Will C-Laurdan Dethrone Laurdan in Fluorescent Solvent Relaxation

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Will C-Laurdan dethrone Laurdan in fluorescent solvent relaxation techniques for lipid membrane studies? Justyna Barucha-Kraszewska, Sebastian Kraszewski, and Christophe Ramseyer Langmuir, Just Accepted Manuscript • DOI: 10.1021/la304235r • Publication Date (Web): 02 Jan 2013 Downloaded from http://pubs.acs.org on January 8, 2013

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Will C-Laurdan dethrone Laurdan in fluorescent solvent relaxation techniques for lipid membrane studies? Justyna Barucha-Kraszewska,* Sebastian Kraszewski, Christophe Ramseyer Laboratoire de Nanomédecine, Imagerie et Thérapeutique, EA4662, Université de FrancheComté, Centre Hospitalier Universitaire de Besançon, 16 Route de Gray, 25000 Besançon, France. Molecular Dynamics; Fluorescent Probe; Lipid Membrane; Excited State; Solvent Relaxation; Water Dynamics; Laurdan; Carboxy-Laurdan; Dye Localization; Effective Position; Development of fluorescence methods involves the necessity of understanding the fluorescent probes behaviour in their ground and excited states. In the case of biological membranes, the position of the dyes in the lipid bilayer and their response after excitation are necessary parameters to correctly analyse the experiments. In the present work, we focus on two fluorescent markers: Laurdan (6-lauroyl-2-(N,N-dimethylamino)naphthalene) and its derivative C-Laurdan (6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene) recently proposed for lipid raft visualization.[H.M. Kim et al., ChemBioChem, 8 (2007) 553] C-Laurdan, by the presence of an additional carboxyl group, has an advantage over Laurdan since it has a higher sensitivity to the membrane polarity at the lipid headgroup region and a higher water

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solubility. This theoretical study, based on Quantum Mechanical (QM) and Molecular Dynamics (MD) simulations in a fully hydrated lipid membrane model, compare the equilibrium and dynamic properties of both probes taking into account their fluorescence lifetimes. C-Laurdan is found to be more stable than Laurdan in the headgroup region of the membrane and also much more aligned with the lipids. This study suggests that, besides the lipid raft imaging, the CLaurdan marker can considerably extend the capabilities of fluorescent solvent relaxation method.


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INTRODUCTION Fluorescence methods are convenient experimental tools to study biological and model membranes.1 Knowing how the hydration influences the mobility of biological membrane is helpful to understand its fundamental functions.2-5 One of the fluorescence methods, which allows to examine the water impact on lipid membrane by probing local hydration and mobility of the phospholipid headgroup of lipid bilayer, is the fluorescent solvent relaxation (SR) technique.6-9 SR method uses fluorescent probes, which report on the level of polarity and viscosity, the parameters that are derived from calibration based on macroscopic measurements in neat solvents. Combination of the results coming from a few differently located probes permits evaluating the gradient of water and its dynamics across the lipid bilayer.10, 11 Solvent relaxation in phospholipid membranes is about 104 times slower than in pure water, therefore SR is very sensitive to the localization of the dye molecule especially within the headgroup region of lipid membrane.8-11 For a correct interpretation of solvent relaxation data, a precise knowledge of the dye chromophore location and stability in lipid membrane is of crucial importance, especially since some markers can slide toward more polar regions just after their excitation.12 Laurdan (6-lauroyl-2-(N,N-dimethylamino)naphthalene, see Figure 1a) molecule is a fluorescent probe widely used for SR experiment, but also in many other fluorescent techniques.13, 14 It has a long hydrocarbon chain (11 carbons) attached to the carbonyl group and quaternary ammonium group. Laurdan is one of the naphthalene derivatives, which probe microfluidity and micropolarity changes within the headgroup region of biomembranes. They are capable measuring numerous important parameters of lipid membranes, such as local pH,15 surfaceabsorbed compound like protein,16 membrane curvature,17 effect of temperature,18 binding of calcium ions 11 and variations of lipid composition.19 Laurdan was also successfully used in the


