Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study

Biochimica et Biophysica Acta 1858 (2016) 2647–2661

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Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study António M.T.M. do Canto a, João R. Robalo a,b, Patrícia D. Santos a, Alfredo J. Palace Carvalho a, J.P. Prates Ramalho a, Luís M.S. Loura c,d,⁎ a

Centro de Química de Évora e Departamento de Química, Escola de Ciências e Tecnologia, Colégio Luís Verney, Rua Romão Ramalho 59, P-7002-554 Évora, Portugal Theory and Bio-Systems Department, Max Planck Institute of Colloids and Interfaces, Wissenschaftspark Golm, D-14424 Potsdam, Germany Faculdade de Farmácia, Universidade de Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, P-3000-548 Coimbra, Portugal d Centro de Química de Coimbra, Largo D. Dinis, Rua Larga, P-3004-535 Coimbra, Portugal b c

a r t i c l e

i n f o

Article history: Received 21 March 2016 Received in revised form 5 July 2016 Accepted 25 July 2016 Available online 27 July 2016 Keywords: Diphenylhexatriene Fluorescence Lipid bilayer Membrane probe Molecular dynamics simulation

a b s t r a c t Fluorescence spectroscopy and microscopy have been utilized as tools in membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Among the latter, 1,6-diphenylhexatriene (DPH) and its trimethylammonium derivative (TMA-DPH) have been extensively used. It is widely believed that, owing to its additional charged group, TMA-DPH is anchored at the lipid/water interface and reports on a bilayer region that is distinct from that of the hydrophobic DPH. In this study, we employ atomistic MD simulations to characterize the behavior of DPH and TMA-DPH in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/cholesterol (4:1) bilayers. We show that although the dynamics of TMA-DPH in these membranes is noticeably more hindered than that of DPH, the location of the average fluorophore of TMA-DPH is only ~3–4 Å more shallow than that of DPH. The hindrance observed in the translational and rotational motions of TMA-DPH compared to DPH is mainly not due to significant differences in depth, but to the favorable electrostatic interactions of the former with electronegative lipid atoms instead. By revealing detailed insights on the behavior of these two probes, our results are useful both in the interpretation of past work and in the planning of future experiments using them as membrane reporters. © 2016 Elsevier B.V. All rights reserved.

1. Introduction All processes of biological relevance involve cell membranes at some point, and therefore phospholipid bilayers, as the fundamental constituents of membranes in all cells, have been a central topic object of study in life sciences [1]. Detailed characterization of lipid bilayer organization and dynamics requires the use of versatile and sensitive techniques, capable of probing a wide range of spatial and temporal scales. In this regard, fluorescence-based methodologies, by offering an array of parameters (fluorescence spectra, lifetimes, anisotropy, quenching, Förster resonance energy transfer (FRET)) and spectroscopic and microscopic approaches (steady-state and time-resolved fluorescence spectroscopy, fluorescence correlation spectroscopy, and an ever-increasing number of optical microscopic techniques, some of which with resolution under the diffraction limit), are on the whole one of the most powerful tools available for the study of membranes [2,3]. However, an unavoidable issue when applying fluorescence to ⁎ Corresponding author at: Faculdade de Farmácia, Universidade de Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, P-3000-548 Coimbra, Portugal. E-mail address: [email protected] (L.M.S. Loura).

http://dx.doi.org/10.1016/j.bbamem.2016.07.013 0005-2736/© 2016 Elsevier B.V. All rights reserved.

the study of lipid bilayers is that the vast majority of lipids are themselves nonfluorescent. For this reason, a large number of lipophilic fluorescent probes have been designed and are commercialized [4,5]. 1,6-diphenylhexatriene (DPH; Fig. 1A) is one of most widely used fluorescent membrane probes. It is a rod-shaped molecule whose fluorescence quantum yield and intensity decay show little sensitivity to the lipid phase, unlike the fluorescence anisotropy, which decreases threefold upon melting of the lipid acyl chains. As a consequence, both DPH and its trimethylamino-derivative, TMA-DPH (Fig. 1B) have been commonly used as probes of membrane fluidity [6,7]. While DPH is mostly employed as a reporter of the highly disordered hydrophobic core of the bilayer, TMA-DPH was synthesized [8,9] with the intention of probing the more ordered shallow regions of the bilayer (glycerol backbone and upper segments of the phospholipid acyl chains), because of the cationic group which purposely acts as an anchor to the water/ bilayer interface. In the absence of direct detailed information at the atomic level regarding these probes' behavior, several fluorescence techniques were used to obtain information about their location and dynamics when inserted in membranes. Anisotropy decays of bilayer-inserted DPH and TMA-DPH have been analyzed to several degrees of model

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A

B

13

8 4

50

52

C

24

27

21

13

D

6

Fig. 1. Structures and numbering of atoms mentioned in the text for DPH (A), TMA-DPH (B), POPC (C) and cholesterol (D). Numbering of DPH atoms follows that of TMA-DPH for the corresponding atoms.

sophistication. Engel and Prendergast [10] observed a significantly higher value of long-time residual anisotropy for TMA-DPH compared to DPH in fluid bilayers, denoting a much more restricted degree of rotational motion for the former, consistent with its location in a more ordered environment. Bimodal angular distributions of the long molecular axis, with a maximum parallel and the other perpendicular (in the bilayer midplane, between the two monolayer leaflets) to the bilayer normal, have been proposed for DPH, but not for TMA-DPH, whose depolarization could be explained without invoking a population of probe with orientation along the bilayer plane [11–13]. On the other hand, quenching of DPH and TMA-DPH fluorescence by a series of nitroxidelabeled lipids was used to estimate the depth of fluorophore location in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) fluid vesicles, in absence and presence of cholesterol, using the parallax method [14]. According to expectations, it was verified that TMA-DPH locates at a larger distance to the bilayer center than DPH (1.09 nm vs. 0.78 nm, respectively) in DOPC vesicles, although to a surprisingly small extent. Addition of cholesterol does not lead to large changes in probe location (1.07 nm and 0.67 nm for TMA-DPH and DPH, respectively). It must be mentioned that these experimental data, valuable as they undoubtedly are, are subject to considerable uncertainty. Namely, the recovery of angular distributions from anisotropy decays is an illpoised inversion problem, prone to be affected by the high level of noise commonly obtained even in the most careful anisotropy decay measurements. In fact, it was demonstrated that because one recovers at most the first three coefficients of the Legendre polynomial series expansion of the orientation distribution function from anisotropy decays, truncation errors may lead to important quantitative disagreement between the actual and recovered orientation distributions [15]. On the other hand, the parallax method for analysis of differential

quenching relies on severe assumptions, as it adapts concepts related to the three-dimensional quenching sphere-of-action model to a twodimensional space and relies on a non-diffusive (‘static’) approach [16, 17]. Notably, the quenching experiments are carried out in presence of a large concentration of quencher lipid (15 mol% of total phospholipid [14]) which certainly affects the host bilayer properties. Even though the probe concentration is usually much lower, the possibility of probe-induced membrane perturbation and resulting biasing of interpretation of the probe fluorescence results is always a reason for concern. For these reasons, a methodology able to simultaneously monitor the probe and lipid molecules independently, and therefore at the same time give information on probe behavior and host bilayer perturbation, would provide a much needed complement to the often complex and indirect interpretation of raw fluorescent data. In recent years, molecular dynamics (MD) simulations have emerged as such a tool to help characterize the behavior of fluorescent membrane probes, able to fill in the gaps left by the incomplete and indirect fluorescence measurements and also provide new insights, with atomic-level detailed information [18–20]. Probably due to its importance, DPH was the first probe to be studied in this way. In a pioneering early report [21], a 72-molecule 1,2-dipalmitoyl-sn-3-glycerophosphocholine (DPPC) bilayer was simulated in the presence of either one or three DPH molecules. Its results challenged the picture of two perpendicularly oriented probe populations, as it was observed that the angle between the long molecular axis and the bilayer normal rarely was N 60°, and no in-plane perpendicular orientations were detected in a 72-molecule fluid 1,2dipalmitoyl-sn-3-glycerophosphocholine (DPPC) bilayer. However, this work was severely restricted by the extremely short time-scale probed (≈250 ps for analysis). A more definite study, employing larger (128molecule) DPPC fluid bilayers, simulated for 50 ns, revealed broad angular

