Phosphoryl transfers are essential chemical reactions in key life processes, including energy production, signal transduction and protein synthesis. They are known for having extremely low reaction rates in aqueous solution, reaching the scale of millions of years. In order to make life possible, enzymes that catalyse phosphoryl transfer, phosphoryl transferases, have evolved to be tremendously proficient catalysts, increasing reaction rates to the millisecond timescale. Due to the nature of the electronic structure of phosphorus atoms, understanding how hydrolysis of phosphate esters occurs is a complex task. Experimental studies on the hydrolysis of phosphate monoesters with acidic leaving groups suggest a concerted mechanism with a loose, metaphosphate-like transition state. Theoretical studies have suggested two possible concerted pathways, either with loose or tight transition state geometries, plus the possibility of a stepwise mechanism with the formation of a phosphorane intermediate. Different pathways were shown to be energetically preferable depending on the acidity of the leaving group. Here we performed computational studies to revisit how this mechanistic shift occurs along a series of aryl phosphate monoesters, suggesting possible factors leading to such change. The fact that distinct pathways can occur in solution could mean that the same is possible for an enzyme active site. We performed simulations on the catalytic activity of β-phosphoglucomutase, suggesting that it is possible for two mechanisms to occur at the same time for the phosphoryl transfer. Curiously, several phosphoryl transferases were shown to be able to catalyse not only phosphate ester hydrolysis, but also the cleavage of other compounds. We modeled the catalytic mechanism of two highly promiscuous members of the alkaline phosphatase superfamily. Our model reproduces key experimental observables and shows that these enzymes are electrostatically flexible, employing the same set of residues to enhance the rates of different reactions, with different electrostatic contributions per residue.

The surface functionalisation of polyethylene and polypropylene by industrial and laboratory scale corona treatments and by laboratory flame treatment was studied. The surface sensitive techniques of X-ray photoelectron spectroscopy (XPS), attenuated total reflection infra-red spectroscopy (FTIR-ATR), contact angle measurement and electron microscopy (SEM and TEM) were employed. Corona and flame treatments resulted in incorporation of oxygen only into the surfaces of both polyethylene and polypropylene, resulting in improved surface wettabilities. A variety of oxygen functional groups were introduced by the two treatments. The industrial and laboratory scale treatments of both polymers were found to be similar in terms of the oxygen concentrations incorporated and surface wettabilities achieved. The presence of significant amounts of chain scission products were indicated on corona treated surfaces, while only minimal quantities were indicted on flame treated surfaces. This was attributed to their volatilisation during flame treatment. Introduction of sulfur dioxide into the flame and corona regions during treatment resulted in significant improvements in surface wettability. Incorporation of sulfur and nitrogen resulted from the presence of sulfur dioxide. A possible mechanism involving the formation of sulfonic acid groups and ammonium sulfonate groups was suggested. An oxidation depth model developed for use with variable take-off angle XPS showed that significantly deeper oxidation occurred in the presence of sulfur dioxide. Corona treatment was more effective in improving surface wettabilities than flame treatment, this being attributed to heat induced functional group reorientation during flame . treatment for polyethylene and to differences in surface chemistry resulting from the two treatments in the case of polypropylene. The surface wettability of poly ethylene was more readily improved than the surface wettability of polypropylene after all the treatments investigated. A method for estimating functional group concentrations using chemical derivatisation and contact angle measurement was developed. Functional group estimates for flame treated polyethylene were found to be in good agreement with chemical derivatisation used in conjunction with XPS measurements.

Cells interact with both tethered and motile ligands in their extra-cellular environment, which mediates, initiates and regulates a series of cellular functions, such as cell adhesion, migration, morphology, proliferation, apoptosis, bi-directional signal transduction, tissue homeostasis, wound healing among others. A fundamental understanding of the thermodynamics of receptor-mediated cell interaction is necessary not only from the aspect of physiology, but also for bioengineering applications, e.g. drug discovery, tissue engineering and biomaterial fabrication. Our models on free energy calculations of receptor mediated cell-matrix interactions supplement computational endeavors based on continuum mechanics. By incorporating conformational, entropic, solvation, steric effect, implicit and explicit interactions of receptors and extra-cellular ligand molecules, we can predict free energy, chemical equilibrium constant of binding, spatial and conformational distributions of biopolymers, adhesion force as functions of a set of key variables, e.g. surface coverage of receptor, interaction distance between cell and substrate, specific binding energy, implicit interaction strength, constraint in ligand’s conformation, size of motile nano-ligand, aggregation of receptors, sliding velocity relative to fluid. Our work has improved understanding of phenomena in cell-matrix interactions at both cellular and the molecular scales. / text

