Showcasing collaborative research from fluorescence
spectroscopists Mariana Amaro and Martin Hof (J. Heyrovský
Institute of Physical Chemistry of the Academy of Sciences of
the Czech Republic) and computational chemists Hugo Filipe,
João P. Prates Ramalho and Luís Loura (Universities of Coimbra
and Évora, Portugal)
As featured in:
Title: Fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids
in model membranes is connected not to lipid mobility but to
probe location
By combining computational and experimental approaches, we
explain the photophysics of NBD, one of the most frequently used
fluorescent membrane labels. We resolve previously published
conflicting results and the long-standing misinterpretation of
fluorescence data of NBD.
See Martin Hof,
Luís M. S. Loura et al.,
Phys. Chem. Chem. Phys.,
2016, 18, 7042.
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Cite this: Phys. Chem. Chem. Phys.,
2016, 18, 7042
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Fluorescence of nitrobenzoxadiazole (NBD)labeled lipids in model membranes is connected
not to lipid mobility but to probe location†
Mariana Amaro,a Hugo A. L. Filipe,bcd J. P. Prates Ramalho,e Martin Hof*a and
Luı́s M. S. Loura*bdf
Nitrobenzoxadiazole (NBD)-labeled lipids are popular fluorescent membrane probes. However, the
understanding of important aspects of the photophysics of NBD remains incomplete, including the
observed shift in the emission spectrum of NBD-lipids to longer wavelengths following excitation at
the red edge of the absorption spectrum (red-edge excitation shift or REES). REES of NBD-lipids in
membrane environments has been previously interpreted as reflecting restricted mobility of solvent
surrounding the fluorophore. However, this requires a large change in the dipole moment (Dm) of NBD
upon excitation. Previous calculations of the value of Dm of NBD in the literature have been carried out
using outdated semi-empirical methods, leading to conflicting values. Using up-to-date density
functional theory methods, we recalculated the value of Dm and verified that it is rather small (B2 D).
Fluorescence measurements confirmed that the value of REES is B16 nm for 1,2-dioleoyl-sn-glycero-3phospho-L-serine-N-(NBD) (NBD-PS) in dioleoylphosphatidylcholine vesicles. However, the observed
shift is independent of both the temperature and the presence of cholesterol and is therefore insensitive
to the mobility and hydration of the membrane. Moreover, red-edge excitation leads to an increased
contribution of the decay component with a shorter lifetime, whereas time-resolved emission spectra of
NBD-PS displayed an atypical blue shift following excitation. This excludes restrictions to solvent
relaxation as the cause of the measured REES and TRES of NBD, pointing instead to the heterogeneous
Received 2nd September 2015,
Accepted 7th December 2015
transverse location of probes as the origin of these effects. The latter hypothesis was confirmed by
DOI: 10.1039/c5cp05238f
of NBD correlated with the measured fluorescence lifetimes/REES. Globally, our combination of theoretical
and experiment-based techniques has led to a considerably improved understanding of the photophysics
www.rsc.org/pccp
of NBD and a reinterpretation of its REES in particular.
molecular dynamics simulations, from which the calculated heterogeneity of the hydration and location
Introduction
a
Department of Biophysical Chemistry, J. Heyrovský Institute of Physical Chemistry
of the Academy of Sciences of the Czech Republic, v.v.i., Dolejskova 3, 182 23
Prague, Czech Republic. E-mail: [email protected]; Fax: +420-286582677;
Tel: +420-266053264
b
Centro de Quı́mica de Coimbra, Largo D. Dinis, Rua Larga, 3004-535 Coimbra,
Portugal. E-mail: lloura@ff.uc.pt; Fax: +351-239827126; Tel: +351-239488485
c
Departamento de Quı́mica, Faculdade de Ciências e Tecnologia, Universidade de
Coimbra, Largo D. Dinis, Rua Larga, 3004-535 Coimbra, Portugal
d
Centro de Neurociências e Biologia Celular, Universidade de Coimbra,
3004-504 Coimbra, Portugal
e
Departamento de Quı́mica and Centro de Quı́mica de Évora, Escola de Ciências e
Tecnologia, Universidade de Évora, Rua Romão Ramalho, 59, 7000-671 Évora,
Portugal
f
Faculdade de Farmácia, Universidade de Coimbra, Pólo das Ciências da Saúde,
Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5cp05238f
7042 | Phys. Chem. Chem. Phys., 2016, 18, 7042--7054
Fluorescence techniques are commonly and widely used to
study the properties of biological membranes. There are various
techniques, both steady-state and time-resolved, that make use
of the high sensitivity to the environment of fluorescence.
These are also powerful tools in membrane biophysics, because
they employ a wide range of chromophores that respond in
specific ways to certain environmental conditions. These extrinsic
fluorophores can be covalently linked to lipids and are commonly used for studies of the organization and dynamics of
membranes.
Within the family of fluorescent lipid analogues, lipids
labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group
are widely used for various applications.1–4 NBD-labeled lipids
are commercially available for all major phospholipids and are
readily incorporated into lipid membranes. The photophysical
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characteristics of NBD, such as its fluorescence quantum yield
and lifetime, depend strongly on the solvent,5 which makes it a
sensitive fluorescent probe. Moreover, NBD-lipids have a good
quantum yield in lipid bilayers while being weakly fluorescent
in water. An interesting application of NBD-lipid analogues in
membranes is the sensing of slow solvent relaxation by the rededge excitation shift (REES) phenomenon.6
REES is a shift in the maximum of a fluorescence emission
spectrum towards higher values of wavelength, which is caused
by shifting the wavelength of excitation towards the red edge of
the excitation spectrum. This behavior is observed for fluorophores only in viscous to moderately viscous polar solvents
where solvation dynamics occurs on the same timescale, or
slower, as fluorescence emission. If the solvent were to reorient
rapidly after the excitation of the dye molecule (i.e., faster than
the fluorescence lifetime of the dye), the fluorescence emission
would always arise from the same solvent-relaxed excited state.
Therefore, the emission would be independent of the wavelength
of excitation. However, in the former case, the photoselection
that is provided by red-edge excitation is not lost because the
reorientation of the solvent is slow and consequently REES is
observed. REES can serve as an indicator of the mobility of the
microenvironment of a probe, as its magnitude is dependent on
the dipolar relaxation dynamics. The interfacial region of a lipid
bilayer is known to be characterized by such nanosecond-scale
slow solvation dynamics.7,8 Therefore, fluorophores that reside
in this region could, in principle, exhibit a REES effect.
