Article
pubs.acs.org/Langmuir
Self-Assembly of X‑Shaped Bolapolyphiles in Lipid Membranes:
Solid-State NMR Investigations
Anja Achilles,† Ruth Bar̈ enwald,† Bob-Dan Lechner,‡ Stefan Werner,‡,§ Helgard Ebert,∥
Carsten Tschierske,∥ Alfred Blume,‡ Kirsten Bacia,‡,§ and Kay Saalwac̈ hter*,†
†
Institut für Physik − NMR, ‡Institut für Chemie − Physikalische Chemie, §ZIK HALOmem, and ∥Institut für Chemie − Organische
Chemie, Martin-Luther-Universität Halle-Wittenberg, D-06120 Halle, Germany
ABSTRACT: A novel class of rigid-rod bolapolyphilic molecules
with three philicities (rigid aromatic core, mobile aliphatic side
chains, polar end groups) has recently been demonstrated to
incorporate into and span lipid membranes, and to exhibit a rich
variety of self-organization modes, including macroscopically
ordered snowflake structures with 6-fold symmetry. In order to
support a structural model and to better understand the selforganization on a molecular scale, we here report on proton and
carbon-13 high-resolution magic-angle spinning solid-state NMR
investigations of two different bolapolyphiles (BPs) in model membranes of two different phospholipids (DPPC, DOPC). We
elucidate the changes in molecular dynamics associated with three new phase transitions detected by calorimetry in composite
membranes of different composition, namely, a change in π−π-packing, the melting of lipid tails associated with the
superstructure, and the dissolution and onset of free rotation of the BPs. We derive dynamic order parameters associated with
different H−H and C−H bond directions of the BPs, demonstrating that the aromatic cores are well packed below the final phase
transition, showing only 180° flips of the phenyl ring, and that they perform free rotations with additional oscillations of the long
axis when dissolved in the fluid membrane. Our data suggests that BPs not only form ordered superstructures, but also rather
homogeneously dispersed π-packed filaments within the lipid gel phase, thus reducing the corrugation of large vesicles.
■
INTRODUCTION
Lipid molecules constitute the main building blocks of cell
membranes, the latter being complex entities that contain many
additional components such as proteins, carbohydrates, and
cholesterol. The organization of all these components within
the membrane determines biological function, and its
molecular-scale study is a major research goal. The selforganization of synthetic amphiphilic molecules within lipid
membranes to form entities with controlled functionality such
as, e.g., synthetic ion channels, has found much interest in
recent years.1−9 Also, facial amphiphilic molecules were shown
to be useful for drug delivery.10 The interactions of different
parts of such synthetic molecules with the lipids within the
membrane, as well as their influence on structure and dynamics
of the lipid molecules, have not been investigated systematically. It is thus of interest in which way the properties of the
membrane as a whole or that of the individual lipid molecules
are changed in the presence of the synthetic molecules. In
addition, the self-assembly of the guest molecules and the
formation of a composite structure with lipids within the bilayer
may lead to membrane domains where the lipids show similar
behavior as previously found for the so-called liquid-ordered
phase in phospholipid-cholesterol systems.11,12
In two recent publications13,14 we have reported on novel
bolapolyphilic molecules (BPs), which can be incorporated into
model lipid bilayers (see Figure 1a). The BPs consist of a rigid
hydrophobic phenylene-ethinylene backbone with two hydro© 2016 American Chemical Society
philic glycerol headgroups attached at both ends. In addition,
two hydrophobic, flexible alkyl chains are connected to the
central phenyl ring, giving the molecules an X-shaped structure.
One of those molecules is the BP B12, which was hypothesized
to self-organize into a honeycomb lattice, where the honeycomb walls are formed by the rod-like π−π-stacked backbone
and its cells accommodate the alkyl side chains as well as
confined lipids.13 The glycerol headgroups are aligned along the
upper and lower edges of the honeycombs. This structural
model was based upon an extensive multimethod approach,13
and included the assignment of phase transitions to different
molecular processes on the basis of integrals from 1H highresolution magic-angle spinning solid-state NMR spectra.
In the present paper, we provide a detailed description and
discussion of these results and report on a variety of advanced,
site-resolved solid-state NMR experiments aimed at fully
characterizing structural and dynamic properties of the
membrane components. Probes of dynamics include the
mentioned simple 1H magic-angle spinning (MAS) NMR line
widths and intensities as well as residual 13C−1H dipolar
couplings from dipolar-shift correlation (DIPSHIFT) dephasing curves15,16 and residual 1H−1H dipolar couplings from
double-quantum (DQ) sideband patterns17−19. These were
Received: October 5, 2015
Revised: January 5, 2016
Published: January 6, 2016
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observation in sufficiently narrow integration ranges. Just above the
main lipid phase transition, all resonances of pure hydrated DPPC
become well-resolved and quantitatively detectable, while their
intensity remains lower for the mixtures. The resolved glycerol
resonance of the pure DPPC preparation at 323 K (50 °C) served as
an external intensity standard, defining 100% of the signal for each
group of resonances. Significant intensity is found in sharp spinning
sidebands, which are also integrated. Deviations of experimental hightemperature data from 100% mobilization demonstrate that the
measurements are subject to systematic errors on the order of 20%,
which can be explained by baseline problems upon integration, and the
external calibration, where a change of sample in a MAS rotor can lead
to setup variations and thus deviations in the absolute spectral
intensities.
