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Low concentrated hydroxyectoine solutions in presence of DPPC lipid bilayers: a

computer simulation study

Jens Smiatek1,2 , Rakesh Kumar Harishchandra3, Hans-Joachim Galla3 , and Andreas Heuer2
1
Institute for Computational Physics, University of Stuttgart, D-70569 Stuttgart, Germany
2
Institute of Physical Chemistry, University of Muenster, D-48149 Munster, Germany
3
Institute of Biochemistry, University of Muenster, D-48149 Munster, Germany

The influence of hydroxyectoine on the properties of the aqueous solution in presence of DPPC
lipid bilayers is studied via semi-isotropic constant pressure (NPT) Molecular Dynamics simulations.
arXiv:1305.3563v1 [physics.bio-ph] 15 May 2013

We investigate the solvent-co-solute behavior in terms of Kirkwood-Buff integrals as well as hydro-


gen bond life times for an increasing hydroxyectoine concentration up to 0.148 mol/L. The observed
preferential exclusion mechanism identifies hydroxyectoine as a kosmotropic osmolyte. Our findings
in regards to the DPPC lipid bilayer indicate an increase of the surface pressure as well as the solvent
accessible surface area in presence of higher hydroxyectoine concentrations. The results are in agree-
ment to the outcome of recent experiments. With this study, we are able to validate the visibility
of co-solute-solute-solvent effects for low and physiologically relevant osmolyte concentrations.
Keywords: Osmolytes, Molecular Dynamics simulations, DPPC lipid bilayers, Kosmotropes, Preferential
Exclusion, Kirkwood-Buff theory

INTRODUCTION network are called kosmotropes (structure makers) while


osmolytes which weaken the water network structure are
called chaotropes (structure breaker). The separation
Osmolytes allow extremophilic microorganisms to re- of co-solutes into these two species is not unique and
sist harsh living conditions [1, 2]. Typical examples straightforward [15]. Interactions and binding properties
for these species are ectoine and hydroxyectoine which between kosmotropes and chaotropes can be predicted
are zwitterionic, strong water binding and low-molecular by the law of matching water affinities [13, 15]. A main
weight organic molecules. The functionalities of these point of this theory is the investigation of the correspond-
molecules in living organisms among others are given by ing hydration free energies which loosely depend among
the protection of protein conformations [15] and the other factors on the molecular charge [15]. One of the
fluidization of lipid membranes [6, 7]. The protective major achievements of the law of matching water affini-
properties become mainly important under environmen- ties is the molecular description of a repulsive behavior
tal stress conditions, e. g. high temperature, extreme for kosmotropic osmolytes from polar surfaces and vice
dryness and salinity [8]. Several studies have identified versa, the attraction of chaotropic agents like urea. The
that several combinations of osmolytes can be found in preferential binding of urea has been validated in recent
biological cells. The concentration of a single osmolyte computer simulations [16] while additional studies have
in these mixtures varies between 0.1 to 1.0 mol/L ([9] observed a kosmotropic behavior for ectoine in terms of a
and references therein). Due to the fact that a lot of preferential exclusion mechanism around Chymotrypsin
osmolytes are not affecting the cell metabolism, specific Inhibitor II [4].
molecules like the ectoines are also commonly called com- It has been stated that kosmotropic co-solutes typically
patible solutes. accumulate in the second or third hydration shell of the
Recent studies were focusing on the molecular function- solvated macromolecule. In regards to their high charg-
ality of the ectoines as well as the analysis of the protec- ing and affinity for water molecules, it is assumed that
tive behavior [3, 4]. The theoretical framework for the this appearance strongly influences the first and the sec-
explanation of co-solute-solute effects has been mainly ond hydration shell of the polar solute. The consequence
established in terms of the preferential exclusion [10] and of this behavior is given by a diminished number of
the transfer free energy model [11]. solute-water hydrogen bonds which is compensated by
These models focus on the strong ordering of the local a significant shrinkage of the solute surface to maintain a
water shell around the osmolytes and the exclusion from constant hydrogen bond surface density. It is commonly
the immediate hydration shell of the solute which re- believed that this shrinkage in size is the molecular rea-
sults in a preferential hydration behavior and a stabi- son for the preservation of native protein conformations
lization of the solutes native structure [10, 11]. Among in presence of kosmotropic co-solutes [13, 15].
these, more refined versions of theories have been in Although the stabilizing effects on proteins in presence
addition published which explicitly rely on the proper- of specific osmolytes have been studied extensively be-
ties of chaotropic and kosmotropic behavior [1215] and fore, less is known about compatible solutes and their
the corresponding interaction with macromolecular sur- interactions with bilayers. A small number of theoretical
faces. Co-solutes which strengthen the water hydrogen studies have focused on sugars like trehalose and their in-
2