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visualization of lipid domains in the model of lipid bilayer and rafts in biological membrane.20, 21 A recent report notifies about the synthesis of a new Laurdan derivative: C-Laurdan (6dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene, see Figure 1b).22 This one is apparently more sensitive to the membrane polarity. It has two orders of magnitude higher water solubility (30 µM) than Laurdan (0.1 µM), and was claimed to not form aggregates in the lipid bilayer. C-Laurdan is supposed to be located at the headgroup region of zwitterionic lipid bilayers. Its chromophore was supposed to be aligned more parallel to the lipid molecules, comparing to Laurdan, due to the presence of carboxyl group attached to it. This chemical modification was argued to change the hydrophobic-hydrophilic balance of the molecule and slightly alter its localization within membrane. Note that other functionalizations may result with the same effect. C-Laurdan molecule is apparently a particularly good marker reflecting differences between fluid and gel phases in lipid membranes (rafts) as revealed by the generalized fluorescence at two-photon fluorescence microscopy,22-25 or even by conventional fluorescence correlation spectroscopy.26 These recent findings on the specific molecular properties of C-Laurdan probe suggest also its usefulness in the fluorescent solvent relaxation technique. It should be a sensitive indicator of the polarity in the lipid headgroup region. Since the fluorescence emission of SR adapted probes is strongly sensitive to the presence of water (which concentration gradually decreases from bulk to the hydrophobic part of the membrane), the question about the exact location of C-Laurdan in the lipid bilayer must be answered.


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Figure 1 Licorice molecular models of Laurdan (a) and C-Laurdan (b). Navi blue part is the dimethylamino electron-donating group and the red subunit corresponds to the carbonyl electron acceptor. Supplementary carboxylic group of C-Laurdan is visible at the top of the molecule. Naphtalene chromophore is marked with transparent green surface. High structural anisotropy of lipid bilayer combined with steep (at molecular level) gradients of hydration, polarity, and electrostatic potentials makes molecular probing in biomembranes very challenging. Since the fluorescent probes always sense the properties of their local environment, an intrinsic anisotropy of this environment influences their many properties including location and orientation of probing dyes, and the fluorescence response leading to a number of nontrivial effects. These effects include static charge–dipole and dipole–dipole interactions of the probing dye with surrounding groups of atoms.27 Molecular Dynamics (MD) simulations and molecular probing are presently seen as the only methods that can allow to provide in a consistent manner the analysis of structure, dynamics, and interactions leading to an appropriate description.28 Especially, a combination of MD simulations with Quantum Mechanical (QM) calculations allows studying both the intermolecular interactions and intra-molecular processes. This two


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theoretical approaches was already proved to correctly reproduce solvent relaxation phenomenon upon electronic excitation of acetone in water,29 and also of Prodan and Laurdan in DOPC lipid bilayer.12 In this work, we carry out an extensive computational analysis of Laurdan and C-Laurdan molecules embedded into DOPC lipid membrane. First, quantum mechanical calculations are performed to obtain adequate molecular parameters (like bond lengths, interatomic angles, partial charges and dipole moments) for fluorescent markers in their ground and excited states. It allows us to create the model of interactions, necessary to study their behavior at molecular level within the lipid bilayer. Second, all atom MD simulations are performed for the dye molecules in their ground and excited states surrounded by fully hydrated DOPC lipid membrane in ambient temperature. The comparative analysis of both markers is performed for answering the question about dyes position in the membrane, their translational and orientational stabilities in bilayer and their dynamical properties after excitation. Finally, we propose for the first time a statistical approach to study the excited probes position in the membrane, which fully takes into account the probes lifetime after excitation.

METHODS Quantum Mechanics All quantum level calculations were performed using the Gaussian 03 software package.30 The ground state equilibrium geometries of Laurdan and C-Laurdan were optimized by density functional theory DFT 31, 32 model with the b3lyp/6-311+G(d,p) basis set. The excited state


