<p>The structural and thermodynamic properties of a crystal-melt interface in</p> <p>elemental magnesium have been investigated using molecular dynamics (MD)</p> <p>simulations with an embedded atom method description of the interatomic potential.</p> <p>Three low index interfacial orientations, (0001), (1101) and (1120), have been studied.</p> <p>From fine-grained atomic density profiles, the structural interfacial widths show obvious anisotropy and the variation of interatomic planar spacing as a function of distance through the crystal-melt boundary is established. Mainly from the coarse-grained density profiles, the effective 10-90 width of the interface region, defined as the intrinsic width, in each orientation has been determined. In addition, the interfacial stresses are obtained from an integration of the interfacial stress profiles and the results show significant anisotropy, which is possibly related to the anisotropy of occupation fraction profiles. Finally, from a determination of the excess energy and interfacial stress of the solid-liquid interface and from previous published results for the interfacial free energy at the melting point, the Gibbs-Cahn integration is employed to derive an estimation of the temperature dependence of the interfacial free energy at non-equilibrium temperatures. All of the crystal-melt interfacial properties for magnesium are compared with simulation data from other elemental metals and alloys, as well as from other model systems such as Lennard- Jones and hard spheres.</p> / Master of Applied Science (MASc)

Pentameric ligand-gated ion channels (pLGICs) are membrane receptors that play a crucial role in every living organism. The pLGIC protein structure forms a pore through the membrane of a cell that can let specific ions pass through, upon activation by endogenous agonists. pLGICs are allosterically modulated by ligands binding at allosteric sites, that either stabilize a certain conformation or change the binding affinity of the endogenous agonist. However, much remains unknown about the exact way in which these modulators bind to and affect pLGICs. An increased understanding could help in the search for novel and/or more effective target drugs. With this masters thesis, I hope to contribute by investigating the modulatory effect of ethanol on the bacterial Gloeobacter ligand-gated ion channel (GLIC). This has been done by performing oocyte electrophysiology recordings and analysis of molecular dynamics simulations, both with and without ethanol, and of four separate variants of GLIC that are either potentiated or inhibited by ethanol. Two possible allosteric sites were discovered in a transmembraneintrasubunit pocket: a potentiating allosteric site close to the M2 helix and residue V242, as well as an inhibitory membrane- and M4 helix-close intrasubunit site. Finally, evidence was found that could support a previously suggested inhibitory allosteric site in the pore around the 9’ hydrophobic gate. / Pentameriska ligandstyrda jonkanaler (pLGICs) är membranreceptorer som utgör vitala delar av varje levande organism. pLGICs proteinstruktur formar en por genom cellmembranet, som kan släppa igenom specifika joner efter aktivering av endogena agonister. pLGICs är allostermodulerade av ligander som binder vid allostera säten och som därigenom antingen stabiliserar en viss form eller förändrar den endogena agonistens bindningsstyrka. Emellertid saknas fortfarande mycket kunskap på detaljnivå om hur dessa modulatorer binder sig till och påverkar kanalerna. En ökad förståelse skulle hjälpa forskningen efter nya och/eller mer effektiva mediciner. Mitt examensarbetehoppas bidra genom att studera hur etanol modulerar den bakteriella ligandstyrda jonkanalen GLIC från Gloeobacter. Det har gjorts genom elektrofysiologimätningar på oocyter och analys av molekulärdynamiksimuleringar, båda av fyra olika GLIC-varianter, som antingen potentieras eller hämmas av etanol, och med eller utan etanol. Två allostera säten upptäcktes i det transmembrana intrasubenhetområdet: ett säte för potentiering nära M2 helixen och aminosyran V242, och ett hämmande säte nära membranet och helix M4. Slutligen hittades tecken som kan styrka existensen av det tidigare föreslagna hämmande allostera sätet i poren kring den hydrophoba porten.

