Molecular modeling and thermodynamics simulation of nucleic acids

Nucleic acids participate in many cellular processes. DNA is responsible for gene heredity and its structure is mainly in double helix, whereas RNA has wide functions in gene transcription and regulation so its structures are varied among species. RNA modifications which are known for their abundance and chemical diversity further increase the conformational variability. Functions of some RNAs closely tie to modifications. For example, modified nucleotides maintain correct tRNA structure so that enzyme and ribosome can recognize the tRNA in protein translation. Few epigenetic modifications are also found in DNA, such as 5-methyl cytidine. More often artificially modified DNA, like locked nucleic acid (LNA), is applied to alter the binding affinity of DNA duplex and triplex. Starting from the structures solved by experiments or modeled by programs, molecular dynamics (MD) simulations are employed to mimic the dynamic process and compute the thermodynamic properties, so that the structure and function of nucleic acids can be better understood. This thesis covers computational studies of both RNA and DNA structures. In paper I, the naturally modified ribonucleotides are parameterized in an additive CHARMM force field. The parameters are targeted on quantum chemistry data. The charge and dihedral parameters are fine-tuned for some molecules to reproduce the experimental conformation. This force field allows wider computational studies on modifications involved RNA molecules. In paper II, the new force field is used in the simulations of four tRNAs. The results show with modifications the structural stability, nucleotide conformation and base pair maintenance are almost better than those without modifications, especially in dihydrouridine loop and anticodon loop. The enhanced stability by magnesium ions is also observed. In paper III, MD simulations combined with electrophoretic mobility shift assay illustrate the LNA effects in DNA helical structures. The results show LNA substitutions in duplex strand or the third strand improve the triplex formation, because LNA pre-organizes the DNA strands to reduce their structural adaption required upon triplex forming. In paper IV, a method is developed to calculate free energy for LNA. The angle energies are transformed to convert the locked ribose to deoxyribose. The protocol can be in one-step or three-step by transforming bonded and nonbonded energies separately. Both protocols solve the reasonable solvation free energy and are expected to be applied in larger systems

How Atomic Level Interactions Drive Membrane Fusion: Insights From Molecular Dynamics Simulations

This project is focused on identifying the role of key players in the membrane fusion process at the atomic level with the use of molecular dynamics simulations. Membrane fusion of apposed bilayers is one of the most fundamental and frequently occurring biological phenomena in living organisms. It is an essential step in several cellular processes such as neuronal exocytosis, sperm fusion with oocytes and intracellular fusion of organelles to name a few. Membrane fusion is a frequent process in a living organism but is still not fully understood at the atomic level in terms of the role of various factors that play a crucial part in completion of membrane fusion. Two major factors that have been identified and studied experimentally are the protein Synaptotagmin and SNAREs. In addition, Ca2+ is known to play a crucial role in this process, however the exact mechanism of action is still unknown. Prime objective of this study is to understand these interactions and the role of Ca2 + in the process at the atomic level by carrying out molecular dynamics simulations. One of the primary calculations to perform is potential of mean force (PMF) between SYT and bilayer to analyze the effect of Ca2+ on their relative affinities. 1-octanol-water partition coefficient (log Kow) of a solute is a key parameter used in the prediction of a wide variety of complex phenomena such as drug availability and bioaccumulation potential of trace contaminants. Adaptive biasing force method is applied to calculate 1-octanol partition coefficients of n-alkanes and extended to other complex systems like ionic liquids, energetic materials and chemical warfare agents. Molecular dynamics simulations show that both domains of SYT-1, C2A and C2B, once calcium bound, insert into the lipid bilayer composed of anionic phospholipids. In contrast, no insertion is observed when the domains do not have bound calcium or when the bilayer is not charged negative. Electrostatic interactions play an important role in this insertion process. Effect of calcium binding to the C2A and C2B domain on the overall electrostatics of the protein was studied by generating the ESP maps. Negative potential on the Calcium binding pocket transforms into positive potential once calcium is attached to those sites. Interaction of this positive potential surface with the negatively charged bilayer acts as a driving force for protein insertion into the bilayer. In addition, adaptive biasing force method has emerged as a powerful tool for prediction of 1-octanol water partition coefficients and is successfully implemented and optimized for n-alkanes and extended to the systems of ionic liquids, energetic materials and chemical warfare agents for which 1-octanol water partition coefficient is either not known or is difficult to measure via experimental methods

Molecular simulations of hybrid cross-linked membranes for H₂S gas separation at very high temperatures and pressure:Binary 90%/10% N₂/H₂S and CH₄/H₂S, ternary 90%/9%/1% N₂/CO₂/H₂S and CH₄/CO₂/H₂S mixtures