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properties of fluorescent dyes were obtained from time dependent density functional theory TDDFT 33-35 with the same basis set as for DFT. The water solvent effect, which is crucial for charges screening, and thus for appropriate dipole moment reproduction, was taken into consideration using the integral equation formalism of the polarizable continuum model IEFPCM, with a dielectric constant ε=78.39.36-39 The dipole moments (both in ground and excited states) were determined from the Mulliken charges distribution, which appears to be the most suitable approach to reproduce the experimental transition dipole value, as discussed in our recent work.12 Supplementary analysis based on the construction of the Hessian matrix (containing the second derivatives of the energy with respect to the geometry) was also performed for further use in the force field parameterization. For additional details on our calculation method and the choices we made, we refer the reader to our previous study.12 Specific geometric and electronic data like bond lengths, angles, dihedrals and charges distribution were extracted from QM simulations. Those parameters are crucial for implementing the force field used in MD. The most important and easily implementable in MD simulation parameter, which distinguishes ground state from the excited one, is the dipole moment of the molecule. The partial electron shift from the donor to acceptor group of chromophore increases significantly the dipole moment value upon electronic excitation. The values of dipole moments obtained for C-Laurdan molecule in ground and first excited states showed more than a twofold increase, from 4.67 D for ground state to 11.04 D for excited state. The same behavior was also obtained for Laurdan, since we got 5.73 D and 12.93 D for its ground and excited states, respectively. Molecular Dynamics


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All MD simulations were carried out in the NPT ensemble (constant Number of particles, Pressure and Temperature) using the NAMD2.640 suite of programs within the CHARMM27 force field.41 This force field is known to provide structural results for hydrated all-atom DOPC that are in good agreement with experiments.42 However, the common force fields, that are specific databases of intra and inter-molecular interactions, include only parameters for frequently studied molecules, i.e. proteins, nucleic acids, and lipids.40, 43-45 Excitable molecules such as fluorescent probes are not very popular for computational analysis despite their relative importance for biological sciences. Thus, for the missing potential parameters, we used our previous QM calculations and we followed the general procedure described by Norrby and Brandt.46 We successfully proved that it gives the molecular models of fluorescent markers accurately reproducing their macroscopic behaviour.12 Solvent water molecules were described by the TIP3P model.47 A constant temperature of 300 K and a constant pressure of 1 atm were ensured by Langevin dynamics and Langevin piston Nosé-Hoover algorithm, respectively.48 Chemical bonds between hydrogen and heavy atoms were constrained to their equilibrium values, which allowed us to employ the integration time step of 2.0 fs. Long-range electrostatic forces were evaluated using the particle mesh Ewald (PME) method.49 In the experimental sample, a single liposomal vesicle contains about 105 of lipid molecules and up to 103 fluorescent probes. MD simulations have a limitation on the number of molecules, which could be implemented. Thus, it is difficult to analyze more than a few dyes in one sample if one wants to keep dye to lipid ratio at reasonable 1:100 level. Therefore, to study the behaviour of the probe in the vicinity and within the lipid membrane, a small DOPC lipid bilayer patch, composed of only 72 lipids (36 per monolayer) and hydrated on each side by 35 Å water slabs (4985 molecules), was used as a numerical model. The lipid bilayer was first generated


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using a pre-equilibrated lipid membrane followed by a very short energetic optimization. Then, a long equilibration run, during more than 25 ns directly at 300 K and 1 atm, was performed. The membrane thickness (distance between choline headgroups) reached 40.2±0.6 Å already after 2 ns and area per lipid (A) is of 62.9±0.9 Å2 at the end of the equilibration, being in the range of experimental values (from 59.4 Å2 50 to 72.2 Å2 51). The size of the complete system was about (50×50×100) Å3, which is enough for the 25 Å long molecule (in its completely stretched form) to freely rotate within lipid bilayer, and to avoid any artificial bias toward vertical orientation of the probe. Simulations were primarily conducted for Laurdan and C-Laurdan molecules in their ground state (S0), and initially located in the bulk water. After 320 ns simulation for C-Laurdan (500 ns for Laurdan), when the dye was already spontaneously incorporated into the membrane core, the first excited (S1) state was induced by a change of charges distribution on the probe mimicking the dipole change in the excitation process. From that moment, the system containing fluorophore continuously kept in the excited state was observed for further 300 ns (also 300 ns in case of Laurdan). This protocol was employed in order to find differences between two stationary defined states. Moreover, for checking the stability of the fluorescent probe locations within phospholipid bilayer and the related dynamic water behavior just after excitation, many short (of 10 ns) supplementary simulations for Laurdan and C-Laurdan (34 and 36 runs with induced exited state, respectively), initially located at different depths in the membrane, were also performed. These initial configurations were taken directly from the main calculation with the molecule in the ground state. A total accumulated simulation time for this study of 2.4 µs was performed. It allowed us to have a well-defined statistical view on the probes behavior embedded into DOPC bilayer, comparing to the 4 ns fluorescence lifetime of naphthalene chromophore.