DIPSHIFT and Recoupled DIPSHIFT. Dipolar modulation curves
were acquired using the pulse sequences described in our previous
publications16,21,22 using Lee−Goldburg homodecoupling at an
effective nutation frequency of 85 kHz (absolute nutation frequency
of 62.5 kHz at 49.1 kHz offset), relying on 13C DP or CP, depending
on which of the two gave a better signal-to-noise ratio for the BP
resonances of interest. It is stressed that we can exclude a bias due to
subensembles of different mobilities, as the 1H integrals demonstrate
that the respective BP is either fully immobilized or fully mobile at
experimental temperatures well above or below relevant phase
transitions. Recoupled DIPSHIFT data, necessary to quantify rather
weak couplings, was taken with N = 4 rotor periods of recoupling.16 In
order to obtain residual 13C−1H dipole−dipole coupling constants
from the measured modulation curves, the data was fitted to analytical
formulas for the powder-averaged signal according to published
procedures, taking into account the specificities of CH, CH2, and CH3
groups (see, e.g., the Supporting Information of ref 21). Note that 13Ccentered DIPSHIFT techniques, as opposed to so-called protondetected local field techniques23 provide only one effective order
parameter for CH2 groups, even in cases where the two protons are
magnetically and dynamically inequivalent.
1
H−1H DQ Spinning-Sideband Analysis. For the neighboring
aromatic protons, the corresponding dipole−dipole coupling constants
were determined by DQ24 spinning sideband analysis17−19 using the
BaBa-xy16 pulse sequence25,26 at 10 kHz MAS and 4 rotor periods of
recoupling. The pattern was fitted to a numerically calculated and
Fourier-transformed powder average of the theoretical time domain
signal, assuming a Gaussian distribution of residual dipole−dipole
couplings Dres. Its standard deviation was usually around 10% of the
average, indicating weak apparent distribution effects.27 Such effects do
not necessarily arise from an actual coupling distribution, but can also
be assigned to changes in the pattern shape arising from couplings to
remote protons19 or from a bias in the assumed isotropic powder
average24 as a consequence of an anisotropic transverse relaxation time
T2 .
Figure 1. (a) Structures of the investigated BP molecules with n = 12
and different hydrophilic headgroups and a sketch showing the
membrane integration of the BPs. Fluorescence images of giant
unilamellar vesicles (GUVs) prepared from (b) DPPC+B12, (c)
DOPC+B12 and (d) DPPC+E12/7 at molar ratios of 10:1. Lipids are
doped with a red dye label, while the BPs exhibit green
autofluorescence. Images in panels b and d are reproduced from ref
13 with permission of John Wiley & Sons.
measured for pure B12 and its mixtures with DPPC (molar
ratios 1:10 and 1:4) and DOPC (molar ratio 1:10). A
headgroup-modified BP (E12/7) was also investigated in a
mixture with DPPC. With these sources of information we
could assign the three additional new phase transitions in the
differential scanning calorimetry (DSC) thermograms of the
B12/DPPC mixtures to one specific molecular process each.
Furthermore, we obtained a coherent picture of the molecular
dynamics and thus also structural aspects of the BP molecules
within the lipid bilayer as well as in the bulk.
■
EXPERIMENTAL SECTION
1
H and 13C−1H MAS NMR. MAS NMR investigations were carried
out on multilamellar vesicle (MLV) preparations made by codissolving
lipid and the respective BP in methanol/chloroform, drying, and
subsequent rehydration by adding 50 wt % of H2O/D2O to the
powder. All data was acquired on a Bruker Avance III instrument with
400 MHz 1H Larmor frequency using a Bruker 4 mm MAS WVT
double-resonance probe head at 5−10 kHz spinning frequency, relying
on a flow of heated air for temperature regulation. Typical 90° pulse
lengths were 3 and 3.2 μs for 1H and 13C, respectively. 13C spectra
were usually taken with a 5 s recycle delay using either direct
polarization (DP) by a 90° pulse or cross-polarization (CP), with 1.5
or 5 ms contact time using spin-lock nutation frequencies of 83 kHz
and 71.5 kHz for 13C and 1H, respectively. SPINAL6420 was used for
1
H dipolar decoupling at a nutation frequency of 83 kHz. Heating
effects due to bearing air friction under MAS and long rf irradiation
were taken into account by a calibration and by sufficiently long
recycle delays, respectively, as tested on the phase transition
temperatures of hydrated pure lipids.
