Dehydroergosterol as an Analogue for Cholesterol: Why It Mimics

Jun 3, 2014 - Although dehydroergosterol (DHE) is one of the most commonly used cholesterol (CHOL) reporters, it has remained unclear why it performs ...
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Dehydroergosterol as an Analogue for Cholesterol: Why It Mimics Cholesterol So Wellor Does It? Mohsen Pourmousa,† Tomasz Róg,† Risto Mikkeli,‡ llpo Vattulainen,†,∥ Lukasz M. Solanko,⊥ Daniel Wüstner,⊥ Nanna Holmgaard List,# Jacob Kongsted,# and Mikko Karttunen*,▽ †

Department of Physics, Tampere University of Technology, Korkeakoulunkatu 3, 33720 Tampere, Finland Department of Applied Physics, Aalto University, P.O. Box 11100, FI-00076 Aalto, Finland ∥ MEMPHYS−Center of Biomembrane Physics, Physics Department, University of Southern Denmark, Odense, Denmark ⊥ Department of Biochemistry and Molecular Biology and #Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark ▽ Department of Chemistry and Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo N2L 3G1, Ontario, Canada ‡

S Supporting Information *

ABSTRACT: Although dehydroergosterol (DHE) is one of the most commonly used cholesterol (CHOL) reporters, it has remained unclear why it performs well compared with most other CHOL analogues and what its possible limitations are. We present a comprehensive study of the properties of DHE using a combination of time-resolved fluorescence spectroscopy, quantum-mechanical electronic structure computations, and classical atomistic molecular dynamics simulations. We first establish that DHE mimics CHOL behavior, as previous studies have suggested, and then move on to elucidate and discuss the particular properties that render DHE so superior. We found that the main reason why DHE mimics CHOL so well is due to its ability to stand upright in a membrane in a manner that is almost identical to that of CHOL. The minor difference in how DHE and CHOL tilt with respect to membrane normal has only faint effects on structural membrane properties, and even the lateral pressure profiles of model membranes with CHOL or DHE are almost identical. These results suggest that the mechanical/elastic effects of DHE on the function of mechanically sensitive membrane proteins are not substantially different from those of CHOL. Our study highlights similar dynamical behavior of CHOL and DHE, which implies that DHE can mimic CHOL in processes with free energies close to the thermal energy.



INTRODUCTION

Because the structures of CHOL analogues are inevitably different from those of CHOL, so are their properties. Several studies have revealed that even seemingly minor changes in sterol structure can lead to major effects on sterol properties in lipid membranes.10,11 In addition, attention has to be paid to other aspects of analogues, such as their quenching properties and lifetime in the case of fluorescent sterol probes. Finding good CHOL analogues remains one of the important challenges in CHOL research and is definitely not a simple feat. Here we focus on DHE, which is one of the most commonly used CHOL analogues. This is largely due to its intrinsic fluorescence,9 which renders it very suitable for visualization of sterol partitioning because it does not require additional moieties covalently attached to CHOL. DHE was discovered over 80 years ago, but its complete structure was confirmed only in 1985.12 DHE differs from CHOL by having two additional double bonds in the steroid

CHOL is one of the most important constituents of cellular membranes. It is fundamental in membrane permeability, lateral lipid organization, signal transduction, and membrane trafficking.1 A commonly used strategy to study how CHOL functions in these and other cellular processes is to employ CHOL reporters, that is, molecules that mimic the properties of CHOL but are easier to study due to features such as intrinsic fluorescence. Reporters should perturb the system as little as possible and be easily detectable in experiments for extended periods of time. CHOL reporters can be divided into two classes. First, there are CHOL binding molecules, which form a complex with CHOL. An example of such is Filipin. It is a fluorescent polyene antibiotic that binds to CHOL and is commonly used to visualize the cellular distribution of free CHOL.2−4 The second class consists of CHOL analogues, which can be photoreactive such as [3H]6-azi-5α-cholesterol,5 spin-labeled such as 3βdoxyl-5α-cholestane,6 or fluorescent such as Bodipy-cholesterol7,8 and DHE.9 © 2014 American Chemical Society