teractions with lipid membranes and monolayers [1723]. date an increase of the surface pressure and the solvent-
For high molar concentrations of trehalose, it has been accessible surface area in agreement to the experimental
found that replacement of water molecules by the forma- results [57]. We emphasize the importance of electro-
tion of additional sugar-membrane hydrogen bonds plays static interactions between co-solutes and solutes for the
a major role [18]. Despite this interpretation, it has been understanding of the observed effects by the calculation
also discussed that the effects observed in sugar-DPPC of the bilayer electrostatic potential.
mixtures can be only systematically explained by an in- The paper is organized as follows. In the next section
terplay of several mechanisms [23]. we shortly introduce the theoretical background. In the
In addition to theoretical studies, experimental findings third section we illustrate the simulation details and the
have indicated a significant broadening of the liquid ex- methodology. The results for the solvent properties and
panded (LE) - liquid condensed (LC) phase transition the DPPC lipid bilayer are presented and discussed in
of monolayers in presence of ectoine and hydroxyectoine the fourth section. We briefly conclude and summarize
[6, 7]. This was mainly indicated by the study of the cor- in the last section.
responding surface pressure area isotherms. A main re-
sult of these studies was the observation of a surface pres-
sure increase for higher hydroxyectoine concentrations. THEORETICAL BACKGROUND
In addition it was supposed that the domain sizes of the
liquid condensed regions significantly shrink in presence Kirkwood-Buff integrals and preferential binding
of hydroxyectoine which corresponds to a variation of parameter
the line tension [6, 2426]. In regrds to the biological
function, the above mentioned effects are of particular The evaluation of statistical mechanics methods on
important for signaling processes and cell repair [2, 6]. the co-solvent and solvent distribution function allows
In regards to the preferential exclusion/binding behav- important insights into the preferential exclusion as
ior of osmolyte-solute-solvent mixtures, computer simu- well as binding behavior in terms of the corresponding
lations allow a detailed study of the corresponding molec- Kirkwood-Buff theory which has been introduced in the
ular mechanisms. A theoretical framework which allows early 1950s [27, 28]. The radial distribution function of
to distinguish between exclusion and binding behavior molecules or atoms around solutes can be expressed
has been established in terms of the Kirkwood-Buff the- by
ory of solutions [2729]. The corresponding analysis has (r)
been therefore successfully applied to the study of urea g (r) = (1)
,
and polyglycine interactions [16]. It has been shown that
the calculation of the Kirkwood-Buff integrals allows the where (r) denotes the local density of at a distance
effective determination of transfer free energies in addi- r around the solute and , the global density in the
tion to the detection of kosmotropic as well as chaotropic bulk phase [30]. The Kirkwood-Buff integral is given by
behavior [4, 16]. the integration of Eqn. 1
Z r=R
In this paper, we study the properties of an aqueous hy-
droxyectoine solution in presence of DPPC lipid bilay- G = lim G (R) = lim 4r2 (g (r) 1)dr
R R r=0
ers via semi-isotropic constant pressure (NPT) all-atom (2)
Molecular Dynamics simulations. The concentration of where the above relation is valid in the limit of R =
hydroxyectoine is low but physiologically relevant [9] with [4, 16, 2729]. Eqn. 2 can be used to calculate the
a maximum value of 0.148 mol/L. We have been in- excess coordination number of molecules or atoms of
spired to use these small concentrations due to recent (hydroxyectoine) around (DPPC) via [4, 27, 28]
experimental findings for aqueous hydroxyectoine-DPPC
Nxs = , G = , lim G (R) (3)
monolayer mixtures [6, 7]. Most of the simulation studies R
usually employ high co-solute concentrations which are which allows to evaluate the preferential binding coeffi-
often above one mole per liter to study pronounced be- cient (R) under the assumption of finite distances R
havior at unphysiological conditions [3, 4, 16]. With this with
study, we are able to validate the observation of effects , xs
at smaller concentrations in agreement to experimental (R) = , (G (R)G (R)) = Nxs (R) N (R)
,
findings. (4)
Our main results include the characterization of hydrox- where the indices represent solvent molecules while
yectoine as a kosmotropic osmolyte which strengthens and are the solute DPPC and the co-solute hydrox-
the water hydrogen bond network. We are further able yectoine. A negative value for the preferential binding
to validate a weakening of DPPC-water hydrogen bond coefficient of Eqn. 4 implies a preferential exclusion of
interactions in presence of hydroxyectoine. In regards hydroxyectoine from the lipid bilayer surface while a pos-
to the DPPC lipid bilayer properties, our results vali- itive value indicates a preferential binding [4, 29].
3