However, an appropriate location within the bilayer is not the
sole condition that a fluorophore has to fulfill in order for REES to
be observed in lipid membranes. In addition, a significant change
in the dipole moment of the dye upon electronic excitation must
occur in order to induce the rearrangement of the dye’s solvent
shell. The difference between the ground- and excited-state dipole
moments of an NBD molecule with a secondary amino group (this
would be an analogue of NBD-labeled lipids) has been estimated to
be 0.8 D.9 Higher values for the change in dipole moment, around
1.8 D, were also reported in 1993 for NBD with a primary and
tertiary amine.5 In the following year, yet another value for the
change in dipole moment of NBD was reported in the literature
to be 3.6 D.10 Despite these (20-year-old) conflicting values, the
magnitude of the increase in dipole moment is, however, very
small. Such a modest change between the ground- and excitedstate dipole moments would, most likely, not cause a dramatic
rearrangement of the solvent shell around the NBD molecule. For
comparison, the typical dyes that are widely used for testing solvent
relaxation, Prodan and Laurdan, display increases in dipole
moment upon excitation of the order of 15 D.11 Moreover, the
Stokes shifts that are displayed by NBD are of a small magnitude
and vary randomly with the solvent polarity,5 which further supports the expectation that NBD is unlikely to be a good chromophore for testing dipolar relaxation. However, there are reports that
REES of NBD-labeled lipids can be used for investigating such
solvent relaxation phenomena in lipid membranes, even though
the reported values of REES have small magnitudes.6,12–15
This work is a contribution to clarifying important questions
regarding the photophysical behavior of NBD and its implications
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Fig. 1 Structures and numbering of selected atoms of NBD-C2 (A), DOPC (B),
Chol (C), and NBD-PSH (D).
for the study of membranes using NBD-labeled lipids. For this
purpose, we employ a combination of theoretical and experimental techniques. Firstly, we address the conflicting values of
the change in the dipole moment of NBD upon excitation. We
update the 20-year-old values using density functional theory
calculations and confirm that the difference between the groundand excited-state dipole moments of NBD is of a small magnitude. We then present a detailed experimental study that is
focused on the NBD-lipid for which a maximum REES effect has
been reported,13 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N-(NBD)
(NBD-PS). With this aim, we obtain REES and time-resolved
emission spectra of NBD-PS in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC/cholesterol (Chol) lipid bilayers (see
Fig. 1 for structures). The effect of membrane rigidity is tested
both by varying the temperature and by the addition of Chol. The
influence of the protonation state of NBD-PS is also experimentally determined in DOPC bilayers at pH values of 5 and 7.
Atomistic molecular dynamics simulations of bilayers that
mimic the above experimental systems are also performed in
order to obtain atomic-level insights. Our theoretical, experimental and simulation findings combine to explain the true
origin of the small REES that is displayed by NBD-lipid analogues
in lipid membranes.
Methods
Quantum chemical calculations
For this theoretical study, we selected N-ethylamino-NBD (NBD-C2,
see Fig. 1A). This molecule has a small alkylamino side chain that
is similar to that of NBD-labeled lipids, is small enough to permit
calculations in a reasonable time, and has the advantage of being
closely related to NBD-C1 and NBD-C3 (for which literature data
have been reported).5,16
Ground-state and first-excited-state geometries were obtained
by density functional theory (DFT) and time-dependent (TD) DFT
methods, respectively, from optimizations using the hybrid
exchange–correlation functional B3LYP17,18 with the 6-31+G(d)
basis set.
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Several solvents covering a range of different polarities were
considered by means of the implicit polarized continuum
(PCM)19,20 and SMD21 models. For each solvent, geometries of
NBD-C2 were optimized in both the ground and excited state.
Frequency analysis was subsequently performed to confirm
that each optimized geometry was an energy minimum by the
absence of imaginary frequencies. For geometry optimization,
an equilibrium formulation that allowed the solvent polarization to be fully equilibrated with the electronic configuration
of the solute was chosen, whereas a non-equilibrium regime
was considered for the calculation of fast vertical electronic
transitions, where only the fast degrees of freedom of the solvent
polarization were allowed to relax.22,23 On the other hand, an
equilibrium approximation was chosen for the fluorescence
calculations.24
The TDDFT method was then used to calculate the low-lying
excited states of the NBD derivative using the hybrid PBE0
functional25 combined with PCM and the hybrid meta-GGA
M0626 functional with the SMD solvent model using two different basis sets: 6-31+G(d), similarly to the optimizations, and the
larger 6-311+G(d,p) basis set. The equations were solved for
20 excited states.
All calculations were performed using the GAMESS-US software package, version May 1, 2012 (R1).27,28
Molecular dynamics simulations
Simulations and analysis of trajectories were carried out using
the GROMACS 4.5.3 package.29,30 The topology of the DOPC
molecule (Fig. 1B) consisted of a united-atom description for
CH, CH2, and CH3 groups based on the parameters presented by
Berger et al.31 for 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine
(DPPC) and by Bachar et al.32 for the acyl chain cis-double bonds.
The structure and topology of Chol (Fig. 1C) were as used by
Höltje et al.33 (available for downloading at the GROMACS web
page34). The simple point charge water model was used.35 Using
standard GROMACS tools, starting structures of fully hydrated
DOPC (128DOPC:6186H2O) and DOPC/Chol (120DOPC:30Chol:
4768H2O) bilayers were built.
Previously,36 we combined the description of DPPC that is
available from Dr D. Peter Tieleman’s group’s web page37 with
that of the NBD fluorophore, as described in detail by Loura
et al.,38 to derive the topology of N-(7-nitrobenz-2-oxa-1,3-diazol-4yl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (NBD-PE).
For this work, we obtained a basic topological description of
1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N-(7-nitro-2,1,3-benzoxadiazol-4-yl) (NBD-PS) by adding a carboxylate group to NBD-PE
at atom C15 (see structure and numbering in Fig. 1D) with
standard GROMACS force field parameters for the additional
bonds, angles and dihedral angles. In particular, the correct
chiral configuration at C15 was maintained by an improper
dihedral angle. The palmitoyl chains were replaced by oleoyl
chains taken from DOPC. Two topologies were built, which
corresponded to the probe in different protonation states.