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distributions of the DPH long axis, and despite a peak being observed for f(θ)sin(θ) (where f is the orientation distribution function) at θ ≈ 25°, significant distribution also occurs around θ ≈ 90° [22]. However, perpendicular orientations do not correlate with center of mass location near the bilayer center. The average center of mass transverse position was determined to lie at 0.75 nm from the bilayer center, in agreement with the aforementioned fluorescence quenching data. Dynamic properties of DPH, such as lateral diffusion and rotational mobility, were also addressed. A subsequent work [23] focused on the effects of DPH on the properties of the host bilayer, concluding that even though significant local ordering effects are observed, DPH has only a small overall perturbing effect on DPPC fluid bilayers. A later report by the same group [15] extended this characterization to DPPC/cholesterol mixtures, revealing that the presence of the latter affects the location and orientation of bilayer-inserted DPH slightly, and that for 20 mol% of cholesterol, the well-known ordering effect of this component is so dominant that the additional role of DPH becomes almost negligible. More recently, in a study of DPH in unsaturated DOPC bilayers, a representative population of DPH molecules was intriguingly found in the acyl-chain region of the bilayer immediately beneath the DOPC/water interface, with a tilt angle of near 90° relative to the bilayer normal [24]. No such orientations were found for molecules close to the bilayer center, however. In contrast with DPH, to our knowledge a sole MD study has been reported for TMA-DPH in fluid DPPC [25], only covering 2 ns of simulation (of which 1 ns was taken as system equilibration). The simulations focused in the atomic positions of the end nitrogen and hydrogen atoms, and, for example, revealed no significant differences in the positions of the H atoms closest to the bilayer center for DPH (0.42 ± 0.35 nm relative to the center) and TMA-DPH (0.28 ± 0.19 nm). The slightly more interior position in TMA-DPH could be viewed as surprising, but the exceedingly small simulation time, together with the scarcity of parameterization details presented in the published work, limit considerably the degree of certainty associated with this observation. In this work, we present extensive (200–400 ns) simulations of bilayers of 1-palmitoyl,2-oleoyl-sn-3-glycerophosphocholine (POPC), both pure and with 20 mol% cholesterol, with inserted TMA-DPH and DPH. Our analysis encompasses both probe (location, orientation, dynamics) and bilayer (area/lipid, thickness, order) properties. Because what is known of the behavior of TMA-DPH is mainly based in interpretation of complex fluorescence results, as well as general intuition, the main motivation for this paper is a complete characterization of this probe inserted in lipid bilayers, using the unique features of MD simulations. DPH is simulated for validation (comparison with literature reports) and to provide a direct comparison and assessment of TMA-DPH differences. 2. Methods MD simulations and analysis of trajectories was carried out using the GROMACS 4.6.3 package [26,27]. The SPC water model was used [28]. For the main set of simulations, the topology of the POPC molecule consisted of an united united-atom description for CH, CH2, and CH3 groups, based on the parameters presented by Berger et al. [29] for 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), adapted to take into account the changed parameters for the double bond in the sn-2 acyl chain, as described elsewhere [30,31]. The unitedatom structure and topology of cholesterol were adapted from those of Höltje et al. [32] (available for download at the GROMACS webpage, http://www.gromacs.org/@api/deki/files/29/=cholesterol.tgz) by changing the atom types from CH2/CH3 to LP2/LP3 to avoid overcondensation of the bilayer, as previously suggested [33,34] and validated by ourselves in previous studies [35,36]. The starting structures were POPC (128 molecules) or POPC:20 mol% cholesterol (96 POPC:24 cholesterol) bilayers [37], fully hydrated with N40 water molecules per phospholipid. For DPH and TMA-DPH, a preliminary topology was obtained using the PRODRG server [38]. The PRODRG output partial atom charges

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were then replaced with values calculated from the optimized geometries obtained from quantum chemical calculations performed at Hartree-Fock level with the 6-31G(d) basis set, followed by a leastsquares fit to the electrostatic potential obtained at the same theory level, according to the Kollman and Singh scheme [39,40]. All quantum calculations were performed using the GAMESS-US package [41,42]. Fig. 1 shows structures and numbering of relevant atoms of the DPH, TMADPH, POPC and cholesterol molecules, while the charges adopted for the probes are displayed in Table 1 (for DPH, the charges agree closely with the previously reported values of Repáková et al. [22] and Hurjui et al. [24]). Bilayers containing probe molecules (either 1 or 2 in each leaflet) were obtained by random insertion inside the bilayer without replacement of phospholipids or cholesterol, with orientation roughly aligned with the bilayer normal. For TMA-DPH, probe molecules were inserted with the charged trimethylamino group pointing to the interface, and a corresponding number of Cl− ions was randomly added to the aqueous medium to ensure electroneutrality. In all systems, unfavorable atomic contacts were removed by steepest descent energy minimization. For each system, a short (100 ps) MD run was then carried out using a 1 fs integration step, followed by a full 200 ns or 400 ns run (for pure POPC or POPC/cholesterol bilayers, respectively) using a 2 fs integration step. Bond lengths were constrained to their equilibrium values, using the SETTLE algorithm [43] for water and the LINCS algorithm [44] for all other bonds. All simulations were carried out under constant number of particles, pressure (1 bar) and temperature (300 K), and with periodic boundary conditions. Pressure and temperature control were carried out using the weak-coupling Berendsen schemes [45], with coupling times of 1.0 ps and 0.1 ps, respectively. Semiisotropic pressure coupling was used. Van der Waals and the real space component of Coulomb interactions were cut-off at 1.0 nm, whereas for long-range electrostatics the Particle Mesh Ewald treatment [46] was applied. The first 50 ns of each simulation in POPC and 100 ns of each simulation in POPC/20 mol% cholesterol were used for equilibration, and the remaining 150 ns (in POPC) or 300 ns (in POPC/cholesterol) were used for analysis. For the sake of verification of the robustness of the results obtained with these simulations, two additional sets of simulations of probefree systems and of bilayers with four inserted probe molecules were carried out. One of them used the same force field description described above, with addition of salt (equal number of sodium and chloride ions) to a physiological ionic strength value (150 mM). In the other set, we employed the all-atom Slipids forcefield for POPC and cholesterol [47–49] together with probe parameters obtained using Amber Tools [50–52] and refined with atomic charges calculated according to the Slipids protocol. Briefly, geometry optimization was performed with the B3LYP functional [53–56] with the cc-pVTZ basis set [57]. The partial charges were then obtained at the same theory level using the restrained electrostatic potential (RESP) method [58]. The solvent was taken into account by the SMD model [59] using the integralequation-formalism polarizable continuum model (IEFPCM). A polarizable medium with ε = 2.04 was considered to model the bilayer environment. We verified that replacement of this medium with a dielectric continuum with ε = 78.4 led to only very minor differences in the calculated charges (e.g., 0.008 and 0.012 in the TMA-DPH nitrogen and methyl atom charges, respectively). The main results of these two sets of simulations are included in the Supplemental Material file, and commented here at the end of the Results and Discussion section. Areas per phospholipid were obtained dividing the instant box area by the number of POPC molecules in each bilayer leaflet (64 and 48 in the absence and in the presence of cholesterol, respectively). Bilayer thickness was defined as the average distance between the POPC P atoms in opposing leaflets. Deuterium order parameters, SCD, were calculated using 
  SCD ¼ 3 cos2 θ −1 =2

ð1Þ

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Table 1 - DPH and TMA-DPH atomic charges. See Fig. 1 for numbering. Atom

