Exploring free-energy landscapes and microscopic interactions of selected small-molecules and proteins wih cell membranes

The present Thesis is devoted to the study of the physical-chemical properties of selected small-molecules (such as amino-acids like tryptophan or hormones like melatonin) and proteins (such as KRAS-4B) absorbed in model lipid bilayers located at physiological environments. Since in such conditions biological membranes composed of phospholipids and cholesterol are surrounded by electrolyte solutions, understanding the interactions of the small molecule or protein with the surrounding phospholipids, cholesterol, water and all sorts of ion species is a topic of great fundamental importance. In particular, the present Thesis has advanced into the analysis of the structural and energetic aspects of an oncogenic protein from the RAS family, characterising the physical conditions that allow such protein to remain anchored to the cell. The findings reported in the Thesis may help to shed light in the understanding of a wide variety of cancers, with direct impact on the design of drugs or treatments useful for curation. The lipids considered in this Thesis include the saturated lipids dimyristoilphosphatidylcholine (DMPC) and dipalmytoilphosphatidylcholine (DPPC), the unsaturated lipids dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine(DOPS) and cholesterol. Classical molecular dynamics simulations and well-tempered metadynamics simulations have been applied in this thesis so that all considered systems have been modelled and simulated at the all-atom level, with systems containing up to 200000 atoms. Using classical molecular dynamics simulations at the microsecond time scale, we studied the microscopic structure and dynamics of the small-molecules and KRAS4B proteins, the latter in the wild-type and mutated (oncogenic) forms. The cell membrane has been always considered in the liquid crystalline phase, what in some cases required to rise the temperature of the system up to 323 K. Structural properties such as the area per lipid and thickness of the membrane, density profiles, deuterium-order parameters, orientational distributions and the extent of water penetration in the membrane have been analysed. Molecular self-diffusion and spectral densities of atomic species reveal a variety of time scales playing a role in membrane dynamics. The physical meaning of all spectral features from lipid atomic sites is analysed and correlated with experimental data. Most relevant have been the location of individual sites of binding of probes at the interface of the membrane. Finally, using reversible work techniques, we estimated the extent of free energy required to form such liaisons. By applying 1-microsecond well-tempered metadynamics simulations, we have performed systematic free energy calculations of probe binding to the membrane and water for the first time. Free energy landscapes unveil specific binding behaviour of small-molecules and proteins at phospholipid membranes. This Thesis provides a general methodology to explore such free energy landscapes at complex biological interfaces which can be extended to study other interactions of interest between molecules, peptides, proteins or drugs and charged head-groups in colloidal chemistry and biology. We further applied this methodology to study the case of a prototypical oncogenic protein (KRAS), being able to produce a wide variety of cancers. Our results from resulting free energy landscapes indicate the existence of specific hydrogen-bonding connections between parts of the protein (hypervariable region and farnesylated tail) that might be responsible of the permanent infection of healthy cells through its anchoring at the interface of the membrane.La presente Tesis doctoral está dedicada al estudio de las propiedades físico-químicas de moléculas pequeñas seleccionadas (por ejemplo aminoácidos como el triptófano u hormonas como la melatonina) y proteínas (como la KRas-4B) absorbidas en membranas celulares formadas por fosfolípidos y ubicadas en entornos fisiológicos. Dado que en estas condiciones, las membranas biológicas compuestas por fosfolípidos y colesterol están rodeadas de soluciones de electrólitos, entender las interacciones de la molécula pequeña o proteína con los fosfolípidos circundantes, el colesterol, el agua y todo tipo de especies iónicas es un tema de gran importancia fundamental. En particular, la presente Tesis se ha avanzado en el análisis de los aspectos estructurales y energéticos de una proteína oncogènica de la familia Ras, caracterizando las condiciones físicas que permiten que esta proteína se mantenga anclada a la célula. Las resultados descritos a la tesis pueden ayudar a dar luz a la comprensión de una gran variedad de cánceres, con un impacto directo en el diseño de medicamentos o tratamientos útiles para su curación. Los lípidos considerados en esta Tesis incluyen los lípidossaturados dimiristoilfosfatidilcolina y dipalmitoilfosfatidilcolina, los lípidos insaturados dioleoilfosfatidilcolina y dioleoilfosfatidilserina y el colesterol. En esta Tesis se han aplicado simulaciones de dinámica molecular clásica y simulaciones de metadinámica bien temperada, de forma que todos los sistemas considerados han estado modelizados y simulados a nivel puramente atómico, con sistemas de hasta 200000 átomos. Utilizando simulaciones clásicas de dinámica molecular (a escala 1 microsegundo), hemos estudiado la estructura y la dinámica microscópicas de las moléculas pequeñas y las proteínas KRas4B, estas últimas en formas pura y mutada (oncogénica). La membrana celular siempre se ha considerado en fase cristalina líquida, cosa que en algunos casos ha requerido aumentar la temperatura del sistema hasta 323 K. Se han calculado propiedades estructurales como por ejemplo el área por lípido y el grosor de la membrana, perfiles de densidad, parámetros de orden del deuterio, distribuciones orientacionals y el alcance de la penetración del agua a la membrana. Los coeficientes de difusión moleculares y las densidades espectrales atómicas revelan una gran variedad de escalas de tiempos que tienen un papel en la dinámica de membrana. El significado físico de todas las características espectrales de los lugares atómicos lipídicos se ha analizado y correlacionado con datos experimentales. El más relevante ha sido el hallazgo de la ubicación de lugares individuales de enlace de las varias sondas (pequeñas moléculas y proteínas) a la interfaz de la membrana. Finalmente, utilizando técnicas de trabajo reversible, se ha podido estimar la cantidad de energía libre necesaria para formar estos enlaces. Mediante la aplicación de simulaciones de metadinámica bien temperada de 1 microsegundo, hemos realizado por primera vez cálculos de energía libre sistemática de la unión de las varias sondas a la membrana y al agua. Las superficies de energía libre muestran un comportamiento específico de los enlaces de moléculas pequeñas y proteínas a las membranas fosfolípidiques. Esta Tesis proporciona una metodología general para explorar superficies de energía libre en interfaces biológicas complejas que se pueden ampliar para estudiar otras interacciones de interés entre moléculas, péptidos, proteínas o fármacos y membranas en Química y Biología coloidal. También hemos aplicado esta metodología para estudiar el caso de una proteína oncogènica prototípica (KRas), que se considera responsable de una gran variedad de cánceres. Nuestros resultados en superficies de energía libre indican la existencia de conexiones específicas de enlace de hidrógeno entre partes de la proteína (región hipervariabley cola farnesilada) que podrían ser responsables de la infección permanente de células sanas a través de su anclaje a la interfaz de la membrana.Postprint (published version