Fig. 1D depicts a single negatively charged NBD-PS molecule
with a protonated carboxyl group. This form, which is expected
to predominate at a pH of 5,13 is designated as NBD-PSH in
7044 | Phys. Chem. Chem. Phys., 2016, 18, 7042--7054
Paper
the text. At neutral pH, the proton on the carboxyl group (H68 in
Fig. 1D) is lost, yielding a probe with double negative charge13
termed NBD-PS. Partial charges of NBD-PS and NBD-PSH entities
with truncated acyl chains were calculated from the optimized
geometries that were obtained from quantum chemical calculations
performed with the B3LYP exchange–correlation functional17,18 and
the 6-31+G(d) basis set, followed by a least-squares fit to the
electrostatic potential that was obtained at the same level of theory,
according to the scheme of Kollman and Singh.39,40 All calculations
were performed using the GAMESS-US package.27,28 The values
that were adopted for NBD-PS and NBD-PSH are displayed in
ESI,† Tables S1 and S2.
Bilayers that contained NBD-PS (two probes in each leaflet)
were obtained by randomly inserting probes in DOPC and
DOPC/Chol bilayers without replacement of phospholipids.
For the systems that contained NBD-PSH, starting configurations were obtained by the addition of a proton to each carboxyl
group in the final structures of the corresponding simulations
for NBD-PS. In the main set of simulations, a corresponding
number of Na+ ions (eight or four in the simulations for NBD-PS
and NBD-PSH, respectively) were randomly added to the aqueous
medium to ensure electroneutrality. Additional simulations were
carried out in the presence of an increased number of Na+ and
Cl ions to produce an ionic strength of 150 mM. Run protocols
for equilibration/production and other simulation options were
as described by Filipe et al.36 For systems with NBD-PS the
duration of the full run was 200 ns, or 300 ns for systems without
or with 20 mol% Chol, respectively. To enable proper equilibration
of the transverse location and orientation of the fluorophore
(see Results section), systems with NBD-PSH were simulated for
300 ns (bilayer without Chol) or 400 ns (bilayer with 20 mol%
Chol). For the visualization of structures and trajectories,
Visual Molecular Dynamics software (University of Illinois) was
used.41 For analysis, the last 150 ns (simulations without Chol)
or 200 ns (simulations with 20 mol% Chol) of each trajectory
were used.
Materials
DOPC, Chol and NBD-PS (ammonium salt) were obtained from
Avanti Polar Lipids, Inc. (Alabaster, AL, USA). NaCl and ethylenediaminetetraacetic acid (EDTA) were obtained from SigmaAldrich (St. Louis, MO, USA) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) from Fluka (Buchs, Switzerland).
Preparation of large unilamellar vesicles
Solutions in chloroform of DOPC or DOPC/Chol (80 : 20 mol%)
and NBD-PS were prepared to obtain a molar ratio of probe to
lipid of 1 : 100. The solvent was then evaporated under a stream
of nitrogen. For thorough removal of the solvent the lipid
films were left under vacuum overnight. HEPES buffer (10 mM,
150 mM NaCl, pH 7.0 (or pH 5.0), 1 mM EDTA) was then added
to the dried lipid film (lipid concentration of 1 mM), which
was left to hydrate for 30 minutes. The resulting suspension
was vortexed for at least 4 min and then extruded through
polycarbonate membranes with a nominal pore diameter of
100 nm (Avestin, Ottawa, Canada). For measurements the
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vesicle suspension was diluted to an overall lipid concentration of 0.5 mM.
Table 1 Calculated and experimental spectral properties of NBD-C2
(wavelengths l and oscillator strengths f) and dipole moments of the
ground (mg) and excited (me) states in selected solvents
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Steady-state fluorescence measurements
Solvent
Steady-state excitation and emission spectra were acquired
using a Fluorolog-3 spectrofluorometer (model FL3-11; Jobin
Yvon Inc., Edison, NJ) equipped with a xenon arc lamp. The
spectra were recorded in steps of 1 nm (bandwidths of 1.5 nm
were chosen for both the excitation and emission monochromators). The temperature in the cuvette holder was maintained
using a water-circulating bath. For the determination of REES,
full emission spectra were obtained under the abovementioned
conditions for various excitation wavelengths at the red edge of
the excitation spectrum (460–525 nm).
Time-resolved fluorescence measurements
Fluorescence decays were recorded on a 5000 U single-photon
counting setup (IBH, Glasgow, U.K.) using a PicoQuant pulsed
diode laser (470 nm peak wavelength, 2.5 MHz maximum
repetition rate) and a cooled Hamamatsu R3809U-50 microchannel plate photomultiplier. A 499 nm cut-off filter was used
to eliminate scattered light. The temperature in the cuvette
holder was maintained using a water-circulating bath. Emission decays were recorded at a series of wavelengths spanning
the steady-state emission spectrum (510–610 nm) in steps of
10 nm (8 nm emission slits). The signal was kept below 1% of
the repetition rate of the light source. Data were collected until
the peak value reached 5000 counts. Fluorescence decays were
n
P
t
fitted to the multi-exponential function IðtÞ ¼
ai exp
ti
i¼1
using IBH DAS6 software. The normalized pre-exponential
ai
factors Ai were calculated according to Ai ¼ P
. Three expon
ai
i¼1
nential components were necessary to obtain a satisfactory fit
of the data. The purpose of the fit was to deconvolve the
instrumental response from the data and should not be overparameterized. The fit simply represents the measured decays;
it is empirical and no physical meaning is attributed to the
derived parameters. The fitted decays, together with the steadystate emission spectrum, were used for the reconstruction of
time-resolved emission spectra by a spectral reconstruction
method.42
Results and discussion
Quantum chemical calculations
The calculated wavelengths of the two transitions with lowest
energy and corresponding oscillator strengths of NBD-C2 (see
Fig. 1A for the structure) are presented in Table 1, together with
experimental values from the literature, for representative apolar
(cyclohexane) and polar (ethanol, water) solvents. Results for
other media and different levels of theory with an indication of
the main orbitals involved in the transitions can be found in
ESI,† Table S3. Although the exact percentage values of the
orbital character of the excited states vary slightly with the
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Property
S0 - S1 absorption Calc. l (nm)
f
Exp. l (nm)
S0 - S3 absorptiona Calc. l (nm)
f
Exp. l (nm)
Emission l (nm)a
Calculated
Experimental
b
Dipole moment (D) mg
me
Dm = mg me
a
Cyclohexane Ethanol
Water
415
0.34
425c,d
309
0.21
305c
477
495c, 499d
12.2
14.34
2.13
429
0.39
482c, 478d
330
0.26
348c
508
566c, 541d
15.39
17.14
1.75
428
0.39
464c, 459d
328
331a
331c
505
537c, 524d
15.14
16.94
1.8
a
SMD-M06/6-311+G(d,p). b PCM-PBE0/6-311+G(d,p). c Values obtained
for NBD-C3, taken from Fery-Forgues et al.5 d Values obtained for NBDC1, taken from Uchiyama et al.16
functional and solvent model employed (r1%), the essential
nature of the transitions is unchanged.
