NBD-Labeled Cholesterol Analogues in Phospholipid Bilayers

Article pubs.acs.org/JPCB

NBD-Labeled Cholesterol Analogues in Phospholipid Bilayers: Insights from Molecular Dynamics Joaõ R. Robalo,†,‡ J. P. Prates Ramalho,†,‡ and Luís M. S. Loura*,§,⊥ Departamento de Química, Escola de Ciências e Tecnologia, Universidade de Évora, Rua Romão Ramalho, 59, 7000-671 Évora, Portugal ‡ Centro de Química de Évora, Universidade de Évora, Rua Romão Ramalho, 59, 7000-671 Évora, Portugal § Faculdade de Farmácia, Universidade de Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal ⊥ Centro de Química de Coimbra, Largo D. Dinis, Rua Larga, 3004-535 Coimbra, Portugal †

ABSTRACT: Nitrobenzoxadiazole (NBD)-labeled sterols are commonly used as fluorescent cholesterol analogues in membrane biophysics. However, some experimental reports have questioned their ability to emulate the behavior of cholesterol in phospholipid bilayers. For the purpose of a detailed clarification of this matter, atomistic molecular dynamics simulations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, containing either cholesterol or one of two fluorescent cholesterol analogues, 22-NBD-cholesterol or 25-NBD-cholesterol, were carried out. It is found that these sterol probes tend to adopt conformations in which their tail-labeled fluorophore is oriented toward the lipid/water interface, with a location similar to that observed in molecular dynamics simulations of other NBD probes. This implies that in these molecules the long sterol axis is no longer aligned with the membrane normal, and preferentially adopts orientations approximately parallel to the bilayer plane. In turn, these stretched conformations, together with NBD-POPC atomic interactions, lead to slowed-down lateral diffusion of both fluorescent sterols, compared to cholesterol. From computation of the deuterium order parameter and acyl chain tilts of POPC chains for varying POPC-sterol distance, it is observed that the local ordering effect of sterol is altered in both fluorescent derivatives. In agreement with reported experimental data, both fluorescent sterols are able to increase the order of POPC at 20 mol % concentration (as some molecules adopt an upright conformation, possibly related to formation of transbilayer aggregates), albeit to a smaller extent to that of cholesterol. Altogether, this study indicates that both 22- and 25-NBD-cholesterol are unable to mimic the most important features of cholesterol’s behavior in lipid bilayers.

■

matrix.13 However, a red-edge excitation shift study suggested that the fluorophore of 25-NBD-Chol is hydrated in fluid disordered bilayers,14 which could reflect a more shallow position. Most worryingly, rapid reduction of NBD with dithionite (consistent with a superficial location) was observed for both reporters, indicating an upside-down orientation of the probes, and NMR order parameters showed much less condensation of lipid chains by both NBD-Chol probes, compared to Chol.9 Additionally, it was reported that 25-NBDChol does not distribute preferentially in the lo phase of raft model mixtures.15 To help clarify whether these probes are adequate mimics of Chol, we use atomistic molecular dynamics (MD). MD simulation is a useful tool to probe membrane structure and dynamics and interaction with foreign molecules. 16 In particular, MD can provide unique insights on the location, orientation, dynamics, and perturbing effect of fluorescent

INTRODUCTION Because of the importance of cholesterol (Chol) in membrane structure and function, as well as the usefulness of fluorescence as a biophysical tool, fluorescent sterols are an important class of membrane probes.1 Among them, two sterols labeled with the nitrobenzoxadiazole (NBD) group, 22-(7-nitrobenz-2-oxa1,3-diazol-4-yl-amino)-23,24-bisnor-5-colen-3β-ol (22-NBDChol) and 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)methyl]amino]-27-norcholesterol (25-NBD-Chol), are commercially available and have found considerable use in membrane biophysics (see structures in Figure 1).2−8 However, several studies, using different experimental approaches, have suggested that 22-NBD-Chol does not mimic Chol correctly.4,9−12 On the other hand, the ability of 25-NBD-Chol to emulate Chol is a controversial issue. Some studies have suggested that 25-NBD-Chol might be an adequate Chol analogue. For example, treatment of cell membranes with Chol-depleting agent β-cyclodextrin affected 25-NBD-Chol’s (but not 22NBD-Chol’s) fluorescence,12 and it was suggested from fluorescence quenching data that the fluorophore of 25-NBDChol located deeply in the acyl chain region of the lipid © 2013 American Chemical Society

Received: June 21, 2013 Revised: October 7, 2013 Published: October 7, 2013 13731

dx.doi.org/10.1021/jp406135a | J. Phys. Chem. B 2013, 117, 13731−13742

The Journal of Physical Chemistry B

Article

Figure 1. Molecular stuctures of POPC, Chol, 22-NBD-Chol, and 25-NBD-Chol and relevant atom numbering. Depicted in dashed lines are the molecular axes considered in this work: POPC’s sn-1; steroid ring system long axis; NBD group’s long and short axes.

membrane probes (such as diphenylhexatriene,17−19 pyrene,20−23 carbocyanine,24−26 and rhodamine27,28 probes; see refs 29 and 30 for reviews) and/or Chol analogues and related molecules31−35 (see ref 36 for a review), including fluorescent Chol derivatives.37,38 In this work, 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) bilayers containing small concentrations of Chol, 22-NBD-Chol, or 25-NBD-Chol were simulated to compare the behavior of these sterols in liquid crystalline bilayers. For the specific purpose of comparison with experimental order parameter results in the presence of probe,9 systems with 20 mol % of sterol were also simulated. Simulation Details. All simulations and analyses were carried out with the GROMACS 4.5 package.39−41 The socalled GROMACS force field (which is based on the GROMOS87 force field,42 with modifications as detailed elsewhere43−47) was used to describe all the interactions, with the following adaptations. The United-Atom (UA) topology of the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) molecule, based on the 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine description by Berger et al.,48 and a POPC bilayer coordinate file were retrieved from Dr. Peter Tieleman’s group webpage (http://moose.bio.ucalgary.ca/index.php?page= Structures_and_Topologies).20 The topology of POPC was then adapted to take into account the changed parameters for the double bond in the sn-2 acyl chain, as described elsewhere.49,50 Chol UA structure and topology were adapted from those of Höltje et al.51 (available for download at the GROMACS webpage, http://www.gromacs.org/index. php?title=Download_%26_Installation/User_contributions/ Molecule_topologies) by changing the molecule types from