Quantitative 1H MAS Spectra. Weight-controlled samples were
prepared in 4 mm MAS rotors using Teflon spacers for reproducible
sample positioning. The recycle delay was at each temperature
adjusted to a value much larger than T1 relaxation time, and the
spectral intensities were corrected for the Curie temperature
dependence, i.e., multiplying them by T/323 K (T in K), allowing
for a quantitative evaluation of the integrals. The aliphatic, aromatic,
and glycerol resonances of immoblized BPs and DPPC in the gel
phase (20 °C) at the given spinning frequency of only 5 kHz are
subject to dipolar line broadening down to the baseline level due to
the well-defined packing and low mobility, precluding their
■
SUMMARY OF PREVIOUS RESULTS
Fluorescence imaging and spectroscopy experiments on giant
unilamellar vesicles (GUVs) prepared from DPPC and B12 at a
10:1 molar ratio revealed a self-assembly of B12 within the
membrane exhibiting a dendritic structure of a snowflake-like
appearance with 6-fold symmetry, as seen in Figure 1b.13 From
orientation-dependent (polarized) fluorescence measurements
an incorporation of B12 in transmembrane orientation could be
deduced. Furthermore, in DSC thermograms of such mixtures,
three new phase transitions appeared in addition to the main
phase transition of the pure lipid at about 42 °C, see Figure 2a
and Figure 3 of ref 13. This was also interpreted as an
indication of phase separation between pure phospholipid and
B12-rich domains, the latter with variable B12 and significant
lipid content. For high temperatures (T ≥ 75 °C), a
homogeneous mixture was assumed and proven with different
methods.14
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evidenced by 31P NMR measurements. Analysis of the 31P
line shape and T2 relaxation time, probing headgroup dynamics,
revealed that the two structural fractions exhibit different
motional amplitudes: one as in the pure DPPC and another
one with notably larger amplitude despite alkyl chain order
persisting up to higher temperatures.
Based on these measurements, a structural model for B12 in
DPPC in the temperature range of phase separation was
derived, including an explanation for the hexagonal structure.
This honeycomb model includes stacked B12 backbones
arranged along honeycomb walls, while the resulting hexagonal
cells are filled by the lateral alkyl chains of B12 as well as those
of DPPC. The DPPC alkyl groups are thus more ordered and
immobilized due to the confinement caused by the B12
molecules surrounding them, raising their main phase
transition. See Figure 12 further below for a sketch.
For a different lipid, the unsaturated DOPC, a different
situation is suggested by fluorescence imaging (see Figure 1c).
The B12 molecules appear to be homogeneously distributed
within the bilayer, showing no macroscopic phase separation. In
addition, structural variation of the hydrophilic headgroups of
the BP leads to different interactions with the lipid DPPC. E12/
7 with its oligo(ethylene oxide) (EO) headgroup (see Figure
1a) is also incorporated in a transmembrane fashion, but again a
nearly uniform distribution of the BP in the bilayer is suggested
by GUV imaging (see Figure 1d). Hence, we either have freely
mobile, individual E12/7 within the bilayer, or only small
complexes of E12/7 and possibly lipid. This difference was
attributed to the ability of a BP headgroup to either form
hydrogen bonds or not, which appears to be a key factor for
macroscopic phase separation within the membrane. The
question of whether we indeed have full solubility or possibly
nanometer-scale phase separation in the latter two cases will be
addressed below.
Figure 2. (a) DSC thermograms of pure DPPC and mixtures with B12
at different molar ratios. Given values for ΔH are the sum of secondary
and main phase transistions of the lipid. The additional thermal
transitions are numbered in Roman. (b) 1H MAS and (c) 13C DP
MAS NMR spectra of a hydrated DPPC-B12 mixture (4:1). The
aromatic 13C resonances are simply numbered according to their
appearance in the spectrum. Panel a reproduced from ref 13 with
permission of John Wiley & Sons.
■
RESULTS AND DISCUSSION
DPPC+B12. The 1H and 13C NMR spectra in Figure 2 of
the DPPC-B12 mixture are well-resolved, enabling site-resolved
measurements. In particular, all B12 core signals are accessible,
which can thus be analyzed in detail. As described above and in
our recent paper,13 temperature-dependent quantitative 1H
NMR spectra were acquired to investigate the phase behavior
between 20 and 75 °C qualitatively. We here discuss these
published results in more detail than was possible in our first
account.13
Mobility from 1H Signal Intensities. As the measured 1H
spectral integrals (including spinning sidebands) are proportional to number of mobile molecules in the sample, the values
for the alkyl chain, the glycerol-2 proton (g2) as well as for the
B12 aromatic rings were analyzed (see Figure 3a). For the pure
DPPC below its main phase transition temperature, only very
low signal intensities are detectable, indicating mostly immobile
lipids. At the main transition (∼40 °C), the integral increases to
the expected maximum, meaning that all lipid molecules
become mobile at this temperature.
For the B12-DPPC mixture, a different picture is observed.
Below the main lipid transition, all lipids as well as the B12
aromatic cores show negligible signal, indicating immobile, thus
well-packed molecules. At the main lipid transition, only a
fraction of the lipid molecules becomes mobile for the different
mixtures, as the alkyl and g2 intensities do not reach the
expected maximum value. Upon increasing the temperature
beyond the main transition, the intensity of the lipid signals
Figure 3. Temperature-dependent proton integrals of different
spectral regions for (a) pure DPPC and the 10:1 mixture with B12,
and (b) the 4:1 mixture. The respective DSC thermograms are also
included in the graphs. The additional thermal transitions are
numbered in Roman. Plots adapted from ref 13 with permission of
John Wiley & Sons.
