simulation which means at or at least
near equilibrium conditions. The area per lipid (APL) was
calculated by dividing the xy plane area of the simulation box
by the 128 lipids per leaflet. We also fitted the time evolution of the
APL to the exponential function y = A1 exp(-x/t1) þ y0, where
y is APL at any given time and y0 is APL at equilibrium conditions
(Figure 1). The values of APL, obtained by fitting and by averaging, do not deviate significantly (Table 2), which indicates that
the system around 400 ns is at (or near) equilibrium. To obtain the
density profiles, the box was divided into 200 slices along the
Langmuir 2010, 26(13), 11118–11126
Golovina et al.
Article
Table 2. APL (nm2) of Dehydrated Bilayer with Trehalose in Vacuum
Model (SE: Standard Error, SD: Standard Deviation)
model
APL [nm2]
exponential
fit ( SE
APL [nm2]
averaged over the
last 50 ns ( SD
APL [nm2] block
averaged: last 50 ns,
5 blocks each
10 ns ( SD
h28-00
v11-00
v11-03
v11-05
v11-10
v11-14
v5-00
v5-03
v5-05
v5-10
v5-14
0.6915 ( 0.0001
0.5151 ( 0.0007
0.5367 ( 0.0001
0.5284 ( 0.0002
0.5848 ( 0.0003
0.6381 ( 0.0002
0.4852 ( 0.00004
0.5114 ( 0.00003
0.5402 ( 0.00002
0.6087 ( 0.00003
0.6288 ( 0.00004
0.693 ( 0.008
0.518 ( 0.004
0.536 ( 0.003
0.530 ( 0.002
0.586 ( 0.002
0.639 ( 0.001
0.485 ( 0.002
0.515 ( 0.008
0.540 ( 0.002
0.607 ( 0.001
0.623 ( 0.001
0.6941 ( 0.005
0.519 ( 0.003
0.537 ( 0.003
0.5306 ( 0.001
0.5861 ( 0.001
0.6395 ( 0.001
0.4848 ( 0.001
0.512 ( 0.001
0.5398 ( 0.001
0.6075 ( 0.001
0.6231 ( 0.001
bilayer normal, and the number of atoms of the given molecule
(POPC, water, or trehalose) was calculated in each slice. To calculate the mass density profiles, the number of each atom in a slice
was multiplied but its atomic weight. The height of the box was
different in different modes (Table 1); therefore, the thickness of
the slices was also different. To compare density profiles between
the models, we used the same thickness of the slices in all simulations. We increased the height of each box (hz) after the simulation
but before the analysis to the maximum value of 17 nm (Table 1).
The density profiles were plotted separately for lipids, water,
trehalose, and P atoms. The profiles are symmetrized between
leaflets. Headgroup orientation is characterized by the cosine of
the angle between the PN vector (the dipole between the phosphate P atom and the choline N atom) and the bilayer normal.
Cosines are used to avoid spurious peaks resulting from Jacobians
of the transformation between internal non-Cartesian and external Cartesian coordinates.45 For characterizations we fit the distributions with several Gaussians. Electrostatic potentials for
lipids, water, and trehalose are calculated separately. Charge distributions for all components were calculated as a function of position along the bilayer normal and integrated twice. All potentials
are defined as zero in the center of the bilayer.
Results
Effect of Dehydration and Trehalose on the APL of POPC
Bilayer. Figure 1 shows the evolution of the APL during the
simulation at intermediate (11.7 waters/lipid) and low (5.4 waters/
lipid) water contents with different amounts of trehalose. Different models need different time to attain near-equilibrium conditions ranging from several tens to a few hundred nanoseconds.
The slowest equilibration was in the models with low concentration of trehalose (less than 1 trehalose per lipid).
The effect of trehalose concentration on the APL is shown in the
inset of Figure 1A for 11.7 waters/lipid and in the inset of Figure 1B
for 5.4 waters/lipid. The APL decreases with water content from
0.692 nm2 in a fully hydrated bilayer to values close to the gel state
(0.515 nm2 at 11.7 waters/lipid) or even in the gel state (0.48 nm2
at 5.4 waters/lipid) (Table 2, Figure 1). The value of APL for the
hydrated POPC is slightly higher than the values found in the literature, and this discrepancy is discussed in our previous paper.24
APL data for dry POPC bilayers are not available in the literature
to the best of our knowledge. At 11.7 waters/lipid the APL slightly
increases at low concentration of trehalose (Figure 1 A, inset).
Considerable;almost linear;growth of the APL is observed at
higher trehalose concentrations. At 5.4 waters per lipid the APL
increases linearly over the whole range of trehalose concentrations
(45) Fixman, M. Simulation of polymer dynamics: I. General theory. J. Chem.