Received: July 12, 2013 Revised: June 2, 2014 Published: June 3, 2014 7345

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ring system that make it slightly fluorescent. The tail of DHE is identical to that of ergosterol with an extra double bond and methyl group compared with CHOL. DHE has been proven to faithfully mimic CHOL in many respects. For example, it codistributes with CHOL, is nontoxic to cultured cells and animals, and is accepted as a substrate for esterification.9,13−16 Meanwhile, it has a lower ability than CHOL to stiffen bilayers17 and order lipids18 around the probe. We use atomistic molecular dynamics (MD) simulations to consider the interaction of POPC lipid bilayers with CHOL and DHE molecules and study membrane organization of DHE by fluorescence spectroscopy. Then, using quantum-mechanical (QM) calculations, we link the experimental and theoretical results. We perform a systematic comparison between DHE and CHOL to characterize the perturbations induced by DHE on membrane properties. In this respect, our work complements previous studies,18,11,19−24 which have shown how atomistic simulations can generate a great deal of added value by unraveling, for example, how probes alter membrane properties or how well they can mimic the properties of the host molecules that they have been designed to replace.

Gromacs for calculating the pressure profile. The practical procedure followed previous studies.41−43 Quantum-Mechanical Calculations. In addition to classical MD, we performed QM electronic structure calculations on DHE. Vertical excitation and emission energies as well as associated transition dipole moments of isolated and solvated DHE were computed using the CAM-B3LYP exchange-functional44 in conjunction with the 6-311+ +G**45,46 basis set on the geometries optimized at the same level of theory. The optimized geometries were confirmed to be energy minima by inspection of the vibrational frequencies. Solvation effects (ethanol) were simulated by the polarizable continuum model (PCM) using the integral equation formalism:47 The solvent was described in terms of its static, ϵ(0) = 24.852, and optical, ϵopt = 1.8526, dielectric constants. The radii of the interlocking spheres were taken from the universal force field (UFF) and scaled by a factor of 1.1. In the calculations of the excitation and emission energies in solvent, nonequilibrium linear response solvation was assumed. The transition moments were calculated in the length representation. All computations were carried out using Gaussian 09.48

SIMULATION DETAILS Classical MD. Atomistic MD simulations of three different membrane systems were performed. The first bilayer was composed of 128 POPC molecules, the second was composed of 128 POPC and 32 CHOL molecules, and the third was composed of 128 POPC and 32 DHE molecules. Chemical structures of POPC, CHOL, and DHE are shown in Figures S1A and S1B. (See Supporting Information (SI).) All three bilayers were hydrated with ∼3500 water molecules. The initial structures were obtained from a previous MD simulation,25 to which an equal number of CHOL molecules were added to each leaflet to obtain a symmetric POPC−CHOL bilayer system. The POPC−DHE system was obtained by transforming the CHOL molecules into DHE molecules. The steepest-descent algorithm was used for energy minimization of the initial structures, and short NVT and NpT simulations were run to pre-equilibrate the systems. The simulations were performed using the Gromacs 426 package. Each system was simulated for 150 ns. Equilibration over a time scale of 50 ns was determined by monitoring the area per lipid, temperature, and energy. The values agreed with previous studies.11,27 The Berger force-field parameters28 were used for POPC molecules. Partial charges were taken from the underlying model description.29 For water we used the SPC model,30 and for CHOL, we used the description of Holtje et al.31 Periodic boundary conditions with the usual minimum image convention were used in all three directions. Bond lengths were preserved using the LINCS algorithm.32 The time step was set to 2 fs, and the simulations were carried out at p = 1 atm and T = 303 K, which is above the main phase-transition temperature of POPC (268 K33). Temperature was controlled using the Nosé−Hoover thermostat.34,35 The temperatures of the bilayer and water were controlled independently, and the Parrinello− Rahman barostat36 was applied semi-isotropically. The time constants of thermostat and barostat were 0.5 and 2.0 ps, respectively. The Lennard-Jones interactions and the real space part of the electrostatic interaction were cut off at 1.0 nm. The particle-mesh Ewald method37,38 was used to evaluate longrange interactions to avoid artifacts.39,40 Lateral pressure was computed using Gromacs 4.0.2, a customized version of