Hydrogen bonds and water relaxation times a significant variation of the surface tension can be ob-
served for increasing physiological hydroxyectoine con-
The formation of lipid bilayers in aqueous solutions is centrations. The surface tension can be calculated by
mainly driven by the hydrophobic effect [31]. The de- Z
tailed molecular mechanism for the hydrophobic effect is = (PN PT (z))dz (7)
still heavily under debate but it is consensus that the for-
mation and cleavage of hydrogen bonds is of main impor- which can be also expressed for the ease of computation
tance [31]. For a detailed investigation of the co-solute- by
solvent properties, we have analyzed the water hydrogen   
bond characteristics in presence of varying hydroxyec- 1 1
= Lz Pzz (Pxx + Pyy ) (8)
toine concentrations. 2 2
We apply the Luzar-Chandler definition of hydrogen
where PN denotes the normal pressure with Pzz as the z-
bonds [32, 33], which restricts a maximum length of 0.35
component of the pressure tensor and PT the transversal
nm between the interacting oxygen and hydrogen atom
pressure given by 1/2(Pxx + Pyy ) as defined by the x-
and an angle of not more than 30 degrees. The value for
and the y-components. The factor 1/2 accounts for two
the life time allows an estimate of the relative strength
interfaces in contact with water [3740]. The box length
for the corresponding hydrogen bond. This argument is
in z-direction is denoted by Lz .
inspired by results of transition state theory which con-
The surface pressure is given by
nects the rate constant kF to the activation free energy
G via = 0 (9)
 