Exploring free-energy landscapes and microscopic interactions of selected small-molecules and proteins with cell membranes

The present Thesis is devoted to the study of the physical-chemical properties of selected small-molecules (such as amino-acids like tryptophan or hormones like melatonin) and proteins (such as KRAS-4B) absorbed in model lipid bilayers located at physiological environments. Since in such conditions biological membranes composed of phospholipids and cholesterol are surrounded by electrolyte solutions, understanding the interactions of the small molecule or protein with the surrounding phospholipids, cholesterol, water and all sorts of ion species is a topic of great fundamental importance. In particular, the present Thesis has advanced into the analysis of the structural and energetic aspects of an oncogenic protein from the RAS family, characterising the physical conditions that allow such protein to remain anchored to the cell. The findings reported in the Thesis may help to shed light in the understanding of a wide variety of cancers, with direct impact on the design of drugs or treatments useful for curation. The lipids considered in this Thesis include the saturated lipids dimyristoilphosphatidylcholine (DMPC) and dipalmytoilphosphatidylcholine (DPPC), the unsaturated lipids dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine(DOPS) and cholesterol. Classical molecular dynamics simulations and well-tempered metadynamics simulations have been applied in this thesis so that all considered systems have been modelled and simulated at the all-atom level, with systems containing up to 200000 atoms. Using classical molecular dynamics simulations at the microsecond time scale, we studied the microscopic structure and dynamics of the small-molecules and KRAS4B proteins, the latter in the wild-type and mutated (oncogenic) forms. The cell membrane has been always considered in the liquid crystalline phase, what in some cases required to rise the temperature of the system up to 323 K. Structural properties such as the area per lipid and thickness of the membrane, density profiles, deuterium-order parameters, orientational distributions and the extent of water penetration in the membrane have been analysed. Molecular self-diffusion and spectral densities of atomic species reveal a variety of time scales playing a role in membrane dynamics. The physical meaning of all spectral features from lipid atomic sites is analysed and correlated with experimental data. Most relevant have been the location of individual sites of binding of probes at the interface of the membrane. Finally, using reversible work techniques, we estimated the extent of free energy required to form such liaisons. By applying 1-microsecond well-tempered metadynamics simulations, we have performed systematic free energy calculations of probe binding to the membrane and water for the first time. Free energy landscapes unveil specific binding behaviour of small-molecules and proteins at phospholipid membranes. This Thesis provides a general methodology to explore such free energy landscapes at complex biological interfaces which can be extended to study other interactions of interest between molecules, peptides, proteins or drugs and charged head-groups in colloidal chemistry and biology. We further applied this methodology to study the case of a prototypical oncogenic protein (KRAS), being able to produce a wide variety of cancers. Our results from resulting free energy landscapes indicate the existence of specific hydrogen-bonding connections between parts of the protein (hypervariable region and farnesylated tail) that might be responsible of the permanent infection of healthy cells through its anchoring at the interface of the membrane.La presente Tesis doctoral está dedicada al estudio de las propiedades físico-químicas de moléculas pequeñas seleccionadas (por ejemplo aminoácidos como el triptófano u hormonas como la melatonina) y proteínas (como la KRas-4B) absorbidas en membranas celulares formadas por fosfolípidos y ubicadas en entornos fisiológicos. Dado que en estas condiciones, las membranas biológicas compuestas por fosfolípidos y colesterol están rodeadas de soluciones de electrólitos, entender las interacciones de la molécula pequeña o proteína con los fosfolípidos circundantes, el colesterol, el agua y todo tipo de especies iónicas es un tema de gran importancia fundamental. En particular, la presente Tesis se ha avanzado en el análisis de los aspectos estructurales y energéticos de una proteína oncogènica de la familia Ras, caracterizando las condiciones físicas