EXPERIMENTAL PROCEDURES Reagents. POPC was purchased from Avanti Polar Lipids (Alabaster, AL). CHOL and DHE were from Sigma Chemical (St. Louis, MO). Lipids were stored in either chloroform or ethanol under nitrogen at −80 °C until use. Absorption Spectra of DHE in Various Solvents. Solutions of DHE in either glycerol or ethanol were gently stirred in the dark for several minutes. For measurements at low temperature, the solution was placed on ice. For measurements, the solution was transferred into a quartz cuvette, and after some equilibration in the dark at the set temperature, absorption of DHE was measured at a Lambda 35 UV/vis spectrophotometer (PerkinElmer). Measurements were done with either pure glycerol or 96% ethanol as references, and the wavelength range was from 280 to 360 nm. Steady-State Excitation and Emission Spectra of DHE. POPC, CHOL, and DHE were mixed in a glass vial. Solvent was evaporated under N2 steam, and lipids were dissolved in distilled water to a final concentration of 25 μM. Large unilamellar vesicles (LUVs) were made of varying amounts of POPC, DHE, and CHOL by extrusion using a 200 nm pore diameter filter and a micro extruder from Avanti Polar Lipids. Steady-state excitation and emission spectra of DHE were measured using an ISS Chronos spectrofluorometer (UrbanaChampaign, IL). For the emission spectra, the excitation wavelength was set to 328 nm, and emission was recorded in a wavelength range of 340−500 nm. Excitation spectra were recorded in a wavelength range of 280−360 nm with fixed emission wavelength of 370 nm. For polarization measurements, the excitation polarizer was set to pass only vertically polarized light, whereas the linear emission polarizer was set in either parallel or perpendicular position to the excitation filter. All spectra were averaged and normalized to the highest intensity at 327 nm. Additionally, all spectra acquired with polarization filters were smoothed using a running average filter (each point averaged by two nearest neighbors). Excitation polarization of DHE in ethanol or glycerol was measured as described for LUVs. The spectrophotometer was equipped with a temperature-controlled sample holder being set to 4 °C where required. Excitation and emission anisotropy were calculated using the well-know relation





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Table 1. Comparison of Different Properties of Pure POPC, CHOL, and DHEa membrane

POPC

area/POPC (nm2) thickness (nm) P−N angle (deg) tilt of palmitoyl (deg) tilt of oleoyl (deg) tilt of sterol ring structure (deg) tilt of sterol tail (deg) number of neighboring carbon atoms of POPC per sterol

0.67 3.76 77.33 32.58 34.69

± ± ± ± ±

POPC−CHOL

0.01 0.06 0.01 0.01 0.01

0.63 4.30 79.31 21.50 24.58 23.44 33.23 87

± ± ± ± ± ± ± ±

0.01 0.06 0.02 0.01 0.01 0.01 0.02 2

POPC−DHE 0.65 4.21 78.17 22.77 25.50 25.00 44.84 88

± ± ± ± ± ± ± ±

0.01 0.05 0.01 0.01 0.01 0.02 0.02 2

a

Standard error was used to report the errors of the mean value of angles, and the standard deviation was used to describe the spread of data of other quantities.

r=

IVV − GIVH IVV + 2GIVH

and τ2. The phase and modulation values are calculated from sine and cosine transforms of the intensity decay, f(t), according to tan ϕc = N/D and mc = ((N + D)2)1/2, where N = J−1∫ 0∞f(t) sin (ωt) dt and D = J−1 ∫ 0∞ f(t) cos (ωt) dt. J normalizes the expression to the total intensity according to

(1)

where G is the grating factor (G factor), defined by G=

IHV IHH

(2)