Molecular dynamics (MD) simulations have previously identified four hybrid inorganic-organic membranes based on POSS or OAPS silsesquioxanes hyper-cross-linked with small PMDA or 6FDA imides, which are able to maintain reasonable CO2/N2 and CO2/CH4 permselectivities at very high temperatures (300 °C and 400 °C) and pressure (60 bar). Experimentally, the polyPOSS-imides are known to degrade above 300 °C while the polyOAPS-imides can resist up to above 400 °C. In the present work, the same four model polyOAPS/POSS-imide networks are further tested for their gas separation abilities of H2S-containing mixtures. Indeed, hydrogen sulfide is a hazardous gas present in many gas feeds, and, within the context of a toxic penetrant under harsh conditions, simulations are a useful task to perform before embarking on difficult experiments. The separations of H2S with respect to N2, CH4 and CO2 by the polyOAPS/POSS-imide matrices were studied at 300 °C, 400 °C and at 60 bar, firstly with H2S as a single-gas in order to obtain its ideal permselectivities, secondly as part of binary 90%/10% N2/H2S and CH4/H2S feeds and thirdly as part of ternary 90%/9%/1% N2/CO2/H2S and CH4/CO2/H2S feeds. They were compared to separations of binary 90%/10% N2/CO2 and CH4/CO2 feeds under exactly the same conditions. At 300 °C, H2S is much more soluble in the networks than the other three penetrants. It is the only one leading to a non-negligible volume swelling at 60 bar, although this does not happen for the mixed-gas feeds due to their low H2S partial pressures. Differences are attenuated at 400 °C because of the decrease in solubilities upon heating. The linear N2 and CO2 move faster than the non-linear CH4 and H2S penetrants, but the diffusion selectivities are moderate. As such, the ideal permselectivities under harsh conditions are mainly governed by the solubility selectivities. With binary 90%/10% N2/H2S, CH4/H2S, N2/CO2 and CH4/CO2 feeds, the transport parameters of the major N2 or CH4 components remain similar to their ideal values, whereas the solubilities of the minor H2S and CO2 components increase. This leads to some of the real separation factors for H2S being different from their ideal permselectivities, and approximately twice as high as those with CO2. In the ternary 90%/9%/1% N2/CO2/H2S and CH4/CO2/H2S mixtures, replacing 1% CO2 by 1% H2S in the feeds leads to small changes but, in pratice, these are not significant enough to make a difference. Under the conditions tested, the ternary separation factors are the same than for the 90%/10% binary mixtures. In all cases, the denser polyPOSS-imides show better sieving properties than the more open polyOAPS-imides. As such, the former should preferably be used in applications up to 300 °C, i.e. in the temperature range below their degradation. However, it is also possible to use the polyOAPS-imides at higher temperatures, since they still manage maintaining separation factors between 2 and 6 for CO2 and H2S at 400 °C, which is outstanding for polymer-based membranes at such high temperatures.</p

The interaction of materials and biology: simulations of peptides, surfaces, and biomaterials

Biomaterials were originally designed to augment or replace damaged tissue in the body, but now encompass a wider range of applications including drug delivery, cancer vaccines, electronic sensor devices, and non-fouling coatings for ship hulls. At the heart of all of these applications is the interface between synthetic materials and biology. Modern techniques for studying this interface are limited to the macro and micro scales. With the advent of high performance computing clusters, molecular simulation is now capable of simulating the interface at the nano-scale. This thesis demonstrates how simulation adds important insights to the understanding of biomaterials. It begins with a comprehensive outline of the theoretical aspects of simulating the interface between water and solid surfaces. After this, small surface-bound biological molecules are modelled to explain experiments showing that they can capture cells on the surface. Finally, a new and practical, scalable technique for controlling biological molecules at the surface is developed. This work advances the field of biomaterials by explaining important processes that occur at the interface of biology and technology

Molecular simulation of adsorptive processes

Tese de doutoramento. Engenharia Química. Faculdade de Engenharia. Universidade do Porto. 200

Ion Pairing in Aqueous Metal Sulfates and Platinum Group Metal Ammonium Solutions