que permiten que esta proteína se mantenga anclada a la célula. Las resultados descritos a la tesis pueden ayudar a dar luz a la comprensión de una gran variedad de cánceres, con un impacto directo en el diseño de medicamentos o tratamientos útiles para su curación. Los lípidos considerados en esta Tesis incluyen los lípidossaturados dimiristoilfosfatidilcolina y dipalmitoilfosfatidilcolina, los lípidos insaturados dioleoilfosfatidilcolina y dioleoilfosfatidilserina y el colesterol. En esta Tesis se han aplicado simulaciones de dinámica molecular clásica y simulaciones de metadinámica bien temperada, de forma que todos los sistemas considerados han estado modelizados y simulados a nivel puramente atómico, con sistemas de hasta 200000 átomos. Utilizando simulaciones clásicas de dinámica molecular (a escala 1 microsegundo), hemos estudiado la estructura y la dinámica microscópicas de las moléculas pequeñas y las proteínas KRas4B, estas últimas en formas pura y mutada (oncogénica). La membrana celular siempre se ha considerado en fase cristalina líquida, cosa que en algunos casos ha requerido aumentar la temperatura del sistema hasta 323 K. Se han calculado propiedades estructurales como por ejemplo el área por lípido y el grosor de la membrana, perfiles de densidad, parámetros de orden del deuterio, distribuciones orientacionals y el alcance de la penetración del agua a la membrana. Los coeficientes de difusión moleculares y las densidades espectrales atómicas revelan una gran variedad de escalas de tiempos que tienen un papel en la dinámica de membrana. El significado físico de todas las características espectrales de los lugares atómicos lipídicos se ha analizado y correlacionado con datos experimentales. El más relevante ha sido el hallazgo de la ubicación de lugares individuales de enlace de las varias sondas (pequeñas moléculas y proteínas) a la interfaz de la membrana. Finalmente, utilizando técnicas de trabajo reversible, se ha podido estimar la cantidad de energía libre necesaria para formar estos enlaces. Mediante la aplicación de simulaciones de metadinámica bien temperada de 1 microsegundo, hemos realizado por primera vez cálculos de energía libre sistemática de la unión de las varias sondas a la membrana y al agua. Las superficies de energía libre muestran un comportamiento específico de los enlaces de moléculas pequeñas y proteínas a las membranas fosfolípidiques. Esta Tesis proporciona una metodología general para explorar superficies de energía libre en interfaces biológicas complejas que se pueden ampliar para estudiar otras interacciones de interés entre moléculas, péptidos, proteínas o fármacos y membranas en Química y Biología coloidal. También hemos aplicado esta metodología para estudiar el caso de una proteína oncogènica prototípica (KRas), que se considera responsable de una gran variedad de cánceres. Nuestros resultados en superficies de energía libre indican la existencia de conexiones específicas de enlace de hidrógeno entre partes de la proteína (región hipervariabley cola farnesilada) que podrían ser responsables de la infección permanente de células sanas a través de su anclaje a la interfaz de la membrana

Binding free energies of small-molecules in phospholipid membranes: aminoacids, serotonin and melatonin

Free energy barriers associated to the binding of small-molecules at phospholipid zwitterionic membranes have been computed at 323 K for a variety of species: tryptophan, histidine, tyrosine, serotonin and melatonin bound to a model membrane formed by di-palmitoyl-phosphatidyl-choline lipids inside aqueous sodium chloride solution. We have computed the radial distribution functions of all species for a variety of membrane and water related sites and extracted potentials of mean force through the reversible work theorem. In all cases but histidine, the molecular probes are able to either be fully solvated by water or be embedded into the interface of the membrane. Our results indicate that binding of all species to water corresponds to free energy barriers of heights between 0.2 and 1.75 kcal/mol. Free energy barriers of association of small-molecules to lipid chains range between 0.6 and 3.1 kcal/mol and show different characteristics: all species but histidine are most likely bound to oxygens belonging to the phosphate and to the glycerol groups. Histidine shows a clear preference to be fully solvated by water whereas the aqueous solvation of serotonin is the less likely case of them all. No free permeation through the membrane of any small-molecule has been observed during the time span of the simulation experiments.Preprin