1 kB T G
kF = = exp (5) where 0 expresses the experimental surface tension of
F h kB T water at 300 K (71.6 mN/m) [41] which allows a direct
comparison with experiments as discussed in Ref. [19].
with the thermal energy kB T , the forward life time F
and the Planck constant h [34]. In terms of the under-
lying statistical analysis of hydrogen bonds [34], Eqn. 5
reveals that longer life times correspond to larger free ac- SIMULATION DETAILS
tivation energies which indicates a strengthening of the
hydrogen bond network. We have performed Molecular Dynamics simula-
In addition to the hydrogen bond analysis, we have tions in explicit SPC/E water [42] with the software
also calculated the dipolar reorientation time of water package GROMACS [4345]. The chemical structure
molecules in presence of hydroxyectoine. The evaluation
of this quantity gives access to an estimation of the trans-
lational solvent entropy. It has been discussed that sol-
vent entropies may play a significant role in contributions
to the solvation free energies and therefore the exposure
of hydrophilic and hydrophobic surfaces [15, 31, 35]. To
study this property, we have investigated the autocor-
relation time for the dipolar orientation ~ between two
water molecules in presence of DPPC lipid bilayers and FIG. 1: Structure of neutral hydroxyectoine.
hydroxyectoine which is given by
of hydroxyectoine ((4S,5S)-2-methyl-5-hydroxy-1,4,5,6-
(t0 ) > exp(t/ ) .
< ~(t)~ (6) tetrahydropyrimidine-4-carboxylic acid) in its neutral
form is presented in Fig. 1. The derivation of the force
It can be seen that the autocorrelation function follows field and the topology of hydroxyectoine is in detail de-
a stretched exponential behavior which is dependent on scribed in Ref. [5] where it has been also found that the
the exponent [36]. The evaluation of the correspond- zwitterionic form in aqueous solution is more stable than
ing times allows a quantitative determination of the the neutral counterpart. We follow this finding by us-
influence of hydroxyectoine on the water dynamics. ing purely zwitterionic molecules in our MD simulations.
The force field for the lipids and the starting structure
with 64 DPPC molecules [46] were modeled with the pa-
Surface pressure rameters presented in Ref. [47].
The Molecular Dynamics simulations have been carried
As it has been found out in recent experiments for out with periodic boundary conditions. The simulation
DPPC monolayers in presence of hydroxyectoine [57], box has initial dimensions of (4.724504.231909.95050)
4

nm3 . We performed simulations with 2, 4, 6 and 8 hy-


NDPPC-OHydroxyectoine
droxyectoine molecules which correspond to effective con- 1.4 NDPPC-OWater
centrations of 0.037, 0.074, 0.111 and 0.148 mol/L. Elec-
1.2
trostatic interactions have been calculated by the Par-
ticle Mesh Ewald sum [48]. The time step was t = 2 1
fs and the temperature was kept constant by a Nose-
Hoover thermostat [49] at 300 K. All bonds have been 0.8

g(r)
constrained by the LINCS algorithm [50]. 0.6
After energy minimization and a 10 ns constant volume
simulation to ensure the conformational equilibration of 0.4
the lipid molecules [23], we conducted a 20 ns equilibra-
0.2
tion run followed by a 30 ns semi-isotropic constant pres-
sure (NPT) data production simulation. A Parrinello- 0
Raman barostat has been used with a rescaling time step 0 0.5 1 1.5 2
of 2 ps. The reference pressure in the x/y- and the z- r [nm]

