28398 |Phys. Chem. Chem. Phys., 2016, 18,28398--28402 This journal is ©the Owner Societies 2016
Cite this: Phys.Chem.Chem.Phys.,
2016, 18,28398
Combined influence of ectoine and salt:
spectroscopic and numerical evidence for
compensating effects on aqueous solutions†
Marc Benjamin Hahn,
ab
Frank Uhlig,*
c
Tihomir Solomun,*
a
Jens Smiatek*
c
and
Heinz Sturm
ad
Ectoine is an important osmolyte, which allows microorganisms to
survive in extreme environmental salinity. The hygroscopic effects
of ectoine in pure water can be explained by a strong water binding
behavior whereas a study on the effects of ectoine in salty solution
is yet missing. We provide Raman spectroscopic evidence that
the influence of ectoine and NaCl are opposing and completely
independent of each other. The effect can be explained by the
formation of strongly hydrogen-bonded water molecules around
ectoine which compensate the influence of the salt on the water
dynamics. The mechanism is corroborated by first principles calcu-
lations and broadens our understanding of zwitterionic osmolytes
in aqueous solution. Our findings allow us to provide a possible
explanation for the relatively high osmolyte concentrations in
halotolerant bacteria.
Compatible solutes or osmolytes like ectoine
1
(Fig. 1) are pro-
duced by halotolerant and halophilic bacteria in order to survive
in extreme saline environments.
2
The underlying mechanism in
thepresenceofectoinecanbeexplained by balanced chemical
potentials inside and outside the cell which permit a change of
the interior salt concentration due to osmosis.
3,4
In fact, recent
experimental and simulation results revealed a strong influence
of ectoine on the local water structure.
5–9
In more detail, it was
shown that ectoine forms around seven strong hydrogen bonds
with its first hydration shell, which explains its pronounced
hygroscopic properties.
5,10
Therefore, ectoine is also often used
as the main ingredient in moisturizers and pharmaceuticals.
11
Further beneficial properties are given by the protection of
enzyme structures against heating, freezing and drying,
12
and
the fluidization of lipid bilayers and membranes under high
pressures.
5,13–15
In summary, ectoine can be regarded as a versatile
osmolyte with potential applicability in many consumables.
16
Although the properties of ectoine in pure water were
studied in detail before, it is yet unclear if the observed effects
remain valid in a salty solution. Previous simulation results
verified that the number of hydrogen bonds with the local water
shell is constant for molar salt concentrations of up to
0.5 mol L
1
.
5
Nevertheless, the presence of high molar salt
concentrations and the possible impact of Hofmeister series effects
remains of specific interest.
17
In detail, previous theories consi-
dered osmolytes like ectoine, hydroxyectoine and trimethylamine
N-oxide (TMAO) as water-structure makers (kosmotropes)
5,6,9,18
whereas some salts among other co-solutes were discussed as
water-structure breakers (chaotropes).
17,19,20
Moreover, it was
also discussed that chaotropes can be interpreted as protein
denaturants and kosmotropes as protein protectants.
21,22
Thus,
in contrast to ectoine, specific anions like I
and ClO
4
are able
to destabilize the native structure of proteins, which is known
as a typical Hofmeister effect.
21–23
In organisms, chaotropes
and kosmotropes usually occur simultaneously.
24
Hence, the
response to environmental changes is given by adaptive
concentration changes in protectants and denaturants. Indeed,
it can be speculated that the influence of ectoine is less affected
Fig. 1 Chemical structure of a zwitterionic ectoine molecule (left), and
snapshot of ectoine and its first hydration shell from molecular dynamics
simulations (right). Carbon atoms shown in green, nitrogen atoms in blue,
oxygen atoms in red, and hydrogen atoms in white. Hydrogen bonds are
symbolized by black, dashed lines.
a
Federal Institute for Materials Research and Testing, D-12205 Berlin, Germany.