Further structural investigations included X-ray diffraction
measurements. Above the main phase transition temperature of
the lipid, intense WAXS peaks were still visible, indicating the
sustained presence of ordered lipid domains. These were
interpreted as a modified gel phase with different packing of the
alkyl chains as compared to a pure lipid gel phase.14 In addition,
the coexistence of lipid domains with tilted and nontilted chains
with respect to the membrane normal was suggested, in which
the nontilted chains are assigned mainly to the B12/DPPC
complexes. Two coexisting structural domains were also
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given temperature above the main lipid transition, as well as by
the gradual in- and decrease of signal intensities on heating in
the DP and CP spectra, respectively. Above 70 °C, only one set
of signals is observed, in agreement with the notion that all
molecules are now mobile, as evidenced from the 1H integrals.
Turning to B12, note first that not all core resonances could
be uniquely assigned. The numbering scheme in Figure 2
simply follows the resonance position; CH (ar3,4,7−10) and
quaternary Cq (ar1,2,5,6) resonances could be distinguished by
CP contact time variation in bulk B12. Relative peak intensities
and comparisons to solution-state spectra allowed us to assign
ar8 and ar10 to the CH resonances of the central ring and of the
outermost ring-CH, respectively, while ar3,4,7,9 represent the
remaining ring-CH resonances. Also, the glycerol headgroup
resonances of B12 (G1−4) were not assigned further. The
multiplicity of the resonances, being higher than expected by
molecular symmetry, suggests a symmetry break as a result of
molecular packing.
An interesting observation is made for the aromatic core of
B12. For the signal ar3, a temperature-dependent change in
chemical shift is evidenced (see Figure 4). Whereas at 50 °C
only one signal at approximately 134.4 ppm is visible, two
signals can be clearly distinguished at 60 °C. The first signal
decreases and finally disappears, while the second one at
approximately 133.7 ppm increases. Therefore, it is suggested
that the two signals reflect the same carbon atom but with
different molecular packing. The change in chemical shift is
very likely due to a different position of the respective carbon in
the vicinity of another aromatic ring, i.e., a packing-related ring
current effect.28,29 As the spectra also indicate different
mobilities, the change in packing obviously provides increasing
motional freedom. By referring again to the DSC thermogram
of the corresponding 1:4 mixture of B12 and DPPC (see Figure
2a), the temperature of the packing alteration indeed fits to the
first additional phase transition. We thus assign transition I to a
change in π−π packing as well as mobility of the B12 cores.
Dynamic Order Parameters. In order to elucidate the
molecular dynamics in the different phases in more detail,
13
C−1 H DIPSHIFT experiments were applied for the
determination of dynamic order paramerers related to different
CH bond directions.30 In general, the DIPSHIFT method
provides the magnitude of 13C−1H dipolar coupling constants
DCH. Dipolar couplings are sensitive to both distance and
internuclear orientation of the involved nuclei. At fixed
distance, they thus probe the extent of averaging by motions
of the respective moiety with a rate much higher than 100 kHz.
This dynamic range is related to the magnitude of the staticlimit coupling constant DCH,stat in units of rad/s (DCH,stat/2π ≈
21 kHz). Since the angle dependence of dipolar coupling
follows the second Legendre polynomial (P2(cos θ)), a
dynamic order parameter
increases further. This supports the assumption of phase
separation and partitioning: some of the lipid molecules are
confined and therefore stay immobilized at the main lipid phase
transition. The comparably wide temperature range over which
the spectral changes take place is attributed to sample
inhomogeneity, as is also apparent from the DSC traces.
For the B12 aromatic cores, the intensity rise is even delayed
by 5−10 K as compared to the immobilized part of the lipid,
which indicates that the phase-separated B12 molecules are part
of a distinct structure. Above all phase transitions (T > 75 °C),
all integrals reach the expected maximum, indicating fully
mobilized molecules and absence of phase separation. These
findings agree well with the IR and fluorescence anisotropy
results presented before,14 where only partial fluidization of the
DPPC chains was observed above the first DSC peak and
fluidization was only complete at high temperature. Fluorescence anisotropy data for B12 showed that it becomes only
mobile at higher temperature at which the additional DSC
peaks were seen.
Closer inspection reveals that the inflection points of the
sigmoidal temperature-dependent signal increases for the
mixtures roughly (within the accuracy of the temperature
calibration of the order of 1−2 K) coincide with the second
(lipid) and third (B12 core) additional phase transition seen in
the DSC thermogram. Thus, two out of the three additional
phase transitions can be assigned on the basis of the 1H
integrals: transition II is accompanied by the mobilization of
lipid molecules that are presumably confined and immobilized
within honeycomb cells, and transition III is related to the
dissolution of the supramolecular structure and the formation
of a homogeneous mixture. The assignment of the first
additional phase transition is not possible on the basis of
these data.
Mobility and Packing from 13C Spectra. To solve this
question, and now turning to new and unpublished data, a
closer look at temperature-dependent 13C spectra is instructive.