CH2/CH3 to LP2/LP3 to avoid overcondensation of the bilayer, as previously suggested.52,53 NBD-Chols were modeled combining this description of Chol with previously published NBD parameters.54,55 Water was modeled with the single point charge model.56 System design was carried out with the VMD package.57 Bilayers containing either 128 POPC molecules (system 0) or 128 POPC molecules and two molecules of either Chol, 22-NBD-Chol, or 25-NBD-Chol (systems A, B, and C, respectively; 64 POPC and one sterol per leaflet) were assembled and hydrated with excess water (>30 H2O/lipid).58 Additionally, for the purpose of direct comparison with the experimental data of Scheidt et al.,9 systems containing 96 POPC and 24 sterol molecules (20 mol %, systems D−F) were also assembled and simulated. All sterols were inserted with hydroxyl groups facing the lipid/water interface and sterol long axis normal to the bilayer plane. These initial configurations underwent an energy minimization (steepest descent) followed by a short run of 10 ps (1 fs time step; remaining parameters as described for the production run) and a 100 ns (for systems 0, A, B, and C) or 300 ns (for systems D, E, and F) production simulation under the NPT ensemble and periodic boundary conditions, using Berendsen coupling schemes59 for both pressure (semi-isotropic, 1 bar, 1.0 ps coupling time) and temperature (298.15 K, 0.1 ps coupling time). Bond length constraint algorithms SETTLE (water bonds)60 and LINCS (other bonds)61 allowed the use of a 4 fs time step.62,63 Cut-offs for both Coulomb and van der Waals interactions were set at 1.0 nm, while long-range electrostatics were conducted by the Particle Mesh Ewald method.64 Bilayer thickness was defined as the average distance between the POPC P atoms in opposing 13732



Article

Figure 2. Time variation of the molecular areas of each species in the bilayer plane for POPC (0), 128 POPC:2 Chol (A), 128 POPC:2 22-NBDChol (B), and 128 POPC:2 25-NBD-Chol (C). In all plots black, red, blue, and green stand for POPC, cholesterol, 22-NBD-Chol, and 25-NBDChol, respectively.

Figure 3. Final snapshots of simulations of POPC (0), 128 POPC:2 Chol (A), 128 POPC:2 22-NBD-Chol (B), and 128 POPC:2 25-NBD-Chol (C). Note that the box in panel C was rotated for better visualization of the sterol molecules.

Chol), and C (128 POPC:2 25-NBD-Chol). The latter figure illustrates that whereas Chol stands mostly upright, with the polar hydroxyl facing the water interface and the hydrophobic ring and tail segments buried inside the membrane, both fluorescent sterols tend to adopt markedly different orientations, presenting both the sterol hydroxyl and the polar fluorophore toward the water medium. Figure 4 shows average locations of selected sterol atoms (see Figure 1 for numbering). Chol atomic positions reflect its well-known location within the membrane, with the hydroxyl group located just below the host lipid ester atoms, and the

leaflets. For calculation of molecular areas, the procedure of Hofsäß et al. was used.65 Molecular volumes were obtained with the 3 V Volume Calculator.66 Analysis used the final 60 ns (systems 0, A, B, and C) or 100 ns (systems D, E, and F) of each production simulation, unless stated otherwise.

■

RESULTS AND DISCUSSION Figure 2 shows that the time traces of area per POPC and sterol are equilibrated early on the course of the simulations, whereas Figure 3 shows the final configurations for systems 0 (128 POPC), A (128 POPC:2 Chol), B (128 POPC:2 22-NBD13733



Article

bilayer plane (θ ≈ 60−80°; Figure 5A). Figure 5B shows that the vector connecting the sterol ring center of mass to that of the NBD moiety is not aligned with the membrane normal, with narrow distributions centered around ∼110° being observed in both probes. This angular distribution, approximately parallel to the bilayer plane, reflects the similar transverse location of the steroid ring and NBD atoms in both derivatives. Because the latter have on average slightly more external locations overall than the former, θ > 90° values are dominant. Regarding the orientation of the NBD group (panels C and D of Figure 5), it is similar to that reported for NBD-PC and NBD-Cn probes.54,68 Namely, the predominance of obtuse short axis angles relative to the bilayer normal indicates that the NO2 atoms are the most external ones (which is also seen in the atom locations of Figure 4). It must be noted that due to the limited sampling arising from two sterol molecules, large emphasis should not be placed upon particular details of these distributions. In particular, bimodal distributions of 25-NBD-Chol’s steroid ring system long axis (Figure 5A) and 22-NBD-Chol’s fluorophore short axis (Figure 5B) arise from differences in the conformations sampled by the two individual probe molecules. This should not distract from the main conclusions, which are as follows: (i) the steroid ring has completely different orientation in Chol and in the fluorescence sterols, being mostly upright in the former, but approximately parallel to the membrane plane in the latter; and (ii) the orientation of the NBD group is similar in both fluorescent sterols, with the long axis along the bilayer plane and the NO2 group facing the water medium. The location and orientation data of Figures 4 and 5 point to important conformational differences between Chol and the fluorescent sterols. To try to gain insight on the molecular origins behind this diversity, we studied the occurrence of H bonds featuring sterol atoms either as donors or as acceptors. For this purpose, an H-bond for a given donor−H−acceptor

Figure 4. Average distance (d) between selected atoms of Chol, 22NBD-Chol, or 25-NBD-Chol and the bilayer center.