The structure and dynamics of ions and ion pairs in solution play an integral part in several biological and chemical processes. Historically the calculation of ion pair association constants from computer simulations has been complicated due to the difficulty in validating metal ion force fields for solution simulations. In this thesis a force field for divalent metal ions in metal sulfate solutions (i.e. Mg2+SO4 2-, Ca2+SO4 2-, Mn2+SO4 2-, Fe2+SO4 2-, Co2+SO4 2-, Ni2+SO4 2-, Cu2+SO4 2- and Zn2+SO4 2-) important in physical and biophysical experiments is produced. Potential of mean force calculations are used to provide ion pair free energy profiles and free energy perturbation calculations are used to calibrate the potential of mean force (PMF) from which association constants for ion pairs can be produced for these metal sulfate solutions. The calibrated free energy profiles result in calculated association constants that are in excellent agreement with available experimental data where available. Consequently the force field has been shown to be accurate for simulations of biophysical and physical systems. Furthermore the method, proposed in this thesis, for calibrating PMFs and calculating detailed association constants from those curves can most likely be used for complex systems that have previously been computationally inaccessible. Next a detailed account of solvation structures and the nature of ion pair formation mechanisms for important metal sulfates in aqueous media are presented. Radial and spatial distribution functions calculated for several ion pair species reveal that the transition from free ions to contact ion pairs involves the loss of between one to two water molecules from the cation depending on the cation size. This is correlated with the experimental hydration numbers calculated for metal sulfate electrolyte solutions at several concentrations using density and ultrasonic velocity measurements. These experiments reveal a decrease in hydration number with an increase in concentration, which can be attributed to the formation of ion pairs. A more complex metal system is the industrially important platinum group metal (PGM) chloro-anion one. Their industrial importance relates to the search for a Green Chemistry Process for the separation of PGM chloro complexes that have been extracted from the mined ore into an acidic aqueous media. This requires a PGM separation process in water. Here an understanding of the hydration structure about the iii PGM chloro-anion complexes and the role that ammonium counter-ions play in disrupting that solvent structure when ammonium PGM salts are formed, is critical in the process design. To this end a solution force field, inclusive of the majority of PGM chloro-anion complexes (i.e. (Y)2[PtCl4]2-, (Y)2[PdCl4]2-, (Y)2[PtCl6]2-, (Y)2[PdCl6]2-, (Y)2[IrCl6]2-, (Y)2[OsCl6]2-, (Y)2[RuCl6]2-, (Y)3[IrCl6]3-, (Y)3[RhCl6]3- and (Y)3[RuCl6]3- , where Y = NH4 +) arising in acidic aqueous media, parameterised from experimental and quantum mechanically derived properties, was developed. Nanosecond atomistic molecular dynamics simulations were then performed for the PGM chloro-anion complexes. Analysis of the solvation structure using radial and spatial distribution functions revealed two distinct solvent structures corresponding to the square planar and octahedral species. The formation of ion pairs disrupts the solvent structure where the hydration shells about the bivalent hexachlorometallates are more affected compared with the trivalent hexachlorometallates and these first shell waters in turn are more affected than those in the bivalent tetrachlorometallates. Finally to inform the design of a separation process transport properties such as diffusion coefficients, ion hydration numbers and water residence times for the PGM chloro-anion complexes were calculated. It is observed that the diffusion rates of PGM chloro-anion complexes are strongly correlated to their ion hydration numbers as calculated by Voronoi tessellation of the simulation cell, such that a larger hydration shell volume results in a slower PGM chloro-anion diffusion rate

The investigation of type-specific features of the copper coordinating AA9 proteins and their effect on the interaction with crystalline cellulose using molecular dynamics studies

AA9 proteins are metallo-enzymes which are crucial for the early stages of cellulose degradation. AA9 proteins have been suggested to cleave glycosidic bonds linking cellulose through the use of their Cu2+ coordinating active site. AA9 proteins possess different regioselectivities depending on the resulting cleavage they form and as result, are grouped accordingly. Type 1 AA9 proteins cleave the C1 carbon of cellulose while Type 2 AA9 proteins cleave the C4 carbon and Type 3 AA9 proteins cleave either C1 or C4 carbons. The steric congestion of the AA9 active site has been proposed to be a contributor to the observed regioselectivity. As such, a bioinformatics characterisation of type-specific sequence and structural features was performed. Initially AA9 protein sequences were obtained from the Pfam database and multiple sequence alignment was performed. The sequences were phylogenetically characterised and sequences were grouped into their respective types and sub-groups were identified. A selection analysis was performed on AA9 LPMO types to determine the selective pressure acting on AA9 protein residues. Motif discovery was then performed to identify conserved sequence motifs in AA9 proteins. Once type-specific sequence features were identified structural mapping was performed to assess possible effects on substrate interaction. Physicochemical property analysis was also performed to assess biochemical differences between AA9 LPMO types. Molecular dynamics (MD) simulations were then employed to dynamically assess the consequences of the discovered type-specific features on AA9-cellulose interaction. Due to the absence of AA9 specific force field parameters MD simulations were not readily applicable. As a result, Potential Energy Surface (PES) scans were performed to evaluate the force field parameters for the AA9 active site using the PM6 semi empirical approach and least squares fitting. A Type 1 AA9 active site was constructed from the crystal structure 4B5Q, encompassing only the Cu2+ coordinating residues, the Cu2+ ion and two water residues. Due to the similarity in AA9 active sites, the Type force field parameters were validated on all three AA9 LPMO types. Two MD simulations for each AA9 LPMO types were conducted using two separate Lennard-Jones parameter sets. Once completed, the MD trajectories were analysed for various features including the RMSD, RMSF, radius of gyration, coordination during simulation, hydrogen bonding, secondary structure conservation and overall protein movement. Force field parameters were successfully evaluated and validated for AA9 proteins. MD simulations of AA9 proteins were able to reveal the presence of unique type-specific binding modes of AA9 active sites to cellulose. These binding modes were characterised by the presence of unique type-specific loops which were present in Type 2 and 3 AA9 proteins but not in Type 1 AA9 proteins. The loops were found to result in steric congestion that affects how the Cu2+ ion interacts with cellulose. As a result, Cu2+ binding to cellulose was observed for Type 1 and not Type 2 and 3 AA9 proteins. In this study force field parameters have been evaluated for the Type 1 active site of AA9 proteins and this parameters were evaluated on all three types and binding. Future work will focus on identifying the nature of the reactive oxygen species and performing QM/MM calculations to elucidate the reactive mechanism of all three AA9 LPMO types