<p>Bacterial endospores have been a topic of research interest over the last several decades given their high resistance to ultraviolet (UV) damage. Unlike vegetative bacterial cells, which form cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) as the major product upon UV irradiation, endospore bacteria form a spore photoproduct (5-(<em>R</em>-thyminyl)-5,6-dihydrothymine or SP) as the major product. Vegetative bacteria cells are subject to regular cell activities and processes such as division and deoxyribonucleic acid (DNA) replication, which are prone to damage from UV exposure. However, in endospores, which have a largely anhydrous inner environment, the DNA remains dormant when bound to spore-specific small acid-soluble proteins (SASP) and dipicolinic acid, making spores highly resistant to radiation, heat, desiccation, and chemical harm. During spore germination, SP lesions in DNA are repaired by a distinctive repair enzyme, spore photoproduct lyase (SPL). In this thesis, molecular dynamics (MD) simulations were carried out to (i) examine how the formation of the SP lesion in DNA affects the global and local structural properties of duplex DNA and (ii) study how this lesion is recognized and repaired in endospore. The first part of this work was focused on designing and developing a structurally and dynamically stable model for dinucleotide SP molecule (TpT), which was subsequently used as an SP patch incorporated into duplex DNA. Computationally, this requires modifications of the bond and nonbonded force field parameters. The stability of the patch and developed parameters was tested via solution-phase MD simulations for the SP lesion incorporated within the B-DNA dodecamer duplex (PDB 463B). The second part involved applying the new SP patch to simulate the crystallographic structure of the DNA oligomer containing SP lesions. Solution-phase MD simulations were performed for the SP-containing DNA oligomers (modeled based on PDB 4M94) and compared to the simulations of the native structure (PDB 4M95). Our analysis of the MD trajectories revealed a range of SP-induced structural and dynamical changes, including the weakened hydrogen bonds at the SP sites, increased DNA bending, and distinct conformational stability and distribution. In the third part of this thesis project, we carried out MD simulations of SP-containing DNA bound with SASPs to examine how the DNA interacts differently with SASP in the presence and absence of the SP lesion. The simulation results suggested decreased electrostatic and hydrogen bonding interactions between SASP and the damaged DNA at the SP site compared to the undamaged DNA-protein complex. In addition, decreased helicity percentage was observed in the SASPs that directly interact with the SP lesion. The last part of this this thesis work focused on the SP-dimer flipping mechanism, as the lesion is likely flipped out to its extrahelical state to be recognized and repaired by SPL. Using steered molecular dynamic (SMD) simulations and a pseudo-dihedral angle reaction coordinate, we obtained possible SP flipping pathways both in the presence and absence of SASP. Collectively, these simulation results lend new perspectives toward understanding the unique behavior of the SP lesion within the DNA duplex and the nucleoprotein complex. They also provide new insights into how the SP lesion is efficiently recognized and repaired during spore germination.</p>

Knowledge of the dissociation constants of the ionizable protons of weak acids in aqueous media is of fundamental importance in many areas of chemistry and biochemistry. The pKa value, or equilibrium dissociation constant, of a molecule determines the relative concentration of its protonated and deprotonated forms at a specified pH and is therefore an important descriptor of its chemical reactivity. Considerable efforts have been devoted to the determination of pKa values by deferent experimental techniques. Although in most cases the determination of pKa values from experimental is straightforward, there are situations where interpretation is difficult and the results ambiguous. It is, therefore, not surprising that the capability to provide accurate estimates of the pKa value has been a central goal in theoretical chemistry and there has been a large effort in developing methodologies for predicting pKa values for a variety of chemical systems by differing quantum chemical techniques. A prediction accuracy within 0.5 pKa units of experiment is the desirable level of accuracy. This is a non-trivial exercise, for an error of 1 kcal/mol in estimates of the free energy value would result in an error of 0.74 pKa units. In this thesis ab initio Car-Parrinello molecular dynamics (CPMD) has been used for investigating the Brϕnsted acid-base chemistry of weak acids in aqueous solution. A key issue in any dissociation event is how the solvating water molecules arrange themselves spatially and dynamically around the neutral and dissociated acid molecule. Ab initio methods have the advantage that all solvent water molecules can, in principle, be con- sidered explicitly. One of the factors that has inhibited the widespread use of ab initio MD methods to study the dissociation reaction is that dissociation of weak acids are rare events that require extremely long simulation times before one is observed. The metady- namics formalism provides a solution to this conundrum by preventing the system from revisiting regions of configuration space where it has been in the past. The formalism allows the system to escape the free-energy minima by biasing the dynamics with a history dependent potential (or force) that acts on select degrees of freedom, referred to as collective variables. The bias potentials, modeled by repulsive inverted Gaussians that are dropped during propagation, drive the system out of any free-energy minima and allow it to explore the configuration space by a relatively quick and