molecule spanning the hydrophobic region of the corresponding lipid monolayer in the direction normal to the membrane plane.31,67 Whereas the hydroxyl groups of both fluorescent sterols are located at a similar depth to that of Chol (slightly deeper for 22-NBD-Chol, slightly more shallow for 25-NBDChol), the NBD moiety of both probes does not stay near the hydrophobic core of the bilayer, and adopts a more external position than that of the −OH group. This interfacial location of the fluorophore is similar to that observed in other NBD probes.54,68 While the locations of the hydroxyl groups of the three sterols are similar, the orientation of the steroid ring systems of the fluorescent sterols is very different from that of Chol, as is clearly visible from the angular distributions of their long axes (defined in Figure 1) relative to the bilayer normal (Figure 5A). Chol’s broad tilt distribution, centered around 30°, reflects relatively unhindered rotational (librational) motion of sterol in this disordered system, and is in very good agreement with that obtained for a 70 POPC:2 Chol bilayer by Zhang et al.69 While Chol’s long axis is mostly oriented along the bilayer normal, the long axes of 22-NBD-Chol and 25-NBD-Chol lie closer to the

Figure 5. Probability density functions P(θ) of the angle between the sterol long axis (A), the vector connecting the ring systems and NBD group’s center of mass (B), the short NBD axis (C), and the long NBD axis (D) and the bilayer normal; in all plots, red, blue, and green lines refer to Chol, 22-NBD-Chol, and 25-NBD-Chol, respectively. 13734



Article

Figure 6. Fractional frequencies of hydrogen bonding between (A) sterol donors and water or POPC acceptors and (B) water donors and sterol acceptors.

triad was registered each time the donor−acceptor distance is less than 0.35 nm and the H-donor−acceptor angle was 1.2 nm (magenta) for systems A, B, and C. The blue curves are the −SCD profiles averaged over all distances. Analysis was carried over the final 50 ns.

Figure 10. Tilt of of POPC sn-1 chains for different ranges of distance R to the closest sterol molecule: R < 0.6 nm (red), 0.6 nm < R < 1.2 nm (green), and R > 1.2 nm (magenta) for systems A, B, and C. The blue curves are the average tilt distributions taking into account all configurations. Analysis was carried over the final 50 ns.

Figure 11. Time variation of the molecular areas of each species in the bilayer plane for POPC loaded with 20 mol % of Chol (left), 22-NBD-Chol (middle), or 25-NBD-Chol (right). Black, red, blue, and green stand for POPC, cholesterol, 22-NBD-Chol, and 25-NBD-Chol, respectively.

relatively rare R < 0.6 nm events. While neither the steroid (Figure 5A) nor the fluorophore (Figure 5D) rigid ring systems have their long axes aligned with the membrane normal (unlike cholesterol), it is possible that partial alignment of the NBD short axis with the bilayer normal (in the planar fluorophore, the nitro group is oriented toward the exterior of the bilayer, Figure 5C) leads to ordering of the lipid acyl chains in this region. Unlike cholesterol, insertion of both fluorescent sterols significantly reduces the order further down the acyl chain. This is understood by noting that no probe atoms reach the deep core region of the bilayer, leaving a void that is occupied by disordered POPC chains. Overall, both probes are able to order the top of nearby POPC acyl chains, while clearly disordering the lower region, and hence are not as efficient as Chol at increasing overall lipid chain order. This effect is also noticeable (albeit less evident) in the angular distributions of POPC long sn-1 axes shown in Figure 10. Whereas the distribution of

chains near Chol molecules is displaced to smaller angles, no significant differences are observed for the fluorescent sterols. 22- and 25-NBD-Chol are membrane probes typically used in small concentrations in experimental fluorescence studies (∼1−2 mol %), and it was for this reason that most of our analysis focused on membranes with a small mole fraction of fluorescent reporters. However, we also simulated bilayers containing an artificially high amount of fluorescent probe (20 mol %), for the sake of comparison with the NMR data of Scheidt et al.9 measured for this composition. Because of the slower dynamics in these ordered systems, a 300 ns simulation time was used. Figure 11 depicts the time evolution of the molecular areas in these bilayers, which indicate that this time window ensures their convergence to the average values given in Table 1. Scheidt et al.9 measured |SCD| profiles for bilayers of POPCd31 (POPC with a perdeuterated sn-1 chain) with and without 13737



Article

Figure 12. Deuterium order parameters (−SCD) of POPC sn-1 (A) and sn-2 (B) chains for pure POPC (black), POPC/20 mol % 22-NBD-Chol (blue), POPC/20 mol % 25-NBD-Chol (green), and POPC/20 mol % Chol (red).

Figure 13. Final snapshots of simulations of POPC bilayers loaded with 20 mol % of Chol (left), 22-NBD-Chol (middle), or 25-NBD-Chol (right). Note that the box in the middle panel was rotated for better visualization of the sterol molecules.

20 mol % Chol) is obtained, while the opposite trend is verified for the bilayer thickness. On the other hand, the sterol molecular area increases from Chol to 22-NBD-Chol to 25NBD-Chol, as expected from their molecular structures. It is also noteworthy that NBD sterols appear to have a maximal ordering effect nearer the top of the acyl chain, compared to Chol. This is clearer in the sn-1 profile: the POPC C atoms for which the enhancement of −SCD is maximal upon addition of either 22-NBD-Chol or 25-NBD-Chol are C39− C41 (the fifth to seventh acyl chain atoms), whereas the corresponding atoms for the POPC/Chol system are C42− C44 (8th to 10th acyl chain atoms). These qualitative differences are related to the different effects of the sterols on nearby POPC atoms in dilute systems A−C (Figure 8), and can be explained taking into account the differences in penetration inside the bilayer among the three sterols, especially of their rigid ring systems. Figure 13 depicts the final snapshots corresponding to the systems with 20 mol % Chol. These snapshots illustrate that while Chol molecules adopt an upright orientation, with the hydroxyl group facing the lipid/water interface, a large proportion of 22-NBD-Chol molecules are oriented approximately parallel to the bilayer plane, keeping both the hydroxyl and the NBD group accessible for interactions with the POPC and solvent charged atoms, as observed in the dilute systems described above, and in agreement with the dithionite quenching results of Scheidt et al.9 An intermediate situation is verified for 25-NBD-Chol, in which a comparatively larger number of molecules have an upright conformation, which, together with the structural rigidity of the sterol and NBD ring systems, explains why this sterol is more efficient at ordering the bilayer than 22-NBD-Chol. One could wonder what is the driving force behind these upright conformations, which are virtually absent in the sterol-dilute systems B and C. Visual inspection revealed that a number of these upright molecules