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Statistical analysis The positions of the marker molecule during simulations x(t ) can be discretized and represented as histograms using a normalized probability distribution P Norm ( xi (t ) ) . To construct a graphical representation for each bin i , occurrences of marker localization ranging from xi (n∆t ) to N

xi ( n∆t ) + ∆x were summed up over all simulation time n∆t :

∑ P(xi (n∆t ) + ∆x ) ; N being the

n =0

total number of trajectory snapshots, ∆x being the bin width of 0.25 Å, and ∆t being a recording time of the simulation trajectory equal to 5 ps. Normalized probability of each bin was then N

obtained as follows: P

Norm

(xi (t )) =

∑ P(xi (n∆t ) + ∆x )

n =0

P

max

; P max being the maximal number of

occurrences in a most populated bin in all dataset. In order to statistically take into account the excited markers relocalisation only within the fluorescent lifetime, we weighted the marker localization occurrences by the picoseconds timeresolved fluorescence FTR (n∆t ) obtained experimentally for the Laurdan and C-Laurdan in

DOPC vesicles, and measured at 440 nm.22 Thus, for each bin i , the occurrences of marker N

localization were defined as

∑ P(xi (n∆t ) + ∆x )FTR (n∆t ) weighted by the exponentially

n =0

decreasing contribution of relocalisation over the fluorescence lifetime.

RESULTS AND DISCUSSION Location and orientation in lipid bilayer


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It is well known, that amphiphilic molecules, such as Laurdan, easily incorporate into lipid membranes.52 In order to validate our Laurdan and C-Laurdan models, and to explore their incorporation behavior, the molecules in their ground states were initially placed into separate systems containing fully hydrated DOPC lipid bilayer, and were placed far from the bilayer membrane (into bulk water, see Figure 2). C-Laurdan is found to be incorporated into the membrane after 40 ns, as shown in Figure 2b. In comparison, Laurdan needs only about 10 ns. This difference could be explained by the more hydrophobic character of Laurdan (lack of the supplementary hydrophilic carboxylic group). C-Laurdan remains in the same lipid layer for the rest of simulation time (0.3 µs). Its chromophore location is found at headgroup region of the lipid bilayer, while its hydrocarbon tail is inserted between phospholipid hydrocarbon chains. By contrast, Laurdan is starting to jump from one lipid layer to the other 100 ns over its incorporation into the membrane. This instability is observed continuously over all 0.5 µs (see Figure 2a). Apparently the long hydrocarbon tail of Laurdan, supposed to better anchor the marker into the membrane comparing for instance to Prodan (having a very short hydrophobic propionyl group), is considerably lowering the flip-flop barrier. To the best of our knowledge this high flipping ability of Laurdan was not yet reported but cannot be excluded. Even if it could be checked with rather simple experiment using for instance supported bilayers, as recently described by Kulakowska et al.,53 the observed flip-flop rate appears to reach already the timescale resolution of the mentioned experimental setup.


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Figure 2 Left hand side: Laurdan (a) and C-Laurdan (b) chromophore location (black curve) as a function of time of unconstrained MD simulation. The positions of lipid ammonium (blue curve), lipid phosphorous (red curve), lipid glycerol (green curve), and center of DOPC membrane (dashed line) are also plotted. These results were obtained with dye molecules in their ground states (S0). Right hand side: Corresponding snapshots extracted from simulations at 200 ns. Chromophore group and probe hydrocarbon tail are represented as red and yellow sticks, respectively. Membrane hydrophobic part is shown as brown lines, lipid heads – as licorice with atom dependent colors, and water – as a blue transparent surface, respectively.


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The location probabilities of Laurdan and C-Laurdan within the membrane are presented in Figure 3 in terms of the chromophore center distance from the bilayer center. Histograms were calculated on the basis of the data issued from long runs of markers in ground and excited states as described in Methods section. To better visualize the most probable values of the dyes locations, a Gaussian deconvolution on each of the histograms was applied. These results are summarized in Table 1. It should be noted that the positions obtained for excited state from such a long simulation could be not accurate, since fluorescent probe has relatively short lifetime (~4 ns for naphthalene derivatives). In reality, a dye does not have enough time to equilibrate after excitation. To answer this issue, we propose here a statistically defined position for excited state, called hereafter effective position.