DPH TMA-DPH

1

2

3

4

5

6

7

8

9

10

11

–0.098 –0.087

0.020 0.039

–0.030 –0.010

0.020 0.039

–0.098 –0.087

0.270 0.253

–0.110 –0.094

0.046 –0.001

–0.020 0.095

–0.020 –0.087

0.046 0.152

Atom

DPH TMA-DPH

12

13

14

15

16

17

18

19

20

21

22

–0.110 –0.215

0.270 0.312

–0.098 –0.040

0.020 –0.044

–0.030 0.159

0.020 –0.044

–0.098 –0.040

– 0.175

– 0.175

– 0.175

– 0.175

where θ is the angle between a C\\D bond and the bilayer normal, and the brackets denote averaging over time and C\\D bonds [60]. In the simulations using a united atom force field, deuterium positions of acyl chain atoms were constructed from the neighboring carbons assuming ideal geometries. -SCD can vary between 0.5 (full order along the bilayer normal) and − 0.25 (full order along the bilayer plane), whereas SCD = 0 denotes isotropic orientation. Lateral diffusion coefficients Dlat were calculated from the twodimensional mean squared displacement (MSD), using the Einstein relation 1 dMSDðt Þ D ¼ lim 4 t→∞ dt

ð2Þ

In turn, MSD is defined by D 2 E ! ! MSDðt Þ ¼  r i ðt þ t 0 Þ− r i ðt 0 Þ

ð3Þ

! where r i is the (x, y) position of the center of mass of molecule i of a given species, and the averaging is carried out over all molecules of this kind and time origins t0. To eliminate noise due to fluctuations in the center of mass of each monolayer, all MSD analyses were carried out using trajectories with fixed center of mass of one of the monolayers, and the final result is averaged over the two leaflets. The electrostatic potential across the bilayer (z coordinate) was calculated by double integration of the charge density:

ψðzÞ−ψð∞Þ ¼ −

1 ε0

Z∞ z

dz0

Z∞

ρðz00 Þdz00

ð4Þ

z0

For this calculation (as well as for mass density profiles), and because the bilayers' centers of mass may fluctuate in time, the positions of all atoms were determined relative to the instantaneous center of mass (z = 0) in all simulations, for each frame. The potential was also averaged and symmetrized for the two bilayer leaflets [61]. Area compressibility moduli, KA, were calculated from the fluctuations of the area per lipid, a [62]: KA ¼

2hai βnL σ 2a

ð5Þ

In this equation, β = 1/kT (where k is the Boltzmann constant), nL is the number of phospholipid molecules, and σ2a is the variance associated with a. Local pressure profiles across the z axis were calculated using a modified version of GROMACS 4.5.5 designed especially for this purpose [63, 64]. Briefly, new trajectories were constructed based on the original ones, but with the bilayer centered in the simulation box. The modified version of GROMACS was then used to reanalyze this centered trajectory and the stress tensors were calculated.

Unless stated otherwise, error estimates from MD data were obtained using the block method described by Flyvbjerg and Petersen [65]. For visualization of structures and trajectories, Visual Molecular Dynamics software (University of Illinois) was used [66].

3. Results and discussion 3.1. Structural and conformational properties of the host lipids Fig. S1 shows time traces of area per lipid, which illustrate that the systems are equilibrated early on the course of the simulations. Averages of the molecular areas are given in Table 2. The value obtained for pure POPC (0.661 nm2) is in good accordance with both experimental (0.65 nm2, T = 298 K [67]; 0.64 nm2, T = 298 K [68]; and 0.68 nm2, T = 303 K [69]) and simulation (0.655 nm2, T = 300 K [70]; 0.68 nm2, T = 310 K [71]) reported data. The ordering effect of cholesterol induces a reduction in the calculated average area per POPC, which is 0.622 nm2 in the absence of probe. Inclusion of probes generally leads to very modest changes in lipid molecular areas, within the estimated uncertainty in all cases. If anything, a very slight probe concentration-dependent reduction is observed in the molecular area in the absence of cholesterol, pointing to a small ordering effect induced by both probes. An opposite effect is apparent in the POPC/cholesterol/DPH simulations. Such small variations (but in the opposite direction, as expected) are also apparent in the bilayer thickness values (Table 2). Although individual differences are not statistically significant, several trends can be observed. Bilayer thickness increases upon insertion of both probes in POPC, decreases upon insertion of DPH in POPC/20 mol% cholesterol, and is unaffected by the presence of TMA-DPH in the latter lipid system. These trends can also be observed in the average atomic positions (bzN), relative to the bilayer center, depicted in Fig. 2. Upon probe insertion and in the absence of cholesterol, the transverse positions of POPC headgroup (N4, P8), glycerol (C13) and middle-acyl chain (C24) atoms are, in average, located further from the bilayer center (higher z) than in the pure phospholipid system. For the systems with 20 mol% cholesterol, this is also verified for TMA-DPH (albeit very slightly), but not for DPH. However, in all cases, the differences relative to the probe-free bilayers are within the statistical uncertainty, confirming the mild effects of both compounds on the bilayer structure. An exception is the position of the O6 (and to a minor extent, also C13) atom of cholesterol, which is significantly more internal in the POPC/cholesterol +4 DPH system. For this system, the most internal atoms (C21 and C27) are not affected, implying that DPH affects the orientation of cholesterol in this system. The average sterol tilt is increased, possibly reflecting a decreased membrane order. The latter can be addressed more directly by calculating deuterium order parameter (SCD) profiles along the POPC acyl chains, as shown in Fig. 3. The profiles for the probe-free systems obtained agreed with both experimental (e.g., [34,72,73]) and simulated (e.g., [34,70,71,74–76]) reported data. Incorporation of probe leads to minor absolute variations

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Table 2 Areas per phospholipid (a), bilayer thickness (BT), and average POPC P8-N4 axis tilt in all studied systems. POPC

No probe +2 DPH +4DPH +2 TMA-DPH +4 TMA-DPH

POPC/20 mol% cholesterol

a/nm2

BT/nm

Avg. P-N tilt/deg

a/nm2

BT/nm

Avg. P-N tilt/deg

0.661 ± 0.017 0.661 ± 0.017 0.658 ± 0.017 0.658 ± 0.017 0.655 ± 0.016

3.66 ± 0.09 3.67 ± 0.09 3.70 ± 0.09 3.68 ± 0.09 3.71 ± 0.09

76.8 ± 1.5 76.5 ± 1.6 77.0 ± 1.5 75.7 ± 1.6 74.4 ± 1.6

0.622 ± 0.015 0.630 ± 0.015 0.635 ± 0.016 0.621 ± 0.015 0.625 ± 0.015

4.28 ± 0.12 4.26 ± 0.11 4.24 ± 0.10 4.30 ± 0.10 4.29 ± 0.10

78.2 ± 2.8 78.4 ± 2.5 78.2 ± 2.5 76.9 ± 2.8 75.4 ± 2.5

(b 0.016 in all cases), which are difficult to appreciate in the profiles of Fig. 3A and B. However, considering now the relative changes in SCD (taking the probe-free system as reference), clear patterns are identified. In the absence of cholesterol (Fig. 3C), both DPH and TMA-DPH display a concentration-dependent ordering effect: while changes in SCD do not exceed 5% for the 2 probe:128 lipid systems, variations between 5% and 10% were observed in the order parameters of the 4 probe:128 lipid bilayers. For equal probe concentration, the ordering effect of TMA-DPH is slightly stronger than that of DPH. On the other hand, in the presence of 20 mol% cholesterol, the qualitative effects induced by the two probes are distinct: TMA-DPH incorporation leads to a slight | SCD | increase, while the opposite holds for DPH (Fig. 3D). For both probes and probe:lipid ratios, relative variations do not exceed 5% in these ordered systems. We also investigated whether DPH and TMA-DPH caused perturbations in the headgroup region of the membrane systems. For this purpose, we calculated the tilt of the P8-N4 axis relative to the bilayer normal (Table 2). While DPH insertion does not affect the orientation of the phospholipid headgroup, TMA-DPH causes a probe concentration-dependent decrease in the average tilt angle. Whereas this slight change lies within the estimated uncertainty, the probability density functions show a noticeable displacement to lower tilts (i.e. more upright headgroup orientations), as shown in Fig. S2. To avoid unfavorable interaction with the positive charge of the trimethylammonium group of TMA-DPH (which has a shallow location within the bilayer, see Fig. 2 and following subsection), the choline groups of nearby POPC molecules surge towards the water phase, thus reducing the P-N tilt relative to the bilayer normal. We carried out calculations of additional bilayer properties (electrostatic and lateral pressure profiles, and isothermal area compressibility