20 mol % of Chol, 22-NBD-Chol, and 25-NBD-Chol. They verified that whereas all sterols induced an increase in the order parameter profiles, the effect of the NBD-labeled sterols is considerably weaker than that of Chol, especially for 22-NBDChol. Figure 12 shows the order profiles obtained in this study in the absence (system 0) and presence (systems D-F) of 20 mol % of each sterol. The profiles corresponding to POPC/ Chol agree well with both experimental and simulation results.53 To compare the relative ordering effect of each probe, Scheidt et al. assumed a linear relationship between average order parameter and molar content of native cholesterol, estimating the cholesterol concentration that has the same lipid condensation effect as 20 mol % of fluorescent sterol, as: equiv % Chol =

−SCD(POPC/20% sterol) − ( −SCD(POPC)) × 20% −SCD(POPC/20% Chol) − ( −SCD(POPC)) (3)

Using this equation, the values 1.4% and 2.7% were obtained for 22- and 25-NBD-Chol, respectively. These values are significantly lower than those that could be calculated from our simulations (6.1% and 10.6%, respectively). However, it must be emphasized that both experimental and simulation estimates are calculated from differences between order parameter values, and it is clear and encouraging that our results reproduce the trend observed by Scheidt et al.,9 with both NBD-Chols inducing a small ordering effect (especially in the case of 22NBD-Chol), when compared to the strong ordering induced by an equal amount of Chol. This trend could also be hinted at from the area/POPC and bilayer thickness data of Table 1 and Figure 11. For the former parameter, the order a(pure POPC) > a(POPC/20 mol % 22NBD-Chol) > a(POPC/20 mol % 25-NBD-Chol) > a(POPC/ 13738



Article

the small length (and therefore limited bending ability) of the alkyl chain that links the fluorophore to the steroid ring system, the implication of the snorkeling of the former group is that the latter becomes highly tilted, and, as such, is expected to lose most of its lipid ordering ability.34 In fact, our work demonstrates that, for low concentrations, both molecules tend to orient the sterol ring system almost parallel to the bilayer plane, unlike Chol, which displays an upright configuration. Both probes order bilayers to a smaller extent than Chol (and in fact induce local disordering near the acyl chains’ ends in POPC bilayers). Fluorescent sterols also diffuse slower than Chol in POPC bilayers, on account of NBD interactions with the host lipid and their almost horizontal conformation in the bilayer. 25-NBD-Chol actually presents no major advantages over 22-NBD-Chol, despite the former’s longer side chain, more similar to that of Chol. Interestingly, for very high sterol concentration (20 mol %), a significant proportion of fluorescent sterols adopts an upright conformation, which is possibly related to transbilayer sterol aggregation. Because of this phenomenon, both fluorescent sterols (especially 25-NBD-Chol, possibly due to its longer chain) are capable of ordering the bilayer for the POPC/20 mol % sterol composition, but still considerably less so than Chol. As recently demonstrated by ourselves, intrinsically fluorescent sterols cholestatrienol and dehydroergosterol display a much closer behavior to that of Chol.38 If convenient photophysical properties (such as long wavelength emission, high photostability, and brightness) are sought, an alternative would be to use BODIPY-Cholesterol. A recent MD study with this probe highlighted the similarity between the behaviors of this sterol and that of Chol, despite the existence of a residual population of probe characterized by a more tilted steroid ring system and a slight upward loop of the BODIPY moiety.37

have the NBD group located inside the lipid bilayer, involved in transmembrane (tail−tail) aggregation of molecules in opposing leaflets, reminiscent of that proposed in published experimental studies that showed spectral alterations (due to excitonic interaction) for large probe concentrations.7,89 Thus, a high sterol mole fraction and the resulting enhanced probability of formation of aggregates could provide a mechanism for the reorientation of sterol molecules to an upright conformation. It is plausible that these transbilayer aggregates form more easily for 25-NBD-Chol than for 22NBD-Chol, due to the former’s longer alkyl chain, and this could be related to the higher fraction of upright molecules and ordering induced by 25-NBD-Chol compared to 22-NBDChol. Because of the small biophysical relevance of these systems with a very high fluorescent label concentration, a detailed characterization of fluorophore location/orientation was not undertaken.

■

CONCLUSIONS Despite their popularity as fluorescent membrane probes, a number of literature studies have suggested that NBD-sterols might differ markedly from Chol regarding both orientation in the bilayer9 and partition between liquid ordered and disordered phases,10,15 and a definite assessment of the utility of these probes was still lacking in the literature. This was the main objective of our work, together with a clarification of whether 25-NBD-Chol behaved significantly better than 22NBD-Chol, as claimed in some reports.12,13 At this point, we would like to comment on the potential limitations of this work. As with all MD simulation studies, ours is dependent on the force fields and the simulation time. The latter is expected to be particularly important in systems D−F, which contain 20 mol % sterol and therefore have slower dynamics. For this reason, we used extended simulations in these systems (300 ns), which may still not suffice for ensuring appropriate sampling of the probe-containing bilayers. Regarding the used force field, it has been remarked recently that commonly used phospholipid and Chol parametrizations, based on the Berger et al.48 and Höltje et al.51 parameters, respectively, do not reproduce experimental order parameters measured for the atoms in the lipid head group and sterol alkyl chains (respectively).53 Although we acknowledge this possibility, this important methodological matter requires specific attention and should be investigated separately, and therefore lies out of the scope of the current paper. While these limitations might affect quantitatively some of the results presented here, the following conclusions can be safely extracted. The results presented here indicate beyond doubt that both NBD-labeled sterols are inadequate analogues of Chol. Because of their polar group located at the tail end, they differ considerably from the parent molecule in orientation and ordering capability. A looping behavior is observed in phospholipids with a modified acyl chain, bearing polar functional groups such as hydroperoxide or aldehyde, in lipid peroxidation products90,91 (which have effects on membrane properties that bear important physiological/pathological consequences, see ref 92 for a recent review), or the NBD fluorophore itself, in NBD-PC fluorescent lipid analogues,54 which tend to bend the altered chain in the direction of the lipid/water interface, thus enabling formation of favorable H bonding or direct electrostatic interactions. This work shows that a similar phenomenon occurs for NBD-tail labeled Chol analogues. In this case, given