E-mail: Tihomir.Solomun@bam.de; Tel: +49 30 8104 3382
b
Free University Berlin, Department of Physics, D-14195 Berlin, Germany
c
Institute for Computational Physics, University of Stuttgart, D-70569 Stuttgart,
Tel: +49 711 685 63757
d
Technical University Berlin, D-10587 Berlin, Germany
†Electronic supplementary information (ESI) available: Details of experimental
and theoretical methods. See DOI: 10.1039/c6cp05417j
Received 4th August 2016,
Accepted 22nd September 2016
DOI: 10.1039/c6cp05417j
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by ordinary monovalent salts like NaCl due to its evolutionary
success in halotolerant microorganisms. Although it is well
known that Cl
is neither a kosmotropic or a chaotropic
anion,
21
it can be assumed that the combined effects of salts
like NaCl and ectoine might impose a significant influence on
the local water structure.
With this article, we present the first Raman spectroscopy
study of the water OH stretching behavior in the combined
presence of ectoine and NaCl in physiologically relevant
concentrations.
12,25
The experimental findings are analyzed in
terms of the water structure around ectoine and its dependence
on the presence of salt. Furthermore, density functional theory
(DFT) and ab initio molecular dynamics (AIMD) simulations
were performed to study the properties of the first solvation
shell around ectoine (as depicted in Fig. 1) in more detail. The
outcomes of our simulations verify a stable hydration shell
whose influence on the global water properties is not affected
by the presence of salt for increasing ectoine concentrations.
Our findings are important for a deeper understanding of
zwitterionic osmolytes like ectoine and their functionality in
aqueous solution. Moreover, we provide a possible explanation
for the high ectoine concentration in halotolerant bacteria.
The detailed description of the experimental procedure and
the simulation protocol can be found in the ESI.†
We start the discussion with the resulting confocal Raman
spectra of ectoine in aqueous solution without salt. The Raman
spectrum of pure water at room temperature and a pressure of
1 atm (black curve in Fig. 2) shows a broad peak at wavenumber
kE3400 cm
1
with a shoulder at 3200 cm
1
. A common
approach for the interpretation of vibrational spectra is given
by a fitting procedure which relies on the use of several
Gaussian functions (or other similar profiles) which are centered
at different positions. Typical examples are presented by the
dashed curves in Fig. 2. In contrast to the underlying interpreta-
tion of the Gaussians, which is still under debate
26–34
we focus on
the spectral features which allow us to disentangle the mutual
influences of ectoine and salt on bulk water properties indepen-
dently. It turns out that the ratio of the intensities for wave-
numbers between k= 3200 cm
1
and 3400 cm
1
is a sensitive
probe for influences of ectoine and NaCl on the water properties.
Therefore, we focus on this ratio for pure water, aqueous NaCl
solutions, aqueous ectoine solutions, and aqueous ectoine
solutions in the presence of salt. In order to distinguish
between the influence of ectoine and the contributions of bulk
water, we also analyzed the Raman spectra of dry ectoine power
(bottom of Fig. 2). It can be observed that the ectoine n(CH)
vibrational frequencies are located at wavenumbers between
k= 2850 cm
1
and 3050 cm
1
. Therefore, the analysis of
experimental data was limited to kZ3050 cm
1
which belongs
to bulk water modes. These n(OH) modes are the most relevant
vibrations for our discussion as they show high sensitivity to
hydrogen bonding effects.
8
This assumption can be also
verified with regard to the results for a one molar aqueous
ectoine solution (red curve in Fig. 2) in comparison to dry
ectoine powder where a clear distinction between water and
ectoine vibrational modes can be seen. In comparison, we also
observe the corresponding OD stretching modes of neat D
2
O
between k= 2300 cm
1
and 2700 cm
1
(blue curve in Fig. 2).
The corresponding smaller bandwidth as well as the shift of
frequencies compared with bulk H
2
O can be attributed to the
larger mass of D
2
O.