Phys. 1978, 69 (4), 1527-1537.
Langmuir 2010, 26(13), 11118–11126
Figure 2. Profiles of the order parameter Smol from C3 to C15 at
different water and trehalose contents: (A) 5.4 waters/lipid, (B)
11.7 waters/lipid.
(Figure 1 B, inset), resulting in similar APL values at 1.4 trehalose/
lipid as in v11 models (Table 2). However, the APL of the fully
hydrated bilayer is not reached in either model (dashed red line in
insets of Figure 1).
Effect of Dehydration and Trehalose on Molecular Order
Parameter. The lipid chain molecular order parameters S mol
(using the usual definition23) were used to quantify the extent of
chain ordering induced by dehydration and trehalose in a single
dry bilayer. Smol was calculated according to the equation
Smol ¼ ð3=2Þ½cos2 θ - 1=2
where θ is the angle between the z-axis of the simulation box and
the molecular axis of the lipid molecule. The latter is defined as the
vector from Cn-1 to Cnþ1 and therefore can be applied to the
united-atom description. Moreover, S mol relates to effective length
of the acyl chain and therefore reflects stiffening or contraction of
the hydrophobic core46 and can be directly compared with the
structural parameters of the bilayer.
The order parameter profiles for the sn-1 palmitoyl chains of
POPC lipids for low (5.4 waters/lipid) and intermediate (11.7
waters/lipid) water contents and different amounts of trehalose
are shown in Figure 2 in comparison with the fully hydrated
bilayer (h28-00). Dehydration causes considerable ordering of the
palmitoyl chain, and the degree of ordering is higher in v5 models
than in v11 models. Trehalose causes concentration-dependent
disordering of the acyl chains at both water contents but does not
reach the degree of disorder of the fully hydrated bilayer (Figure 2).
The deuterium order parameter profile of sn-2 oleoyl chain is
different from that of sn-1 palmytoil chain due to the bend in the
sn-2 chain after C2 and alignment of the double bond almost
parallel to the bilayer normal.47 This unique orientation of the cis
double bond in membranes causes the characteristic dip at C10.
However, after correction for this geometric factor, molecular
order parameters are identical in both chains.47 Therefore, both
chains in POPC behave similar in terms of statistical fluctuations,
and we can use sn-1 to characterize the degree of ordering in the
bilayer core.
For better comparison at intermediate and low water contents,
the integrated order parameter (the sum of all order parameters
from C3 to C15) was plotted against trehalose content (Figure 3A).
(46) Seelig, A.; Seelig, J. Effect of a Single Cis Double-Bond on Structure of a
Phospholipid Bilayer. Biochemistry 1977, 16 (1), 45-50.
cevic, N. Molecular order in cis and trans unsaturated
(47) Seelig, J.; Waespe-Sar
phospholipid bilayers. Biochemistry 1978, 17 (16), 3310-3315.
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Figure 3. Integrated order parameter (sum of Smol from C3 to C15).
(A) Dependence on trehalose content for intermediate v11 (11.7
waters/lipid, black line) and low v5 (5.4 waters/lipid, red line) water
contents; dashed line is integrated order parameter for the fully
hydrated bilayer h28-00 (28.5 waters/lipid). (B) Correlation between
integrated order parameter and area per lipid (points represent the
data for all the models).
Surprisingly, water content influences the integrated order parameter only in models without trehalose. In the presence of sugar
the degree of disordering is determined by trehalose content
rather than hydration level (Figure 3A).
The APL and order parameters are intimately linked.46 Figure 3 B
shows the correlation between APL and the integrated order
parameter of the palmitoyl chain for all 11 models. In a log-log
scale the negative correlation is almost linear. Because of the small
range, we refrain from determining a scaling exponent. The order
parameter is more sensitive to changes of APL in or near the gel
state rather than in a fluid state of the bilayer.
Mass Density Profiles of POPC. Mass density profiles
change with dehydration (Figure 4, Table 3). The peak-to-peak
separation (PP) is partly determined by the hydrophobic thickness of the bilayer. A considerable increase of PP with dehydration results from the extension of the acyl chains in the gel state.
Dehydration is also accompanied by the increase of peak density
(Dmax) and peak sharpness originating probably from immobilization. The terminal methyl trough (Dmin) is deeper in v11-00 and
v5-00 than in the fully hydrated bilayer (Figure 4, Table 3),
indicating more highly ordered hydrocarbon chains as compared
to the fluid phase.48 The more pronounced methyl trough at low
water contents together with the gradual decrease of order parameter toward the end of acyl chains (Figure 2) show that bilayer
dehydration causes acyl chain immobilization without interdigitation. The appearance of a region of constant density within the
hydrophobic part of the bilayer (Figure 4B,C) correlates with the
plateau in order parameter profiles (Figure 2) and originates from
relatively ordered methylene groups of the acyl chains.49 All
structural changes relate to the transition from the fluid to the
gel state of the acyl chains under dehydration.
