Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes

RESEARCH ARTICLE Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes Huixia LuID ☯ , Jordi Martı ´ID ☯ * Department of Physics, Technical University of Catalonia-Barcelona Tech. Barcelona, Catalonia, Spain ☯These authors contributed equally to this work. *[email protected] Abstract The characterization of interactions between melatonin, one main ingredient of medicines regulating sleeping rhythms, and basic components of cellular plasma membranes (phospholipids, cholesterol, metal ions and water) is very important to elucidate the main mechanisms for the introduction of melatonin into cells and also to identify its local structure and microscopic dynamics. Molecular dynamics simulations of melatonin inside mixtures of dimyristoylphosphatidylcholine and cholesterol in NaCl solution at physiological concentration have been performed at 303.15 K to systematically explore melatonin-cholesterol, melatonin-lipid and melatonin-water interactions. Properties such as the area per lipid and thickness of the membrane as well as selected radial distribution functions, binding free energies, angular distributions, atomic spectral densities and translational diffusion of melatonin are reported. The presence of cholesterol significantly affects the behavior of melatonin, which is mainly buried into the interfaces of membranes. Introducing cholesterol into the system helps melatonin change from folded to extended configurations more easily. Our results suggest that there exists a competition between the binding of melatonin to phospholipids and to cholesterol by means of hydrogen-bonds. Spectral densities of melatonin reported in this work, in overall good agreement with experimental data, revealed the participation of each atom of melatonin to its complete spectrum. Melatonin self-diffusion coefficients are of the order of 10 −7 cm 2 /s and they significantly increase when cholesterol is addeed to the membrane. Introduction Cell membranes are biological structures composed of hundreds of different classes of lipids, sterols and proteins, acting as boundaries of cells [1]. The composition of a membrane can affect its fluidity and structure, so that addition of different molecules to the membrane may be able to change substantially its properties [2,3]. Furthermore, the human cell membrane acts as an external selective container of the cell elements, so it is very important to know its structural and dynamical properties concerning new, external molecules appearing at the PLOS ONE | https://doi.org/10.1371/journal.pone.0224624 November 7, 2019 1 / 20 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Lu H, Martı ´J (2019) Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes. PLoS ONE 14(11): e0224624. https://doi.org/10.1371/ journal.pone.0224624 Editor: Dennis Salahub, University of Calgary, CANADA Received: July 9, 2019 Accepted: October 17, 2019 Published: November 7, 2019 Copyright: ©2019 Lu, Martı ´. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by a doctoral grant from the China Scholarship Council (CSC) awarded to H.L. (grant number 201607040059), a grant from the Spanish Ministry of Economy and Knowledge (grant FIS2015-66879-C2-1-P), awarded to J.M., to computational resources provided by the Barcelona Supercomputing Center (grants FI-2018-2-0018 and FI-2018-3-0023) awarded to both authors, and a grant from the Spanish Ministry of Science, Innovation and interface of membrane bilayer systems. For instance, recent studies have shown that the role of some proteins and their interactions with components of plasma membranes is extremely important to understand the mechanisms of protein anchoring at the membrane that can lead to oncogenesis [4]. In this work we have focused our efforts on the study of the binding of a small molecule, the neurohormone melatonin (MEL) [5,6] at a simplified model cell membrane. This is a process that aims to improve our understanding of the basic mechanisms of molecular binding and crossing of biological membranes by small solutes and the interactions with their surroundings. Nevertheless, reproducing cell membranes of mammalians using realistic computational methods is a highly difficult task [7]. In particular, all-atom simulations involve the computation of interactions between Nparticles, where Nis of the order of 10 5 , so that for a single run computational times scale as N(N−1) and make the simulation a challenging task, often requiring the use of high-performance computational facilities. Given the cost of such realistic calculations involving a wide variety of components, well beyond the scope of the present work, we must assume some simplifications. One of most usual is to consider a single class of lipids. In the present work, we have considered a model membrane