Molecular dynamics of di-palmitoyl-phosphatidyl-choline biomembranes in ionic solution: adsorption of the precursor neurotransmitter tryptophan

Microscopic structure of a fully hydrated di-palmytoil-phosphatidyl-choline lipid bilayer membrane in the liquid-crystalline phase has been analyzed with all-atom molecular dynamics simulations based on the recently parameterized CHARMM36 force field. Within the membrane, a single molecule of the a-aminoacid tryptophan (precursor of important neurotransmitters such as serotonin and melatonin) has been embedded and its structure and binding sites to water and lipids have been explored. In addition, properties such as radial distribution functions, hydrogen-bonding, energy and pressure profiles and the potentials of mean force of water-tryptophan and lipid-tryptophan have been evaluated. It has been observed that tryptophan usually has a tendency to place itself close to the lipid headgroups but that it can be fully hydrated during short time intervals of the order of a few nanoseconds. This would indicate that, for tryptophan, both hydrophobic forces as well as the attraction to polar sites of the lipids play a significant role in the definition of its structure and binding states.Postprint (author's final draft

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

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.Postprint (author's final draft

Long-lasting salt bridges provide the anchoring mechanism of oncogenic kirsten rat sarcoma proteins at cell membranes

RAS proteins work as GDP-GTP binary switches and regulate cytoplasmic signaling networks that are able to control several cellular processes, playing an essential role in signal transduction pathways involved in cell growth, differentiation, and survival, so that overacting RAS signaling can lead to cancer. One of the hardest challenges to face is the design of mutation-selective therapeutic strategies. In this work, a G12D-mutated farnesylated GTP-bound Kirsten RAt sarcoma (KRAS) protein has been simulated at the interface of a DOPC/DOPS/cholesterol model anionic cell membrane. A specific long-lasting salt bridge connection between farnesyl and the hypervariable region of the protein has been identified as the main mechanism responsible for the binding of oncogenic farnesylated KRAS-4B to the cell membrane. Free-energy landscapes allowed us to characterize local and global minima of KRAS-4B binding to the cell membrane, revealing the main pathways between anchored and released states.Postprint (published version

Cellular absorption of small molecules: free energy landscapes of melatonin binding at phospholipid membranes

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.Peer ReviewedPostprint (published version

BOUNDEDNESS OF HIGHER-ORDER MARCINKIEWICZ-TYPE INTEGRALS

Let A be a function with derivatives of order m and D γ A ∈Λ β (0 < β < 1, |γ| = m). The authors in the paper proved that if Ω ∈ L s (S n−1 ) (s ≥ n/(n − β)) is homogeneous of degree zero and satisfies a vanishing condition, then both the higher-order Marcinkiewicz-type integral

BOUNDEDNESS OF HIGHER-ORDER MARCINKIEWICZ-TYPE INTEGRALS

Let A be a function with derivatives of order m and D γ A ∈Λ β (0 < β < 1, |γ| = m). The authors in the paper proved that if Ω ∈ L s (S n−1 ) (s ≥ n/(n − β)) is homogeneous of degree zero and satisfies a vanishing condition, then both the higher-order Marcinkiewicz-type integral

Finite-temperature violation of the anomalous transverse Wiedemann-Franz law

The Wiedemann-Franz (WF) law links the ratio of electronic charge and heat conductivity to fundamental constants. It has been tested in numerous solids, but the extent of its relevance to the anomalous transverse transport, which represents the topological nature of the wave function, remains an open question. Here we present a study of anomalous transverse response in the noncollinear antiferromagnet Mn$_{3}$Ge extended from room temperature down to sub-Kelvin temperature and find that the anomalous Lorenz ratio remains close to the Sommerfeld value up to 100 K, but not above. The finite-temperature violation of the WF correlation is caused by a mismatch between the thermal and electrical summations of the Berry curvature, rather than the inelastic scattering as observed in ordinary metals. This interpretation is backed by our theoretical calculations, which reveals a competition between the temperature and the Berry curvature distribution. The accuracy of the experiment is supported by the verification of the Bridgman relation between the anomalous Ettingshausen and Nernst effects. Our results identify the anomalous Lorenz ratio as an extremely sensitive probe of Berry spectrum near the chemical potential.Comment: 9 pages,6 figures, Supplemental Material include