In the presence of trehalose peak-to-peak separation (PP)
decreases in both v5 and v11 models (Table 3). With intermediate
water content (v11) PP reduces to the level of the fully hydrated bilayer at lipid:trehalose = 1:1 and remains there at higher
(48) McIntosh, T. J.; Simon, S. A. Area per Molecule and Distribution of Water
in Fully Hydrated Dilauroylphosphtidylethanolamine Bilayers. Biochemistry
1986, 25, 4948-4952.
(49) Nagle, J. F.; Wiener, M. C. Relations for lipid bilayers. Connection of
electron density profiles to other structural quantities. Biophys. J. 1989, 55,
309-313.
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Golovina et al.
Figure 4. Mass density profiles of different components (POPC:
black; water: blue; P atoms: red) in fully hydrated bilayer h28-00
(A), at intermediate water content v11-00 (B), and at low water
content v5-00 (C). The mass density profile of water in each leaflet
of dehydrated bilayers can be represented as two Gaussian distributions (gray lines), which are referred to as outer and inner
water according to the positions within the leaflets. The center of
the bilayer is at z = 0.
trehalose concentration (Table 3). At low water content (v5) the
peak-to-peak separation levels off at lipid:trehalose=1:1 without
reaching the value for the fully hydrated bilayer (Table 3).
The regions of constant density between headgroups and terminal methyls disappear in dehydrated bilayers in the presence of
trehalose (Figure 5). The depth of the terminal methyl trough
decreases and in the model with intermediate water content reaches the value of that of the hydrated bilayer at trehalose/lipid =
1:2 (Table 3). At low water content the depth of the trough
decreases gradually and does not reach the value of the hydrated
bilayer even at the highest trehalose concentration (Table 3).
Overall, the structural changes indicate a concentration dependent fluidizing of dehydrated bilayers by trehalose. However, the
lipid density profiles remain different at the interface (Figure 5).
The distance between the headgroup peak and the outer edge of
the bilayer (Wouter), defined as the distance over which the headgroup density drops from 90% to 10%, decreases with dehydration from ≈0.7 nm (fully hydrated bilayer) to ≈0.5 and ≈0.4 nm in
v11-00 and v5-00, respectively (Table 3, Figure 4). Trehalose
causes a concentration-dependent increase of Wouter. It levels off
around the fully hydrated value of 0.7 nm at trehalose:lipid= 1:2
for low water content v5 (Table 3). At intermediate water content
(v11) Wouter is wider than in the fully hydrated bilayer at all
trehalose concentrations (Figure 5, Table 3) This is particularly
visible in models v11-10 and v11-14 where the peak positions are
similar to h28-00, but interfaces are considerably extended outward of the leaflets creating wings in the density profiles absent in
the fully hydrated bilayer (Figure 5). Therefore, the structure of
the dry bilayer with trehalose remains different from the hydrated
bilayer in spite of acyl chain fluidizing.
Mass Density Profiles of Water, Trehalose, and Phosphorus
(P) Atoms. Water density profiles in a fully hydrated bilayer can
be divided into two zones: the interbilayer bulk water (plateau)
and the lipid hydration shell. The density of water gradually decreases from around 1000 kg/m3 in the interbilayer space to zero in
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Table 3. Structural Parameters of Dehydrated Bilayer with Trehalose Derived from Density Profiles
5.4 waters/lipid
11.7 waters/lipid
3
3
trehalose/box
PP [nm]
Wouter [nm]
Dmin [kg/m ]
Dmax [kg/m ]
PP [nm]
Wouter [nm]
Dmin [kg/m3]
Dmax [kg/m3]
fully hydrated
00
80
128
256
360
2.83
4.01
3.91
3.65
3.26
3.23
0.7
0.4
0.6
0.7
0.7
0.7
670
480
575
570
630
650
972
1311
1176
1119
1092
1018
2.83
3.83
3.73
3.57
2.74
2.92
0.7
0.5
0.8
1.1
1.2
1.0
670
525
612
670
670
670
972
1222
1093
1002
957
984
Figure 6. Lipid (solid line), water (dashed line), and total (dotted
line) potential profiles in hydrated (h28-00, black line) and dehydrated (v11-00, red line; v5-00, blue line) bilayers. The center of the
bilayer is at z = 0.