The M06 functional performs better than the PBE0 functional for all solvents that were studied (ESI,† Table S3). It is
also interesting to note that for both functionals the S0 - S3
transition is predicted with higher accuracy than the S0 - S1
transition. There is an improvement in the calculated values of
wavelengths when a basis set of higher quality is employed.
Also notable is the increased accuracy of calculations for solvents
with low dipole moments such as cyclohexane compared with
polar solvents. The lower degree of concordance with experimental data in protic solvents probably stems from the use of an
implicit solvent model in our calculations.
The results show a significant red shift of the transition with
lowest energy in solution relative to the results in the gas phase.
This red shift tends to be more pronounced with an increase in
solvent polarity, which is in agreement with the experimentally
measured wavelengths. Because the S0 - S1 transition is mainly
of HOMO - LUMO character, this means that the size of the
HOMO–LUMO gap is reduced by the presence of the solvent.
This can be attributed to an increase in the stabilization of the
more polar excited state (Table 1) by the solvent.
The calculated absorption spectra of NBD-C2 in the gas
phase and in solution (Fig. 2) resemble closely the experimental
excitation spectra of NBD lipid probes (see the example in the
following subsection). The calculated maximum molar absorption coefficients e also agree well with the experimental results
of Fery-Forgues et al.5 and correctly predict both the order of
magnitude of e (B2–3 104 M1 cm1) and its increasing trend
in more polar solvents (for example, measured values of e of
19 200, 23 100 and 28 000 M1 cm1 were reported in acetone,
dimethylsulfoxide, and water, respectively). The S0 - S1 transition is responsible for the absorption band with lowest energy
and can be essentially assigned to a HOMO - LUMO transition
(ESI,† Fig. S1). The S0 - S2 transition is forbidden with a
negligible calculated oscillator strength. The next dipole-allowed
transition corresponds to a transition to the S3 state and can be
mostly assigned to a HOMO - LUMO+1 transition. The contour
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Fig. 2
Paper
Calculated electronic absorption spectra of the NBD derivative in the gas phase and in solution calculated at the TD-SMD-M06/6-311+G(d,p) level.
Fig. 3 Representation of the charge transfer that occurs upon the two
transitions with lowest energy of NBD-C2 in a solution. Magenta and blue
isosurfaces represent negative (loss of electrons) and positive (gain of
electrons) values, respectively.
plots in ESI,† Fig. S1 and transition wavelengths in Table 1
agree closely with a previous theoretical study of NBD involved
in a Pt complex, for which the molecular orbitals that were
involved in the electronic transitions with lowest energy were
highly localized on the NBD group.43
A more illustrative picture of the rearrangement of charge
that occurs upon S0 - S1 and S0 - S3 transitions is given
in Fig. 3. The difference between the electron densities of the
states involved in the transitions is depicted, which reveals
more clearly the regions of the NBD chromophore that lose or
gain electrons. The traditional assignment of the two transitions with lowest energy of NBD, which was originally proposed
by Heberer and Matschiner44 on the basis of empirical data and
is often found in the literature,5,9 characterizes the transition
with lowest energy as an intramolecular charge transfer band,
whereas the second lowest transition is assigned as a p - p*
transition. In contrast to this simple view, our results indicate
that both S0 - S1 and S0 - S3 transitions are accompanied by a
shift in electron density from the alkylamino moiety towards
the nitro group and therefore both transitions possess significant charge-transfer character.
The emission energies that correspond to the S1 - S0
transition, which were calculated by considering as the reference structure the optimized excited-state geometry and including solvent effects, are shown in Table 1 and ESI,† Table S4.
Similarly to the case of the absorption process, the M06 functional also performs slightly better than the PBE0 functional,
with additional accuracy when the basis set of higher quality
is employed. Again, although the calculated wavelengths are
7046 | Phys. Chem. Chem. Phys., 2016, 18, 7042--7054
systematically lower than the measured values (especially for
solvents of high polarity), a qualitative trend of an increase in
wavelength in more polar solvents is apparent, which is in
agreement with the experimental data.
The calculated values of the dipole moment for both the
ground state and the first excited state (see Table 1 for cyclohexane, ethanol and water and ESI,† Table S5 for all studied
systems and levels of theory) are weakly dependent on the basis
set and the calculated changes in the dipole moment (Dm) are
almost insensitive to an improvement in the basis set. In addition, the dependence on the functional that is used is weak when
comparing the values that were obtained with the B3LYP and
PBE0 functionals for the same basis set and solvent model. On
the other hand, the values seem to be slightly dependent on the
solvent model that is used (PCM or SMD).
For all the studied cases, the calculated dipole moment of
the excited state was larger than for the ground state and the
calculated values of the dipole moments of both electronic
states in solution are consistently higher than those in the gas
phase (ESI,† Table S5) and increase with the solvent polarity. The
change in the magnitude of the dipole moment Dm in solution,
however, tends to decrease with an increase in solvent polarity.
In addition to the variation in the magnitude, a modest change
in the direction of the dipole moment vector was also observed
upon excitation (ESI,† Fig. S2).
The calculated changes in the magnitude of the dipole
moment Dm for the ground (S0) and first excited state (S1) were
found to be between 1.5 and 2.2 D, which agrees with some
previous calculations that were carried out for related NBD
derivatives5,19 and conflicts with both the lower (0.8 D) and
higher (3.6 D) values that were reported by Paprica et al.9 and
Mukherjee et al.,10 respectively. We must stress that these
values from the literature are quite old and were obtained by
outdated semi-empirical approaches and therefore the values
that are reported here represent a much needed reassessment
of Dm of the NBD fluorophore. The fact that such low (B2 D)
values of Dm are consistently obtained for different environments and levels of theory leads to serious reservations regarding the interpretation of the REES of NBD probes in terms of
restricted dipolar relaxation of the environment surrounding the
fluorophore upon excitation of the latter. To clarify this matter,
we carried out fluorescence experiments that focused on NBD-PS
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because it is reported to be the NBD-lipid analogue for which
the highest REES has been observed.13
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Fluorescence spectroscopy
Four different systems were prepared in order to investigate the
ability of NBD-PS to indicate dipolar relaxation of its environment in lipid bilayers. Large unilamellar vesicles (LUVs) of DOPC
and DOPC/Chol (80 : 20 mol%) containing 1 mol% NBD-PS were
prepared in HEPES buffer at pH values of 7 and 5 to study the
effect of protonation of the carboxylate of NBD-PS.13,45,46
The excitation and emission spectra of NBD-PS (Fig. 4) were
found to be identical for all the studied systems (DOPC, pH 7,
T = 5, 10, 15, 20 1C; DOPC, pH 5, T = 15 and 20 1C; DOPC/Chol,
pH 7 and pH 5, T = 20 1C). To determine the REES, full emission
spectra were obtained for various excitation wavelengths at the
red edge (460–525 nm) of the lowest-energy band of the excitation spectrum of NBD-PS and a REES was found to occur in the
studied systems (Table 2).
