Free energy calculations are essential to unveil mechanisms at the atomic scale such as binding of small solutes and their translocation across cell membranes, eventually producing cellular absorption. Melatonin regulates biological rhythms and is directly related to carcinogenesis and neurodegenerative disorders. Free energy landscapes obtained from well-tempered metadynamics simulations precisely describe the characteristics of melatonin binding to specific sites in the membrane and reveal the role of cholesterol in free energy barrier crossing. A specific molecular torsional angle and the distance between melatonin and the center of the membrane along the normal to the membrane Z-axis have been considered as suitable reaction coordinates. Free energy barriers between two particular orientations of the molecular structure (folded and extended) have been found to be of about 18 kJ/mol for z-distances of about 1–2 nm. The ability of cholesterol to expel melatonin out of the internal regions of the membrane towards the interface and the external solvent is explained from a free energy perspective. The calculations reported here offer detailed free energy landscapes of melatonin embedded in model cell membranes and reveal microscopic information on its transition between free energy minima, including the location of relevant transition states, and provide clues on the role of cholesterol in the cellular absorption of small molecules.

The characterization of the microscopical forces between the essential α-amino-acid tryptophan, precursor of the neurotransmitter serotonin and of the hormone melatonin, and the basic components of cell membranes and their environments (phospholipids, cholesterol, ionic species, and water) is of central importance to elucidate their local structure and dynamics as well as the mechanisms responsible for the access of tryptophan to the interior of the cell. We have performed nanosecond molecular dynamics simulations of tryptophan embedded in model zwitterionic bilayer membranes made by di-palmitoyl-phosphatidyl-choline and cholesterol inside aqueous sodium-chloride solution in order to systematically examine tryptophan-lipid, tryptophan-cholesterol, and tryptophan-water interactions under liquid-crystalline phase conditions. Microscopic properties such as the area per lipid, lipid thickness, radial distribution functions, hydrogen-bonding lengths, atomic spectral densities, and self-diffusion coefficients have been evaluated. Our results show that the presence of tryptophan significantly affects the structure and dynamics of the membrane. Tryptophan spends long periods of time at the water-membrane interface, and it plays a central role by bridging a few lipids and cholesterol chains by means of hydrogen-bonds. The computed spectral densities, in excellent agreement with experimental infrared and Raman data, revealed the participation of each atomic site of tryptophan to the complete spectrum of the molecule. Tryptophan self-diffusion coefficients have been found to be in between 10⁻⁷ and 10⁻⁶ cm²/s and strongly depending of the concentration of cholesterol in the system.

KRas proteins are the largest family of mutated Ras isoforms, participating in a wide variety of cancers. Due to their importance, large effort is being carried out on drug development by small-molecule inhibitors. However, understanding protein conformational variability remains a challenge in drug discovery. In the case of the Ras family, their multiple conformational states can affect the binding of potential drug inhibitors. To overcome this challenge, we propose a computational framework based on combined all-atom Molecular Dynamics and Metadynamics simulations in order to accurately access conformational variants of the target protein. We tested the methodology using a G12D mutated GTP bound oncogenic KRas-4B protein located at the interface of a DOPC/DOPS/cholesterol model anionic cell membrane. Two main orientations of KRas-4B at the anionic membrane have been determined. The corresponding torsional angles are taken as reliable reaction coordinates so that free-energy landscapes are obtained by well-tempered metadynamics simulations, revealing local and global minima of the free-energy hypersurface and unveiling reactive paths of the system between the two preferential orientations. We have observed that GTP-binding to KRas-4B has huge influence on the stabilisation of the protein and it can potentially help to open Switch I/II druggable pockets, lowering energy barriers between stable states and resulting in cumulative conformers of KRas-4B. This may highlight new opportunities for targeting the unique meta-stable states through the design of new efficient drugs.

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 cm²/s and they significantly increase when cholesterol is addeed to the membrane.

The interactions at the atomic level between small molecules and the main components of cellular plasma membranes are crucial for elucidating the mechanisms allowing for the entrance of such small species inside the cell. We have performed molecular dynamics and metadynamics simulations of tryptophan, serotonin, and melatonin at the interface of zwitterionic phospholipid bilayers. In this work, we will review recent computer simulation developments and report microscopic properties, such as the area per lipid and thickness of the membranes, atomic radial distribution functions, angular orientations, and free energy landscapes of small molecule binding to the membrane. Cholesterol affects the behaviour of the small molecules, which are mainly buried in the interfacial regions. We have observed a competition between the binding of small molecules to phospholipids and cholesterol through lipidic hydrogen-bonds. Free energy barriers that are associated to translational and orientational changes of melatonin have been found to be between 10–20 kJ/mol for distances of 1 nm between melatonin and the center of the membrane. Corresponding barriers for tryptophan and serotonin that are obtained from reversible work methods are of the order of 10 kJ/mol and reveal strong hydrogen bonding between such species and specific phospholipid sites. The diffusion of tryptophan and melatonin is of the order of 10${}^{-7}$ cm${}^2$/s for the cholesterol-free and cholesterol-rich setups.