The first project in this work used classical molecular dynamics to study the ice recrystallization inhibition potential of a series of carbohydrates and alcochols, using the hydration index, partial molar volumes and isothermal compressibilities as parameters for measuring their cryogenic efficacy. Unfortunately, after 8 months of testing, this work demonstrates that the accuracy and precision of the density extracted from simulations is not sufficient in providing accurate partial molar volumes. As a result, this work clearly demonstrates that current classical molecular dynamics technology cannot probe the volumetric properties of interest with sufficient accuracy to aid in the research and development of novel cryoprotectants.The second project in this work used molecular dynamics simulations to evaluate the Gibbs free energy change of substituting CO2 in sI clathrate hydrates by N2,CH4, SO2 and H2S flue gas impurities under conditions proposed for CO2 capture (273 K, 10 bar). Our results demonstrate that CO2 substitutions by N2 in the small sI cages were thermodynamically favored. This substitution is problematic in terms of efficient CO2 capture, since the small cages make up 25% of the sI clathrate cages, therefore a significant amount of energy could be spent on removing N2 from the flue gas rather than CO2. The thermodynamics of CO2 substitution by CH4, SO2 and H2S in sI clathrate hydrates was also examined. The substitution of CO2 by these gases in both the small and large cages were determined to be favorable. This suggests that these gases may also disrupt the CO2 capture by sI clathrate hydrates if they are present in large concentrations in the combustion flue stream. Similar substitution thermodynamics at 200 K and 10 bar were also studied. With one exception, we found that the substitution free energies do not significantly change and do not alter the sign of thermodynamics. Thus, using a lower capture temperature does not significantly change the substitution free energies and their implications for CO2 capture by sI clathrate hydrates.

The three-dimensional structure of a protein, formed as a result of amino-acid sequences folding into compact domains, is regarded as a key factor in its biological function. How and why proteins fold into specific topologies, remain the key focus of scientific research in the field of biophysics. By stripping down complex reactions down to the most basic elements, biophysicists aim to develop simplified models for biological phenomena such as antibody discrimination, viral fusion or self-assembly. Focusing on small model peptide systems, rather than the full proteins from which they were derived, was hoped to result in accurate structural measurements and provide a more transparent comparison between simulation and experiment. The aim of this research was therefore to investigate how accurate these models were when compared against experiment. Furthermore, while breaking down the complex biological phenomena into simple models, there was also a conscious effort to ensure that the models were representative of real biological systems, and a major focus was therefore aimed at determining whether any meaningful biomedical insight may be extrapolated from such models. Peptides found in hormones (human chorionic gonadotropin, luteinizing hormone), viruses (HIV) and amyloid diseases (transthyretin) were selected in order to probe a variety of questions in relation to the aforementioned biological phenomena. Namely, how the primary sequence influenced the three-dimensional structure (and thus its biological function), how its environment could influence such a confirmation, and how these systems aggregated. This doctoral study has made use of a combination of computer simulations and experimental techniques to investigate a selection of biologically relevant peptides; utilising classical atomistic molecular dynamics (MD) simulations to characterise the free-energy landscapes of the chosen peptides, and compare these findings with the secondary structure content predicted by spectroscopic methods such as circular dichroism and infrared spectroscopy. The peptide systems studied within, were found to be characterised by rugged free-energy landscapes unlike their protein counterparts (defined by singular, deep minima). Furthermore, these landscapes were found to be highly plastic and sensitive to changes in the local environment.