direction was 1 bar and a compressibility of 4.5 105


bar1 was used. The solvent accessible surface area tot FIG. 2: Pair radial distribution function g(r) for nitrogen
in DPPC (NDP P C ) and oxygen in hydroxyectoine (red line),
was calculated by the sum of spheres centered at the respectively oxygen in water (blue line) for a hydroxyectoine
atoms of the studied molecule, such that a spherical sol- concentration of 0.148 mol/L.
vent molecule can be placed in closest distance and in
agreement to van-der-Waals interactions by following the
constraint that other atoms are not penetrated [51]. be validated by the smaller occurrence of hydroxyectoine
Hydrogen bonds have been defined as present if the dis- at 0.3 nm which roughly corresponds to the peak of the
tance between the interacting atoms is less than 0.35 nm first hydration shell. Thus it can be concluded that direct
and the interaction angle is not larger than 30 degrees. interactions between hydroxyectoine and DPPC in terms
The hydrogen bond density which is calculated for the of hydrogen bonds are less important for these concen-
DPPC lipid bilayers is given by the number of hydro- trations as it was also stated in [7].
gen bonds divided by the hydrophilic solvent accessible To further investigate the hydration behavior of DPPC,
surface area HB =< NHB /HP L >. we have calculated the water hydrogen bond density at
the DPPC hydrophilic solvent accessible surface area
HL . We have found a nearly constant value of HB =
RESULTS 7.43 0.01 nm2 for the hydrogen bond density aver-
aged over all hydroxyectoine concentrations. The re-
Solvent properties sults show no significant deviations concerning higher
hydroxyectoine concentrations such that it can be as-
The average position and the distribution of hydrox- sumed, that the concentration of the co-solutes does
yectoine in front of the DPPC lipid bilayer can be easily not affect the overall water hydrogen bond density at
determined by the evaluation of the pair radial distribu- the DPPC surface. In addition, we have evaluated the
tion function (rdf). Due to the fact, that the nitrogen number of direct contacts between hydroxyectoine and
of DPPC is slightly positively charged (+0.55e) and the DPPC in terms of hydrogen bonds where we have found
oxygens in the carboxy group of hydroxyectoine are neg- a value of < NHB >= 0.6 0.5 for the highest hy-
atively charged (0.87e), we have decided to evaluate droxyectoine concentration. All other values for lower
the rdf between these two atoms due to favorable elec- concentrations are vanishing or nearly identical. Com-
trostatic interactions. Furthermore we have calculated pared to the number of hydrogen bonds between water
for a comparisoon the rdf for nitrogen in DPPC and oxy- and DPPC and their contributions to the total energy,
gen in water molecules. The results for a hydroxyectoine it can be concluded that the influence of the DPPC-
concentration of 0.148 mol/L are shown in Fig. 2. It can hydroxyectoine hydrogen bonds is nearly negligible. Re-
be seen that the pair radial distribution function g(r) garding the water-replacement theory and compared to
between DPPC and hydroxyectoine reveals a significant the results of trehalose-DPPC systems [18], it can be
appearance of the compatible solute at a distance of 0.35 concluded that the reduction of the number of water
to 0.85 nm which is in good agreement to previous as- molecules in front of the DPPC lipid bilayer by hydrox-
sumptions [5]. Comparing the pair radial distribution yectoine as it has been assumed for proteins [13] is a mi-
function for the nitrogen and water oxygen to determine nor effect which has not been detected in our simulations.
the position of the first hydration shell reveals that a Despite the additionally proposed diminished solvent ac-
large amount of compatible solutes are accumulated at cessible surface area [13], instead we have observed an
the second hydration shell of the lipid bilayer. This can increasing surface area for the lipid bilayer. We will dis-
5

cuss this point in more detail in the next section. 3.35

For a detailed investigation of the preferential exclu-


3.3
sion behavior, we have calculated the corresponding
Kirkwood-Buff integrals [16, 2729]. The results for the 3.25

F [ps]
preferential binding parameter (Eqn. 4) are shown in
Fig. 3. It can be clearly seen that the preferential binding 3.2

WaterWater hydrogen bonds


3.15
0.01
3.1
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14
0
cHydroxyectoine [mol/L]

-0.01 58

56
-0.02
(r)

54
-0.03

F [ps]
52
DPPCWater hydrogen bonds
-0.04
50
c=0.037 mol/L
-0.05 c=0.074 mol/L
48
c=0.111 mol/L
c=0.148 mol/L
-0.06 46
0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14
cHydroxyectoine [mol/L]
r [nm]

FIG. 3: Preferential binding coefficient (R) for distances FIG. 4: Water-water hydrogen bond forward lifetimes F
r up to 0.55 nm for all four concentrations in presence of (top) and DPPC-water hydrogen bond life times (bottom)
hydroxyectoine. for increasing hydroxyectoine concentrations.