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +351 239488485. Fax: +351 239827126. E-mail: lloura@ ff.uc.pt. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors acknowledge funding by FEDER, through the COMPETE program, and by FCT (Fundaçaõ para a Ciência e a Tecnologia, Portugal), project reference FCOMP-01-0124FEDER-010787 (FCT PTDC/QUI-QUI/098198/2008). J.R.R. acknowledges FCT and the same project for a research grant.

■

REFERENCES

(1) Wüstner, D. Fluorescent Sterols as Tools in Membrane Biophysics and Cell Biology. Chem. Phys. Lipids 2007, 146, 1−25. (2) Mukherjee, S.; Chattopadhyay, A. Monitoring Cholesterol Organization in Membranes at Low Concentrations Utilizing the Wavelength-Selective Fluorescence Approach. Chem. Phys. Lipids 2005, 134, 79−84. (3) Slotte, J. P. Effect of Sterol Structure on Molecular Interactions and Lateral Domain Formation in Monolayers Containing Dipalmitoyl Phosphatidylcholine. Biochim. Biophys. Acta 1995, 1237, 127−134. (4) Slotte, J. P.; Mattjus, P. Visualization of Lateral Phases in Cholesterol and Phosphatidylcholine Monolayers at the Air/Water Interface − A Comparative Study with Two Different Reporter Molecules. Biochim. Biophys. Acta 1995, 1254, 22−29.

13739



Article

(5) Kheirolomoom, A.; Ferrara, K. W. Cholesterol Transport from Liposomal Delivery Vehicles. Biomaterials 2007, 28, 4311−4320. (6) Slotte, J. P. Lateral Domain Formation in Mixed Monolayers Containing Cholesterol and Dipalmitoylphosphatidylcholine or Npalmitoylsphingomyelin. Biochim. Biophys. Acta 1995, 1235, 419−427. (7) Mukherjee, S.; Chattopadhyay, A. Membrane Organization at Low Cholesterol Concentrations: A Study Using 7-Nitrobenz-2-oxa1,3-diazol-4-yl-Labeled Cholesterol. Biochemistry 1996, 35, 1311− 1322. (8) Rukmini, R.; Rawat, S. S.; Biswas, S. C.; Chattopadhyay, A. Cholesterol Organization in Membranes at Low Concentrations: Effects of Curvature Stress and Membrane Thickness. Biophys. J. 2001, 81, 2122−2134. (9) Scheidt, H. A.; Müller, P.; Herrmann, A.; Huster, D. The Potential of Fluorescent and Spin-Labeled Steroid Analogs to Mimic Natural Cholesterol. J. Biol. Chem. 2003, 278, 45563−45569. (10) Loura, L. M. S.; Fedorov, A.; Prieto, M. Exclusion of a Cholesterol Analog from the Cholestrol-Rich Phase in Model Membranes. Biochim. Biophys. Acta 2001, 1511, 236−243. (11) Ishii, H.; Shimanouchi, T.; Umakoshi, H.; Walde, P.; Kuboi, R. Analysis of the 22-NBD-Cholesterol Transfer Between Liposome Membranes and its Relation to the Intermembrane Exchange of 25Hydroxycholesterol. Biointerfaces 2010, 77, 117−121. (12) Ostašov, P.; Skýkora, J.; Brejchová, J.; Olżyńska, A.; Hof, M.; Svoboda, P. FLIM Studies of 22- and 25-NBD-Cholesterol in Living HEK293 Cells: Plasma Membrane Change Induced by Cholesterol Depletion. Chem. Phys. Lipids 2013, 167−168, 62−69. (13) Chattopadhyay, A.; London, E. Parallax Method for Direct Measurement of Membrane Penetration Depth Utilizing Fluorescence Quenching by Spin-Labeled Phospholipids. Biochemistry 1987, 26, 39− 45. (14) Chattopadhyay, A.; Mukherjee, S. Red Edge Excitation Shift of a Deeply Embedded Membrane Probe: Implications in Water Penetration in the Bilayer. J. Phys. Chem. B 1999, 103, 8180−8185. (15) Shaw, J. E.; Epand, R. F.; Epand, R. M.; Li, Z.; Bittman, R. Correlated Fluorescence-Atomic Force Microscopy of Membrane Domains: Structure of Fluorescence Probes Determines Lipid Localization. Biophys. J. 2006, 90, 2170−2178. (16) Lyubartsev, A. P.; Rabinovich, A. L. Recent Development in Computer Simulations of Lipid Bilayers. Soft Matter 2011, 7, 25−39. (17) Repáková, J.; Č apková, P.; Holopainen, J. M.; Vattulainen, I. Distribution, orientation and dynamics of DPH probes in DPPC bilayer. J. Phys. Chem. B 2004, 108, 13438−13448. (18) Repáková, J.; Holopainen, J. M.; Morrow, M. R.; McDonald, M. C.; Č apková, P.; Vattulainen, I. Influence of DPH on the structure and dynamics of a DPPC bilayer. Biophys. J. 2005, 88, 3398−3410. (19) Franová, M.; Repáková, J.; Č apková, P.; Holopainen, J. M.; Vattulainen, I. Effects of DPH on DPPC-cholesterol membranes with varying concentrations of cholesterol: from local perturbations to limitations in fluorescence anisotropy experiments. J. Phys. Chem. B 2010, 114, 2704−2711. (20) Hoff, B.; Strandberg, E.; Ulrich, A. S.; Tieleman, D. P.; Posten, C. 2H-NMR Study and Molecular Dynamics Simulation of the Location, Alignment and Mobility of Pyrene in POPC Bilayers. Biophys. J. 2005, 88, 1818−1827. (21) Repáková, J.; Holopainen, J. M.; Karttunen, M.; Vattulainen, I. Influence of pyrene-labeling on fluid lipid membranes. J. Phys. Chem. B 2006, 110, 15403−15410. (22) Curdová, J.; Č apková, P.; Plásek, J.; Repáková, J.; Vattulainen, I. Free pyrene probes in gel and fluid membranes: perspective through atomistic simulations. J. Phys. Chem. B 2007, 111, 3640−3650. (23) Loura, L. M. S.; do Canto, A. M. T. M.; Martins, J. Sensing hydration and behavior of pyrene in POPC and POPC/cholesterol bilayers: a molecular dynamics study. Biochim. Biophys. Acta 2013, 1828, 1094−1101. (24) Gullapalli, R. R.; Demirel, M. C.; Butler, P. J. Molecular dynamics simulations of DiI-C18(3) in a DPPC lipid bilayer. Phys. Chem. Chem. Phys. 2008, 10, 3548−3560.