J=

IVV and IVH imply that the sample is excited using vertically polarized light pulses, while the intensity decay of the sample is measured through a polarizer oriented vertically and horizontally to the sample, respectively. Similar definitions are used for IHV and IHH. Time-Resolved Fluorescence Spectroscopy of DHE. Fluorescence lifetimes of DHE were measured using the frequency-domain method at the ISS spectrofluorometer with an LED emitting at 300 nm as excitation source and a 400 nm long pass cutoff filter to reduce light scattering. During the measurement, sample and reference were alternately excited with λex = 300 nm being sine-wave modulated with a modulation frequency ω varying from 200 to 450 MHz. Because of the finite lifetime of the probe, emission is delayed relative to the modulated excitation light causing a phase shift (ϕ) and amplitude modulation (m). They were used to extract the fluorescence lifetime (τ) using tan ϕ = ωτ and m = ((1 + ω2τ2)1/2)−1. One of the requirements of the proper lifetime measurement is to use a reference compound with similar lifetime as the sample. To do so, we used 2,5-diphenyl-oxazole (PPO). The lifetime of this reference is 1.4 ns in ethanol compared with 0.75 ns, as reported for DHE by Smutzer et al.49 We used the following parameters for that reference: excitation of PPO, 280−350 nm; emission, 330−480 nm; number of iterations for each point: 150; start 200 MHz, stop 450 MHz; frequencies, 15. We measured DHE’s lifetime at 25 °C because the lifetime reference value for PPO was determined at this temperature. To analyze the data, we used the Vinci Beta 1.7 Build 51 software delivered with the ISS spectrofluorometer. The program has an implemented lifetime fitting to estimate the lifetime(s) of the measured sample from the amplitude modulation and phase shift. That information is converted into lifetime and fractional amplitude of single- or multistep decay process. For a two-component model, the decay is modeled by f (t ) = A1 exp( −t /τ1) + A 2 exp(−t /τ2)

∫0

n

f (t ) dt = A1τ1(1 − e−n / τ1) + A 2 t 2(1 − e−n / τ2) (4)

which for n → ∞ becomes A1τ1 + A2τ2. Thus, we end up with ⎛ A ωτ A 2 ωτ2 ⎞ 1 1 ⎟ /(A1τ1 + A 2 τ2) N=⎜ + 2 2 1 + ω 2τ22 ⎠ ⎝ 1 + ω τ1

(5)

⎛ Aτ A 2 τ2 ⎞ 1 1 ⎟ /(A1τ1 + A 2 τ2) D=⎜ + 2 2 1 + ω 2τ22 ⎠ ⎝ 1 + ω τ1

(6)

A least-squares type analysis is used to estimate values for A1 and A2 and τ1 and τ2 by minimizing χ2, being proportional to the squared distance of measured and calculated phase shift (ϕ,ϕc) and modulation amplitude (m,mc), respectively. The analysis has been performed over phase and modulation separately for each frequency. DHE in liposomes was always fitted to a biexponential model. (See eq 3.) The amplitudeweighted average lifetime is finally calculated using τav =

A1τ12 + A 2 τ22 A1τ1 + A 2 τ2

(7)



RESULTS FROM ATOMISTIC SIMULATIONS DHE Is Almost as Good as CHOL in Condensing Membranes. We defined membrane thickness as the distance between the opposing POPC headgroup phosphorus atoms in the two monolayers, and the area per POPC was computed by considering the total area divided by the number of POPC molecules in a single leaflet. Table 1 shows the results. The presence of either sterol leads to condensation and an increase in thickness. This agrees with previous simulations and experiments.18,27,50 Comparison of the condensing and thickening effects in Table 1 shows that the effect of CHOL is only slightly stronger than that of DHE: For POPC−DHE, the area per lipid is ∼3% larger than that of POPC−CHOL, and the thickness is 2% less than that of POPC−CHOL. The fact that DHE has a lower condensing effect than CHOL has also been reported by experiments.18 Our results also compare well with those obtained by Loura et al.20 The error bars in their simulations are one order of magnitude larger than ours,

(3)

Here f(t) is the measured fluorescence decay kinetics (as extracted from the amplitude and frequency modulation) and A1 and A2 are the fractional amplitudes of the first and second decay component, respectively. The associated lifetimes are τ1 7347