In order to assign the intensity of the modes, we calculated
the area Aunder the Gaussians below and above 3300 cm
1
where the positions with maximum values can be attributed to
collective (CM) and non-collective (NM) modes of bulk water
stretching, respectively.
8
The corresponding spectra of pure
H
2
O can be best fitted by Gaussian functions centered at
wavenumbers k= 3050 cm
1
and 3215 cm
1
for the collective
modes and k= 3412 cm
1
, 3560 cm
1
and 3630 cm
1
,respectively
for the non-collective modes. For D
2
O, the equivalent peaks are
located at k= 2286 cm
1
and 2367 cm
1
(collective modes) and
2456 cm
1
, 2536 cm
1
and 2623 cm
1
(non-collective modes).
Indeed, it was discussed that all above-mentioned frequencies at
specific wavenumbers belong to OH-vibrational modes of water.
35
The results for the ratio CM/NM
H
2
O
=(A
3050
+A
3215
)/(A
3412
+A
3560
+
A
3630
)(H
2
O) and CM/NM
D
2
O
=(A
2286
+A
2367
)/(A
2456
+A
2536
+A
2623
)
(D
2
O) for various solution compositions in terms of different
ectoine and NaCl concentrations are presented in Fig. 3.
It can be seen that under the influence of NaCl ((H
2
O)NaCl),
the collective behavior of H
2
O as given by the ratio CM/NM
decreases by about 23% 4% (from CM/NM = 0.56 for c=0M
to CM/NM = 0.43 for c= 1 M, error estimated from error in
slopes of linear fits) for increasing salt concentrations (iden-
tical system with D
2
O: 41% 7%). In contrast, ectoine leads to
an increase of the relative influence of the collective modes
Fig. 2 Raman spectra in terms of OH (OD) stretching mode regions of:
(a) water, (b) 1 M ectoine solution (H
2
O), (c) D
2
O and (d) dry ectoine
powder. For clarity, the spectra are shifted vertically. The spectra (a) and (b)
are normalized with respect to the area of the OH stretching bands (after
appropriate fitting as described in the ESI†). The residual fit for water is
marked with an asterisk. The spectra (b) and (d) are additionally normalized
with respect to the area of the ectoine CH stretching. The analysis of the
data is limited to kZ3050 cm
1
. The weak ectoine NH stretching
at around 3200 cm
1
(about 1.5% of the water contribution in 1 M
ectoine solution) accounts in part for the proportionality to the ectoine
concentration, but only for H
2
O and not D
2
O due to the frequency shift in
the OD vibrations. This contribution has been accounted for in the data
of Fig. 3.
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((H
2
O) ectoine) by 9% 2% (D
2
O: 14% 2%). Hence, it can be
concluded that ectoine enhances the collective modes of water,
in contrast to NaCl which weakens the relative contribution of
the collective modes. Interestingly, the finding for pure ectoine
solution remains even valid in the presence of a 0.4 M NaCl
concentration ((H
2
O) ectoine + 0.4 M NaCl) as the data in Fig. 3
and Table 1 reveal. The increase of the CM/NM ratio with higher
ectoine concentrations points at an identical value for the slope
compared to the pure ectoine solution. The only difference is a
constant shift due to the presence of salt. Identical results with a
corresponding constant shift can be also obtained for ectoine +
0.2 molar NaCl solution (data not shown).
In order to understand these effects, we have studied
the properties of the local hydration shell around ectoine in
more detail. We computed the theoretical Raman spectrum of
ectoine in a small water cluster, and recorded the experimental
Raman spectrum of hydrated ectoine powder. For the computa-
tional and the experimental details, we refer the reader to the
ESI.†A similar approach was also described in ref. 8. The
numerical and the experimental results are shown in Fig. 4.