(25) Muddana, H. S.; Gullapalli, R. R.; Manias, E.; Butler, P. J. Atomistic simulation of lipid and DiI dynamics in membrane bilayers under tension. Phys. Chem. Chem. Phys. 2011, 13, 1368−1378. (26) Ackerman, D. G.; Heberle, F. A.; Feigenson, G. W. Limited perturbation of a DPPC bilayer by fluorescent lipid probes: a molecular dynamics study. J. Phys. Chem. B 2013, 117, 4844−4852. (27) Kyrychenko, A. A molecular dynamics model of rhodaminelabeled phospholipid incorporated into a lipid bilayer. Chem. Phys. Lett. 2010, 485, 95−99. (28) Skaug, M. J.; Longo, M. L.; Faller, R. Computational studies of Texas Red-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine − model building and applications. J. Phys. Chem. B 2009, 113, 8758− 8766. (29) Loura, L. M. S.; Ramalho, J. P. P. Fluorescent Membrane Probes’ Behavior in Lipid Bilayers: Insights from Molecular Dynamics Simulations. Biophys. Rev. 2009, 1, 141−148. (30) Loura, L. M. S.; Ramalho, J. P. P. Recent Developments in Molecular Dynamics Simulations of Fluorescent Membrane Probes. Molecules 2011, 16, 5437−5452. (31) Smondyrev, A. M.; Berkowitz, M. L. Molecular Dynamics Simulation of the Structure of Dimyristoylphosphatidylcholine Bilayers with Cholesterol, Ergosterol and Lanosterol. Biophys. J. 2001, 80, 1649−1658. (32) Ollila, O. H. S.; Róg, T.; Karttunen, M.; Vattulainen, I. Role of Sterol Type on Lateral Pressure Profiles of Lipid Membranes Affecting Membrane Protein Functionality: Comparison Between Cholesterol, Desmosterol, 7-Dehydrocholesterol and Ketosterol. J. Struct. Biol. 2007, 159, 311−323. (33) Vainio, S.; Jansen, M.; Koivusalo, M.; Róg, T.; Karttunen, M.; Vattulainen, I.; Ikonen, E. Significance of Sterol Structural Specificity. Desmosterol cannot replace Cholesterol in Lipid Rafts. J. Biol. Chem. 2006, 281, 348−355. (34) Aittoniemi, J.; Róg, T.; Niemela, P.; Pasenkiewicz-Gierula, M.; Karttunen, M.; Vattulainen, I. Tilt: Major Factor in Sterols’ Ordering Capability in Membranes. J. Phys. Chem. B 2006, 110, 25562−25564. (35) Róg, T.; Stimson, L. M.; Pasenkiewicz-Gierula, M.; Vattulainen, I.; Karttunen, M. Replacing the cholesterol hydroxyl group with the ketone group facilitates sterol flip-flop and promotes membrane fluidity. J. Phys. Chem. B 2008, 112, 1946−1952. (36) Róg, T.; Pasenkiewicz-Gierula, M.; Vattulainen, I.; Karttunen, M. Ordering Effects of Cholesterol and its Analogues. Biochim. Biophys. Acta 2009, 1788, 97−121. (37) Hölttä-Vuori, M.; Uronen, R.; Repakova, J.; Salonen, E.; Vattulainen, I.; Panula, P.; Li, Z.; Bittman, R.; Ikonen, E. BODIPYCholesterol: A New Tool to Visualize Sterol Trafficking in Living Cells and Organisms. Traffic 2008, 9, 1839−1849. (38) Robalo, J. R.; do Canto, A. M. T. M.; Carvalho, A. J. P.; Ramalho, J. P. P.; Loura, L. M. S. Behavior of Fluorescent Cholesterol Analogues Dehydroergosterol and Cholestatrienol in Lipid Bilayers: a Molecular Dynamics Study. J. Phys. Chem. B 2013, 117, 5806−5819. (39) Berendsen, H.; van der Spoel, D.; van Drunen, R. GROMACS: a Message-Passing Parallel Molecular Dynamics Implementation. Comput. Phys. Commun. 1995, 91, 43−56. (40) van der Spoel, D.; Lindhal, E.; Hess, B.; Groenhof, G.; Mark, A.; Berendsen, H. GROMACS: Fast, Flexible and Free. J. Comput. Chem. 2005, 26, 1701−1718. (41) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theory Comput. 2008, 4, 435−447. (42) van Gunsteren, W. F., Berendsen, H. J. C. Gromos-87 manual; Biomos BV: Nijenborgh 4, 9747 AG Groningen, The Netherlands, 1987. (43) van Buuren, A. R.; Marrink, S. J.; Berendsen, H. J. C. A molecular dynamics study of the decane/water interface. J. Phys. Chem. 1993, 97, 9206−9212. (44) Mark, A. E.; van Helden, S. P.; Smith, P. E.; Janssen, L. H. M.; van Gunsteren, W. F. Convergence properties of free energy calculations: α-cyclodextrin complexes as a case study. J. Am. Chem. Soc. 1994, 116, 6293−6302. 13740