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Figure 3 Location probability histograms for Laurdan (a) and C-Laurdan (b) chromophore groups in their ground S0 (gray plot) and excited S1 (red plot) states, issued from long MD runs. Effective position upon excitation, based on many short-time MD runs, is marked by blue-stars line. Probabilities are shown as a function of the distance between the chromophore and the bilayer centers.

Table 1 Dyes locations in DOPC bilayer. Distances (in Ångstroms) from the bilayer center, obtained by Gaussian deconvolution of data shown in Figure 3. Found two significant peaks are reported with calculated normalized amplitudes (indicated by ). Laurdan S0 lower significant peak

10.8

C-Laurdan

EFF

S1

S0

.

12.8

12.1

EFF

S1

.

12.1

higher significant peak

11.9

12.0

14.2

14.1

13.0

14.1

weighted mean

11.4

12.0

13.6

13.1

13.0

13.2

To retrieve the effective location probability of the dyes in the membrane during their excited state many repetitive simulations for each particular position are needed, instead of one long simulation run. Basing on the previous histograms calculated for the ground state (grey plots in Figure 3) one needs to perform as many short-time simulations in the excited state as different locations in ground state occur. Moreover, to be statistically consistent, the excitation run should be performed more than once for each initial location. Initial snapshots of the system with the dye subjected to excitation at given depths were extracted at different times from previous long MD run with the marker in its ground state. We decided to fit a number of these excitations accordingly to the location probability shown in Figure 3. Since each of these short MD runs are


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relatively expensive in terms of computational power, we assumed that the most probable location (11.4 Å for Laurdan and 13.1 Å for C-Laurdan) deserves a maximum of independent MD runs, here chosen as six. For less probable locations we decreased gradually this number of MD runs, getting for instance for C-Laurdan five MD runs for initial chromophore location at 12 Å and 14 Å from bilayer center, four MD runs at 11 Å and 15 Å, and so on, up to one MD run at 6, 7, 8 and 18 Å, respectively. Every single initial configuration in the ground state was excited by changing the charges distribution from ground state to excited one at time "zero" (t=0). Relative relocations of the excited dye were next weighted by the corresponding experimental picoseconds time-resolved fluorescence spectra (see Methods section). The relative probe relocation is thus taken into account only in the range of possible excitation lifetime, through the exponential decrease of location occurrences over time. Obtained separated relocation contributions from each MD run were then gathered together to the histogram shown in Figure 3 (blue-stars line). We believe that such an approach gives more appropriate estimation of marker delocalization after excitation than we recently proposed basing on long time onemolecule MD run only.12 From the long MD runs of 300 ns, two mean positions in ground and excited states were found for both dyes with a significant probability (see Table 1). C-Laurdan in the ground state (S0) is found to be located at mean position 13.1 Å from the bilayer center what is about 1.7 Å more outside than mean position calculated for Laurdan (11.4 Å). In the first excited state (S1) the positions of Laurdan and C-Laurdan, calculated from these long simulations, are roughly the same: 13.6 Å and 13.2 Å, respectively. However, the statistical calculation of the effective position for Laurdan reveals a smaller shift toward the more polar region of the lipid bilayer (0.6 Å) after excitation, comparing to the well-equilibrated location for excited state retrieved