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acyl-chain-ordering effect in POPC liposomes as compared with CHOL.18 Distributions of the tilt angles of the palmitoyl and oleoyl acyl chains of POPC are shown in Figure 2A, and the corresponding mean values are given in Table 1. The tilt angle of the oleoyl acyl chain is higher than that of palmitoyl due to the double bond in the oleoyl acyl chain. Figure 2A also shows that both CHOL and DHE decrease the tilt angles of both acyl chains. As Table 1 shows, CHOL decreases the average tilts of the palmitoyl and oleoyl chains by ∼34 and ∼29°, respectively. For DHE, the respective reductions are slightly less, ∼ 30 and ∼26°. Similar qualitative decrease in tilt angle has been reported in simulations of POPC−CHOL and POPC−DHE systems,20 DPPC−CHOL systems,1,11 DMPC−CHOL bilayers,51 and DPPC−CHOL and DOPC−CHOL systems.21 To study ordering of the lipid head groups, we measured the angle of the phosphorus−nitrogen (P−N) vector with respect to the bilayer normal (z axis). The results are shown in Table 1, and they agree well with previous simulations of POPC25 and POPC−CHOL systems.50 The presence of sterols makes the P−N vector slightly more inclined to the bilayer plane, and this effect is stronger in POPC−CHOL as compared with POPCDHE. CHOL Orients Slightly More Vertically than DHE in Membranes. A sterol’s tilt angle with respect to membrane normal correlates with its ordering and condensing capability:22,24,52 A larger angle and larger fluctuations are indications of decreased ordering capability and may even allow sterol flip-flop.23 The characteristic tilt angle originates from the atomic-level interactions and provides a physically meaningful and experimentally accessible quantity to characterize sterol ordering. We measured the tilt angles for both the ring structures and tails. For the ring structure, we measured the angle between the z-axis and the vector connecting the C3 methyl group to C17. (See Figure S1B in the SI for atom numbering.) Distributions are shown in Figure 2B, and the corresponding mean values are given in Table 1. The mean value of DHE’s tilt angle is 7% larger than CHOL’s. Our measurements compare well with other simulations: The average tilt angles of CHOL in CHOL− DOPC and CHOL−DPPC systems have been found to be

Figure 1. Order parameter Smol (eq 8) of the palmitoyl acyl chains in pure POPC (solid line), POPC−CHOL (dashed line), and POPC− DHE systems (dash-dotted line).

which can be attributed to their shorter analysis time (25 ns) compared with ours (100 ns). Order and Conformation of Acyl Chains Is Almost the Same for the Two Sterols. The subtle differences in the structures of CHOL and DHE are not well reflected in the density profile. To quantitatively evaluate the order of lipid chains, we used the molecular order parameter 1 Smol = (8) 2 where θn is the instantaneous angle between the bilayer normal and the nth segmental vector, that is, (Cn−1,Cn+1), the vector linking the (n − 1)th and the (n + 1)th carbon atoms along the hydrocarbon chain. The angular brackets denote both ensemble and time averages. The results are shown in Figure 1. Both sterols increase Smol at all depths. On average, POPC carbon atoms are ∼6% less ordered in POPC-DHE than in POPC−CHOL, and the difference is ∼8% for carbons 4 to 10. (See Figure S1 in the SI for atom numbering.) Our results are in agreement with experiments of Scheidt et al. that also show that DHE has lower

Figure 2. (A) Normalized distribution of the tilt angle of palmitoyl and oleoyl chains in the three different systems. The angle is defined to be the one between the z axis and the vector connecting the two ends of POPC tails, that is, C50 to C34 for palmitoyl and C52 to C15 for the oleoyl chain. (B) Normalized distribution of the tilt angles of CHOL and DHE ring structures and CHOL and DHE tails. The tilt angle of the sterols’ ring is defined to be the one between the z axis and the vector connecting the two ends of the sterols’ ring structure, that is, C3 and C17, and the tilt angle of the sterols’ tail is defined to be the one between the z axis and the vector connecting C17 to C25. 7348

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Figure 3. (A) Angle between the vector connecting C20 to C21 and the plane formed by three methyl groups C18, C13, and C20 in CHOL (solid black line) and DHE (dashed black line). The graph in red relates to the extra methyl group C29 and is the angle between the vector connecting C24 to C29 and the plane formed by three methyl groups C18, C13, and C24. For atom numbering, see Figure S1B in the SI. Inset: Schematic illustrations of CHOL (left) and DHE (right). The sterols are divided into two main parts, a ring system and a tail. The other methyl groups are budded out of the body of sterols in the way, which is shown. The extra methyl group of DHE (C29) is shown in red. (B,C) Two-dimensional radial distribution functions (RDFs) of the center of mass (COM) of CHOL and DHE versus different components of systems. (D) Lateral pressure for pure POPC, POPC−CHOL, and POPC−DHE. The horizontal axis corresponds to the bilayer normal. Inset: At the water−membrane interface (z ≈ 4.8 nm) and the headgroup region (z ≈ 5.1 nm), the attractive and repulsive contributions to the local pressure are, respectively, ∼720 bar and ∼360 bar greater in the POPC−CHOL membrane when compared with POPC−DHE membrane.