made with cholesterol and only one type of phospholipid, dimyristoylphosphatidylcholine (DMPC), extensively studied in the literature from the experimental and also computational points of view [8–10] and that belongs to the class of phosphatidylcholines, basic components of lecithin, a substance forming egg yolk and soy. Plenty of experimental and computational work on mixtures of cholesterol and melatonin at phosphatidylcholine membranes has been published to analyze the joint effects of the two species (see for instance [11,12]), allowing us to ensure the reliability of our simulations since, as we will show below, the force field employed in the present work has revealed to be very successful in describing the physical properties of a DMPC membrane. The benefits of MEL in the human body have recently drawn much attention in different fields. MEL is a natural hormone secreted by the pineal gland well known to regulate biological rhythms [13], to induce sleep [14], and that can also contribute to protect the organism from Alzheimer disease [15]. MEL is reported to induce/promote complex antioxidative and DNA repair systems which make it a very good candidate for curing several dermatoses associated with substantial oxidative damage. It helps for preventing skin cancer, skin photoand radioprotection and also works as an inducer of repair mechanisms of human skin recovery from environmental damage [16,17]. In recent years, the community of biologists, dermatologists and physicists have published plenty of works on MEL and studied how it can affect the human body [18–21]. For instance, MEL was found to have a significant effect on reducing cholesterol absorption and causing greatly decreases in total cholesterol in membrane bilayers and concentrations of cholesterol in the liver [22]. MEL is not only important for humans, but also for plants and animals. In particular, MEL works as a multifunctional signaling molecule which regulates broad aspects of responses to environmental changes [23]. In summary, the present study is devoted on the analysis of the structure and dynamics of melatonin and it follows previous works where tryptophan (the precursor of MEL) [24,25] and other similar solutes were simulated [26]. In order to investigate these relevant effects of MEL on human body and more specifically on plasma membranes, we have focused our attention to the characterization of the structure and transport processes of MEL at the atomic scale with the aid of molecular dynamics (MD) simulations at the time-scale of hundreds of nanoseconds. Methods There exist plenty of experiments and simulations already performed on DMPC and on similar phospholipids such as dipalmytoilphosphatidylcholine (DPPC), usually including the study Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes PLOS ONE | https://doi.org/10.1371/journal.pone.0224624 November 7, 2019 2 / 20 Universities, Ref. number PGC2018-099277-B-C21 to J.M. The funders had no role in study design, simulations, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. of the influence of cholesterol [11,12,27–29]. These results will allow us to validate our methods and results, as it will be described in the beginning of Section “Results and Discussion”. Further, the transition temperature from gel to liquid crystal phase of DMPC has been determined and it is close to 297 K [30], what allows us to perform computer simulations able to be equilibrated in a reasonable short time. When transition temperatures are higher (case of DPPC, T = 314 K), we have previously observed that getting an equilibrated bilayer membrane will require much longer simulation runs. As a general fact, DMPC membranes have been satisfactorily reproduced in a wide variety of simulations and, in particular, the force field employed in the present work has revealed to be very successful in the reproduction of the structural characteristics of the DMPC membrane, as we will show below. A realistic model of DMPC-cholesterol membrane bilayers in a sodium chloride solution has been generated with the well-known CHARMM-GUI web-based tool [31,32]. Cholesterol-free system was composed of 204 DMPC (C 36 H 72 NO 8 P) lipids molecules, approximately 10250 TIP3P [33] water molecules (allowing flexible bonds through harmonic springs), with 21 sodium and 21 chlorine ions of physiological concentration, along with one single MEL molecule (C 13 H 16 N 2 O 2 , obtained from pdb 4QOI through PDB Reader plugin of CHARMM-GUI website-based tool). From information previously obtained [25], only two significant cholesterol (C 27 H 46 O) percentages should be considered: 30, and 50% such that the harvested data should be compared with the results obtained for the case with 0% cholesterol. MEL locates at the interface of membrane bilayer in all three cases in the beginning of