Figure 5. Mass density profiles of POPC (black), water (external
dark blue and internal light blue), and trehalose (red) in bilayer
leaflets at intermediate 11.7 waters/lipid (left column) and low
5.4 waters/lipid (right column) water contents and different trehalose concentrations (Table 1). For comparison, the density profile
of POPC in a fully hydrated bilayer 28. Five waters/lipid (h28-00)
is presented in all graphs as gray lines. Vertical line is the position of the outer edge of the lipid interface defined at 10% of the
maximal lipid density at peak position. The center of the bilayer is
at z = 0.
the membrane core (Figure 4A). We estimate the depth of water
penetration into the bilayer core as the position from the bilayer
center, where water density is around 1% from the density of bulk
water (1000 kg/m3). Water penetrates toward the acyl chains up to
≈0.6 nm, which is slightly deeper than measured in experiment.50
Water density profiles in leaflets of dehydrated bilayers (v11-00
and v5-00 models) can be fitted by two Gaussians (Figure 4B,C).
We identify them as outer and inner water, respectively. Inner
water associates with the positions of P atoms, whereas the outer
water is shifted to the outer edge of the interface. The inner
distribution is wider than the outer in both dehydrated systems
(50) Kucerka, N.; Tristram-Nagle, S.; Nagle, J. F. Structure of Fully Hydrated
Fluid Phase Lipid Bilayers with Monounsaturated Chains. J. Membr. Biol. 2005,
208, 193-202.
Langmuir 2010, 26(13), 11118–11126
(Figure 4B,C). At intermediate water content (v11) a considerable part of the outer water is outside of the interface (Figure 5B).
The penetration depth of water toward the chains decreases from
≈0.6 nm in h28-00 to ≈1.2 nm and ≈1.5 nm in v11-00 and v5-00,
respectively.
Trehalose distribution depends on concentration. When the
number of trehalose molecules is less than the number of lipids, all
trehalose is within the interface region (Figure 5). For trehalose:
lipid g 1:1, some trehalose molecules are outside of the outer edge
of the bilayer. Trehalose penetrates as deep into the bilayer as
water. The presence of trehalose changes the water distribution
within the bilayer. The effect depends on trehalose concentration.
The outer water distribution moves out of the interface when
excess trehalose forms the layer outside of the lipid boundary
(Figure 5). The inner water distribution becomes wider in the presence of trehalose but its position does not change with concentration. Water molecules penetrate deeper into bilayer in dehydrated bilayer with trehalose than in the fully hydrated bilayer.
The depth of penetration gradually increases with the increase of
trehalose in both v5 and v11 models and finally reaches ≈0.5-0.7 nm
at the highest trehalose concentration, which is similar to that in a
fully hydrated bilayer (Figure 5).
Lipid Potential and PN Vector Orientation. Lipid electrostatic potential and orientation of the dipole vector connecting the
phosphorus and nitrogen atoms (PN vector) are closely related.24
As in most simulations the values of partial charges are fixed, only
the distribution of charges in the simulations can change the
electrostatic potential. The dominant contribution comes from
the headgroup charges such that the most notable influence on the
electrostatic potential of the lipid is the orientation of the dipole
between the phosphorus and the choline nitrogen.
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Figure 7. Potentials of POPC (black), water (red), trehalose (green),
and total potential (blue) in the models at 11.7 waters/lipid (v11, open
symbols, dashed lines) and at 5.4 waters/lipid (v5, closed symbols,
solid lines) at different trehalose:lipid ratio. Inset: potential profiles of
lipid (black), water (red), and trehalose (green) and total potential
(blue) in v11-10 model as an example. z = 0 is the center of the bilayer.
In our vacuum model dehydration causes a decrease of lipid
potential proportionally to water loss (Figure 6). The presence of
trehalose does not increase the lipid potential in dehydrated
bilayer. Instead, the lipid potential decreases further and drops
under 4 V at high trehalose contents in both v5 and v11 models
(black solid and dashed lines, respectively, in Figure 7).
The PN vector orientation is characterized by the angle θ with
respect to the bilayer normal. In a fully hydrated POPC bilayer
the distribution of cos θ for PN orientations can be fitted with one
Gaussian function with an average θ=75°.24 This value is close to
ones reported for previous simulations25,51,52 and found in experiments.53 The distribution of cos θ in the dehydrated bilayer in the
vacuum model is more complex. In the v11-00 bilayer the distribution can be fitted by two Gaussian functions and in the v5-00 model
by four Gaussians (Figure 8). This is probably the result of slow
motions, when different PN vector orientations are not completely
averaged out within the time of observation (the last 50 ns).
When cos θ > 0, the PN vector points outward (projection on
the bilayer normal is positive), resulting in a positive lipid potential.