moduli KA), which are shown in the Supplemental Material file, Figs. S3–S5. From the electrostatic profiles (Fig. S3), it is apparent that DPH-induced changes in the profiles are minimal (taking into account that the probe/lipid ratios, 4 DPH:128 POPC and 4 DPH:96 POPC:24 cholesterol, are relatively high). Given that DPH is an apolar probe, with low individual atomic charges, this slight effect is most probably the result of the probe-induced bilayer condensation, which increases charge densities along the membrane plane and thus the potential difference between the center of the membrane and bulk water. In the TMA-DPHcontaining systems, this effect is compounded by the changes in transverse charge distributions arising from the probes, leading to a larger increase in membrane potential relative to bulk water. Fig. S4 shows the values of KA calculated for all systems. The value obtained for pure POPC, (299 ± 41) mN/m, agrees with the experimental range reported by Binder and Gawrisch [77] ((180–330) mN/m), as well as the values obtained by Rawicz et al. [78] for a series of related phosphatidylcholines (in the 210–270 mN/m range). It appears that inclusion of probe (as well as 20 mol% cholesterol) generally leads to increased KA values, consistent with the observed effects on area per lipid and order parameters. Nevertheless, because the large relative uncertainties (stemming mostly from the uncertainty associated with σ2a), the confidence intervals of the systems without and with inserted probes (with the sole marginal exception of POPC/20 mol% Cholesterol/4 DPH) generally overlap, implying that the variations are not significant. Lateral pressure profiles, shown in Fig. S5, display relatively minor changes for the systems with inserted probes relative to those in the absence of probe. The profile for pure POPC agrees very well with other simulation studies [71,79]. On the whole (with the possible exception of the electrostatic profiles in the presence of TMA-DPH, namely in the presence of cholesterol), the extent of probe-induced perturbation of host lipid properties

POPC

Chol

Probe

/nm 2.0

1.5

1.0

0.5

0.0 N4

P8 POPC

C13 +2DPH

C24 +4DPH

POPC bilayers

C50 +2TMA

C52 +4TMA

O6 POPC/Chol

C13 +2DPH

C21

C27

+4DPH

COM +2TMA

N (TMA)

+4TMA

POPC/Chol bilayers

Fig. 2. POPC (N4-C52), cholesterol (Chol; O6-C27) and probe (center of mass of ring system (COM), TMA-DPH N19) average transverse atomic positions bzN, relative to the bilayer center.

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A

B

C

D

0.25 0.20 0.15 0.10 0.05 0.00 Δ|S CD | 10%

5%

0%

-5% 0

2

4

No probe

6

8

10

12

14 atom

+2DPH

0

2

4

6

+4DPH

8

10

12

+2TMA

14

atom

+4TMA

Fig. 3. Top: deuterium order parameter (|SCD |) profiles of the POPC sn-1 acyl chain in the absence (A) and in the presence of 20 mol% cholesterol (B). Bottom: changes Δ|SCD | in the deuterium order parameter values in the absence (A) and in the presence of 20 mol% cholesterol (B), relative to the respective values in the absence of probe.

is modest, given the relatively high concentrations of DPH and TMADPH in our simulations (1.5–3.5 mol%). This finding is in line with the work of Franová, Repáková and coworkers [15,23] who found small overall DPH-induced effects on both DPPC and DPPC/20 mol% cholesterol fluid bilayers, including no appreciable changes in the experimentally measured main transition temperature and enthalpy. Indeed, the fact that DPH and TMA-DPH report correctly on phospholipid phase transitions (as verified in numerous historical works, and more recently by Nelson et al. in DPPC membranes [25]) agrees with the lack of major disturbance of membrane properties. In comparison, other probes, such as NBD-PC, produce much more pronounced changes, including considerable broadening of DPPC phase transition for probe concentrations similar to those addressed here [80].

3.2. Location and conformation of DPH and TMA-DPH The different effects of DPH and TMA-DPH on the POPC headgroup orientation could possibly result from differences in the location of the two probes. From simple visual inspection of simulation snapshots (Fig. S6), it is apparent that both DPH and TMA-DPH are positioned inside one of the bilayer leaflets, with orientation mostly aligned with the bilayer normal. These visual indications are confirmed in the calculations, which show that the geometrical center of the DPH fluorophore (defined as midpoint of the C3-C16 axis, see Fig. 1) is located inside the hydrocarbon core region of the bilayer for both probes. Our results agree with the experimental results of Kaiser and London [14], in that the DPH fluorophore is located deeper in the phospholipid bilayer (bz N = 0.68 nm in our POPC simulations, 0.78 nm in Kaiser and London's experiments in DOPC) than that of TMA-DPH ((bz N = 0.90 nm in our POPC simulations, 1.09 nm in Kaiser and London's experiments in DOPC), albeit by a relatively small extent (0.22 nm in our simulations, 0.31 nm in Kaiser and London's experiments). Similar comments could be made regarding our simulations in POPC/20 mol% cholesterol (bz N = 0.82 nm and 1.09 nm for DPH and TMA-DPH, respectively) and Kaiser and London's experiments in DOPC/33 mol% cholesterol (bz N = 0.67 nm and 1.07 nm for DPH and TMA-DPH, respectively). Our larger b z N in POPC/cholesterol only reflects the increased order and bilayer thickness of this bilayer, and is not indicative of a more external probe location. For each lipid system, the difference between the location of the two probes, though small, was still significant (see error bars in Fig. 2), at variance with the data of Nelson et al. [25].

As clarified in our work, the small magnitude of vertical fluorophore displacement following attachment of the trimethylammonium moiety in TMA-DPH stems from the fact that the TMA nitrogen atom is positioned near the glycerol region of the bilayer (~ 1.5 nm in POPC, ~1.8 nm in POPC/20 mol% cholesterol), much deeper than the location of the POPC choline nitrogen (~ 1.9 nm in POPC, ~ 2.2 nm in POPC/ 20 mol% cholesterol). Consequently, and in agreement with Kaiser and London's discussion [14], the TMA group only manages to pull the DPH fluorophore towards the interface by a small extent, and the two probes report essentially the same region in the bilayer. The transverse location of the TMA group lies between those of the POPC phosphate (shallower than TMA) and POPC ester/POPC carbonyl/cholesterol hydroxyl (deeper than TMA) electronegative oxygen atoms. Radial distribution functions of these atoms (Fig. 4) reveal that they are often found in close vicinity of the TMA group (unlike water oxygen atoms, because of the limited water penetration in this region of the bilayer), helping to stabilize the positive charge of the latter.

12

Ester/carbonyl Phosphate

A

g (r )

Water

8

4

0 12

Ester/carbonyl Phosphate

B

g (r )

Water

8

Cholesterol 4

0 0.0

0.5

1.0

1.5

2.0 r /nm

Fig. 4. Radial distribution functions g(r) of different groups of oxygen atoms around the N19 atom of TMA-DPH, averaged for all probe molecules in both simulations (2- and 4inserted probes) for (A) POPC and (B) POPC/20 mol% cholesterol.

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ρ/kgm 1000

A

C

B

D

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800 600 400 200 0 -3 ρ/kgm 1000 800 600 400 200 0 0.0

1.0

z /nm 0.0

2.0

POPC

Water

1.0

2.0

Cholesterol

3.0

z /nm

Probe x 20

Fig. 5. Mass density profiles across the bilayer normal direction for the POPC +4 DPH (A), POPC +4 TMA-DPH (B), POPC/20 mol% cholesterol +4 DPH (C) and POPC/20 mol% cholesterol +4 TMA-DPH (D) systems.