coefficient (r) at lipid bilayer distances up to 0.55 nm


is negative for all hydroxyectoine concentrations. The yectoine are obvious and can be identified by a strength-
amount of exclusion increases with the concentration of ening of the water hydrogen bond network. In addition,
the compatible solutes. This also clearly states that hy- we have observed a saturation plateau of life times for
droxyectoine is preferentially excluded from the DPPC hydroxyectoine concentrations c 0.074 mol/L. The re-
bilayer surface due to energetic reasons [29]. Hence, the verse behavior can be also observed for DPPC-water hy-
observed behavior is in good agreement to the predicted drogen bonds which means decreasing life times for in-
behavior for kosmotropic osmolytes [13]. creasing hydroxyectoine concentrations until a saturation
In the framework of recent theories, it has been stated plateau is reached. Thus, it can be concluded that the
that highly charged ions can be interpreted as kos- hydration properties of DPPC bilayers in terms of ener-
motropes (structure maker) [13]. These molecules tend getic contributions are slightly disturbed in presence of
to strengthen the water-water structure by increasing hy- low molar hydroxyectoine solutions. These findings are in
drogen bond lifetimes. The influence of chaotropic so- good agreement to the law of matching water affinities
lutes like urea on the dynamics of water-water hydro- which states that kosmotropic agents weaken the hydra-
gen bonds has been investigated in Ref. [16]. It was tion behavior of polar solute surfaces [13]. Nevertheless,
found that urea in contrast to kosmotropes decrease the pronounced influence of the DPPC lipid bilayer on
the strength of the water hydrogen bond network. We the water dynamics can be also observed in terms of long
have analyzed the corresponding characteristics for hy- lifetimes as it was also discussed in a recent publication
droxyectoine in terms of the hydrogen bond transition [52]. It was mentioned that in close vicinity to the bi-
state theory and the orresponding statistical analysis layer, a significant decrease of water diffusion coefficients
[34]. The corresponding forward life times of hydrogen can be validated.
bonds for water-water interactions and water-DPPC hy- Finally we have calculated the he hydrogen bond life
drogen bond interactions are shown in Fig 4. It can be times for hydroxyectoine and water where we have found
clearly seen that hydroxyectoine leads to an increase of a nearly constant values of < F >= 4.27 0.22 ps for
the hydrogen bond life times in presence of higher con- all concentrations. To illustrate the importance of hy-
centrations. Due to the fact that the forward life times droxyectoine on the water dynamics and therefore the
are proportional to log(F ) G , an increase of the life entropic contributions in terms of hydration behavior, we
times also indicates a higher activation free energy bar- have also analyzed the water dipole reorientation times
rier G . Hence the kosmotropic properties of hydrox- (Eqn. 6). It has been stated that the general entropy
6

9.3 concentrations is a significant effect.

9.2
DPPC lipid bilayer properties
Relaxation time [ps]

9.1

9 In recent experiments for DPPC monolayers in pres-


ence of an aqueous hydroxyectoine concentration [57],
8.9
a broadening of the liquid expanded (LE) - liquid con-
8.8 densed (LC) phase transition was observed. This finding
for hydroxyectoine concentrations around 0.1 mol/L has
8.7 been validated in terms of surface pressure-area diagrams
and has been compared to urea solutions where this be-
8.6
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 havior was found to be absent.
cHydroxyectoine [mol/L] In order to prove the experimental results, we have cal-
culated the surface pressure for varying hydroxyectoine
concentrations according to Eqns. 8 and 9 for concen-
FIG. 5: Increase for the dipolar autocorrelation time in pres- trations that are comparable to the experiments. The
ence of hydroxyectoine.
results for the surface pressure in presence of hydroxyec-
toine are shown in Fig. 6. It becomes obvious that the

of the water is significantly influenced by the number 80


of intermolecular hydrogen bonds and the values for the
78
reorientation times [31, 35]. By the evaluation of the
water dipole autocorrelation function for each concen- 76
tration, we have identified a value for in Eqn. 6 of
[mN/m]