The total lipid potential is the sum of the z-projections of all orientations of the PN vector. Increasing populations of PN vectors with
cos θ < 0 will decrease the lipid potential due to increase of the
negative z-projection. We estimate the portion of inward oriented
PN vectors, producing negative z-projection of the PN vector, as
the area under the cumulative curve at -1 < cos θ < 0 (Figure 9,
inset as an example). Indeed, we have found a negative correlation
between lipid potential and inward oriented PN vectors (Figure 9).
The data were fitted by a Boltzmann distribution (blue line).
Goodness of fit is calculated as adjusted R2 = 0.857 98.
Trehalose, Water, and Total Potentials. The water potential decreases with dehydration to a greater extent than the lipid
potential (Figure 6). Thus, the water potential does not compensate for the lipid potential, and the total potential becomes slightly
positive in v11-00 models and even more so in v5-00. The water
(51) Pandit, S. A.; Bostick, D.; Berkowitz, M. L. Molecular dynamics simulation of a dipalmitoylphosphatidylcholine bilayer with NaClþ. Biophys. J. 2003, 84,
3743-3750.
(52) Mukhopadhyay, P.; Monticelli, L.; Tieleman, D. P. Molecular dynamics
simulation of a palmitoyl-oleoyl phosphatidylserine bilayer with Naþ counterions
and NaCl. Biophys. J. 2004, 86 (3), 1601-1609.
(53) Seelig, J. [2H] Hydrogen and [31P] phosphorus nuclear-magnetic-resonance
and neutron-diffraction studies of membranes. Biochem. Soc. Trans. 1978, 6, 40-42.
11124 DOI: 10.1021/la100891x
Golovina et al.
Figure 8. Distributions of PN vector orientations (cos θ) in dehydrated models without trehalose (v11-00 and v5-00, gray lines) in
comparison with the fully hydrated bilayer (h28-00, black line). The
distributions were fitted with two (A, v11-00) and four (B, v5-00)
Gaussian functions (in separate colors). Red: sum of all fitted curves.
Figure 9. Correlation between population of inward oriented PN
vectors (cos θ < 0) and lipid potential. Data represent all the
models: black stars are models without trehalose and with different
water contents (indicated by arrows); red circles are models with
different trehalose concentration at water content of 11.7 waters/
lipid; blue triangles are models with different trehalose concentration and 5.4 waters/lipid. The data are fitted by Boltzmann function (blue line). Goodness of fit is calculated as adjusted R2 =
0.857 98. The population of inward oriented PN vectors is calculated as area under the cumulative curve of PN distribution over
cos θ. The way of calculation is shown in the inset for v5-10 as an
example. In the inset the distribution of PN vector orientations
with fitted Gaussian functions are shown in color. The cumulative
curve is shown as a gray line. The area under the gray line at cos θ < 0
represents the total number of PN vectors oriented inward the bilayer. Because the total lipid potential is the sum of the z-projections of all orientations of the PN vector, increasing populations
of PN vectors with -1 < cos θ < 0 will decrease the lipid potential
due to increase of the negative z-projection.
potential becomes less negative with trehalose, and this effect is
concentration dependent (Figure 7).
Trehalose also causes a negative potential itself (model v11-10
as example in Figure 7 inset) on top of the negative potential
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Golovina et al.
of water. The absolute value of the trehalose potential slightly
increases with increasing of trehalose:lipid ratio (Figure 7). Water
and trehalose potentials together cannot compensate for even the
decreased lipid potential, and the total potential remains slightly
positive in all cases except v5-14 (Figure 7).
Discussion
In this paper we present results of MD simulations of a single
POPC bilayer at intermediate (11.7 waters/lipid) and low (5.4
waters/lipid) hydration. The separation of lipid bilayers was
provided by a 4 nm layer of empty space, which we call vacuum.
The necessity of separation of dehydrated lipid bilayers became
evident when we failed to obtain the gel state in MD simulations
of POPC bilayer stacks even at 5.4 waters/lipid,24 while experimentally a gel state of dehydrated PC bilayers is well established.
We expect that the inconsistency between MD simulations and
experimental data results largely from periodic boundary conditions. There are, however, a number of secondary effects like the
system size (which limits fluctuations and therefore changes fluctuation dependent quantities), the force field, and several others.