Article

(45) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926−935. (46) van Buuren, A. R.; Berendsen, H. J. C. Molecular Dynamics simulation of the stability of a 22 residue alpha-helix in water and 30% trifluoroethanol. Biopolymers 1993, 33, 1159−1166. (47) Liu, H.; Müller-Plathe, F.; van Gunsteren, W. F. A force field for liquid dimethyl sulfoxide and liquid properties of liquid dimethyl sulfoxide calculated using molecular dynamics simulation. J. Am. Chem. Soc. 1995, 117, 4363−4366. (48) Berger, O.; Edholm, O.; Jähnig, F. Molecular Dynamics Simulations of a Fluid Bilayer of Dipalmitoylphosphatidylcholine at Full Hydration, Constant Pressure and Constant Temperature. Biophys. J. 1997, 72, 2002−2013. (49) Bachar, M.; Brunelle, P.; Tieleman, D. P.; Rauk, A. Molecular dynamics simulation of a polyunsaturated lipid bilayer susceptible to lipid peroxidation. J. Phys. Chem. B 2004, 108, 7170−7179. (50) Martinez-Seara, H.; Róg, T.; Karttunen, M.; Reigada, R.; Vattulainen, I. Influence of cis double-bond parametrization on lipid membrane properties: How seemingly insignificant details in forcefield change even qualitative trends. J. Chem. Phys. 2008, 129, 105103. (51) Höltje, M.; Förster, T.; Brandt, B.; Engels, T.; von Rybinski, W.; Höltje, H. Molecular Dynamics Simulations of Stratum Corneum Lipid Models: Fatty Acids and Cholesterol. Biochim. Biophys. Acta 2001, 1511, 156−167. (52) Tieleman, D. P.; MacCallum, J. L.; Ash, W. L.; Kandt, C.; Xu, Z. T.; Monticelli, L. Membrane protein simulations with a united-atom lipid and all-atom protein model: lipid−protein interactions, side chain transfer free energies and model proteins. J. Phys.: Condens. Matter 2006, 18, S1221−S1234. (53) Ferreira, T. M.; Coreta-Gomes, F.; Ollila, O. H. S.; Moreno, M. J.; Vaz, W. L. C.; Topgaard, D. Cholesterol and POPC segmental order parameters in lipid membranes: solid state 1H-13C NMR and MD simulation studies. Phys. Chem. Chem. Phys. 2013, 15, 1976−1989. (54) Loura, L. M. S.; Prates Ramalho, J. P. Location and Dynamics of Acyl Chain NBD-Labeled Phosphatidylcholine (NBD-PC) in DPPC Bilayers. A Molecular Dynamics and Time-Resolved Fluorescence Anisotropy Study. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 467− 478. (55) Loura, L. M. S.; Carvalho, A. J. P.; Ramalho, J. P. P. Direct Calculation of Förster Orientation Factor of Membrane Probes by Molecular Simulation. J. Mol. Struct.: THEOCHEM 2010, 946, 107− 112. (56) Berendsen, H.; Postma, J.; van Gunsteren, W.; Hermans, J. Intermolecular Forces; Reidel: Dordrecht, The Netherlands, 1981. (57) Humphrey, W.; Dalke, A.; Schulten, K. VMD − Visual Molecular Dynamics. J. Mol. Graphics 1996, 14, 33−38. (58) Murzyn, K.; Róg, T.; Jezierski, G.; Takaoka, Y.; PasenkiewiczGierula, M. Effects of Phospholipid Unsaturation on the Membrane/ Water Interface: a Molecular Simulation Study. Biophys. J. 2001, 81, 170−183. (59) Berendsen, H.; Postma, J.; DiNola, A.; Haak, J. Molecular Dynamics with Coupling to an External Bath. J. Chem. Phys. 1984, 81, 3684−3690. (60) Myamoto, S.; Kollman, P. SETTLE an Analytical Version of the SHAKE and RATTLE Algorithms for Rigid Water Models. J. Comput. Chem. 1992, 13, 952−962. (61) Hess, B.; Bekker, H.; Berendsen, H.; Fraaije, J. LINCS: a Linear Constraint Solver for Molecular Simulations. J. Comput. Chem. 1997, 18, 1463−1472. (62) Feenstra, K.; Hess, B.; Berendsen, H. Improving efficiency of Large Timescale Molecular Dynamics Simulations of Hydrogen-Rich Systems. J. Comput. Chem. 1999, 20, 786−798. (63) Anézo, C.; de Vries, A. H.; Höltje, H. D.; Tieleman, D. P.; Marrink, S. J. Methodological Issues in Lipid Bilayer Simulations. J. Phys. Chem. B 2003, 107, 9424−9433. (64) Essman, U.; Perela, L.; Berkowitz, M.; Darden, T.; Lee, H.; Pedersen, L. A Smooth Particle Mesh Ewald Method. J. Chem. Phys. 1995, 103, 8577−8592.