0.860.05 which validates a stretched exponential behav- 74


ior in agreement to recent results and theories [36]. The 72
corresponding relaxation times for each concentration
are presented in Fig. 5. We clearly observe an increase of 70
the relaxation times in presence of hydroxyectoine which
68
can be related to diminished entropic contributions. The
direct connection between the entropy and the relaxation 66
dynamics has been discussed in Ref. [53] in which it has 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16
been shown that the relation = 0 exp(S/kB ) with cHydroxyectoine [mol/L]
the entropy S is valid for several measurable relaxation
times. Furthermore a plateau value for hydroxyectoine FIG. 6: Surface pressure calculated by the relation = 0
concentrations of c 0.074 mol/L in agreement to the with 0 = 71.6 mN/m. The presence of hydroxyectoine leads
results of Fig. 4 can be also observed. Thus, a strong wa- to a pressure difference of 6 mN/m for the highest concentra-
ter structure influence effect can be observed in presence tion.
of hydroxyectoine.
To summarize the results of this subsection, we have val- presence of the osmolyte leads to a significant increase of
idated that hydroxyectoine is repelled from DPPC lipid the surface pressure [6]. The corresponding Pearson cor-
bilayer surfaces in terms of a preferential exclusion behav- relation coefficient is given by r = 0.93 which validates
ior. This effect is in good agreement to recent theories a linear dependence between osmolyte concentration and
concerning kosmotropic behavior for osmolytes. In a re- surface pressure. Although we have studied bilayers, the
cent study [5] we were able to indicate a net accumulation evident properties of Fig. 6 are in good agreement to the
of roughly 8-9 water molecules due to electrostatic inter- experimental findings in Refs. [57] for DPPC monolay-
actions around hydroxyectoine. Combined with these re- ers. The direct connection between the surface pressure
sults, the analysis of the hydrogen bond life times and the and the surface tension in regards to Eqn. 8 also al-
dipole reorientation times clearly reveals the kosmotropic lows to validate an identical behavior as it has been ob-
behavior of hydroxyectoine. It has to be noted that the served for trehalose-DPPC mixtures [19]. In regards to
dynamic properties have been calculated by considering the presence of unfavorable environmental conditions for
the complete number of water molecules, regardless if extremophilic organisms, the observed effect is advanta-
they interact with hydroxyectoine, DPPC or with them- geous due to the fact that it leads to a fluidization of
selves. We are therefore confident that the change of lipid membranes. It has been discussed in Ref. [2] that
global water dynamics in presence of low hydroxyectoine the enhanced flexibility of membranes facilitates cell re-
7

pair and signal transport. eral trend is obvious although a deviation for the concen-
As an additional property, we have calculated the solvent tration c = 0.111 can be identified which can be related
accessible surface area (SASA). It can be assumed that to a slight asymmetric potential distribution compared
a less rigid bilayer coincides with an increasing solvent to the other concentrations. However, these findings are
accessible surface area. The results are shown in Fig. 7. in agreement to a recent publication [54], where it has
It can be seen that a small increase of the total SASA been found that electrostatic contributions play a signif-
tot for higher hydroxyectoine concentrations is evident. icant role for the internal organization of lipid bilayers.
Although the overall amount of this increase compared to It can be assumed that the strong zwitterionic charges
the total area is small ( 1.5 nm2 compared to 43 nm2 ), of hydroxyectoine interact with the nitrogen atoms and
the general behavior follows a monotonous increase with the phosphate groups in DPPC. Due to the fact that the
a Pearson correlation coefficient of r = 0.97. We have electrostatic potential has been calculated by integrating
also calculated the ratio of the hydrophilic SASA HL , over the charges within a slice, we propose that the accu-
where only the polar regions of the molecules are taken mulation of charged groups at the interface accounts for
into account to the total SASA tot . The results are this observation. This reason has been also discussed in
shown in the inset of Fig. 7. A linear increase for this a recent publication [55], where the strong dependence
quantity can be also identified. The observed behavior of the electrostatic potential on the local ordering and
can be brought into agreement with the increased sur- the packing fraction has been pointed out. Although the
face pressure as shown in Fig. 6, due to the fact that the authors have remarked, that the molecular origin for the
hydrophilic regions form the interface with the hydrox- varying potential remains controversial, a strong depen-
yectoine solution. We therefore propose that this effect dence between local ordering of the lipid molecules and
accounts for an increased fluidization of the lipid bilayer the resulting electrostatic potential has been proposed.
in agreement to recent experiments [7]. We are therefore confident that the observed behavior
In contrast to the proposed decreased solvent accessi- validates the ordering effect of lipid bilayers in presence
ble surface for polar protein surfaces in presence of kos- of low hydroxyectoine concentrations. Hence, it has to
motropes [13], we have observed the opposite trend for be stated that only a combination of many effects may
DPPC bilayers. The difference between molecular com- explain the observed characteristics as it has been also
plex surfaces like membranes with a more flexible surface proposed in Ref. [23].
area compared to a single molecular surface may be re-
sponsible for this observation. 0.5
Finally we have calculated the electrostatic potential
0
Electrostatic potential [V]