For a more detailed discussion we refer the reader to a recent
contribution by Poger et al.54
When the water layer in the interbilayer space is absent, the
interfaces of neighboring bilayers come into close contact, which
might have two consequences. First, water of the hydration shell is
shared by both bilayers. Second, interfaces of adjacent bilayers
can interpenetrate and cause self-spacing. The first factor would
result in an effective almost doubling of water content, and 5.4
waters/lipids in fact can be considered close to 10.8 waters per
lipid. However, even the further decrease of water content to
2 waters/lipid did not result in formation of the gel phase. We have
shown that interpenetration and self-spacing of two adjacent
bilayers in dehydrated stack bilayers may be one of the main reasons of the absence of a gel state of lipid acyl chains at low water
content.24 The headgroups overlap is considered as the main
problem in APL calculations in stack bilayers at water contents
less than 12 waters/lipid in X-ray experiments.28
To prevent overlap, the interfaces have to be separated. There
are two requirements for the separating medium: it should not
interact with bilayers, and the separation has to be large enough to
cancel or at least significantly weaken the interactions between
bilayer interfaces. We have found that in our case 4 nm (2 nm
from each leaflet) of empty space (vacuum) between bilayers is
enough to exclude interactions; the actual value for the vacuum
layer will depend on the details of the simulations, most notably
the cutoff and the implementation of the electrostatics. The
ensemble NPT was converted to NhzpxyT, where the constant
pressure of 1 atm was maintained only in the xy-plane, while in the
z-direction the height of the box was fixed and was big enough to
prevent lipid polar group interactions via PBC. In such a model
the absence of interactions between bilayers along the z-axis and
maintaining interactions in the xy-plane results in a stable single
bilayer (400 ns of simulation) in (or near) a gel state both at 5.4
and 11.7 waters/lipid. The (near) gel state of dehydrated POPC
bilayers has been concluded from low APL (0.485 nm2 for v5-00
and 0.515 nm2 for v11-00, Table 2) accompanied by an increased
order parameter for all carbon atoms (Figure 2), an increased PP
separation, and more pronounced methyl trough (Table 3,
Figure 4).
(54) Poger, D.; Mark, A. E. On the Validation of Molecular Dynamics
Simulations of Saturated and cis-Monounsaturated Phosphatidylcholine Lipid
Bilayers: A Comparison with Experiment. J. Chem. Theory Comput. 2010, 6 (1),
325-336.
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Our dehydrated single bilayer model is different from the one
proposed by Leekumjorn and Sum,30 where dehydrated DPPC
(10 waters/lipid) bilayers lost structural integrity within 30 ns of
simulation. Formation of nonbilayer structures of dehydrated
DPPC is not supported by experimental data. It is well established
that three methyl groups on the nitrogen of PC lipids contribute a
steric component to prevent this lipid from forming nonbilayer
structures.32 The lamellar phase reappears even in PE lipids at
sufficiently low hydration.32 The reported formation of nonbilayer structures of DPPC at 10 waters/lipid30 probably results
from an unusual setup of MD simulations. These authors fixed
the size of the xy area of a dehydrated bilayer to the value of
hydrated bilayers (APL 0.645 nm2) under zero lateral compressibility (NPzAT ensemble). Such conditions prevent lateral interactions between lipids resulting in destabilization of the bilayer. In
our model, using the NPxyhzT ensemble, we allow lipid interactions within xy-plane in a dehydrated bilayer. As a result, we
obtained a bilayer in a gel state, which was stable within 400 ns.
The aim of this work was to study the water replacement
hypothesis (WRH) in atomic details. WRH is based on four main
statements: (1) dehydrated membranes are in a gel state; (2) rehydration of the membrane in the gel state causes leakage, which
is detrimental for membrane integrity; (3) trehalose interacts with
dehydrated lipids and prevents gel phase formation; and (4)
rehydration of membranes in fluid state does not cause leakage.
Using empty space between lipid lamellae, we obtained dehydrated single bilayers in a gel state, which fits the first statement of
the WRH. This model can be further used to study the validity of the
third statement of the WRH, claiming that trehalose increases the
spacing between lipids and promote fluid state of a dehydrated
bilayer. Here, we call a bilayer dehydrated if there is no interbilayer water present. This occurs when the water content is below
the size of the hydration shell (around 12 waters/lipid).
We have shown that trehalose increases the APL of dehydrated
POPC bilayers (at both 11.7 and 5.4 waters/lipid) in a concentration-dependent manner (Figure 1, Table 2). Increasing APL
correlates with decreasing order parameter (Figure 3), PP separation and depth of the methyl trough (Table 3). These structural
data are consistent with gradual concentration-dependent fluidization of bilayer core resulted from interactions between lipids
and trehalose. Therefore, our model confirms the third statement
of the WRH as well.
However, a dehydrated bilayer in the presence of trehalose is
structurally different from a fully hydrated one in spite of a similar
degree of fluidization of the membrane core (Figure 5, Table 3).