Student Number : 0002483R - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Isothermal titration calorimetry was used to investigate the forces that drive ligand binding to the active site of rGST M1-1. In an attempt to gain insight into the recognition of non-substrate ligands by GSTs, this study also investigates interactions between rGST M1-1 and ANS, a non-substrate ligand. At 25 °C, complex formation between rGST M1-1 and GSH, GSO3 -, and S-hexylglutathione is characterised by a monophasic binding isotherm with Kd values of 38.5 mM, 2.1 mM and 0.2 mM, respectively. One molecule of each ligand is bound per monomer of rGST M1-1. Binding of these ligands is enthalpically favourable and entropically unfavourable with a resultant favourable Gibbs free energy, overall. The effects of temperature and buffer ionisation on the energetics of binding were studied. The enthalpic and entropic contributions for all three ligands exhibited temperature dependence over the temperature range investigated (5-30 °C). The Gibbs free energy showed negligible changes with increasing temperature due to enthalpy-entropy compensation. The temperature dependence of the binding enthalpy yielded heat capacity changes of – 2.69 kJ/mol/K and –3.68 kJ/mol/K at 25 °C for GSH and S-hexylglutathione binding and –1.86 kJ/mol/K overall for GSO3 -. The linear dependence of DH on temperature for GSO3 - binding to rGST M1-1 suggests the formation of a more constrained complex which limits the fluctuations in conformations of the mu-loop at the active site. The non-linear dependence of DH on temperature for GSH and Shexylglutathione binding to the enzyme suggests the formation of a complex that samples different bound conformations due to the mobility of the mu-loop even after ligand is bound. Calorimetric binding experiments in various buffer systems with different ionisation enthalpies suggest that the binding of GSH to rGST M1-1 is coupled to the deprotonation of the thiol of GSH while GSO3 - binding to rGST M1-1 is independent of the buffer ionisation. At 25 °C, the rGST M1-1#1;ANS association is represented by a monophasic binding isotherm with one molecule of ANS bound per monomer of rGST M1-1. The interaction is both enthalpically and entropically driven with a Kd value of 27.2 mM representing moderate affinity. The effect of temperature on the interaction was investigated over the temperature range of 5-30 °C. The linear dependence of the binding enthalpy on temperature indicates that no significant structural changes occur upon binding of ANS to the enzyme (DCp = -0.34 kJ/mol/K). The change in heat capacity associated with the interaction can be attributed to the burial of the polar sulphonate group of ANS and the exposure of the anilino and naphthyl rings to solvent as well as the possibility of weak electrostatic interactions between ANS and residues at the active site. The effect of ethacrynic acid, GSH, GSO3 - and S-hexylglutathione on the fluorescence of ANS was investigated in order to obtain some idea as to the location of the ANS binding site on rGST M1-1. ANS was displaced by GSO3 -, S-hexylglutathione and ethacrynic acid, while no displacement occurred upon binding of GSH to the active site of rGST M1-1. Displacement studies and molecular docking simulations indicate that ANS binds to the H-site of rGST M1-1 and the possibility of a second binding site for the molecule cannot be ruled out.

A study of how the matrix environment impacts the structure and IR spectra of N-acetylglycine conformers. The conformational composition of this compound is determined according to an analysis of the FTIR spectra of N-acetylglycine isolated in low temperature argon matrices. Bands of three N-acetylglycine conformers are identified based on the spectra: one major and two minor. The structure of all observed conformers is stabilized by different intramolecular hydrogen bonds. The Gibbs free energies of the conformers were calculated (CCSD(T)/CBS method), and these energy values were used to calculate conformer population at a temperature of 360 K, of which 85.3% belonged to the main conformer, and 9.6% and 5.1% to the minor conformers. We also determined the size and shape of the cavities that form when the N-acetylglycine conformers are embedded in the argon crystal during matrix deposition. It is established that the most energetically favorable cavity for the planar main conformer is the cavity that forms when 7 argon atoms are replaced. At the same time, bulky minor conformers were embedded into cavities that correspond to 8 removed argon atoms. We calculated the complexation energy between argon clusters and conformers, and the deformation energy of the argon crystal and the N-acetylglycine conformers. The matrix-induced shifts to the conformer oscillation frequency are calculated. Published by AIP Publishing.