-0.5
444.5 0.194
-1
0.1935

-1.5
HL/tot

444 0.193

0.1925 -2

443.5 0.192
-2.5
tot [nm2]

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14


cHydroxyectoine [mol/L]

-3
c=0.0 mol/L
443 -3.5 c=0.037 mol/L
c=0.074 mol/L
-4 c=0.111 mol/L
c=0.148 mol/L
442.5 -4.5
0 0.5 1 1.5 2 2.5 3 3.5 4
r [nm]
442
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14
cHydroxyectoine [mol/L] FIG. 8: Electrostatic potential for the DPPC lipid bilayer
in presence of varying hydroxyectoine concentrations.

FIG. 7: Total solvent accessible surface area for the DPPC


lipid bilayer in presence of an increasing hydroxyectoine con-
centration. Inset: Ratio of the hydrophilic HL to the total
solvent accessible surface area tot . SUMMARY AND CONCLUSION

for the DPPC lipid bilayer in presence of varying hy- Molecular Dynamics simulations of DPPC lipid bilay-
droxyectoine concentrations. The results are presented ers in presence of hydroxyectoine have been performed.
in Fig. 8. It can be seen that for higher hydroxyec- We have analyzed the properties of the aqueous solu-
toine concentrations an increase of the DPPC electro- tion for low concentrations of hydroxyectoine. Our re-
static potential can be additionally observed. The gen- sults have clearly revealed that the presence of hydrox-
8

yectoine results in a strengthening of the water hydrogen motropic co-solute results in an increased surface pres-
bond network due to increased forward life times. Due sure for DPPC bilayers which accounts for the experi-
to the fact that the life times are related to the acti- mentally observed broadening of the LE/LC phase tran-
vation free energy barriers, we propose that the water sition.
hydrogen bond network is strengthened in presence of
hydroxyectoine. Although we have simulated very low
concentrations of hydroxyectoine, all observed effects are
ACKNOWLEDGMENTS
clearly visible. Thus, we were able to show that specific
cosolute-water-solute effects can be even observed at low
physiological concentrations in agreement to recent ex- The authors thank Davit Hakobyan and Oliver Rub-
perimental findings. Furthermore we have shown that ner for enlightening discussions and helpful remarks. Fi-
the presence of hydroxyectoine leads to a energetic de- nancial support by the Deutsche Forschungsgemeinschaft
crease of the hydrogen bond network between DPPC and (DFG) through the SFB 858 and the transregional col-
water molecules in terms of slightly decreased forward life laborative research center TRR 61 is gratefully acknowl-
times. These findings are in good agreement to previous edged.
literature results for kosmotropic properties in solution.
Summarizing the results for the co-solute-solvent inter-
actions, it can be concluded that hydroxyectoine can be
interpreted as a typical kosmotropic osmolyte.
Electronic address: smiatek@icp.uni-stuttgart.de
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