minimization. Sketches of the backbone structure of MEL, DMPC and cholesterol are represented in Fig 1. The NAMD2 software package [34] with the recently reparameterized CHARMM36m force field [35–38] was used in all MD simulations at a fixed temperature of 303.15 K and at the fixed pressure of 1 atm (NPT), in order to make sure all simulations were performed at the liquid crystal phase [39]. As usual in such kind of simulations [24] the temperature was regulated with a Langevin thermostat [40] with damping coefficient of 1 ps −1 , whereas Fig 1. Backbone structures. Sketches of molecular structures of MEL, DMPC and cholesterol. Part of HydrogenCarbon bonds not shown. The highlighted sites of MEL (C3, C4, H15, H16, N1, N2, O1 and O2) and of DMPC (O1, O2, O6 and O8) will be referred in the text by the same labels. https://doi.org/10.1371/journal.pone.0224624.g001 Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes PLOS ONE | https://doi.org/10.1371/journal.pone.0224624 November 7, 2019 3 / 20 the pressure was controlled by a Nose ´-Hoover Langevin piston [41] with Langevin dynamics [42] at an oscillation period of 50 fs. All MD simulations were operated in NPT conditions. After 150 ps NPT relaxation and 100 ns equilibration periods, several production runs were generated and statistically meaningful trajectories of more than 100 ns were recorded in all cases. The simulation boxes had different sizes because of different cholesterol concentrations. For instance, the size of cholesterol-free system was of 79 Å × 79 Å × 85 Å. A time step of 2 fs was used and periodic boundary conditions were applied. All bonds involving hydrogens were set to fixed lengths, allowing fluctuations of bond distances and angles for the remaining atoms. During the calculation of spectral densities all bonds (including those involving hydrogens) were left flexible. The cutoff for the Van der Waals interactions was of 12 Åand a switching function was employed starting at 10 Å. Coulomb forces were computed using the particle mesh Ewald method [43], with a grid space of 1 Å. Every time step electrostatic interactions were updated. The usual periodic boundary conditions in all directions of space were taken. Results and discussion Physical characteristics of the membranes In order to explore the phase-diagram states of the model systems being simulated as well as to efficiently characterize the ordering inside the hydrated lipid bilayer, a procedure already employed in previous works [25,28,44,45] was used. A deuterium order parameter S CD was defined for each CH 2 group of the DMPC lipid tails as follows: SCD ¼1 2ð3<cos 2yCD >1Þ;ð1Þ with θ CD standing for the angle between the direction normal to the surface of the membrane and a CH-bond. S CD can be also obtained from 2 H NMR experiments [46]. The averaged results are shown in Fig 2 for both tail chains of all DMPC lipids at the three cholesterol concentrations considered in this work. The results for the cholesterol-free case were previously tested [28] and are in good agreement with both simulation [47,48] and experimental works [49,50], confirming the liquid crystal phase was represented well in the three systems adopted in the present work. We should note that as cholesterol concentration increases in the system, the tendency to higher ordering increases too, which is represented by profiles of S CD having larger maxima (around *0.45 for the cholesterol-rich setups versus 0.25 for the cholesterolfree system), a tendency which was already observed by Petrache et al. [50]. The area per lipid is definitely a relevant output from most molecular simulation of plasma membranes. We have calculated the area per lipid considering the membrane surface along the XY plane divided by the number of lipids and cholesterol [51]. For continuous MD production runs, area per lipid as a function of simulation time is reported in Fig 3 whereas their averaged values together with the averaged thickness of membranes are reported in Table 1. Area per lipid decreases as cholesterol concentration increases. We obtained a value of around 62 Å 2 for a cholesterol-free system and smaller values down to 40 Å 2 for the system with a concentration of cholesterol of 50%. These results are in excellent agreement with other computational works [50,52] where the value for pure DMPC is of about 60 Å 2 at 303 K. According to the review of Nagle et al. [53], values of area per lipid of pure DMPC membranes (303 K) can be obtained from multiple methods (neutron scattering, X-ray and NMR) and were reported to be between 59 and 62 Å 2 at the liquid phase. In our case, the change in the area per lipid has been observed to be more marked when the concentration of cholesterol was above 20% (not reported). This is consistent with the observed fact that DMPC membranes in this work Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes PLOS ONE | https://doi.org/10.1371/journal.pone.0224624 November 7, 2019 4 / 20 experienced the phase transition point from a liquid-disordered phase (cholesterol-free system) to a liquid-ordered phase (systems of cholesterol 30% and 50%) [29,54]. Thickness of the membrane may provide additional clues about the influence of cholesterol on the mechanical properties of plasma membranes, such as rigidity and capability of allowing the movement of species in and out of the cell. We have obtained the thickness of the membrane Δzby computing the distance between phosphorus atoms (P) of the DMPC head groups from both layers. The results of the thickness of the membrane are in good agreement with those reported by Kučerka et al. [52] by means of X-ray and neutron scattering. These authors reported a value of 36.7 Åat 303 K for the DMPC membrane at a cholesterol-free system. In the present work we observe a tendency to larger bilayer thickness as cholesterol concentration increases. As it was pointed out in the case of the binding of tryptophan at DPPC-cholesterol membranes [25], at higher cholesterol percentages, the values of Aare smaller: the larger the cholesterol contents the more compressed are the bilayer structures. This eventually can increase the rigidity of the membrane, extending the lipid tails and producing larger bilayer thickness. In summary, the increase of the rigidity of the membrane is a fact already observed by several authors from both experimental and computational sides, such as Drolle et al. [11] or Choi et al. [12] for cholesterol-melatonin mixtures in phosphatidylcholine membranes. In their studies these Fig 2. Order parameter. |S CD | for the (sn1, sn2) acyl tails of DMPC at three different cholesterol concentrations. https://doi.org/10.1371/journal.pone.0224624.g002 Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes PLOS ONE | https://doi.org/10.1371/journal.pone.0224624 November 7, 2019 5 / 20 authors found out the effect of melatonin reducing the thickness of the membrane and enhancing its fluidity, a compensating effect of the condensation introduced by cholesterol. In the present work we only considered a single melatonin molecule what did not allow us to explore the joint effects of melatonin and cholesterol on the thickness of the membrane. The penetration of MEL in the membrane along its normal direction is also a relevant feature. We report in Fig 4 the Z-axis position of MEL from the center of the bilayer (i.e. z= 0) using the last meaningful 80 ns of each production trajectory in all three cases, namely, concentrations of cholesterol set at 0%, 30% and 50%. Red symbols represent averaged positions of phosphorus (P) atoms of the head groups of DMPC along the direction normal to the membrane (Z-axis); green symbols stand for the Z-axis distance between the center of mass of MEL Fig 3. Physical characteristics of the membranes. Area per lipid of systems with different cholesterol contents: 0% (black line), 30% (blue line), 50% (red line) as a function of simulation time. The green dashed lines indicate the average values for the last 150 ns at each concentration. https://doi.org/10.1371/journal.pone.0224624.g003 Table 1. Area per lipid and thickness of the membrane for all three cases studied in this work. Aand Δzat different cholesterol concentrations. Estimated errors in parenthesis. Percentage of cholesterol A(Å 2 )Δz(Å) 0% 61.8(1.2) 34.9(0.6) 30% 42.1(0.5) 44.3(0.3) 50% 40.2(0.3) 44.7(0.3) https://doi.org/10.1371/journal.pone.0224624.t001 Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes PLOS ONE | https://doi.org/10.1371/journal.pone.0224624 November 7, 2019 6 / 20 and the center of DMPC bilayers, as a function of simulation time. It stands clearly that the thickness of the DMPC bilayer membrane is increased when cholesterol is present in the membrane, in good agreement with the results of thickness reported in Table 1. At 0% and 30% cholesterol systems, MEL stays in the internal region of DMPC bilayers most of the time, with a few occasional visits to the interface of the membrane, i.e. surroundings of the P atoms; however, when more cholesterol is added into the system (i.e. at 50%), MEL can diffuse into water bulk and become fully solvated by water or the head groups of DMPC without changing the size of system too much, indicating the competition between water and lipids to solvate MEL. These results are in good agreement with those of Drolle et al. that reported the preferential location of MEL in a DPPC bilayer at distances around z= 1.2 nm (see Fig 8 of Ref. [11]), i.e. at the crossover region between lipid head groups and the fatty acid chains. In the case of 50%, Fig 4 shows that MEL has the ability of either being adsorbed by the the head groups of DMPC