Spectra for the 1:4 mixture of B12 and DPPC were taken with
two excitation methods, DP and CP (see Figure 4). With such
spectra we are able to distinguish different mobilities, namely,
the more mobile carbons being mainly excited by DP and the
immobile ones being excited more efficiently by CP. The
acquired spectra indeed look different for different temperatures and the respective excitation methods. For the lipid
signals (not shown), phase separation and coexistence is
evidenced by the appearance of an additional set of signals for a
SCH = DCH,exp /DCH,stat
(1)
is defined. Its value can be used to distinguish between different
motional geometries.30,31 Note that, from our data (modulation
curves reaching again maximum intensity at maximum
modulation time), we can exclude the presence of significant
“intermediate” motions with rates in a range between 1 and 100
kHz.16,32 Slower motions cannnot be detected with this
method.
We first focus on the pure B12 substance, for which
DIPSHIFT measurements were performed at two different
temperatures, T = 30 and 90 °C (data for the lower
Figure 4. 13C MAS spectra of the aromatic spectral region for the 1:4
B12-DPPC mixture at different temperatures, using two excitation
schemes (DP: upper, CP: lower spectra). Two peaks with chemical
shifts differing by about 0.7 ppm are observed for resonance ar3, one
converting into the other upon increasing the temperature.
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temperature not shown). From the dephasing curves (for an
example, see Figure 5a), different residual couplings were
determined that indicate distinct motional amplitudes within
the B12 molecule. From the corresponding dynamic order
parameters SCH ≈ 0.6 for all aromatic ring carbons with directly
bonded protons, and SCH ≈ 1 for quaternary ring carbons (see
Figure 5b), relevant conclusions can be drawn. The CH dipolar
tensors for the two carbon types (see the inset) differ in their
sensitivity to different motional models. Comparing the SCH
values to predictions according to n-site jumps, diffusion on/
within a cone, etc.,30,31 we conclude on fast two-site 180° jumps
around the molecular long axis. This is a well-known motif for
para-substituted and well-packed phenyl rings.28,33
In the 1:4 mixture of B12 and DPPC, the B12 resonances
were sufficiently intense to also investigate the dynamics of B12
in the mixture, whereas this was not possible for the 1:10
mixture due to low signal-to-noise ratios. We discuss data for
two temperatures, one in the region of phase separation (T =
45 °C), and one above all phase transitions (T = 75 °C). Again,
order parameters were determined from the DIPSHIFT
experiments, the results for which are also shown in Figure
5b. As can be seen for the lower temperature, there is no
significant difference in the order parameters of the B12
carbons in the mixture as compared to pure B12. This means
that all aromatic carbons show the same motional behavior in
the mixture as in the B12 crystal, i.e., fast 180° flips of the
aromatic rings. Also, the order parameters of the alkyl chains as
well as the glycerol carbons did not change in the mixture for
the low temperature as compared to the bulk substances. This,
along with the fact that all 13C resonances are rather narrow,
indicates that the B12 molecules are well packed and ordered
within the separated supramolecular structure.
At the high temperature, all carbon atoms exhibit strongly
reduced order parameters. All lipid molecules are then in the
mobile liquid-crystalline phase, and also the B12 molecules
show increased mobility. On the basis of suitable motional
models (diffusion on or within a cone), we find that our data is
compatible with uniaxial rotation of the aromatic rings about
the molecular long axis, with additional wobbling motions that
reduce the order parameter further. Here, also the alkyl chains
and glycerol carbons show increased molecular amplitudes
(lower SCH) due to the much increased motional freedom.
These observations support the above interpretation of free
dissolution of the B12 in the fluid membrane.
In addition to the SCH values characterizing the dynamics of
the B12 cores via C−H coupling directions, we also measured
homonuclear residual dipolar couplings by 1H−1H DQ
spinning sideband analysis.17−19 These provide dynamic order
parameters SHH characterizing the dynamics of the fairly
isolated aromatic 1H pairs of the para-substituted phenyl rings.
Notably, SHH is not sensitive to axial rotation, but only to the
additional fluctuations (wobbling) of the molecular long axis
(see again the inset of Figure 5b).
These measurements were performed for high temperatures
between T = 65 and 80 °C for both mixtures of DPPC and
B12, i.e., the range within which a fraction of the fast-rotating
B12 molecules are observed. Note that π-packed B12 cores
exhibiting only the fast yet restricted 180° flips are not
observable under the given experimental conditions.
From the measured sideband patterns (for an example, see
the inset of Figure 6), the residual dipolar coupling was
determined and hence SHH calculated. The “static-limit”
(rather: perfect-rotation) reference coupling of 8.3 kHz was
estimated from the average distance of two aromatic ring
protons of about 2.44 Å. In this way, S ≈ 0.6−0.8 were found,
as shown in Figure 6. It can be seen that the values are nearly
constant for the different temperatures, thus the motional
amplitude is not significantly altered upon heating.