DPH

TMA-DPH

z /nm 1.5 0.5 -0.5 -1.5 -2.5 z /nm 1.5 0.5 -0.5 -1.5 -2.5 z /nm

1.5 0.5 -0.5 -1.5 -2.5 z /nm 1.5 0.5 -0.5 -1.5 -2.5 0

50

100

150

t /ns 0

50

100

150

t /ns

Fig. 6. Time traces of the instant position of atoms C3 (red) and C16 (blue) relative to the bilayer center, for the 4 individual molecules in the POPC +4 DPH (left panels) and POPC +4 TMADPH (right panels) simulations. The dotted vertical lines indicate the beginning of the time range used for analysis (t N 50 ns).

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Another view into the location of membrane-inserted DPH and TMA-DPH is provided by mass density plots (Fig. 5). The slightly more external location of the latter is apparent, which leads to an increased exposure of the fluorophore to water. This can be quantified by the average water density sensed by the fluorophore b ρ(water)Nfluorophore (taken as the whole molecule for DPH, and atoms 1–16 in Fig. 1 for TMA-DPH), calculated according to [81] Z ρfluorophore ðzÞρwater ðzÞdz Z ρfluorophore ðzÞdz

h ρðwaterÞifluorophore ¼

ð6Þ

The values obtained, 55.4 kg/m3 for POPC +4 TMA-DPH (compared to 24.7 kg/m3 for the POPC +4 DPH system), and 28.7 kg/m3 for POPC/ 20 mol% cholesterol +4 TMA-DPH (compared to 8.4 kg/m3 for the POPC + 4 DPH system), are all low in absolute terms, reflecting the deep location of both probes in the studied membrane systems. Another noticeable difference between the density profiles of DPH and TMA-DPH across the bilayers is the higher probability of finding the former near the bilayer center (z = 0). This observation led us to investigate the possibility of probe translocation between the bilayer leaflets. Figs. 6 and 7 depict the time traces of the instant transverse locations of the opposite C atoms of the probes' fluorophores (C3 and

C16 in Fig. 1) for the simulations with 4 inserted molecules, in POPC and POPC/20 mol% cholesterol, respectively. From these figures, it is clear that DPH is able to change its bilayer leaflet during the timescale of our simulations. These hopping events were much more frequent in the disordered POPC bilayer (Fig. 6). On average, each DPH molecule translocates once every 50 ns in this system (18 events in 900 ns, taking into account the analysis time range of both POPC +2DPH and POPC +4DPH trajectories). These results agree qualitatively with those of Repáková et al. [22] in their simulations of DPH in fluid DPPC, where a total of 4 changes of leaflet were observed in 400 ns of simulation, i.e. one translocation event every 100 ns. In ordered POPC/20 mol% cholesterol, translocations were rare. Only 4 events were observed in 1800 ns, taking into account the analysis time range of both POPC/20 mol% cholesterol +2 DPH and POPC/20 mol% cholesterol +4 DPH trajectories. This suggests that translocation is slower in this system by close to an order of magnitude compared to pure POPC. In the DPPC/20 mol% cholesterol simulation study of Franová et al. [15], no changes of leaflet were reported, possibly due to the shorter trajectories (60 ns). No hopping events were observed for TMA-DPH in any of our simulations. It appears that the presence of the charged trimethylammonium group prevents the occurrence of translocation in a time scale of, at the very least, hundreds of nanoseconds, in agreement with the behavior described by Lentz [7], according to whom no translocation of TMA-

DPH

TMA-DPH

z /nm 1.5 0.5 -0.5 -1.5 -2.5 z /nm

1.5 0.5 -0.5 -1.5 -2.5 z /nm

1.5 0.5 -0.5 -1.5 -2.5 z /nm 1.5 0.5 -0.5 -1.5 -2.5 0

100

200

300

t /ns 0

100

200

300

t /ns

Fig. 7. Time traces of the instant position of atoms C3 (red) and C16 (blue) relative to the bilayer center, for the 4 individual molecules in the POPC/20 mol% cholesterol +4 DPH (left panels) and POPC/20 mol% cholesterol +4 TMA-DPH (right panels) simulations. The dotted vertical lines indicate the beginning of the time range used for analysis (t N 100 ns).

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et al. [24] reported the existence of a significant population of highly tilted DPH molecules located in the ordered upper acyl chain region of DOPC bilayers, just below the lipid ester/carbonyl atoms (z ~ 1.2 nm). Our results show qualitative agreement with this observation, to the extent that our most probable location for highly tilted DPH molecules (z ~ 1.0 nm) is more external than the overall average center of mass location (z ~ 0.7 nm). In our study, this was also apparent in the POPC/ 20 mol% cholesterol system (the most probable location for highly tilted confirmations is for z ~ 1.4 nm, while the center of mass average location is z ~ 0.8 nm). A reason behind this phenomenon might be that flipping motions of DPH in shallow locations inside the membrane only affect host lipid molecules located in the same bilayer leaflet. Conversely, because of the ~1.4 nm length of the DPH long axis, vertical flips of more deeply located probe molecules necessarily affect lipids located in both bilayer leaflets, with increased conformational requirements and entropic cost. At variance with DPH, highly tilted conformations of TMA-DPH are very rare (~1%) in POPC, and wholly absent in POPC/20 mol% cholesterol.

DPH in lipid vesicles was detectable in 24 h. In any case, the plots of Figs. 6 and 7 demonstrate that TMA-DPH spans the whole hydrophobic region of the membrane. C3 is often found near the bilayer midplane, more so in POPC than in POPC/20 mol% cholesterol. The average location in the former system, (0.34 ± 0.03) nm, actually agrees well with the aforementioned location ((0.28 ± 0.19) nm) of the TMA-DPH end H atom in fluid DPPC, as calculated by Nelson et al. [25]. However, we could not confirm the corresponding result obtained by these authors for DPH ((0.42 ± 0.35) nm), which would point to a possibly more external location of this probe. This particular observation is probably affected by the previously commented simulation limitations of the aforementioned study. In most instances, translocation of DPH proceeds by vertical translation of the molecule, with the previously most internally located phenyl ring becoming the most externally located one in the opposite leaflet. However, in roughly a quarter of these events (5 out 18 in POPC, 1 out of 4 in POPC/20 mol% cholesterol), DPH translocation is accompanied by rotation around the long axis of the molecule (flip-flop), which at one point is oriented parallel to the bilayer plane. The corresponding probe configuration is reminiscent of the population oriented perpendicular to the bilayer normal, between the two leaflets, as proposed in early fluorescence anisotropy studies (see Introduction). As shown in Fig. 8A, highly tilted (with long axis tilt relative to the bilayer normal θ N 80°) orientations are observed for a sizeable fraction of DPH molecular configurations in POPC (~ 4%) and considerably less so in POPC/ 20 mol% cholesterol (~0.1%). However, in our simulations, these configurations always represent transient states, with the molecule rapidly (most often within ~ 100–200 ps) realigning with its long axis with the bilayer normal. Additionally, these full rotations around the long axis are not necessarily accompanied by translocation, and are more frequently observed far from the bilayer center. Fig. 9 shows the probability density functions of the distance z between the bilayer midplane and the fluorophore geometrical center for the highly tilted configurations of DPH. A clear maximum is observed for z ~ 1.0 nm in POPC and z ~ 1.4 nm in POPC/20 mol% cholesterol (the latter reflecting the more ordered, thicker bilayer). Despite the high DPH local density at z ~ 0 (Fig. 5A), and the reduced order and density in this region of the bilayer, the relative frequency of highly tilted configurations in the bilayer center is not increased compared to more shallow locations. These observations agree with the results of Repáková et al. [22] and Franová et al. [15], who found that no distinct correlation between orientation and location existed in fluid DPPC and DPPC/cholesterol bilayers (respectively), and, in particular, orientations with θ ~ 90° were not especially favored near z = 0. More recently, Hurjui

0.04 P (θ) 0.03

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3.3. Probe rotational and lateral diffusion As described in the introduction, both DPH and TMA-DPH have found wide use as reporters of membrane fluidity, assessed from their fluorescence anisotropy, which in turn is highly dependent on probe rotational dynamics. For this reason, we calculated rotational autocorrelation functions C(t), as defined below: C ðt Þ ¼ hP 2 ð cos θðξÞÞi