(65) Hofsäß, C.; Lindahl, E.; Edholm, O. Molecular Dynamics Simulations of Phospholipid Bilayers with Cholesterol. Biophys. J. 2003, 84, 2192−2206. (66) Voss, N.; Gerstein, M. 3V: Cavity, Channel and Cleft Volume Calculator and Extractor. Nucleic Acid Res. 2010, 38, 555−562. (67) Smondyrev, A. M.; Berkowitz, M. L. Structure of DPPC/ Cholesterol bilayer at low and high cholesterol concentrations: molecular dynamics simulation. Biophys. J. 1999, 77, 2075−2089. (68) Filipe, H. A. L.; Moreno, M. J.; Loura, L. M. S. Interaction of 7Nitrobenz-2-oxa-1,3-diazol-4-yl-Labeled Fatty Amines with 1-Palmitoyl, 2-Oleoyl-sn-glycero-3-phosphocholine Bilayers: A Molecular Dynamics Study. J. Phys. Chem. B 2011, 115, 10109−10119. (69) Zhang, Z.; Lu, L.; Berkowitz, M. L. Energetics of cholesterol transfer between lipid bilayers. J. Phys. Chem. B 2008, 112, 3807−3811. (70) Chattopadhyay, A. Chemistry and biology of N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes. Chem. Phys. Lipids 1990, 53, 1−15. (71) Oghalai, J. S.; Tran, T. D.; Raphael, R. M.; Nakagawa, T.; Brownell, W. E. Transverse and lateral mobility in outer hair cell lateral wall membranes. Hear. Res. 1999, 135, 19−28. (72) Christova, Y. T.; James, P. S.; Cooper, T. G.; Jones, R. Lipid diffusion in the plasma membrane of mouse spermatozoa. Changes during epididymal maturation, effects of pH, osmotic pressure and knockout of the c-ros gene. J. Androl. 2002, 23, 384−392. (73) Pucadyil, T. J.; Mukherjee, S.; Chattopadhyay, A. Organization and dynamics of NBD-labeled lipids in membranes analyzed by fluorescence recovery after photobleaching. J. Phys. Chem. B 2007, 111, 1975−1983. (74) Grzybek, M.; Kubiak, J.; Łach, A.; Przybyło, M.; Sikorski, A. F. A raft-associated species of phosphatidylethanolamine interacts with cholesterol comparably to sphingomyelin. A Langmuir-Blodgett monolayer study. PLoS One 2009, 4, e5053. doi:10.1371/journal.pone.0005053. (75) Lindahl, E.; Edholm, O. Molecular dynamics simulation of NMR relaxation rates and slow dynamics in lipid bilayers. J. Chem. Phys. 2001, 115, 4938−4950. (76) Kochy, T.; Bayerl, T. M. Lateral diffusion coefficients of phospholipids in spherical bilayers on a solid support measured by 2Hnuclear-magnetic-resonance relaxation. Phys. Rev. E 1993, 47, 2109− 2116. (77) Bockmann, R.; Hac, A.; Heimburg, T.; Grubmuller, H. Effects of Sodium Chloride on a Lipid Bilayer. Biophys. J. 2003, 85, 1647−1655. (78) Lantzsch, G.; Binder, H.; Heerklotz, H.; Wendling, M.; Klose, G. Surface Areas and Packing Constraints in POPC/C12EOn Membranes. Biophys. Chem. 1996, 58, 289−302. (79) Konig, B.; Dietrich, U.; Klose, G. Hydration and Structural Properties of Mixed Lipid/Surfactant Model Membranes. Langmuir 1997, 13, 525−532. (80) Kučerka, N.; Tristram-Nagle, S.; Nagle, J. F. Structure of fully hydrated fluid phase lipid bilayers with monounsaturated chains. J. Membr. Biol. 2005, 208, 193−202. (81) Ollila, S.; Hyvönen, M. T.; Vattulainen, I. Polyunsaturation in lipid membranes: dynamic properties and lateral pressure profiles. J. Phys. Chem. B 2007, 111, 3139−3150. (82) Greenwood, A. I.; Tristram-Nagle, S.; Nagle, J. F. Partial Molecular Volumes of Lipids and Cholesterol. Chem. Phys. Lipids 2006, 143, 1−10. (83) Zhang, Y.; Luo, Y.; Deng, Y.; Mu, Y.; Wei, G. Lipid interaction and membrane perturbation of human islet amyloid polypeptide monomer and dimer by molecular dynamics simulations. PLoS One 2012, 7, e38191. doi: 10.1371/journal.pone.0038191. (84) Martins do Canto, A. M. T.; Palace Carvalho, A. J.; Prates Ramalho, J. P.; Loura, L. M. S. Effect of Amphipathic HIV Fusion Inhibitor Peptides on POPC and POPC/Cholesterol Membrane Properties: A Molecular Simulation Study. Int. J. Mol. Sci. 2013, 14, 14724−14743. (85) Loura, L. M. S.; Fernandes, F.; Fernandes, A. C.; Prates Ramalho, J. P. Effects of fluorescent probe NBD-PC on the structure, dynamics and phase transition of DPPC. A molecular dynamics and 13741



Article

differential scanning calorimetry study. Biochim. Biophys. Acta 2008, 1778, 491−501. (86) Seelig, J.; Waespesarcevic, N. Molecular Order in cis and trans Unsaturated Phospholipid Bilayers. Biochemistry 1978, 17, 3310−3315. (87) Klose, G.; Madler, B.; Schafer, H.; Schneider, K. Structural Characterization of POPC and C12E4 in their Mixed Membranes at Reduced Hydration by Solid State 2H NMR. J. Phys. Chem. B 1999, 103, 3022−3029. (88) Gurtovenko, A.; Anwar, J. Interaction of Ethanol with Biological Membranes: the Formation of Non-Bilayer Structures within the Membrane Interior and their Significance. J. Phys. Chem. B 2009, 113, 1983−1992. (89) Loura, L. M. S.; Prieto, M. Dehydroergosterol structural organization in aqueous medium and in a model system of membranes. Biophys. J. 1997, 72, 2226−2236. (90) Wong-ekkabut, J.; Xu, Z.; Triampo, W.; Tang, I. M.; Tieleman, D. P.; Monticelli, L. Effect of lipid peroxidation on the properties of lipid bilayers: a molecular dynamics study. Biophys. J. 2007, 93, 4225− 4236. (91) Jarerattanachat, V.; Karttunen, M.; Wong-ekkabut, J. Molecular Dynamics Study of Oxidized Lipid Bilayers in NaCl Solution. J. Phys. Chem. B 2013, 117, 8490−8501. (92) Volinsky, R.; Kinnunen, P. K. J. Oxidized phosphatidylcholines in membrane-level cellular signaling: from biophysics to physiology and molecular pathology. FEBS J. 2013, 280, 2806−2816.

13742


NBD-Labeled Cholesterol Analogues in Phospholipid Bilayers

Recommend Documents