Figure 5. (a) 13C−1H DIPSHIFT (left) and recoupled DIPSHIFT
(right) dephasing curves for aromatic resonance ar3 of B12 in the 1:4
mixture with DPPC; (b) dynamic order parameters SCH = Dres/Dstat for
all resolved B12 resonances as well as expected values for specific
motional models for B12 (dashed lines). Error intervals are estimated
from the experimental signal-to-noise ratio and the standard deviation
over repeated experiments. See Figures 2c and 9 and the main text for
the peak assignments. The inset shows schematically the different
dipolar tensors probed.
Figure 6. 1H−1H order parameters SHH for two B12 aromatic
resonances in 1:4 and 1:10 mixtures with DPPC as a function of
temperature. The error bars indicate the dipolar distribution width σ
from the fit; the actual experimental error is smaller and apparent from
the deviations from the trend lines. Inset: DQ spinning sideband
pattern for the 1:4 mixture taken at 65 °C including a fit.
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bilayers with proteins41 and simulations with other additives,42
revealed no significant change in the lipid order parameters
upon guest insertion into the lipid bilayer. Therefore, it may be
assumed that the presence of the B12 molecules does not
change the packing density of the lipids.
An alternative explanation for the unchanged order
parameters could be that lipid motions are not sensitive to
the organization of the lipids within the membrane. In different
studies concerning several membrane morphologies43,44 as well
as interactions with different proteins45−48 and other guest
molecules,49 it was shown that the apparent lipid dynamics
does not necessarily reflect the molecular order/disorder in the
system, i.e., order and dynamics are not always correlated in a
simple way. This is because the measured dynamic order
parameter is an ensemble-averaged value that may not change if
the individual order parameters within the ensemble change in
the opposite direction.46 Such an inhomogeneous scenario may
not be resolved by the given method due to insensitivity to
distributions. Of course, fast exchange between interacting and
noninteracting lipid molecules in the sample would also lead
one to conclude on the apparently unchanged geometry of lipid
dynamics.47 Since our experiments are subject to experimental
noise and intrinsically reflect an average over motions with rates
of at least a few hundred kilohertz, we cannot exclude an effect
of B12 on adjacent lipids that only leads to a moderate coupling
distribution or is subject to exchange on a comparably short
time scale, respectively.
DOPC+B12. B12 was also studied in membranes of an
unsaturated model lipid, namely DOPC, but, for substance
availability reasons, only in a 1:10 molar ratio. The fluorescence
images (Figure 1c) suggest a homogeneous distribution of the
B12, therefore, we have no indication of macroscopic phase
separation. Hence, it is interesting to compare the dynamic
properties of this mixture with the phase-separated case.
Dynamic Order Parameters. Order parameters SCH for the
lipid resonances from DIPSHIFT experiments (not shown)
actually afforded virtually the same “no-result” as discussed
above for the case of B12 in DPPC membranes (Figure 7),
suggesting again weak specific interactions. As 13C detection of
B12 is unfeasible for sensitivity reasons, we only show results
based upon temperature-dependent 1H−1H DQ sideband
patterns, thus discussing order parameters SHH from the
measured homonuclear residual dipolar coupings in the
temperature range between 5 and 65 °C. The results are
shown in Figure 8, where the inset also shows a typical
measured sideband pattern.
We observe a somewhat reduced order parameter for all
measured temperatures, with a trend toward decreasing values
upon heating. As the mixture of DOPC and B12 is
homogeneous, the lipid molecules are expected to be in the
mobile liquid-crystalline phase typical for the pure membrane.
Therefore, uniaxial rotation of the B12 can be taken for granted,
and reduced SHH values are again explained by additional
wobbling motions, i.e., a molecular tilt accompanied by
diffusion on or within a cone. The reorientation angle is thus
estimated to be in the range of 35−45° with respect to the
membrane normal. The angle (motional amplitude) obviously
increases somewhat upon heating, reducing SHH.
It is further notable that the order parameters of the B12
protons in the 1:10 mixture with DOPC are comparable with
those of the mixture with DPPC at the same molar ratio, i.e.,
the geometry of motion of B12 molecules is rather similar.
Again, we take this as an indication of rather comparably weak
Interestingly, the order parameter for the 1:10 mixture is
systematically lower than that for the 1:4 mixture. This points
to a larger motional amplitude for the B12 molecules in the
1:10 mixture as compared to the 1:4 mixture of B12 and DPPC.
As the orientation of relevant dipolar coupling tensor is parallel
to the molecular long axis, the orientation of the tensor
represents molecular orientation with respect to the membrane
normal. Depending on the assumed motional model (rotation
on or within a cone), the reorientation angle of the molecules
with respect to the membrane normal can be estimated to
about 20−30° and 35−45° for the 1:4 and 1:10 mixtures,
respectively.30,31 The notably smaller angle for the 1:4 mixture
reflects a reduced motional freedom of the B12 molecules,
suggesting a crowding effect of the dissolved B12 on its own
dynamics.
Finally, we address the question of whether the B12
molecules only influence their own dynamics or also that of
the lipid molecules. We thus used the DIPSHIFT methods to
also determine the SCH of the lipid carbons in the pure hydrated
lipid and in the mixtures (see Figure 7). It is obvious that the
order parameters of the lipid molecules are not altered in the
mixture. This indicates no significant change of amplitude of
the local lipid dynamics, meaning that the lipid molecules in the
liquid-crystalline phase are not detectably restricted by the
presence of the B12 molecules.