Even at trehalose concentration of 1.4 trehalose/lipid the APL
does not reach the value of the fully hydrated bilayer in both v11
and v5 models. At this concentration trehalose has a “saturation”
effect on membrane phase transition according to calorimetric
data.55 The order parameter also remains higher than in the hydrated bilayer (Figures 2 and 3). Contrary to this, some experiments
show overfluidization of dry DPPC bilayers in the presence of
trehalose (DPPC:trehalose=1:2).6,7 According to 2H NMR data,
acyl chains are much more disordered in dehydrated DPPC:
trehalose mixtures (1:2) above the phase transition than in a fluid
state of hydrated DPPC bilayer. The authors call this new type of
fluid state a λ-phase.7 On the other hand, Quinn et al.29 did not
find any structural evidence of a new fluid state designated as
λ-phase studying dehydrated trehalose/DPPC (2:1) mixture by a
real time X-ray diffraction. In our model acyl chains remain more
(55) Tsvetkov, T. D.; Tsonev, L. I.; Tsvetkova, N. M.; Koynova, R. D.;
Tenchov, B. G. Effect of trehalose on the phase properties of hydrated and
lyophilized dipalmitoylphosphatidylcholine multilayers. Cryobiology 1989, 26,
162-169.
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ordered in dehydrated POPC (both at 11.7 and 5.4 waters/lipid) at
1.4 trehalose/lipid at 310 K in comparison with the fully hydrated bilayer, which is in a fluid phase at the same temperature
(Figure 3). This is consistent with the lower APL in comparison
with the fully hydrated bilayer (Table 2). Therefore, our model
showed the trend of increasing APL of dehydrated bilayer by
trehalose, but we failed to obtain the structural characteristics
similar to that of the hydrated bilayer even in the excess of trehalose, when sugar forms a phase outside of the lipid headgroup area
(Figure 5).
More structural differences between fully hydrated POPC and
dehydrated POPC/trehalose mixtures are observed in the headgroup region. The average orientation of the PN vector is shifted
parallel to the membrane interface with dehydration and remains
like that with trehalose (Figures 8 and 9) consistent with NMR
and neutron diffraction experiments. NMR spectra of selectively
deuterated headgroups of PC lipids have shown that dehydration
results in the choline group aligning more closely with the bilayer
surface.56,57 Neutron diffraction indicated a spatial limit for PN
vector reorientation during bilayer dehydration of 12°.56 67% w/v
of trehalose at 293 K was shown to cause the shift of PN to a more
parallel orientation than in the hydrated POPC bilayer without
trehalose.58 A similar effect of trehalose on PN orientation might
be expected in a dehydrated bilayer.
The shift of the average PN orientation in dehydrated bilayer
results in the increased proportion of inward oriented PN vectors
(Figure 9), which decreases the z-component of the lipid dipole
and, consequently, the lipid potential (Figure 6). The absolute
value of water potential decreases with dehydration (Figure 6)
and decreases further with trehalose (Figure 7) probably due to
the migration of some water molecules from the phosphate group
to trehalose (Figure 5). Although trehalose creates a negative
potential, both negative potentials of trehalose and water cannot
compensate for the decreased lipid potential, and the total potential remains slightly positive in dehydrated bilayer at all trehalose
concentrations, while the total potential in hydrated POPC
bilayers (h28-00) is negative, -0.46 V.24
The value of the total potential of hydrated POPC bilayer is
in agreement with the experimental data on dipole potential
(400 mV)59 but higher than that in bilayers (220-280 mV).60
The values of the dipole potential obtained in MD are usually
higher than experimental values (around 600 mV).61 This is likely
rooted in the simulation assumption that the dielectric constant is
fixed at ε = 1. Simulations of dry DOPC bilayers have shown the
decrease of the total potential from 500 mV at 16 waters/lipid to
-300 mV at 11.4 waters/lipid and þ400 mV at 5.4 waters/lipid.27
This is in agreement with our observation of changing the total
potential with dehydration. In the model here the total potential
was -460, þ120, and þ200 mV at 28.5, 11.7, and 5.4 waters/lipid,
respectively. Because experimentally the dipole potential can be
obtained for fully hydrated bilayer only, we cannot compare our
(56) Bechinger, B.; Seelig, J. Conformational changes of the phosphatidylcholine headgroup due to membrane dehydration. A 2H-NMR study. Chem. Phys.
Lipids 1991, 58, 1-5.
(57) Ulrich, A. S.; Watts, A. Molecular response of the lipid headgroup to
bilayer hydration monitored by 2H-NMR. Biophys. J. 1994, 66, 1441-1449.
(58) Bechinger, B.; Macdonald, P. M.; Seelig, J. Deuterium NMR studies of the
interactions of polyhydroxyl compounds and of glycolipids with lipid model
membranes. Biochim. Biophys. Acta 1988, 943, 381-385.