Membrane rigidity. Lowering the temperature of the system
decreases the overall mobility within the bilayer.8 The increase in
viscosity reduces the experienced dipolar relaxation dynamics8 and
is therefore expected to increase the magnitude of the REES effect.
Emission spectra of NBD-PS in LUVs of DOPC were obtained at
temperatures of 5, 10, 15, and 20 1C. In these systems, the values of
REES were found to be 16 nm and were insensitive to variations in
temperature (Table 2 and Fig. 5).
The addition of Chol to a lipid bilayer is another factor that
is known to condense the bilayer and decrease the hydration
and mobility of the membrane.47,48 The slower dynamics of
Fig. 4 Normalized fluorescence excitation (Exc.) and emission (Em.) spectra
of NBD-PS embedded in DOPC bilayers (at 1 mol%). Illustrative examples for
a pH of 7 at 5 and 15 1C and a pH of 5 at 15 1C.
Table 2 Summary of the magnitude of the red-edge excitation shift
(REES) obtained for NBD-PS in the various studied systems
Sample
pH
T (1C)
REES magnitude (nm)
DOPC
7
5
10
15
20
16
17
16
17
DOPC/Chol (80 : 20)
5
7
15
20
19
17
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the lipid head group region should create an increase in the
magnitude of the REES. However, the experimental results show
that there is no difference between the DOPC and DOPC/Chol
systems at 20 1C (Table 2).
The set of experiments that was described above was designed
to study the influence of the mobility of the environment of the
chromophore on the REES of NBD-PS. Measurements of the
fluorescence anisotropy of head-labelled NBD-lipid analogues
show that the movement of the chromophore is influenced by
the physical state of the membrane.2,49–52 Therefore, it is known
that NBD can sense the mobility of its microenvironment. However, our results demonstrate that the measured REES is in fact
not sensitive to the overall mobility of the lipid, in particular the
mobility of the head group. This is not what is expected from a
fluorophore that is used to test dipolar relaxation.8 Even though
there is in fact a measurable REES for NBD-PS, it is insensitive
to the actual dipolar relaxation phenomenon that has been
suggested to be its causal factor.
The effect of protonation. It has been hypothesized that the
higher magnitude of the REES that was observed for the NBD-PS
lipid analogue compared with other analogues could be due to its
net charge of 2.13 The hypothesis states that the higher charge
of NBD-PS can promote the formation of a hydration shell. If this
hypothesis were correct, the REES should be reduced when the
pH is lowered to 5,46 at which point NBD-PS becomes protonated
and its net charge is reduced to 1.
The experimental results (Table 2 and Fig. 5) show that there
is a small but significant difference in the REES of NBD-PS at the
two different values of pH. However, the REES is higher (19 nm)
in the protonated state at a pH of 5 than in the non-protonated
state at a pH of 7 (16 nm). This result cannot be explained by the
hypothesis of stabilization of the solvation shell by the net
charge of NBD-PS in the membrane. Our hypothesis for the
origin of the REES effect, which is formulated below, and the
molecular dynamics simulations that are presented in the next
section will elucidate the origin of the higher REES that is
observed for protonated NBD-PS.
Time-resolved emission spectra (TRES). Measurements of
time-dependent shifts in fluorescence can also be used to test
dipolar relaxation in lipid membranes. Dipolar relaxation of
the environment surrounding the chromophore causes a timedependent shift in TRES that can be observed by time-resolved
fluorescence measurements. TRES are reconstructed from a set of
fluorescence decays that are dependent on the emission wavelength.53 The unique advantage of TRES over REES is that it
independently reveals information on the polarity and dynamics
of the solvation shell of the dye.
The fluorescence decays of NBD-PS clearly do not show considerable changes with an increase in the wavelength of detection
(Fig. 6A). Fitting of the decays using a typical multi-exponential
function (see Methods) reveals that only small changes occur when
scanning the emission spectra (Fig. 6B and C). The main occurrence
is an increase in the contribution of the shorter-lived component as
one moves towards the red side of the emission spectra.
The TRES of NBD-PS in DOPC bilayers (Fig. 7) display a most
unusual blue shift with time after excitation of the fluorophore.
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Fig. 5 Position of the emission maxima of NBD-PS for different red-edge excitation wavelengths. Examples of the red-edge excitation shift (REES) for
NBD-PS in LUVs of DOPC (pH 7) at 5, 10 and 15 1C (A) and in LUVs of DOPC (15 1C) at pH values of 5 and 7 (B).
The magnitude of the shift is small (E570 cm1) but it is
clearly present. The observation of a blue shift in the TRES is very
unusual, because a dye used to test solvent relaxation should
always display a shift towards the red side. A blue shift in the
TRES cannot be explained by the physics of dipolar relaxation of
the environment of the fluorophore, as it is thermodynamically
impossible. For comparison, the magnitude of the Stokes shift of
Laurdan (a known dye used to test solvent relaxation with a
transition dipole moment that is estimated to be at least 15 D11)
in LUVs of DOPC is of the order of 4000 cm1.8,48
Time-resolved measurements of the fluorescence of NBD-PS
in DOPC at a pH of 5 also revealed an increase in the contribution
of the shorter-lived decay component when increasing the detection wavelength. The blue shift in the TRES following excitation
was also present and its magnitude was smaller (E400 cm1) in
comparison with that at a pH of 7 (ESI,† Fig. S3). Moreover, the
fluorescence decays of NBD were clearly faster at a pH of 7 than at
a pH of 5 at all the detection wavelengths (ESI,† Fig. S4).
In summary, the experimental results that are reported here
show that: (1) REES is insensitive to the dynamics of dipolar
relaxation; (2) the protonation of NBD-PS results in a higher
Fig. 7 Selected time-resolved emission spectra (TRES) of NBD-PS in LUVs of
DOPC at a pH of 7 and 20 1C. The grey vertical line is positioned at the maxima
of the first generated TRES and is meant to be a guide for the eye. TRES were
generated from 0.01 ns to 30 ns on a logarithmic timescale of 100 steps.
REES; (3) TRES display a blue shift with time after excitation.
All these results contradict what would be expected for a
fluorophore used to test the solvent relaxation phenomenon.