24.71 and 19.8°,42 respectively. In our CHOL−POPC, we measured 23.4°. Loura et al. found the same qualitative effect of CHOL and DHE on POPC membranes.20 Because their definition of tilt angle was different from ours, no quantitative comparison between our results with theirs can be made. To compare the orientations of CHOL and DHE in more detail, we studied the angle between the z axis and the vector connecting the two ends of the sterols’ tails, that is, C25 to C17. The results are shown in Figure 2B, and the corresponding mean values are shown in Table 1. The mean values for CHOL and DHE are 33.23 ± 0.01 and 44.84 ± 0.01°, respectively. DHE’s tail is 35% more tilted than CHOL’s. As previously shown, the tails and ring systems of sterols have different orientations in the membrane. Next, we analyzed the orientations of the methyl groups attached to the sterols’ tails: Does the C21 methyl group have a cis or trans conformation relative to the C18 methyl group of the ring system? In other words, is the C21 methyl group oriented toward the rough or the flat side of CHOL? (See Figure S1B in the SI for the atom numbering.) We computed the dihedral angle between the methyl groups C18−C13−C20−C21. These groups are not consecutive, and the desired angle is the one between the plane formed by the three methyl groups C13, C20,

and C21 and the plane formed by C18, C13, and C20. If this angle is less (greater) than 90°, C18 and C21 are (not) on the same side with respect to the ring structure. Similar analysis can be performed for C29 methyl group of the sterols’ tails using the dihedral angle between C18−C13−C24−C29. The results are shown in Figure 3A: The methyl groups protrude from the sterol body, as shown in the schematic pictures in the inset of Figure 3A. In DHE, C29 is on the same side as C18 and C19. The C21 methyl group is on the opposite side in both CHOL and DHE. In summary, DHE’s ring system and tail are 7 and 35% more tilted than CHOL’s, respectively. Despite the large difference in the orientation of sterols’ tails, the fact that membrane properties remain almost identical in both CHOL and DHE implies that the orientation of the ring system of sterols is much more important than the orientation of tails. Lateral Organization of CHOL and DHE with Phospholipids Is Qualitatively Similar. Figure 3B shows the 2-D RDFs for the centers of masses for CHOL−CHOL and DHE−DHE, and Figure 3C shows those for sterol ring structures versus the sn-1 and sn-2 chains of POPC. The peaks in the POPC−CHOL system are more pronounced in comparison with POPC-DHE: CHOL molecules are more 7349

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with the fact that a POPC lipid has a structure that has the characteristics of both DPPC and DOPC lipids; that is, it has one fully saturated and one unsaturated acyl chain. Therefore, one can conclude that the presence of double bonds in the acyl chains of lipids lessens the differences between the effect of sterols on the bilayer pressure profile. Is the change observed here relevant? Figure 3D indicates that the pressure profiles have the same peaks at exactly the same distances from membrane center. Furthermore, the quantitative differences are minor as they range from 0 to 360 bar: By considering the effect of this difference on a membrane-embedded MScL (large mechanosensitive channel) protein, a simple calculation shows that 360 bar would correspond to roughly the thermal energy. This is consistent with the difference observed between different lipids for MScL.42,57 The free-energy difference between the open and closed states of the MScL is ∼50kBT, and thus we can conclude that there is no reason to expect significant differences between CHOL and DHE on membrane protein function based on their effects on membrane elasticity. Rotational Autocorrelation of Sterols Can Be Described by a Sum of Two Exponentials: DHE Rotates with Almost the Same Speed As CHOL. The rotational correlation time is related to the average lifetime and the steady-state anisotropy of the fluorescent sample by the Perrin equation.58 The rotational correlation time can be computed using the autocorrelation function (ACF)

ordered, but the positions of the peaks are approximately the same in both CHOL and DHE systems. As seen in Figure 3C, both palmitoyl and oleoyl acyl chains have similar RDFs with the same peak positions. The only difference is that the peaks are more pronounced in the case of the POPC−CHOL system. Comparison of the peak positions (see Figure 3B,C) lets us conclude that both CHOL and DHE avoid being located in adjacent positions. Rather, they prefer to be surrounded by POPC molecules. This is in agreement with previous studies.53−55 In particular, Martinez-Seara et al.55 showed that only at high CHOL concentrations (>30 mol %) does the occurrence of close contacts (