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from long MD run (2.2 Å). Effective position after excitation obtained for C-Laurdan is the same as for the ground state. This means that C-Laurdan is extremely stable in the membrane during the excitation process. Moreover, all C-Laurdan locations (for ground state, for effective position, and for the excited state calculated from long time simulation) have the same value (~13 Å with a distribution at half maximum of only ±2 Å), which emphasizes the very high stability of this molecule in the membrane. The shift toward polar region, occurring for Laurdan after excitation, clearly indicates his lower stability in comparison to C-Laurdan molecule. Another argument confirming the exceptional stability of C-Laurdan location is that the flip-flop phenomenon from one leaflet to another was not observed during all MD runs of nearly 1 µs, contrary to Laurdan. Finally, the effective position of C-Laurdan is 1 Å more outside in the membrane than of Laurdan, suggesting that this new probe can be successfully used to monitor other membrane region than commonly used SR probes. Effective position calculation being a steady view on probes location cannot, however, retrieve the dynamics of fluctuations in probes location just after their excitation. Thus, we decided to scan the positions of Laurdan and C-Laurdan after excitation from all short-time simulations (34 for Laurdan and 36 for C-Laurdan, respectively), which have been prepared for effective position investigation. Results presented in Figure 4, show maps of dyes location probability as a function of time just after excitation (time t=0). These translocations of a given dye from all the series of simulations were summed up and normalized by the number of samples indicating the most probable location. Obtained in such a way normalized histograms were gathered interval by interval (250 ps each) to obtain a statistically relevant three-dimensional plot. Both dyes, when are located below 12 Å from bilayer center, move to the more outside region during a first nanosecond after excitation. Moreover, dynamic changes in position of C-Laurdan (mainly


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between 11 and 14 Å) remain small with respect to Laurdan, which broadly oscillates between 9 and 15 Å.

Figure 4 Relocation probabilities of Laurdan (a) and C-Laurdan (b) molecules just after their excitation within DOPC bilayer. Results are plotted as a function of time (x-axis), and distance from the bilayer center (y-axis). The unoccupied locations are colored blue whereas the most occupied are represented by red color.


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The presence of carboxyl group in C-Laurdan also strongly influences the orientation of its chromophore. Results of chromophore inclination angle over 0.3 µs are shown in Figure 5. MD simulations clearly suggest that Laurdan is oriented more perpendicularly to the normal axis of the bilayer. In contrast, C-Laurdan is oriented almost parallel to the lipid molecules. The addition of a hydrophilic group onto the Laurdan molecule stabilizes and aligns the chromophore in the lipid bilayer. This can be benefic for fluorescence experiment, since the molecule takes fewer places in the membrane, thus disturbing it less than other probes.

Figure 5 Tilt angle of Laurdan (green-circles line) and C-Laurdan (red-stars line) chromophores with respect to the normal of DOPC bilayer, calculated over 300 ns of unconstrained MD simulations. Water dynamics after dye excitation Solvent relaxation process is directly linked to the polar solvent rearrangement due to the dipole moment change after excitation of the fluorescent molecule. Relaxation time constant describes the solvent mobility of the dye surrounding. It is a main parameter, which is obtained directly from SR experiment.8 A general relaxation time measured experimentally in pure water is on the


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picoseconds timescale,54 and can be already retrieved using molecular simulations.55 In the membrane, strong interactions between water and lipid headgroups slow down the solvent relaxation time to a few nanoseconds.8 Water molecules reorientation times can also be extracted from MD, after the marker excitation. Figure 6 shows the projection of the total water dipole vector (coming from different water molecules neighboring the dye) on the instantaneous dye dipole vector as a function of time just after excitation. Such a procedure allows tracking the cyclic orientations and disorientations of water molecules, which we believe are the reproduction of solvent relaxation phenomenon, induced by drastic change in probe dipole upon excitation. This procedure was recently successfully used for Laurdan, since we shown that the complete oscillation cycle of water rearrangement can directly be linked to the experimentally observed relaxation time constants (τi).12 We assume that one cycle of water orientation and disorientation, with respect to marker dipole, can directly be read from plots shown in Figure 6. This cycle appearing over time as colored spot finishes by water disorientation (blue or black zones). Thus, visually inspecting plots, one can roughly estimates τi value. However, we wish to warn the reader that this arbitrary examination should only be viewed as a qualitative analysis.


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Figure 6 Dynamic changes of water dipole coming from water molecules neighboring CLaurdan molecule embedded into lipid membrane. Initial positions of C-Laurdan were chosen at the most probable depths of 12 Å (a), 13 Å (b), and 14 Å (c) from bilayer center. Maps are plotted as a function of time (x-axis) and distance from chromophore center (y-axis). Blue areas indicate the lack of the water organization, green and yellow regions witness a progressive arrangement, while red zones correspond to the most organized water molecules vs. dye dipole vector. Maps on the right show zoomed part of the given map from the left. Please note different scales of right insets. The most representative results for C-Laurdan during excitation, with molecule located at the most probable distances 12, 13 and 14 Å from bilayer center, are presented in Figure 6a, 6b and