Fig. 6 Fluorescence decays of NBD-PS in DOPC (pH 7, 20 1C) at selected emission wavelengths spanning the emission spectrum of NBD-PS (A). The
inset shows a magnification of the first 6 ns after excitation for better visualization of the short-lived components. Lifetimes ti (B) and normalized preexponential factors Ai (C) retrieved by fitting the decays to a 3-exponential decay model for all measured decays of NBD-PS in DOPC (pH 7, 20 1C). The
fitting model is the simplest representation of the data; it is empirical and no physical meaning is attributed to the derived exponential parameters.
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The quantum chemical calculations had already demonstrated
that the change in the magnitude of the dipole moment upon
electronic excitation is small. This indicated that solvent
reorientation should not be so significant as to induce a considerable REES. Both theory and experiment point to an alternative cause of the REES that was observed for the NBD-lipid
analogues embedded in lipid membranes. The key to explaining
the photophysical origin of the observed REES lies in the timeresolved data.
The time-resolved data display not only a blue shift in the
TRES (Fig. 7) but also an increase in the contribution of the shortlived component to the fluorescence decay when the detection
wavelength moves towards the red edge of the emission spectrum
(Fig. 6). In Table 7 of the work of Fery-Forgues et al.5 it is shown
that the presence of water molecules reduces the fluorescence
lifetime of NBD and causes a red shift in absorption and
emission spectra. Moreover, it is also shown that a low water
content results in a long fluorescence lifetime and a blue shift in
the emission. The formation of intermolecular hydrogen bonds
between NBD and water has been suggested to be a cause of the
quenching of the fluorescence of NBD.54 In addition, it has
previously been found that NBD probes are located within a
relatively wide range of depths within lipid bilayers.36,55–57 It is
known that the depth profile of the water gradient is steep in
lipid bilayers and changes dramatically in the interfacial region.
If the chromophore of NBD-PS probes a range of environments
with significantly different water contents, then the photophysical
origin of the REES and the unusual TRES can be explained in a
simple and elegant manner.
Let us first consider the time-resolved results. The increase in
the contribution of the short-lived component to the fluorescence
decays at longer wavelengths of detection can be explained by the
selective detection of NBD-PS molecules that are more hydrated.
These fluorophores display a rapidly decaying red-shifted fluorescence.5 Hydrogen bonding between NBD and water molecules
can effectively quench the fluorescence of NBD,54 which causes a
reduction in the decay lifetime. The fact that molecules in more
hydrated environments will fluoresce quickly and emit in the red
region is precisely the cause of the blue shift that is exhibited in
the TRES. At longer times after excitation only the longer-lived
blue-emitting components remain, which causes the observed
shift towards higher energies (Fig. 7).
To explain the steady-state REES phenomenon, one has to
think again about the photoselection that occurs during the
experiment. In this case, it is the excitation wavelength that is
shifted to the red region. This results in selective excitation of
the more hydrated NBD-PS molecules. These not only exhibit a
red shift in absorption but also a red shift in emission.5 Therefore,
such photoselection in excitation also results in a selective shift
towards the red region in the emission spectra. These species emit
in the red region not because of the slow dynamics of their
environment but because of their highly hydrated surroundings.5
Therefore, the observed red-edge shift would not be due to a
transient effect of the dipolar relaxation phenomenon, but
rather would be the effect of selective excitation of more hydrated
NBD molecules.
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This hypothesis can explain both the REES and the unusual
TRES that were obtained experimentally but is based on the
premise that the NBD-lipids are located within a wide range
of depths in the interfacial region of the lipid membrane.36,55
The steep water gradient within this region creates substantial
changes in the fluorescence properties of NBD as one goes
deeper into the bilayer. Changing the excitation wavelength in a
REES experiment selectively excites the shallower, more hydrated
fluorophores, which in turn emit at longer wavelengths.
Molecular dynamics (MD) simulations
We performed MD simulations to support our hypothesis and
obtain molecular-level insights specifically regarding the NBD-PS
lipid analogue and its location in lipid bilayers. NBD-PS was
simulated in DOPC and DOPC/Chol (80 : 20 mol%) systems both
under conditions of an unprotonated (termed NBD-PS below,
which is representative of the experiments at a pH of 7) and a
protonated carboxylate group (termed NBD-PSH below, which
represents the measurements at a pH of 5).
Areas per lipid. The overall areas per lipid molecule were
determined by dividing the instant box area by the number of
lipid molecules in each monolayer, including those of NBD-PS
or NBD-PSH. For the systems with Chol, separation of the molecular areas of the phospholipid and the sterol was carried out
using the method of Hofsäss et al.,58 with the further assumption that NBD-PS and DOPC are not notably dissimilar in molecular area and volume. The molecular areas are rather invariant
over the duration of the simulations (ESI,† Fig. S5). This is an
indicator that the phospholipid molecules are rapidly equilibrated. However, because the main focus of these simulations is
the behavior of the NBD probes, one should look at the changes
over time in the transverse position of the fluorophore for a more
stringent test of convergence. The location of NBD is equilibrated
rapidly in the bilayers that contain NBD-PS (ESI,† Fig. S6A and C)
and, following protonation of the serine carboxyl groups, the
fluorophore adopts a more internal location in each system
(Fig. S6B and D, ESI†). Although this internalization is mostly
achieved relatively early, there is at least one case of a probe
molecule that takes a very long time to reach its new equilibrium location (namely that indicated by the red curve in ESI,†
Fig. S6D, which is only internalized after E175 ns). For this
reason, the simulations of NBD-PSH were carried out over a longer
duration than the corresponding ones of NBD-PS, as described in
the Methods section. In any case, it appears that in the last 150 ns
of the simulations in the absence of Chol, as well as in the last
200 ns of the simulations with Chol, the positions of the fluorophore vary non-systematically, which justifies the use of these
time intervals for analysis. The final structures in all simulations (ESI,† Fig. S7) illustrate that the location of the NBD group
is in general more internal in the systems where the serine
carboxyl is protonated (ESI,† Fig. S7C and F) compared with
those where the latter is charged (Fig. S7B and E, ESI†).