ð7Þ

where θ(ξ) is the angle between the probe long axis at times ξ and t + ξ, and P2(x) = (3x2–1)/2 is the second Legendre polynomial. Averaging is performed over ξ, which assuming a sufficiently ergodic trajectory, is an approximation of the ensemble average. Because the electronic transition dipole of DPH probes is oriented along the probe long axis [6], C(t) is expected to be proportional to the actual anisotropy decay r(t) (the proportionality constant being r(0), the fundamental anisotropy [82]). Fig. 10 shows the C(t) curves obtained for both probes in POPC and POPC/20 mol% cholesterol. To improve statistics, these curves were averaged over the 6 individual probe decays in both 2- and 4-probe molecule simulations. For all systems, finite residual values of C(t) are observed at long times, that is, these functions appear to have nonzero limits C∞ as t → ∞. This is common for probes embedded in lipid bilayers, and may arise from “wobbling-in-cone”-type rotation [83]. Experimentally,

A

B

C

D

0.02 0.01 0.00

0.04 P (θ) 0.03 0.02 0.01 0.00 0

20

40

60 θ/deg 80

0

20

40

60 θ/deg 80

Fig. 8. Probability density functions P(θ) of the fluorophore long axis tilt relative to the bilayer normal for DPH (A, B) and TMA-DPH (C, D), in POPC (A,C) and POPC/20 mol% cholesterol (B,D).

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P(Z ) 0.20

A

B

0.10

0.00 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

z/ nm

z /nm

Fig. 9. Probability density functions P(z) for highly tilted conformations (θ N 80°) of DPH in POPC (A) and POPC/20 mol% cholesterol (B).

this hindered rotation is reflected in a residual component r∞ of the anisotropy decay, and a probe order parameter may be calculated from S = (r∞/r(0))1/2 [84,85], or, from our simulation results, estimated by S = C1/2 ∞ . From simple observation of Fig. 10, it is clear that i) for a given probe, C∞ is higher in presence of 20 mol% cholesterol, because of the increased bilayer order induced by the latter; and ii) for a given system, the decay of C(t) is generally slower and C∞ is higher for TMA-DPH compared to DPH. Table 3 show best fit values for analysis of the C(t) curves with a double exponential decay equation, with rotational correlation times ϕ1 and ϕ2, pre-exponential factors β1 and β2, and offset C∞:

Mitchell and Litman [89] for DPH in POPC/30 mol% cholesterol (0.738 at 20 °C, 0.674 at 30 °C) and with those of van Langen et al. [87] for TMA-DPH in POPC/25 wt.% cholesterol (0.67 at ~20 °C). While the order parameter and residual anisotropy are related to the maximal amplitude of angular tilt displacements, the dynamics of rotation are also of interest and may be compared with literature values. From the experimental anisotropy decay function, and applying the rotational diffusion model to rotation around an axis perpendicular to the symmetry axis of rod-like probes such as DPH or TMA-DPH [11, 83], the rotational diffusion coefficient D⊥ may be calculated as:

C ðt Þ ¼ β1 expð‐t=ϕ1 Þ þ β2 expð‐t=ϕ2 Þ þ C ∞

D⊥ ¼ −

ð8Þ

Also shown in the table are the calculated S = C1/2 ∞ and r∞ = r(0) × C∞ (taking r(0) = 0.362 as the fundamental anisotropy for the DPH fluorophore [86]). The estimated probe order parameters in the absence of cholesterol agree reasonably well with experimental values measured by Prendergast et al. [9] at ~300 K (0.33 and 0.59 for DPH and TMA-DPH, respectively), as well as with those obtained by Mitchell and Litman [13] for DPH (0.346 at 20 °C, 0.262 at 30 °C) and van Langen et al. [87] for TMA-DPH (0.54 at ~ 20 °C), all in POPC bilayers. Our higher values for DPH probably reflect increased ordering induced by the larger probe concentration in our simulations (if we only considered the 2 DPH system in this calculation, S = 0.36 would be obtained, closer to the experimental values). Additionally, the calculated limiting anisotropies are close to those measured in DPPC (~ 0.05 and 0.14 for DPH and TMA-DPH, respectively [9]) and DMPC (~ 0.06 and 0.14 for DPH and TMA-DPH, respectively [88]) vesicles both above their phase transition temperatures (at 45 °C and 25 °C, respectively). Turning our attention to the systems with cholesterol, our values are in reasonable accordance (though somewhat higher) with the experimental data of 1.0 C (t ) 0.8

 1 ∂r  6r ð0Þ ∂t t¼0

ð7Þ

In large part because of the partial derivative at t = 0 in the above equation, values of D⊥ are somewhat dependent on the model used to describe r(t) (or C(t) in our simulations). Using the proportionality between r(t) and C(t), we can calculate D⊥ from the fitting parameters of Eq. (6): D⊥ ¼ ðβ1 =ϕ1 þ β2 =ϕ2 Þ=6

ð8Þ

The resulting values, also shown in Table 3, are of the order of magnitude of the experimental estimates. Quantitative comparison is limited because of the small number of sampled probe molecules and the dependence of D⊥ on the mathematical model used to describe r(t). Using DPH in POPC and a Brownian rotational diffusion model for time-resolved fluorescence anisotropy analysis, Mitchell and Litman [13] obtained D⊥ = (0.096 ± 0.007) ns−1 and (0.156 ± 0.010) ns−1 for T = 20 °C and T = 30 °C, respectively. The same authors also observed a decrease, albeit smaller than that estimated from our results (~ 25% in those authors' experimental data) upon adding 30 mol% cholesterol [68]. Curiously, for TMA-DPH, no significant decrease of D⊥ was observed from pure POPC to POPC/20 mol% cholesterol, in qualitative agreement with the data of van Langen et al. [66], whose absolute values (0.09 in pure POPC, 0.12 in POPC/25 wt.% cholesterol, T ~ 20 °C) are higher than ours by a factor of ~2–3.

TMA-DPH/POPC/cholesterol

0.6 DPH/POPC/cholesterol

0.4 TMA-DPH/POPC

0.2 DPH/POPC

0.0 0

20

40

60

80

100

120 t /ns

Fig. 10. Rotational autocorrelation curves C(t) for the fluorophore long axis of DPH and TMA-DPH in POPC and POPC/20 mol% cholesterol.

Table 3 Parameters obtained from analysis of the fluorophore long axis rotational auto-correlation functions. Lipid system

POPC

Probe

DPH

TMA-DPH

POPC/20 mol% cholesterol DPH

TMA-DPH

β1 ϕ1/ns β2 ϕ2/ns C∞ S r∞ D⊥/ns−1

0.38 ± 0.01 0.45 ± 0.03 0.45 ± 0.02 3.6 ± 0.1 0.18 ± 0.03 0.42 ± 0.04 0.06 ± 0.01 0.16 ± 0.02

0.184 ± 0.002 0.76 ± 0.05 0.488 ± 0.002 19.1 ± 0.2 0.326 ± 0.004 0.571 ± 0.004 0.118 ± 0.002 0.05 ± 0.01

0.319 ± 0.007 1.2 ± 0.1 0.136 ± 0.007 23 ± 2 0.55 ± 0.02 0.74 ± 0.01 0.197 ± 0.005 0.043 ± 0.005

0.10 ± 0.02 0.5 ± 0.2 0.266 ± 0.004 20.3 ± 0.5 0.64 ± 0.03 0.80 ± 0.02 0.23 ± 0.01 0.04 ± 0.02