It can be seen that the experimental as well as theoretical Raman
spectra are dominated by peaks at wavenumbers k=3440cm
1
and 3490 cm
1
, respectively. The corresponding visual representa-
tion of the mode can be obtained from the theoretical modeling
and involves OH stretching of three water molecules bridging the
COO
and NH
+
groups of ectoine. Hence, it can be assumed that
the resulting binding behavior to the carboxyl group (COO
)
mostly dominates the water properties at these wavenumbers
and explains the hygroscopic properties of ectoine.
5
Electronic
structure calculations
5,36
suggested that the zwitterionic form
of ectoine in water at pH = 7 is more stable than the neutral one
(DE= 10.81 kcal mol
1
),
5
which is consistent with our Raman
spectroscopy measurements of a 1 M ectoine solution which
do not show any bands around 1750 cm
1
expected in the
presence of a protonated COOH group. The zwitterionic structure
of ectoine involves a half-chair conformation with the COO
group in an axial position,
37
giving rise to a large dipole
moment between the COO
and the pyrimidinium groups of
the molecule.
5
In addition, the structure of ectoine and its
conformation found in solution (half chair with axially oriented
carboxyl-group and zwitterionic properties) is also highly stable
Fig. 3 Ratio of the intensity between the collective and the non-
collective OH (D) stretching modes for different solute concentrations.
(H
2
O) ectoine represents a solution with increasing amount of ectoine in
the absence of salt, (H
2
O) ectoine + 0.4 M NaCl denotes an aqueous 0.4
molar NaCl solution with increasing concentrations of ectoine and (H
2
O)
NaCl depicts the results for an aqueous salt solution in the absence of
ectoine and increasing concentrations of NaCl. The nomenclature is also
valid for the results in terms of D
2
O. For clarity, the data for (H
2
O) ectoine
in the presence of 0.2 M NaCl, linearly increasing with ectoine concen-
tration and lying between the 0.0 M NaCl and 0.4 M NaCl data, are
omitted.
Table 1 Results from linear fits to the data displayed in Fig. 2, slope Mand
intercept CM/NM
c=0
, and correlation coefficients R
2
. The composition of
the systems is explained in the caption of Fig. 3. The errors of the slopes
are all smaller than 0.01, and the errors in the intercept CM/NM
c=0
are
below 0.005, except for (D
2
O) NaCl, where errors are 0.015, and
0.016, respectively
System MCM/NM
c=0
R
2
(H
2
O) ectoine 0.050 0.551 0.959
(H
2
O) ectoine + 0.4 M NaCl 0.051 0.499 0.850
(H
2
O) NaCl 0.122 0.555 0.978
(D
2
O) ectoine 0.032 0.290 0.887
(D
2
O) NaCl 0.123 0.314 0.984
Fig. 4 Experimental Raman spectrum obtained by direct hydration
of ectoine powder (above) and theoretical Raman spectrum (below)
corresponding to the OH stretching mode of the ectoine–water complex.
The visual representation of the corresponding configuration is shown
attherightpartofthefigure,withthesamecolor-codingasshown
in Fig. 1.
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and dominant in ectoine crystalline states.
37
Based on these
findings, one can assume that the strong coordination of water
molecules to the NH
+
and the COO
group might result in the
formation of a strengthened local water structure involving
multiple hydrogen bonds within the ectoine–water complex in
agreement with the visual representation of the mode shown in
Fig. 4. Moreover, in agreement with previous findings,
5,8
it is
known that the lifetimes of hydrogen bonded water molecules
to the carboxyl group are significantly longer compared with
bulk water properties.