The average area per lipid (ESI,† Table S6) that was calculated
for DOPC, a = 0.684 0.015 nm2, agrees well with both experimental data (0.691 nm2 at T = 288 K;59 0.674 nm2 at T = 303 K60)
and simulations with different force fields (0.671 0.006 nm2
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at T = 323 K;61 0.673 0.004 nm2 at T = 293 K and
0.680 0.005 nm2 at T = 303 K62). The presence of Chol leads
to a decrease in the area per lipid and an increase in the bilayer
thickness (calculated as the average difference between the
transverse locations of the P atoms in DOPC in opposing
leaflets), which is the well-known Chol condensation effect.63
By comparing the values in the presence and absence of probes,
it becomes apparent that the inclusion of either NBD-PSH or
(especially) NBD-PS also induces slight (smaller than the statistical
uncertainty) bilayer condensation in both the DOPC and DOPC/
Chol (80 : 20) systems. This is also shown in an increase in the
bilayer thickness (ESI,† Table S6).
Atomic locations. A slight increase in the separation between
DOPC atoms and the bilayer midplane is also observed in the
average transverse positions of Fig. 8 (please refer to Fig. 1 for the
atom numbering). However, the most striking data shown in this
figure concern the locations of probe atoms, namely those of the
lipid head group and the fluorophore. Contrastingly different
average conformations are observed for the two protonation
states of the probe. For NBD-PSH, the NBD group has a similar
location to that of the DOPC glycerol backbone. This is observed
by comparing DOPC C13 with the positions of the NBD-PSH N1,
O12 and nitro (N6, O7, O8) atoms. On the other hand, the head
group atoms of NBD-PSH have more internal locations than the
corresponding DOPC atoms (compare the NBD-PSH N1, P18 and
C23 atoms with the DOPC N4, P8 and C13 atoms, respectively).
The protonated NBD-serine moiety adopts a position near that of
the DOPC glycerol and in the process pulls the phosphate group
inwards. This situation is reminiscent of another recently
Paper
reported monoanionic head-labeled NBD probe, NBD-PE.36,56 In
NBD-PE, the fluorophore as a whole had an even more internal
location (around or above that of the phospholipid C13 atom).
This is also observed in other NBD probes such as acyl-chainlabelled NBD-PC,55 fatty amine-NBD-Cn,57 and NBD-Chol.64 The
sole difference between the NBD-PE and NBD-PSH head groups is
the COOH group in the latter. This means that the presence of
a carboxyl group, even if in a protonated state, causes a slight
displacement (E0.3 nm) of the fluorophore towards a more
external region. However, this is still a small effect compared
with the displacement that is observed for the bianionic NBD-PS.
For NBD-PS, very similar positions are obtained for corresponding probe and DOPC lipid head group atoms (compare the
NBD-PS N1, P18 and C23 atoms with the DOPC N4, P8 and C13
atoms, respectively). The location of the N atom is only slightly
more external compared with the P atom. On the other hand,
both fluorophore and carboxyl atoms have remarkably external
locations. The fluorophore of NBD-PS, uniquely for NBD probes,
protrudes into the aqueous medium beyond the locations of the
more external host lipid atoms. These considerations essentially
apply to systems both without (Fig. 8A) and with Chol (Fig. 8B).
Fluorophore distribution and accessibility to water. A less
detailed but more illustrative picture of this effect is given by
the mass density profiles (Fig. 9). Although the fluorophore of
NBD-PSH has a transverse distribution that is similar to that of
the DOPC head group (and thus displays peaks in density around
the same location, Fig. 9B and D), it clearly has a more external
location in the unprotonated probe NBD-PS (Fig. 9A and C).
For the latter, increased exposure to the solvent is evident.
Fig. 8 Average transverse locations of selected atoms in the systems with no Chol (A) and 20 mol% Chol (B).
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Fig. 9 Mass density profiles across the normal direction of the bilayer in the (A) DOPC/NBD-PS, (B) DOPC/NBD-PSH, (C) DOPC/Chol/NBD-PS, and
(D) DOPC/Chol/NBD-PSH systems. For better visualization, the profiles of NBD-PS/NBD-PSH, the NBD fluorophore and sodium ions are multiplied by
20, 20, and 50, respectively.
A quantitative measure of the solvation of the fluorophore
(the average water density as sensed by the fluorophore) may
be calculated from the densities r of water and the fluorophore
according to:65
Ð
hrðwaterÞifluorophore ¼
rfluorophore ðzÞrwater ðzÞdz
Ð
rfluorophore ðzÞdz
(1)
This yields values of hr(water)ifluorophore of 328 kg m3 and
352 kg m3 for NBD-PSH in DOPC and DOPC/Chol, respectively, compared with 644 kg m3 and 642 kg m3 for NBD-PS in
the corresponding systems. The lack of significant variation in
hydration between the DOPC and DOPC/Chol compositions agrees
with the identical REES that was measured in the two systems. For
comparison, we previously obtained values of E170–180 kg m3
for NBD-PE and NBD-PC in fluid POPC (1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine) and DPPC (1,2-dipalmitoyl-sn-glycero-3phosphocholine), respectively.36 These values confirm that on
average the NBD fluorophore of head-group-labeled phosphatidylserine experiences a more polar environment than that of
head-group-labeled phosphatidylethanolamine or acyl-chainlabeled phosphatidylcholine. This is especially notable for
the unprotonated form, which is expected to predominate at
neutral pH, which senses an average water density that is equivalent to almost two-thirds of the value of bulk water. Taking
into account the reduction in the fluorescence lifetime of NBD
in the presence of increasing amounts of water,5,54 the increase
in hydration of the fluorophore of NBD-PS relative to that of
NBD-PSH is in full accordance with the shorter average lifetime
that is measured at a pH of 7 compared with that at a pH of 5
(ESI,† Fig. S4). In addition, the TRES that are measured at a pH
of 5 show that the spectrum at the initial stage of relaxation
is already more blue-shifted compared with that at a pH of 7
(ESI,† Fig. S3), which agrees with the decrease in hydration of
NBD-PSH that is obtained in the MD simulations. This difference
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in the initial stage of the relaxation process is the main reason
why the overall magnitude of the blue shift is smaller at a pH of 5.
In Fig. 9 it can be seen, in addition to the average values,
that the distributions of the fluorophore are also remarkably
wide. This implies that NBD experiences a considerable degree of
heterogeneity of water density. In effect, the local water density
varies from E200 kg m3 to almost the value of bulk water for
NBD-PS and from essentially zero to 4900 kg m3 for NBD-PSH.
This means that in relative terms the variation between extremes
of polarity is larger for NBD-PSH than for NBD-PS, which agrees
with the higher REES measured at a pH of 5 compared with
that at a pH of 7. As previously commented for NBD-PE and
NBD-PC,36 a wide variation in the local concentration of water
surrounding the fluorophore is probably the main reason for
the heterogeneity of decay that is most often found in the
fluorescence decays of NBD lipid analogues.