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In addition to the above discussion, which centered on spectroscopic literature studies employing nanometer-sized membrane systems, more recent works have addressed the behavior of DPH probes in giant unilamellar vesicles using fluorescence microscopy techniques. One such study by Haluska et al. [90] used a long chain derivative of TMA-DPH (Lc-TMA-DPH), with a (CH2)22 spacer between the biphenyl and the trimethylamino moieties. It is probable that due to its hydrophobicity, the DPH fluorophore of this probe will still reside in the hydrocarbon region of the bilayer. Because it is not directly linked to a charged group, we speculate that its rotational dynamics should resemble closer those of DPH than those of TMA-DPH. This agrees with the relatively lower order parameter values measured in this study (~0.15 and ~0.3 in the absence and in the presence of 20 mol% cholesterol, respectively) compared to ours. The significant relative increase in order parameters upon adding cholesterol is in accordance with our simulations. The observation by those authors that the depolarization of Lc-TMA-DPH in the liquid-ordered phase is better described by a “probe-on-cone” model (where the fluorophore long axis tilt is confined to a fixed value), at variance with the behavior in liquid-disordered domains, where a broad angular distribution is inferred, also agrees with our data. In fact, we verified that incorporation of 20 mol% cholesterol leads to significantly more narrow angular distributions of the long axes of both probes (Fig. 8). In another study, equimolar mixtures of DOPC, cholesterol and chicken egg yolk sphingomyelin, which display liquid ordered/liquid disordered phase coexistence, were addressed by polarimetric two-photon microscopy using, among other probes, TMA-DPH [91]. Similarly, these authors' results point to reduced angular freedom of TMA-DPH for cholesterol-enriched bilayers or domains, thus in agreement with our simulations. In conclusion, our rotational diffusion calculations seem to predict the order of magnitude of both probe order parameters and rotational diffusion coefficients correctly, as well as the main results of generally slower, more hindered rotations of TMA-DPH compared to DPH and of both probes in POPC/20 mol% compared to POPC. In some cases, our results provide slightly high estimates for the former and low ones for the latter parameter. This hints at a somewhat excessive order in our simulations. Although this could reflect unsolved force field issues, our good comparison of lipid structural properties in the absence of probe suggests an alternative explanation, possibly related to the higher probe concentration in our simulations and/or limitations resulting from the low number of number of sampled molecules. Lipid and probe lateral diffusion coefficients Dlat, calculated for both bilayer compositions, are displayed in Fig. 11. The significance of MSD plots and accurate calculation of lateral diffusion in membranes remains, to a great extent, a controversial problem. It depends largely on the available time window [92,93]. Sampling problems are more important in lateral diffusion than in some other properties, because it

D lat/ 8

involves large-scale motions of whole molecules rather than limited range/segmental motions (like those involved in lipid acyl chains or probe long axis orientation). For relatively short times, lipid diffusion (as perceived by MSD variation) is mainly due to conformational changes of the hydrocarbon chains rather than diffusion of the entire molecule [92], and therefore its meaning and its relationship to experimental observables are somewhat questionable. Taking this into account, our values for POPC agree well with the pulsed field gradient NMR data of Filippov et al. [94]. From these authors' plot of POPC/cholesterol data as a function of composition and temperature (Fig. 6B of their work), we estimate an increase in Dlat of POPC between 7.8 × 10−8 cm2/s and 9.5 × 10−8 cm2/s in the pure system as T varies from 298 K to 303 K, and a corresponding one from 5.7 × 10−8 cm2/s to 7.2 × 10−8 cm2/s in POPC/20 mol% cholesterol. According to our simulations, the rate of diffusion of the latter component is very similar to that of POPC. Concerning the probes, even refraining from a quantitative discussion, it seems clear that DPH diffuses faster than POPC and cholesterol for both compositions. A faster lateral diffusion of DPH compared to phospholipid has been reported by Repáková et al. [22] in fluid DPPC bilayers. Conversely, TMA-DPH diffusion occurs at a rate similar to that of the host lipid species. A previous MD simulation study [22] considered the lateral diffusion of DPH in fluid DPPC in detail. These authors observed that DPH molecules spend long periods of time “rattling” in well-defined voids where they are surrounded by DPPC molecules. Occasionally, however, once a reasonably large density fluctuation takes place, DPH carries out lateral “jumps” of ~1 nm from one void to another, in a typical time scale of ~200–300 ps. We carried out a similar analysis for our POPC/4 DPH system (Fig. S7). A similar behavior to that described by Repáková et al. [22] is observed: each molecule spends a considerable amount of time (~5–10 ns) in a relatively restricted region of the bilayer plane, before undergoing a fast, longer-ranged motion. Overall, each molecule is able of significant lateral motion (of the order of ~10 nm from its initial location) in the 200 ns time range of the simulations. An identical analysis was carried out for TMA-DPH (Fig. S8). It is apparent that, compared to DPH, the lateral motion of TMA-DPH is more confined, lacking the large-range displacements of the former. Overall, after 200 ns of simulation, each probe ends up within ~5 nm of the original location. From our quantum chemical structure calculations, together with a van der Waals molecular surface method estimation, we obtained molecular volumes of 0.238 nm3 and 0.302 nm3 for DPH and TMA-DPH, respectively. The difference, resulting from the additional trimethylamino group of TMA-DPH is therefore not very large (~ 20%) and would not justify per se the observed differences in probe diffusion, also noting that the two molecules have similar rod-like shapes and that several literature studies have pointed to a lack of dependence of lateral diffusion on the size of the hydrophobic region of solutes of similar structure and

A

2 -1

10 cm s

2657

B

30

20

10

0 POPC

DPH

TMA-DPH

POPC

Cholesterol

DPH

TMA-DPH

Fig. 11. Lateral diffusion coefficients (Dlat) for the lipid and probe species in POPC (A) and POPC/20 mol% cholesterol (B).

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varying chain length [95–97]. On the other hand, while the additional trimethylamino group of TMA-DPH is located slightly more externally compared to the diphenyl moiety of both probes (which have similar transverse distributions), its average position lies still within the bilayer region where the lateral pressure is negative (approximately z b 1.75 nm and z b 2.0 nm in the absence and in the presence of 20 mol% cholesterol, see Fig. S5), and therefore we expect that no significant hindrance of lateral diffusion of TMA-DPH compared to that of DPH will result from lateral pressure effects. In this way, we believe that the slower rate of translational diffusion of TMA-DPH (comparable to the lipid species) arises mostly from the establishment of probe-lipid interactions, involving the trimethylamino group of the former and the phosphate and ester/carbonyl groups of the latter (Fig. 4). 3.4. Simulations in presence of salt and with an altenative force field description As described in the Methods section, we also carried out additional sets of simulations. In one of them, we kept the Berger lipids/HF 631G(d) probe topologies used in the main set of simulations, but added sodium and chloride ions to a physiological (150 mM) ionic strength. It should be stressed that possibly owing to the relatively small simulation boxes (namely in the direction normal to the bilayer plane) that may be presently addressed in atomistic MD, as well as to uncertainty in the force field parameters of common ions, the number of cations in close vicinity of the bilayer is overestimated in the conventional MD simulation protocols [98–100]. In turn, this leads to an overestimation of ion-induced alterations of host lipid properties. For example, although it has been verified both experimentally and by MD simulation that addition sodium chloride induces ordering of PC membranes, an extent of bilayer ordering similar to that predicted by ~0.1 M NaCl in the MD study of Böckmann et al. [70] is only observed experimentally for salt concentrations in excess of 1 M [101]. Fig. S9 in the Supplemental Material file shows order parameter profiles and respective variations relative to the probe-free systems. Addition of salt induces considerable ordering of the bilayer systems (probably overestimated, as commented above). Insertion of both probes lead to additional ordering of POPC bilayers, especially for atoms nearer the center. Conversely, modest effects are observed upon probe incorporation in the POPC/20 mol% cholesterol systems. We also looked at the time traces of transverse locations of the opposite C atoms of the probes' fluorophores in both POPC (Fig. S10) and POPC/ 20 mol% cholesterol (Fig. S11). DPH molecules are still capable of translocation to the opposite leaflet. DPH flip-flop events (rotation with inversion of the relative position of the C3 and C16 atoms) are also observed, both accompanying translocation and without change of bilayer leaflet. The qualitative behavior of DPH is therefore identical to that displayed in the absence of salt. However, compared to the systems with no added salt, the frequency of translocation/flip-flop events is approximately halved, probably as a consequence of the increased bilayer order. Identically to the systems with no added salt, no TMA-DPH translocation or flip-flop events were observed. It is also apparent that, apart from the translocation and flip-flop events observed for DPH but not for TMA-DPH, the locations of the fluorophores of both probes are very similar, identically to the systems with no added salt. Regarding dynamical properties, rotational autocorrelation curves of the fluorophore long axis, depicted in Fig. S12, show increased hindrance for rotation in the presence of salt, without qualitative changes in the main results observed in the salt-free trajectories: rotation of TMA-DPH is significantly more impeded than that of DPH for each lipid composition; and rotation of each probe is slower in the presence of 20 mol% cholesterol than in pure POPC. On the other hand, lateral diffusion coefficients calculated for the systems with physiological ionic strength, depicted in Fig. S13, are not statistically different from those calculated in the absence of salt, with the sole exception of DPH in POPC. The diffusion coefficient of this probe in this system is