5,8
Thus, it can be concluded that the
hydrogen bonds of water molecules with ectoine are energeti-
cally more stable compared to intermolecular water hydrogen
bonds. Hence, it can be assumed that water molecules which
interact with the strongly bound water molecules at ectoine are
mostly important for the increase of the collective modes over
the non-collective modes. In order to understand the influence
of ectoine in more detail, we performed ab initio molecular
dynamics simulations and focused on the resulting water
properties in comparison to pure water. The details of the
simulation protocol are described in the ESI.†
Two distinct sets of ab initio molecular dynamics simula-
tions were performed. One set of simulations contained three
molecules of ectoine in aqueous solution (at 1.6 mol L
1
concentration), whereas the other simulation setup is repre-
sented by water molecules only. We performed constant energy
NVE simulations with effective temperatures in the range
between 320 K and 344 K. Fig. 5 presents the radial distribution
functions between the oxygen atoms of water molecules at
different temperatures in the presence of ectoine and in
comparison to pure water. It can be clearly seen that ectoine
leads to a stronger binding of water molecules at short distances
due to higher peak values at r= 2.7 Å. Hence, the corresponding
potential of mean force differences at r=2.7Å,aregivenby
DPMF
322K
ERTlog(g(r)
ect+H
2
O
/g(r)
H
2
O
)E0.8 kcal mol
1
with
the molar gas constant R, which verifies a stronger attraction
between water molecules in the presence of ectoine. Moreover, it
can be seen that higher temperatures diminish the number of
water molecules in the first hydration shell due to lower peak
values at r= 2.7 Å as one would expect. In addition, the resulting
differences between the radial distribution functions in the
presence of ectoine and for pure water at longer distances
rZ4 Å are negligible. Hence, it can be assumed that the
presence of ectoine mainly influences the first hydration shell
around water molecules which indicates the strengthening
of local interactions. These results are also in agreement
with previous simulation and experimental findings
8,9
which
verified longer relaxation times of water molecules, longer
lifetimes of hydrogen bonds between water molecules, and also
a modified dielectric constant of water in the presence of
ectoine. The question now arises why a local effect has such a
big influence on the global water properties. Indeed, due to the
high concentration of ectoine in solution, most water molecules
are in direct or indirect contact with an ectoine molecule.
In detail, for 1.6 M ectoine solution roughly 80% of all water
molecules are located within a distance of rE8 Å around
ectoine which roughly corresponds to the second hydration
shell. Hence, for ectoine concentrations which resemble physio-
logical conditions,
2
the amount of bulk water is rather low which
explains the strong influence of ectoine on global water
properties.
In conclusion, the experimental and simulation results
revealed a strengthening of local interactions between water
molecules in the presence of ectoine. We observed a significant
amount of strongly bound water molecules to the COO
and
NH
+
groups of ectoine. The corresponding vibrational modes
are verified by experimental and calculated Raman spectra.
With regard to the relatively high ectoine concentration, which
is roughly comparable to physiological concentrations, we
conclude that this local effect also drastically influences the
global properties of water. The Raman spectroscopic measure-
ments indicated that ectoine increases the ratio of collective
vibrational behavior over non-collective modes for water
molecules whereas monovalent salts like NaCl decrease this
ratio. Our findings indicate that the direct effects of salt and
ectoine are independent and mutually compensate each other for
comparable concentrations. This finding explains the relatively
high osmolyte concentrations in halophilic bacteria which survive
in extremely saline environments.
Our results reveal a strong influence of ectoine on the local
water structure, which can be attributed to the specific electronic
and conformational properties of zwitterionic ectoine. Indeed,
our study verifies the superb properties of hygroscopic co-solutes
and provides a more detailed understanding of the highly
optimized evolutionary strategies that provide the possibility for
organisms to survive under extreme environmental conditions.
We greatly acknowledge helpful discussions with Johannes
Zeman and Anand Narayanan Krishnamoorthy. This work was
supported by the German Science Foundation (DFG) under
contract number STU 245/4-1, the cluster of excellence ‘Simulation
Technology’ (EXC 310) and the SFB 716. M. B. H. also acknowl-
edges support from the MIS-project of the BAM.
Fig. 5 Radial distribution functions between the oxygen atoms of water
molecules at different temperatures in pure water (magenta line: T=336K,
light blue line: T= 320 K) and in a 1.6 molar ectoine solution (red line:
T= 344 K, dark blue line: T=322K).
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