This finding is notable in relation to the effect of, e.g.,
cholesterol in model membranes. Because of its small
headgroup and primarily hydrophobic nature, cholesterol is
thought to be inserted preferably below the lipid head groups
(umbrella model), leading to the well-known condensing and
ordering effects.34−37 The headgroups of phospholipids
generally exhibit a comparably large volume, leading to
significant motional freedom of the alkyl chains already in the
pure lipid.38,39 As the B12 molecules are inserted into the lipid
bilayer, cross the whole membrane with their well-matched
hydrophobic core length, do not have a large headgroup, and
can adapt to the membrane by a slight tilt (see above), the area
per lipid is probably not changed and therefore the motional
freedom of the lipid molecules in the mixture is not restricted.
Such a behavior was also found in other experiments, which
indicated that the fast local dynamics of lipid molecules is only
affected by additives if they cause a change in the lipids’ packing
density.40 Other investigations, including NMR studies of
Figure 7. Order parameters SCH of the DPPC carbons in the pure lipid
and in the 4:1-mixture with B12 at 75 °C.
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Figure 10. Temperature-dependent proton integrals for different
spectral regions for the 1:4 mixture of E12/7 and DPPC. The DSC
thermogram is also included in the graph.
Figure 8. Order parameters SHH for the main B12 aromatic resonance
in a 1:10 mixture with DOPC as a function of temperature. The error
bars indicate the dipolar distribution width σ from the fit. Inset: DQ
spinning sideband pattern taken at 65 °C including a fit.
of the lipid, the aromatic resonances of E12/7 indicate a mobile
fraction of only about 10% in the 1:4 mixture. This suggests
that the two components are largely independent of each other,
in contrast to the mixture of B12 with the same lipid.
Dynamic Order Parameters. Turning to SCH, at the
measured temperatures most E12/7 resonances showed rather
restricted mobility, thus most residues could be characterized
by normal rather than recoupled DIPSHIFT (Figure 11a).
Only one of the methyl and one of the EO residues required
recoupling due to low order parameters at the chains’ termini.
interactions with the different parts of the lipid, considering that
the B12 concentration effect in the DPPC mixture (Figure 6) is
more significant.
DPPC+E12/7. For the headgroup-modified E12/7 molecules in mixture with DPPC, a homogeneous distribution was
indicated in the fluorescence images (see Figure 1d). Also in
DSC measurements, no high-temperature phase transition was
found, suggesting again an absence of macroscopic phase
separation in a 1:4 mixture. However, in our 13C NMR spectra,
the E12/7 resonances, notably also the aliphatic side chains as
well as the EO terminal chains, could only be detected by CP
excitation (see Figure 9). This indicates rather strong dipolar
couplings and a lack of substantial mobility in the mixture.
Mobility from 1H Signal intensities. 1H spectral integrals
expectedly showed mobile lipid molecules (i.e., narrow signals)
above the lipid main phase transition, with a rather sharp and
quantitative discontinuous change at the transition temperature
(see Figure 10). However, despite the quantitative mobilization
Figure 11. (a) 13C−1H DIPSHIFT (left) and recoupled DIPSHIFT
(right) dephasing curves for different resonances of E12/7 in the 1:4
mixture with DPPC, (b) dynamic order parameters SCH = Dres/Dstat for
all resolved E12/7 resonances as well as an expected value for the
aromatic cores according to a π-flip model (dashed line). See Figures
2c and 9 for the carbon resonance assignments; the label “EOn” refers
to specific signals of the (EO)7OCH3 chain.
Figure 9. (a) Aliphatic and (b) headgroup regions of the 13C MAS
NMR spectrum of the 1:4 mixture of E12/7 and DPPC taken at 50
°C, including resonance assignment. Note that different excitation
schemes were used (DP: upper, CP: lower spectra). The label “EOn”
refers to specific carbon resonances of the (EO)7OCH3 chain.
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■
Inspection of the results shown in Figure 11b reveals lower
order parameters, thus somewhat higher motional amplitude, of
the aromatic core of E12/7 as compared to B12 molecules in
both the crystalline state and in the phase-separated mixture
(see also Figure 5b). The values are, however, not compatible
with free rotation, so we conclude on a somewhat less defined
π−π packing structure.
So despite of the lack of evidence for macroscopic phase
separation in fluorescence imaging and DSC experiments, the
NMR results unambiguously demonstrate that a high fraction
of E12/7 molecules is still immobilized in the mixture with
DPPC even at the highest studied temperatures. This points to
small micro- or even nanodomains, which we imagine to be
π−π packed fibrils that are too small to be visible in microscope
images and do not melt/disintegrate within the studied DSC
temperature range. The fibril hypothesis is motivated by the
lack of corrugation that is typical for GUVs made of pure
DPPC in the gel phase (Figure 1), suggesting that the fibrils
separate small patches of DPPC along line defects. See Figure
12b for a sketch of the structural model.