efficient sampling. The the- sis deals with a detailed investigation of the Brϕnsted acid-base chemistry of weak acids in aqueous solutions by the CPMD-metadynamics procedure. In Chapter 1, current approaches for the theoretical estimation of pKa values are summarized while in Chapter 2 the simulation methodology and the metadynamics sampling techniques used in thisstudy are described. The potential of the CPMD-metadynamics procedure to provide estimates of the acid dissociation constant (pKa) is explored in Chapter 3, using acetic acid as a test sys- tem. Using the bond-distance dependent coordination number of protons bound to the dissociating carboxylic groups as the collective variable, the free-energy profile for the dissociation reaction of acetic acid in water was computed. Convergence of the free-energy profiles and barriers for the simulations parameters is demonstrated. The free-energy profiles exhibit two distinct minima corresponding to the dissociated and neutral states of the acid and the deference in their values provides the estimate for pKa. The estimated value of pKa for acetic acid from the simulations, 4.80, is in good agreement with the experiment at value of 4.76. It is shown that the good agreement with experiment is a consequence of the cancellation of errors, as the pKa values are computed as the difference in the free energy values at the minima corresponding to the neutral and dissociated state. The chapter further explores the critical factors required for obtaining accurate estimates of the pKa values by the CPMD-metadynamics procedure. It is shown that having water molecules sufficient to complete three hydration shells as well as maintaining water density in the simulation cell as close to unity is important. In Chapter 4, the CPMD-metadynamics procedure described in Chapter-3 has been used to investigate the dissociation of a series of weak organic acids in aqueous solutions. The acids studied were chosen to highlight some of the major factors that influence the dissociation constant. These include the influence of the inductive effect, the stabilization of the dissociated anion by H-bonding as well as the presence of multiple ionizable groups. The acids investigated were aliphatic carboxylic acids, chlorine-substituted carboxylic acids, cid and trans-butenedioic, the isomers of hydroxybenzoic acid and phthalic acids and its isomers. It was found that in each of these examples the CPMD-metadynamics procedure correctly estimates the pKa values, indicating that the formulism is capable of capturing these influences and equally importantly indicating that the cancellation of errors is indeed universal. Further, it is shown that the procedure can provide accurate estimates of the successive pKa values of polypro tic acids as well as the subtle deference in their values for deterrent isomers of the acid molecule. Changes in protonation-deprotonation of amino acid residues in proteins play a key role in many biological processes and pathways. It is shown that CPMD simulations in conjunction with metadynamics calculations of the free energy profile of the protonation- deprotonation reaction can provide estimates of the multiple pKa values of the 20 canonical α-amino acids in aqueous solutions in good agreement with experiment (Chapter 5). The distance-dependent coordination number of the protons bound to the hydroxyl oxygen of the carboxylic and the amine groups is used as the collective variable to explore the free energy profiles of the Brϕnsted acid-base chemistry of amino acids in aqueous solutions. Water molecules, sufficient to complete three hydration shells surrounding the acid molecule were included explicitly in the computation procedure. The method works equally well for amino acids with neutral, acidic and basic side chains and provides estimates of the multiple pKa values with a mean relative error with respect to experimental results, of 0.2 pKa units. The tripeptide Glutathione (GSH) is one of the most abundant peptides and the major repository for non-protein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thioldisulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influences the redox couple and hence the pKa value of the cysteine residue of GSH is critical to its functioning. In Chapter 6, it has been reported that ab initio Car-Parrinello Molecular Dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. It is shown that the free-energy landscape for the protonation - deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared to the isolated cysteine amino acid. The dissociation constants of weak acids are commonly determined from pH-titration curves. For simple acids the determination of the pKa from the titration curves using the Henderson-Hasselbalch equation is relatively straightforward. There are situations, however, especially in polypro tic acids with closely spaced dissociation constants, where titration curves do not exhibit clear inflexion and equivalence stages and consequently the estimation of multiple pKa values from a single titration curve is no longer straightfor- ward resulting in uncertainties in the determined pKa values. In Chapter 7, the multiple dissociation constant of the hexapeptide glutathione disulfide (GSSG) with six ionizable groups and six associated dissociation constants has been investigated. The six pKa values of GSSG were estimated using the CPMD-metadynamics procedure from the free-energy profiles for each dissociation reaction computed using the appropriate collective variable. The six pKa values of GSSG were estimated and the theoretical pH-titration curve was then compared with the experimentally measured pH-titration curve and found to be in excellent agreement. The object of the exercise was to establish whether interpretation of pH-titration curves of complex molecules with multiple ionizable groups could be facilitated using results of ab initio molecular dynamics simulations.