approximately half of the value obtained in the absence of salt, but is nevertheless still significantly higher than that of the host lipid (at variance with TMA-DPH). This quantitative difference may stem either from the increased bilayer order or underestimated statistical uncertainty associated with the small number of probe molecules. However, the basic features we identified in the absence of salt are still observed for 150 mM ionic strength, namely the diffusion rate of TMA-DPH is identical to that of the host lipids, whereas that of DPH is significantly higher. Overall, while these results are qualitatively similar to those obtained in the absence of salt, enhanced ordering and reduced probe dynamics are observed. While our main set of simulations combines the widely used Berger lipid topology [29] with probe charges obtained from HF 631G(d) calculation (similarly to the previous DPH bilayer studies of Repáková et al. [22,23]), it could be argued that a more accurate description of the behavior of both probes could be achieved from more computationally demanding all-atom simulations. For the sake of verification, we carried out an additional set of additional simulations employed a different, all atom topology, based in the Slipids force field for POPC and cholesterol and an AMBER-GAFF description [50–52] for the probes (which is fully compatible with Slipids [47–49]; see Fig. S14 for atomic charge schemes). Figs. S15 and S16 in the Supplemental Material file show the time variations of the z coordinates of atoms C3 and C16 relative to the bilayer center, for the 4 individual molecules in the POPC + 4 DPH (left panels) and POPC + 4 TMA-DPH (right panels) simulations, in the POPC and POPC/20 mol% cholesterol systems, respectively. From inspection of these data, it is apparent that the behaviors of both probes are not significantly altered by the changes in parameterization. DPH is still able to translocate to the opposing leaflet in the time scale of the simulations, as well as to flip around its long axis (most frequently in POPC than in POPC/cholesterol, for both types of motion). At variance, no such events are observed for TMA-DPH. Focusing on the average values, some subtle differences may be found (see Table S1). To some extent, they arise from an increased bilayer thickness (by ~0.08 nm) in the Slipids simulations. Taking this into account, one may conclude that the fluorophore in DPH, which is displaced a further ~0.05 nm from the center in the Slipids simulations, has an unchanged position relative to the water/lipid interface (which is taken as the average location of the POPC P atoms). On the other hand, TMA-DPH is predicted to lie ~ 0.2 nm further from the center in the Slipids simulations, and therefore ~0.15 nm closer to the POPC/water interface, when compared to the Berger/HF 6-31G(d) simulations. In any case, the locations of the fluorophores of the two probes are still very similar, and the N atom of TMA-DPH is still located more deeply than both the choline N and the phosphate P atoms of POPC. 4. Concluding remarks Our study sheds light on the behavior of TMA-DPH as a membrane probe, by reporting the first extensive MD simulation of this compound in both pure phospholipid and mixed phospholipid/cholesterol bilayers. While MD has been previously used to address the location, orientation, dynamics and probe-induced effects on the bilayer of the parent molecule DPH, we also included the latter in our study, for the purpose of validating our parameterization and simulation protocols, and also for a more direct comparison between the two probes, which are actually often used together in experimental studies. The trimethylammonium group of TMA-DPH is identical to the terminal (and most external) charged choline N(CH3)3 group of phosphatidylcholine. One could reasonably expect a similar location of this probe group in the membrane/water interface. However, our simulations show that the trimethylammonium group of TMA-DPH actually has a deeper preferential transverse location, between those of the POPC phosphate and carbonyl/ester (or cholesterol hydroxyl)

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groups. This position maximizes favorable interactions with the electronegative oxygen atoms of the latter. In turn, the tethering of the trimethylammonium group to this region of the bilayer implies that the fluorophore of TMA-DPH is only slightly (by ~ 0.3–0.4 nm) more external in membranes than that of DPH. Both rotational and lateral diffusion of DPH and TMA-DPH point to slower, more hindered motions of the latter. Most probably, these clear differences cannot be solely explained taking into account the fluorophore location within the bilayer. DPH is an apolar molecule, unable of specific interaction with phospholipid, sterol or water molecules. For this reason, its molecular motions such as diffusion along the bilayer plane, rotation around the long axis, or translocation to the opposing bilayer leaflet do not involve the energetic cost associated with the loss of such interactions. At variance, the above mentioned stable positioning of the trimethylammonium group of TMA-DPH constitute an efficient deterrent of such motions, as they would necessarily break favorable contacts with electronegative lipid atoms, even if transiently. An important message of our study is that the widespread notion that DPH fluorescence reports the environment of the bilayer core, while TMA-DPH probes that of the membrane/water interface, is exaggerated, given that their average fluorophore depth is quite similar. While a similar note of caution could already be inferred from the Kaiser and London [14] differential fluorescence quenching study, it is our belief that, given the approximations involved in these experiments and their analysis (see Introduction section), a direct confirmation using a non-perturbing method such as MD simulation was required. A similar MD calibration or confirmation has been recently reported for another methodology used for analysis of fluorescence quenching data (Distribution Analysis method), with other fluorescent membrane probes [102]. While fluorescence quenching only reports on location, and fluorescence anisotropy only informs on rotational diffusion (and somewhat indirectly in both cases), our DPH/TMA-DPH simulations can give direct insight on both accounts. They confirm both the similar transverse location and the substantial difference in rotation mobility of the two probes. From our results, it is clear that the generally higher fluorescence anisotropy of TMA-DPH, compared to DPH in the same membrane system, has more to do with the above mentioned motional restrictions (arising from favorable interaction of the trimethylammonium group with lipid atoms) than with differences in the order of the lipid region probed by the fluorophore. In fact, the more restricted mobility of TMA-DPH and its more upright conformation in the membrane act as an extrinsic source of local order, and our simulations show that TMA-DPH is more efficient at ordering the bilayer than DPH. In other words, the measured difference in fluorophore anisotropy stems mainly from the structural differences between the two probes, rather than from intrinsic differences in the environments they are reporting on, which is not what is generally intended when using probes as reporters. This similar fluorophore position in the bilayer is also important to consider when planning or interpreting experiments involving phenomena that depend strongly on transverse location, such as fluorescence quenching or FRET. The differences perceived between DPH and TMA-DPH in such experiments may not necessarily correlate to the location of the foreign quencher or FRET donor/acceptor under study. In these circumstances, a great deal of caution is required, to verify whether specific interactions (e.g. with the charged group of TMA-DPH) might supersede the small difference in probe position. While this work focused on the comparison of location, conformation and dynamics of the two probes in bilayer systems, MD may also be employed to address other important questions related to behavior of these widely used fluorescent reporters. In particular, future studies will concern the free energy profiles of the probes across the bilayer systems, with the purposes of determination of the kinetic parameters describing the different processes of probe/bilayer interaction (insertion, translocation, desorption) and rationalization of the differences between the probes regarding partition to systems of varying lipid order [6,7].

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Transparency document The Transparency document associated with this article can be found, in online version.

Acknowledgments The authors acknowledge funding by FEDER, through the COMPETE program, and by FCT (Fundação para a Ciência e a Tecnologia, Portugal), project reference FCOMP-01-0124-FEDER-010787 (FCT PTDC/QUI-QUI/ 098198/2008). J.R.R. and P.D.S. acknowledge grants under this same project. L.M.S.L. acknowledges the Laboratory for Advanced Computing at University of Coimbra for computing resources, and additional funding by Fundação para a Ciência e Tecnologia (Portugal), project reference UID/QUI/00313/2013.

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbamem.2016.07.013.

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