Modelación molecular de rodopsinas humanas mutantes asociadas a retinosis pigmentaria

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Física Aplicada. Fecha de lectura; 18-12-2013En este trabajo, se realiza una nueva aplicación combinada de modelación por homología, dinámica molecular clásica, teoría de funcionales de la densidad, deformaciones de la densidad electrónica y el método híbrido mecánica cuántica/mecánica molecular para evaluar propiedades geométricas, electrónicas y fotoquímicas en rodopsinas mutantes portadoras de las sustituciones M207R y S186W asociadas a Retinosis Pigmentaria (RP). Nuestros resultados para las rodopsinas bovina y humana normales, usadas como referencia, concuerdan adecuadamente con los resultados experimentales. En los mutantes, se encontró una geometría inadecuada del retinal para la reacción de fotoisomerización, una red puentes de hidrógeno del núcleo proteínico perturbada, y un corrimiento espectral hipsocrómico; esto último debido a características estructurales de la cadena del retinal, efectos del contraión e influencia del estado de protonación de la base de Schiff. Se describe, también por primera vez para el cromóforo retinal, el sistema electrónico π conjugado y sus perturbaciones en mutantes de manera explícita, una función cuadrática para la dependencia directa de las energías verticales de excitación con la alternancia de longitud de enlace, y se propone una vía para la generación fotosensibilizada de oxígeno singlete en rodopsinas que pudiera estar relacionada con la etiopatogenia de la RP y el daño fotoquímico retinian

Structure and dynamics studies of membrane and non-membrane proteins using NMR and MD simulations

The protein structural knowledge is essential in defining molecular recognition rules that power the understanding of basic biological phenomenon. The structures of most proteins are determinable due to advancement in technology and method development. Nuclear Magnetic Resonance (NMR) is one of the most versatile tools designed for this purpose. Proteins are flexible entities and dynamics play key role in their functionality therefore structures alone may provide only partial view on their functions. The experimental techniques have been used to study protein thermodynamics, but computer simulations have evolved to become the most convenient way to obtain the complete picture of protein dynamics. The central aim of this research is to study the structure and DNA binding dynamics of homologous pairing protein 2 (HOP2). In the first phase, the structure of N-terminal domain of HOP2 was investigated using NMR. It was identified with winged-helix DNA-recognition structural motifs. Furthermore, the DNA binding properties of this protein was investigated by NMR chemical shift perturbation method. It was found to bind to double-stranded DNA with considerable affinity, where structural motifs helix 3 (H3) and wing 1 (W1) were responsible for DNA recognition. Additionally, the site directed mutagenesis studies suggested H3 as the major contributor in DNA recognition. In the second phase, the DNA binding dynamics of HOP2 was investigated using classical MD simulations. Complexes of protein HOP2 and its mutants with DNA were constructed and then simulated using software GROMACS. Simulation results revealed the atomic level interactions between HOP2 and DNA, where H3 and W1 motifs engaged with DNA at major and minor grooves respectively. The effects on DNA binding due to point mutations in W1 and H3 were also observed. These effects were accessed in terms of changes in complex stability, binding free energy, and total number of interactions. The simulation results we obtained suggested that the motif W1 is also important as H3 in DNA binding. The NMR experimental and simulation protocol designed in this work will be useful in studying structure and dynamics of protein-protein or protein-ligand systems