The selection of the forcefield is a crucial issue in any MD related work and there is no clear indication as to which of the many available forcefields is the best for protein analysis. Many recent literature surveys indicate that MD work may be hindered by two limitations, namely conformational sampling and forcefields used (inaccuracies in the potential energy function may bias the simulation toward incorrect conformations). However, the advances in computing infrastructures, theoretical and computing aspects of MD have paved the way to carry out a sampling on a sufficiently longtime scale, putting a need for the accuracies in the forcefield. Because there are established differences in MD results when using forcefields, we have sought to ask how we could assess common mobility segments from a protein by analysis of trajectories using three forcefields in a similar environment. This is important because, disparate fluctuations appear to be more at flexible regions compared to stiff regions; in particular, flexible regions are more relevant to functional activities of the protein molecule. Therefore, we have tried to assess the similarity in the dynamics using three well-known forcefields ENCAD, CHARMM27 and AMBERFF99SB for 61 monomeric proteins and identify the properties of dynamic residues, which may be important for function. The comparison of popular forcefields with different parameterization philosophy may give hints to improve some of the currently existing agnostics in forcefields and characterization of mobile regions based on dynamics of proteins with diverse folds. These may also give some signature on the proteins at the level of dynamics in relation to function, which can be used in protein engineering studies. Nanosecond level MD simulation(30ns) on 61 monomeric proteins were carried out using CHARMM and AMBER forcefields and the trajectories with ENCAD forcefield obtained from Dynameomics database. The trajectories were first analyzed to check whether structural and dynamic properties from the three forcefields similar choosing few parameters in each case. The gross dynamic properties calculated (root mean square deviation (RMSD), TM-score derived RMSD, radius of gyration and accessible surface area) indicated similarity in many proteins. Flexibility index analysis on 17 proteins, which showed a notable difference in the flexibility, indicated that tertiary interactions (fraction of nonnative stable hydrogen bonds and salt bridges) might be responsible for the difference in the flexibility index. The normalized subspace overlap and shape overlap score taken based on the covariance matrices derived from trajectories indicated that majority of the proteins show a range between 0.3-0.5 indicating that the first principal components from these proteins in different combinations may not match well. These results indicate that although dynamic properties in general are similar in many proteins. However, flexibility index and normalized subspace overlap score indicate that subspaces on the first principal component in many proteins may not match completely. The number of proteins showing a better correlation is higher in CHARMM-AMBER combinations than the other two. The structural features from trajectories have been computed in terms of fraction of secondary structure, hydrogen bonds, salt bridges and native contacts. Although secondary structures and native contacts are well preserved during the simulations, the tertiary interactions (hydrogen bonds) are lost in many proteins and may be responsible for the difference in the some of properties among forcefields. Comparison of simulation results to experimental structures in terms of Root mean square fluctuations, Accessible surface area and radius of gyration indicates that the simulations results are on par with the ones derived from experimental structures. We have tried to assess the flexibility in the proteins using normalized Root mean square fluctuations (nRMSF), which for a residue is the ratio of RMSF from simulation to that of crystal structure. We have selected a threshold for this nRMSF to indicate the mobile regions in a protein based on secondary structure analysis. Based on the threshold of nRMSF and conformational properties (deviation in the dihedral angles), we have classified the residue and evaluated the properties of rigid hinge residues and corresponding mobile residues in terms of residue propensity, secondary structure preference and accessible surface area ranges. Since the rigid dynamic residues represent the inherent mobility, they might be important for function. Therefore, we have tried to assess the functional relevance considering the dynamic mobile residues from each protein from each forcefield simulation with the residues important for the function (taken from literature and databases). It is observed that some residues found to be mobile from the simulation are found to match with the experimental ones, although in many cases the number of these mobile residues is higher compared to the experimental ones. In summary, an analysis of protein simulation trajectories using three forcefields on a set of monomeric protein has shown that the gross structural properties and secondary structures from many proteins remain similar, but there are differences as may be seen from flexibility index. However correlation in parameters from CHARMM and AMBER force field is better compared to other two combinations. The differences seen in some of structural properties may arise mainly due to the loss of few tertiary interactions as indicated by the fraction of native hydrogen bonds and salt bridges. Based on the nRMSF, mobile segments obtained from the simulations were identified, and some of the mobile segments are found to match the functionally important residues from the experimental ones. Our work indicates that there are still some differences in the properties from the simulations, which indicates that care must be exercised when choosing a forcefield, especially assessing the functionally relevant residues from the simulations.

<p>  </p> <p>Experimental and computational techniques are used to study physico-chemical phenomena occurring in water on which hydrophobic interactions play a role. In particular, hydrophobic self-aggregation, including host-guest binding, and the affinity of ions to oil/water interfaces are investigated. Raman multivariate curve resolution (Raman-MCR) spectroscopy was the experimental technique used to unveil intermolecular interactions through the analysis of solute-correlated (SC) vibrational spectra. Molecular simulations, including molecular dynamics (MD) simulations, quantum-mechanical calculations, or a combination of both, were carried out to assist with the molecular-level interpretation of the experimental SC spectra.</p>