As to the EO headgroups, the order parameters reveal an
increasing flexibility toward the chain ends. Thus, larger parts of
these terminal chains are not subject to motional restrictions to
the same extent as the glycerol headgroups of B12 (see also
Figure 5b). This difference is likely due to different ability to
form hydrogen bonds and therefore due to a different mode of
interaction of the E12/7 headgroups with each other as well as
with the lipid as compared to B12. The observed higher
thermal stability of the E12/7 superstructure is in tune with the
known high-temperature insolubility of poly-EO in water50 and
the related peculiarities in the phase behavior of oligo-EObased surfactants.51
Finally, we note that in Figure 9a two methyl signals of the
alkyl side chains of E12/7 (denoted al12) are visible. Therefore,
we also report two order parameters for these groups (see
Figure 11b). A possible explanation is that there are two
different packing modes for the two lateral alkyl chains of the
E12/7 in the fibrillar structure (realized, e.g., in a double
strand). As another alternative that we cannot ultimately
exclude, the signal could of course also arise from a more
ordered minority fraction of the lipid in interaction with the
E12/7.
Article
SUMMARY
In this work, the dynamics, structural aspects, and thus
interactions of bolapolyphilic molecules in two lipid membrane
model systems, DPPC and DOPC, were investigated by means
of solid-state NMR and complementary techniques such as
fluorescence microscopy and DSC. For the unsaturated lipid
DOPC in the 10:1 mixture with the bolapolyphile B12, a
homogeneous mixture was found, in which the local dynamics
of lipid molecules in the mobile liquid-crystalline phase was
virtually unaffected. This was also found for DPPC lipid in
interaction with B12 at high temperatures. In both cases,
measurements of dynamic order parameters indicated free
rotation of the B12 molecules within the fluid bilayer. Focusing
on fluctuations of the B12 molecular long axis and considering
diffusion on- or within-a-cone models, our data suggests an
opening angle in the range of 20−45° that is mainly dependent
on the B12 concentration in the membrane. This lead us to the
conclusion that the B12 molecules interact comparably weakly
with the fluid membrane components.
For the B12/DPPC mixtures, three new transitions appeared
in the DSC traces above the main lipid transition, accompanied
by macroscopic phase separation with snowflake-like appearance as seen in fluorescence images. We have previously
explained this by a honeycomb superstructure with 6-fold
symmetry, see Figure 12c. On the basis of the finding that the
B12 cores in this structure undergo 180° flips in the same way
as in the bulk crystal, the B12-rich mixed phase was shown to
feature well-packed aromatic B12 cores that are presumably
stabilized by π−π interactions. We could assign the additional
phase transitions of this phase on the basis of our NMR results
to one molecular process each. The first one corresponds to a
change in aromatic π−π packing of the B12 molecules
accompanied by an increase in their mobility. The second
additional phase transition is assigned to a mobilization of the
still ordered lipid tails and B12 side chains that are presumably
confined by B12-rich filaments within a hexagonal honeycomb
arrangement. During the third transition, all molecules in the
mixture become mobile, leading to a homogeneous distribution
of B12 within the mobile liquid-crystalline lipid matrix.
For another bolapolyphile (E12/7) with oligo(ethylene
oxide) rather than glycerol headgroup in mixtures with
DPPC, fluorescence microscopy also suggested a homogeneous
distribution within the membrane. However, the NMR results
demonstrated E12/7 to be organized in a rather rigid selfassembled structure not involving lipid molecules. These
findings are compatible with a nanometer-scale filamentous
structure (Figure 12b), which can explain the intriguing finding
that GUVs seen in fluorescence microscopy are regular rather
than corrugated spheres, the latter being the common finding
for DPPC vesicles in the gel phase. Nanofilaments can reside in
defect lines and thus reduce local corrugation. The same
phenomenon can also explain the spherical appearance of
DPPC vesicles with B12, i.e., the macroscopic snowflake-like
aggregates likely coexist with filaments.
The present work served to elucidate the molecular
interactions and processes underlying the self-organization of
two synthetic bolapolyphilic molecules in lipid membranes.
Based upon these insights, future research will be devoted to
further varying the lateral and headgroup substituents, and
studying mixtures, in order to explore whether other new,
preferably well-defined supramolecular structures can be
obtained. For example, controlled and dispersed fibrillar
Figure 12. Sketch of (a) the structural model of the BPs forming
either (b) π-packed fibrils (E12/7) or (c) hexagonal honeycomb
superstructures (B12); in panels b and c, the view is along the πconjugated rods, i.e., parallel to the membrane normal.
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structures can be used to fine-tune membrane properties such
as their elasticity. The design of possibly guest-selective
synthetic membrane channels is another attractive long-term
goal.
■
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AUTHOR INFORMATION
Corresponding Author
*Electronic address: [email protected];
URL: www.physik.uni-halle.de/nmr.
Notes
The authors declare no competing financial interest.
■
■
ACKNOWLEDGMENTS
We thank the Deutsche Forschungsgemeinschaft (DFG) for
funding in the framework of the Forschergruppe 1145.
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