Another way to analyze the solvation of the fluorophore is to
examine the hydrogen-bonding interactions that are formed
between NBD and water atoms. NBD can act as a H-bonding
donor to water via its amino group (N1H14 in Fig. 1D) or as a
H-bonding acceptor from water to one of its oxygen or nitrogen
atoms. In the following, a H-bond for a given donor–H–acceptor
triad was registered each time the donor–acceptor distance was
less than 0.35 nm and the H–donor–acceptor angle was o301.
As shown in ESI,† Fig. S8, NBD forms a considerable number of
H-bonds with water, especially as an acceptor. This agrees with
previous simulations of NBD lipids36,55,57,64,66 and is largely
to be expected, because the transverse distribution of the NBD
group extends into regions of high accessibility to water. However,
this does not provide insight regarding the degree of heterogeneity of solvation among individual NBD fluorophores. For
the latter purpose, we counted the average number of H-bonds
formed by NBD as a function of the transverse location of the
fluorophore’s centre of mass (Fig. 10). H-bonding from water
to acceptor atoms in NBD increases when the fluorophore is
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also visible in the mass density plots shown in ESI,† Fig. S10.
In addition, in most systems the location of the NBD fluorophore also becomes, in absolute terms, more external by
B0.1 nm. Therefore, the position of the fluorophore remains
practically invariant in relation to the lipid/water interface
and its exposure to water is virtually unaltered. Therefore,
the above discussion concerning the heterogeneity of the location of NBD and its relationship to the measured REES is
equally valid in the absence and presence of physiological salt
concentrations.
Conclusions
Fig. 10 Number of NBD-to-water (green) or water-to-NBD (red) hydrogen bonds (n) as a function of the transverse location of the fluorophore’s
center of mass relative to the bilayer midplane (z) for the (A) DOPC/NBD-PS,
(B) DOPC/NBD-PSH, (C) DOPC/Chol/NBD-PS, and (D) DOPC/Chol/NBD-PSH
systems. The dotted blue line represents the average location of the DOPC
phosphorus atom in each system, whereas the black curve is the mass
distribution profile r(z) of NBD.
located closer to the aqueous medium, as expected. Significantly,
the relative increase is more pronounced for the protonated form.
When the fluorophore of NBD-PSH is located most internally, the
average number of H-bonds is very low (o1 in the absence of
Chol and close to zero in the presence of Chol). Although the
maximum extent of H-bonding is not as high as for NBD-PS, the
factor of the increase in the average number of H-bonds from
the least solvated to the most solvated population is higher for
NBD-PSH than for the unprotonated probe. Another illustrative
way to analyze this effect is to examine specifically the fraction of
configurations of the fluorophore for which no NBD/water
H-bonds are detected (ESI,† Fig. S9). These dehydrated configurations are consistently rare for NBD-PS, even at its most internal
locations. However, their occurrence is significant for NBD-PSH
in internal locations (values of B0.4–0.5) and, moreover, the
fraction undergoes a pronounced decrease to essentially zero for
external locations. This is in accordance with the longer average
fluorescence lifetime that is measured for NBD-PSH and reinforces
the observations regarding the increased degree of heterogeneity
of solvation of the protonated probe, which we assign as the
probable cause of its increased REES.
The simulations that are described above concern systems to
which no ions were added beside those that were necessary to
counterbalance the negatively charged probes. This allows a
direct comparison with our previous MD simulations of NBD
lipids, which were carried out under the same conditions. To
confirm whether the addition of physiological salt concentrations would alter the relative location of the NBD moiety, we
also performed simulations at an ionic strength of 150 mM
identically to the experimental fluorescence data. We observed
that the addition of NaCl orders the bilayer to some extent,
which is an effect that has been observed in earlier studies.67–69
For example, in the NBD-PS/DOPC system the bilayer thickness
increases non-significantly from 3.74 0.10 nm in the absence
of salt to 3.87 0.09 nm at an ionic strength of 150 mM. This is
7052 | Phys. Chem. Chem. Phys., 2016, 18, 7042--7054
In this article, we present a multifaceted study of the behavior of
NBD-labeled lipids. Our DFT calculations of electronic structure
confirm the experimental trends of increases in absorption and
emission wavelengths for more polar solvents and provide good
agreement with experimental absorption spectra. They clarify the
traditional view that the transition of NBD with lowest energy
has more pronounced charge-transfer character compared with
the second-lowest transition by showing that both transitions are
actually associated with electron displacements from the amino
group in NBD to the heterocyclic ring and nitro group. More
importantly, they confirm that the magnitude of the variation in
the dipole moment of the fluorophore between the ground and
first excited states is probably too small (B2 D) to be responsible
for the REES of NBD-labeled lipids. In accordance, fluorescence
measurements using the NBD probe for which a maximum
extent of REES has been reported (NBD-PS) show that this
phenomenon is insensitive to both temperature and the presence of Chol and is therefore insensitive to both the mobility
and the hydration of the lipid. TRES of NBD-PS display an
unusual blue shift following excitation. Moreover, red-edge
detection leads to an increase in the contribution of the shortlived decay component and an overall decrease in the average
fluorescence lifetime. These observations are not consistent with
a REES that is caused by dipolar relaxation of the solvent but
instead with red-edge selective excitation of the more shallowly
located and therefore more hydrated fluorophore population
(which display a red shift in emission and decay at earlier times,
thus shifting the time-resolved emission spectra towards shorter
wavelengths). This hypothesis is corroborated by MD simulations, which show a heterogeneous transverse location of NBD. A
deeper average location and larger extent of heterogeneity of
transverse distribution (which was observed at lower pH) correlate with the experimentally measured longer average lifetime
and increase in REES, respectively. Our combination of methodologies leads to a globally consistent view of the complexity
in decay and REES effect of NBD probes as being caused by
heterogeneity of both the polarity that is experienced by the
fluorophore and the NBD–water hydrogen-bonding efficiency,
which in turn are caused by the wide distribution of NBD that
encompasses large variations in local water density. This study
is thus an important contribution to the understanding of the
photophysics of NBD, which is one of the most frequently used
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fluorescent membrane labels. It resolves previously published
conflicting results and the long-standing misinterpretation of
fluorescence data of NBD.
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Acknowledgements
Financial support from the Czech Science Foundation via grant
P208/12/G016 (M. H. and M. A.) is acknowledged. Moreover,
M. H. acknowledges the Praemium Academie Award from the
Academy of Sciences of the Czech Republic. H. A. L. F. and
L. M. S. L. acknowledge the Laboratory for Advanced Computing
at the University of Coimbra for computing resources, and
funding by Fundação para a Ciência e Tecnologia (Portugal),
project reference UID/QUI/00313/2013.
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