(59) Brockman, H. Dipole potential of lipid membranes. Chem. Phys. Lipids
1994, 73, 57-79.
(60) Clarke, R. J. The dipole potential of phospholipid membranes and methods
for its detection. Adv. Colloid Interface Sci. 2001, 89-90, 263-281.
(61) Berkowitz, M. L.; Bostick, D. L.; Pandit, S. Aqueous solutions next to
phospholipid membrane surfaces: insights from simulations. Chem. Rev. 2006,
106, 1527-1539.
11126 DOI: 10.1021/la100891x
Golovina et al.
results on dehydrated bilayers with experimental data. However,
some support comes from data by Luzardo et al.62 where a
concentration-dependent decrease of the lipid potential of DMPC
monolayers in the presence of trehalose has been observed.
The inversion of the sign of the total dipole potential in dry
bilayers with and without trehalose might provide a new mechanism of membrane protection against dehydration by some
proteins. Late embryogenesis abundant (LEA) proteins have
been proposed to contribute toward desiccation tolerance, but
the actual mechanism of action is unclear.63 Yeast HSP 12 was
first identified as a putative heat shock protein but later has been
classified as a LEA-like protein.64 Membrane protection by HSP
12 against dehydration was only observed with positively charged
liposomes and not with either neutral or negatively charged
liposomes.65 Therefore, inversion of the sign of total dipole potential of a bilayer during dehydration from negative to slightly
positive might provide the conditions for interactions with the
LEA-like proteins and thus stabilize dehydrated membranes.
The data presented in this study show that the separation of
dehydrated bilayers by empty space in MD simulations provides
a means to study of the mechanisms of membrane protection against desiccation by different compounds. This model is
experimentally relevant for two reasons. First, POPC bilayer is
stable in a gel state at a low water content at ambient temperature,
which is in agreement with all experimental data on PC dry bilayers.
Second, the area per lipid of dry POPC increases when trehalose is
added. Although there is no direct experimental data on APL values
of a dry bilayer with trehalose, the decrease of the phase transition
temperature Tm of dry PC bilayer in the presence of trehalose is
commonly used as an indicator of the increased APL.66
Area per lipid, related order parameter, and density profiles are
the only structural data which are influenced by the model. Other
structural data (PN vector orientation and potential) were similar
in both stack and vacuum models, so the comparison with the
experiments, which were carried out on multilamellar liposomes,
is still valid for such data.
Our vacuum model partly validates the WRH by showing that
dehydration causes the decrease of APL and gel-state formation,
and trehalose increases APL and fluidizes the core of the dry
bilayer. However, the detailed structure of dry POPC bilayer in
the excess of trehalose is different from a fully hydrated bilayer,
particularly in the headgroup region.
Acknowledgment. This work was partly supported by project
no. 10195 from the Dutch Foundation for Technological Research STW (E.A.G.) and partly by the NATO Science Program
(NATO collaborative linkage grant LST.CLG.980168 (A.V.G.
and F.A.H.). Computer resources were provided by the Research
Computing Center of Moscow State University. The supercomputer “Chebyshev” was used for all modeling studies.
(62) Luzardo, M. C.; Amalfa, F.; Nunez, A. M.; Diaz, S.; Lopez, A. C. B.;
Disalvo, E. A. Effect of trehalose and sucrose on the hydration and dipole potential
of lipid bilayers. Biophys. J. 2000, 78, 2452-2458.
(63) Chakrabortee, S.; Boschetti, C.; Walton, L. J.; Sarkar, S.; Rubinsztein,
D. C.; Tunnacliffe, A. Hydrophilic protein associated with desiccation tolerance
exhibits broad protein stabilization function. Proc. Natl. Acad. Sci. U.S.A. 2007,
104 (46), 18073-18078.
(64) Mtwisha, L.; Brandt, W.; McCready, S.; Lindsey, G. G. HSP 12 is a LEAlike protein in Saccharomyces cerevisiae. Plant Mol. Biol. 1998, 37, 513-521.
(65) Sales, K.; Brandt, W.; Rumbak, E.; Lindsey, G. The LEA-like protein HSP
12 in Saccharomyces cerevisiae has a plasma membrane location and protects
membranes against desiccation and ethanol-induced stress. Biochim. Biophys.
Acta 2000, 1463, 267-278.
(66) Hoekstra, F. A.; Wolkers, W. F.; Buitink, J.; Golovina, E. A.; Crowe, J. H.;
Crowe, L. M. Membrane stabilization in the dry state. Comp. Biochem. Physiol.,
Part A: Mol. Integr. Physiol. 1997, 117 (3), 335-341.
Langmuir 2010, 26(13), 11118–11126