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6c, respectively. For C-Laurdan located at 12 Å from bilayer center (Figure 6a), two complete cycles of water reorganization are determined after 5 and 10 ns of excitation. Zoom into very short time scales less than 150 ps allows detecting a third, very fast cycle, which is shorter than 50 ps. The water rearrangement takes place mainly at distances up to 10 Å from the center of chromophore. The most probable position of C-Laurdan in the bilayer was found about 13 Å from membrane center. Surprisingly, the water reorganization around the dye located at that particular position during excitation is very extensive and prolonged up to 10 ns after excitation (see Figure 6b). The range of influence exceeds also the distance of 15 Å from the chromophore center. Very first water rearrangement cycle is found during first 10 ps after excitation. Second and third water reorganizations occur around 0.3 - 0.9 ns and 1.1 - 1.5 ns after excitation. Finally, for C-Laurdan, which was initially located at 14 Å from the bilayer center, only two water reorganization changes are detected (see Figure 6c). The first and the strongest water response is observed up to 9 ps after excitation. The second one is occurring in the next 25 ps, and any other influence is recorded. The impact of the change in the dye dipole moment upon excitation reaches the distance of 6.5 Å from the chromophore center (first reorganization) and only 5 Å for second water rearrangement. We are aware that only repeatedly performed simulations could give the quantitative view on occurring phenomena. However, these examples show how sensible is SR method, and C-Laurdan probe in particular, to the presence of solvent molecules. Extremely small changes in chromophore location of only 1 Å within the membrane can drastically change the water response, thus perturbing the experimental results. Analyzing all 36 short MD runs with different initial locations of C-Laurdan molecule allows us to find between two and four relaxation time constants, as reported in Table 2. The fastest rearrangement cycle occurs within the first 50 ps after excitation and may be attributed to the energy dissipation by


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rotations of water molecules. This relaxation time is hard to confirm experimentally due to lower time resolution of usually used equipments. The next relaxation cycle occurs during the first nanosecond and could be related with additional water translations. The last relaxation times (one or two) arise at later times. We suppose that they are induced by lipids reorganization after the probe excitation, causing subsequent impacts on water molecules.17 Even if our data cannot directly support this explanation of physical meaning of relaxation times, it is consistent with such an interpretation.

Table 2 Ranges of relaxation time constants (τi) of C-Laurdan in DOPC bilayer model gathered across multiple simulations. τ1 [ns]

τ2 [ns]

τ3 [ns]

τ4 [ns]

0.01 – 0.03

0.1 – 0.9

2.0 – 10.0

7 – more than 10 ns

CONCLUSIONS The combination of Quantum Calculations (QM) and Molecular Dynamics (MD) simulations was used to characterize two fluorescent probes Laurdan and C-Laurdan in the DOPC bilayer. QM calculations allowed obtaining the necessary parameters for modelling these dyes in ground and excited states. Passive diffusion of the markers from bulk water to the membrane, and their equilibrium positions, were recorded in MD. The analysis of the chromophore orientation in the lipid membrane shows that the presence of carboxyl group in C-Laurdan causes more parallel orientation in comparison to Laurdan. The stability of the dye position in the lipid bilayer, which is a crucial parameter for fluorescent solvent relaxation experiments, plays in favor of CLaurdan. Using a statistical approach of effective position after excitation, and taking into


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account the probes lifetime, we determined that Laurdan upon excitation should be find at about 12 Å from bilayer center, while C-Laurdan is located more outside at around 13 Å. This method shows also smaller shift of the excited marker toward the more polar regions of lipid bilayer, comparing to the previously proposed analysis. Moreover, our results show correct solvent relaxation time scale, which is in the range of nanoseconds for probe embedded into membrane. We believe that this work confirms a hypothesis about usefulness of the newly synthetized CLaurdan molecule for studies of the lipid headgroup region employing fluorescent solvent relaxation approach. However, we are aware that to clearly highlight the superiority of CLaurdan in solvent relaxation technique, the study must be confirmed by experiments. This is actually in progress.


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AUTHOR INFORMATION

Corresponding Author *Email: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Computations have been performed on the supercomputer facilities of the Mésocentre de calcul de Franche-Comté.

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Will C-Laurdan Dethrone Laurdan in Fluorescent Solvent Relaxation

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