either to stay for long periods of time in the water bulk, so we can expect that it is not difficult for a small molecule like MEL to cross the free energy barrier between the two states (solvated by water or solvated by the head groups of DMPC). This latter aspect will be addressed with more details below. Fig 4. Z-axis position of MEL. Penetration of MEL inside DMPC bilayer (green symbols indicate the position of the center of mass of MEL whereas red symbols stand for the position of phosphorus atoms atoms in each layer) at 0%, 30% and 50% cholesterol concentrations. https://doi.org/10.1371/journal.pone.0224624.g004 Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes PLOS ONE | https://doi.org/10.1371/journal.pone.0224624 November 7, 2019 7 / 20 Radial distribution functions of melatonin around DMPC, water and cholesterol A direct route to the characterization of the local structure of each atomic species of the system is usually obtained by means of normalized radial distribution functions (RDF) g AB (r) for two different species Aand B(see Eq (2) of Ref [25]). Among the wide variety of possible RDF that could be computed, we have considered only six relevant RDF based on the first coordination shells of ‘H15’ and of ‘H16’ of MEL. The remaining RDF indicate low maxima at distances significantly longer than the typical hydrogen-bonding values or show too noisy profiles which indicate that the corresponding local structures are not stable enough. The selected g(r)s are reported in Fig 5 for three cholesterol percentages (0, 30, 50%). All six g(r)s show some fluctuations in their profiles, especially at the coordinates of r= 3 Å and beyond, i.e. those corresponding to second coordination shells. We could observe a neat first coordination shell in every case, located around 1.8-2.0 Åthat should be essentially attributed to hydrogen-bonds (HB) between MEL and the remaining species, cause such distance is the signature of typical oxygen-hydrogen HB in water [55]. Interestingly, the largest peak in all RDF is, by far, the one appearing at the MEL-cholesterol association, centered at 1.9 Åwhen the concentration of cholesterol is of 30%. Further, when we raised the concentration to 50% such band decreased dramatically and its position was shifted to about 2.2 Å. The interaction of MEL with cholesterol in DPPC bilayers, already reported by Choi et al. [12] produced a fluidizing effect on the membrane for a melatonin concentration high enough, opposite to the condensing effect of cholesterol. In all the remaining cases, HB lengths are around 1.9 Å. The height of each maxima (related to the intensity of the HB) depended strongly of the concentration of cholesterol, as follows: (1) in the case of MEL-water association, strong HB were observed between ‘H16’ and water at 0% and 30% although at 50% they were much weaker; (2) both ‘H15’ and ‘H16’ were able to form HB with the DMPC sites ‘O1’ (or ‘O2’, both sites sharing the negative charge); (3) finally, MEL can establish HB between both ‘H15’ and ‘H16’ hydrogens with the DMPC sites ‘O6’ (or ‘O8’) in all three percentages of cholesterol. These findings of HB association between MEL and DMPC are in good agreement with those from Severcan et al. [56] who, by means of Fourier transform infrared spectroscopy, observed the existence of hydrogen bonding between the hydrogen in the N-H group of the furanose ring of MEL (labeled ‘H16’ in the present work) and the carbonyl (C = O) and phosphate (PO2) groups in DPPC membranes. From our findings we have observed both HB of ‘H15’ and ‘H16’ of MEL with the phosphate group of DMPC (‘O1’) and also with the more internal C = O groups (‘O6’ and ‘O8’). Thus, the novelty here is the hydrogen bond association of ‘H15’ with the two well-known acceptor groups in phosphatidyl-cholines indicated above, together with the already reported association of ‘H16’. This fact allows MEL to be adsorbed deeper than tryptophan [25] at the membrane lipid bilayer with two selected donors (‘H15’ and ‘H16’) as well as through ‘H15-O Chol.’ bridges, which provides a variety of structures as it will be described in full details below. Results on MEL located close to the lipid head groups in studies of MEL inside DOPC and DPPC membranes were found by Drolle et al. [11] by means of small-angle neutron diffraction and MD simulations as well. Estimation of Helmholtz free energy differences From a general perspective, the calculation of the Helmholtz or Gibbs free energy differences for binding processes or for configurational changes is a difficult task and it requires a considerable amount of computer time and a precise knowledge of the hypersurface of potential energy of the system [57]. This can be explored by means of methods such as metadynamics Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes PLOS ONE | https://doi.org/10.1371/journal.pone.0224624 November 7, 2019 8 / 20 [58,59], hybrid quantum mechanics/molecular mechanics methods [60] or transition path sampling [61–64]. However, a usual way to obtain free energy estimations is through the socalled potential of mean force (PMF), which is an estimation of the Helmholtz free energy difference between two particles (1, 2). Computing the reversible work required to move the two tagged particles from infinite separation to a relative separation r, PMF can be directly Fig 5. Radial distribution functions. Selected radial distribution functions for hydrogens of MEL (‘H15’ and ‘H16’) with oxygens of water (‘OW’); DMPC (‘O2’ (representing ‘O1&O2’) and ‘O6’ (representing ‘O6&O8’)) and cholesterol, belonging to hydroxyl group (‘O Chol’). https://doi.org/10.1371/journal.pone.0224624.g005 Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes PLOS ONE | https://doi.org/10.1371/journal.pone.0224624 November 7, 2019 9 / 20 with MEL in a remarkable way, affecting the vibrational motions of their atomic components. This is in good agreement with the fact reported above (see section 3.1) of the hydrogen-bonding of MEL to cholesterol, especially at the 30% concentration. Conclusion We present all-atom molecular dynamics simulations of a bilayer membrane made up with the zwitterionic phospholipid DMPC at three cholesterol concentrations (0%, 30% and 50%) including a single MEL molecule, all in aqueous NaCl ionic solution at 303 K and at the fixed pressure of 1 atm. We adopted the most recent version of the CHARMM36m force field and the simulation length of each system reached the scale of hundreds of nanoseconds. Our main interest was focused on the local structure and angular distributions of MEL, especially when associated to DMPC and cholesterol molecules. After this, Helmholtz free energy differences of MEL binding onto different membrane bilayer interfaces has been evaluated through potentials of mean force. A one-dimensional reaction coordinate based on radial atomic distances for selected atoms was considered. As a general fact, MEL is able to establish hydrogenbonds with water, cholesterol and DMPC lipids. The strongest association has been observed between species ‘H15’ of MEL and the oxygen pertaining to the hydroxyl group of cholesterol; between ‘H15’ and the carbonyl groups in DMPC and between ‘H16’ and the carbonyl groups of DMPC, always when cholesterol was present in the system. However, some binding between ‘H15’ and ‘H16’ and the phosphate group in DMPC has been also observed. The order of magnitude of the Helmholtz free energy differences has been of 1 kcal/mol, with values between 0.5 and 2.5 kcal/mol. The typical hydrogen-bond distances have been found between 1.8 and 2.0 Å. Two relevant MEL structures have been observed from angular distributions: “folded” and “extended” configurations. We defined three different dihedral angles to account for the two preferential angular configurations. One of them (dihedral ψin Fig 6) has revealed to be a meaningful order parameter (i.e. may act as reliable reaction coordinate) to describe the dynamics of MEL, with preferential angles of *1.42 rad and *2.96 rad, as defined in Fig 6. From our results, we suggest that introducing cholesterol into the system could help MEL change from its folded configuration to extended configuration more easily, using hydrogenbonds between MEL-DMPC and MEL-cholesterol. The self-diffusion coefficient of MEL obtained from slopes of MSD at long times was found to be of the order of 10 −7 cm 2 /s and the presence of cholesterol in the system has an influence on it. As cholesterol concentration increases the membrane tends to become progressively thicker and area per lipid is significantly reduced resulting that the diffusion of MEL increases a factor four. Spectral densities computed in this work are in overall good agreement with experimental Raman and infrared data [71–73] and have revealed the degree of participation of each atomic site of MEL to complete its whole molecular spectrum giving some clues to understand the microscopic origin of molecular vibrations and also giving evidence of the good reliability of the model we adopted in the present work. Acknowledgments The authors acknowledge the use of computer resources from the “Barcelona Supercomputing Center-Red Espanola de Supercomputacion” through projects FI-2018-2-0018 and FI-2018-30023. Author Contributions Data curation: Jordi Martı ´. Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes PLOS ONE | https://doi.org/10.1371/journal.pone.0224624 November 7, 2019 16 / 20 Formal analysis: Huixia Lu, Jordi Martı ´. Funding acquisition: Jordi Martı ´. Investigation: Huixia Lu, Jordi Martı ´. Project administration: Jordi Martı ´. 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