Os sistemas biológicos macromoleculares são conhecidos por serem sistemas interagentes. Essas interações são fundamentais para processos como comunicação celular, especificidade de reações enzimáticas e regulação da expressão gênica. Os métodos disponíveis atualmente para estimar a afinidade das interações biomoleculares podem ser divididos, basicamente, em dois grupos: métodos rápidos que estimam a energia livre de ligação através de aproximações de campo de força (por exemplo, docking); e os métodos que são baseados em ensembles de Dinâmica Molecular (DM) para calcular as energias livres de ligação de maneira mais rigorosa, porém, com custo computacional mais elevado. O objetivo deste trabalho é aprimorar e validar um método menos custoso para o cálculo da energia livre de ligação. Para isso, simulações atomísticas de Monte Carlo (MC) dos ligantes no sítio de ligação são usadas para gerar ensembles termodinâmicos. Depois disso, as energias livres de ligação são calculadas usando uma combinação de energias e entropias estimadas através de uma estratégia de aproximação de primeira ordem. Dois algoritmos de amostragem foram avaliados no cálculo de energia de ligação. O primeiro algoritmo amostra graus de liberdade de translação e rotação randômicas do centro de massa do ligante no sítio de ligação, além de variações randômicas nos ângulos de torção envolvendo átomos pesados (não hidrogênio). O segundo amostra graus de liberdade rotacional e translacional do centro de massa, além de deslocamentos atômicos individuais para cada átomo do ligante. Além disso, diferentes modelos para calcular as contribuições polares para interação intermolecular foram utilizados. Comparações entre as energias livres de ligação calculadas com baixo custo computacional e as experimentais disponíveis na literatura para o sistema modelo utilizado, lisozima do vírus T4, mostraram uma correlação considerável (r=0,64 para N=27). Esses dados também apresentaram resultados interessantes quando comparados com outras metodologias, tais como LIE, MM-PBSA e MM-GBSA. Assim, a abordagem utilizada para a determinação das energias de interação mostrou-se eficiente em termos de tempo computacional e para comparação com dados de energia livre de ligação determinados experimentalmente. / Macromolecular biological systems are widely known by its interaction properties. Those interactions play fundamental roles in processes such as cellular communication, specificity of enzymatic reactions and regulation of gene expression. The methods currently available to estimate the affinity of biomolecular interactions can be divided basically into two groups: fast methods that estimate the free energy of binding through force field approximations (e.g., docking); and methods that are based on Molecular Dynamics (DM) ensembles to calculate binding free energies more rigorously, however, with higher computational cost. The objective of this work is to improve and validate a less costly method for calculating binding free energy. For this, atomistic Monte Carlo (MC) simulations of ligands at the binding site are used to generate thermodynamic ensembles. Thereafter, the binding free energies are calculated using a combination of energies and entropy estimated through a first-order approximation strategy. Two sampling algorithms were evaluated in the calculation of the binding energy. The first one samples the degrees of freedom from translation and rotation of the center of mass of the binder at the binding site, as well as random variations in the torsion angles involving heavy atoms (non-hydrogen). The second one samples the rotational and translational degrees of freedom of the ligand center of mass, as well as individual atomic displacements for each atom of the ligand. In addition, different models to calculate the polar contributions for intermolecular interaction were used. Comparisons between the binding free energies calculated with low computational cost and the experimental ones available in the literature for the system used, T4 virus lysozyme, resulted in acceptable correlation values (r=0.64 for N=27). Those data also showed interesting results compared to different methodologies such as LIE, MM-PBSA and MM-GBSA. Therefore, the used approach for determining the binding energies was efficient in terms of computational time and for comparison with free energy data determined experimentally.

As interações moleculares, em especial as de caráter não-covalente, são processos-chave em vários aspectos da biologia celular e molecular, desde a comunicação entre as células ou da velocidade e especificidade das reações enzimáticas. Portanto, há a necessidade de estudar e criar métodos preditivos para calcular a afinidade entre moléculas nos processos de interação, os quais encontram uma gama de aplicações, incluindo a descoberta de novos fármacos. No geral, entre esses valores de afinidade, o mais importante é a energia livre de ligação, que normalmente é determinada por modos computacionalmente rápidos, porém sem uma forte base teórica, ou por cálculos muito complexos, utilizando dinâmica molecular, onde mesmo com um grande poder de determinação da afinidade, é muito custoso computacionalmente. O objetivo deste trabalho é avaliar um modelo menos custoso computacionalmente e que promova um aprofundamento na avaliação de resultados obtidos a partir de simulações de docking molecular. Para esta finalidade, o método de Monte Carlo é empregado para a amostragem de orientações e conformações do ligante do sítio ativo macromolecular. A avaliação desta metodologia demonstrou que é possível calcular grandezas entrópicas e entálpicas e analisar a capacidade interativa entre complexos proteína-ligante de forma satisfatória para o complexo lisozima do bacteriófago T4. / The molecular interactions, especially the ones with a non-covalent nature, are key processes in general aspects of cellular and molecular biology, including cellular communication and velocity and specificity of enzymatic reactions. So, there is a strong need for studies and development of methods for the calculation of the affinity on interaction processes, since these have a wide range of applications like rational drug design. The free energy of binding is the most important measure among the affinity measurements. It can be calculated by quick computational means, but lacking on strong theoretical basis or by complex calculations using molecular dynamics, where one can compute accurate results but at the price of an increased computer power. The aim of this project is to evaluate a computationally inexpensive model which can improve the results from molecular docking simulations. For this end, the Monte Carlo method is implemented to sample different ligand configurations inside the macromolecular binding site. The evaluation of this methodology showed that is possible to calculate entropy and enthalpy, along analyzing the interactive capacity between receptor-ligands complexes in a satisfactory way for the bacteriophage T4.

When the states of a system can be described by the extrema of a high-dimensional function, the characterisation of its complexity, i.e. the enumeration of the accessible stable states, can be reduced to a sampling problem. In this thesis a robust numerical protocol is established, capable of producing numerical estimates of the total number of stable states for a broad class of systems, and of computing the a-priori probability of observing any given state. The approach is demonstrated within the context of the computation of the configurational entropy of two and three-dimensional jammed packings. By means of numerical simulation we show the extensivity of the granular entropy as proposed by S.F. Edwards for three-dimensional jammed soft-sphere packings and produce a direct test of the Edwards conjecture for the equivalent two dimensional systems. We find that Edwards’ hypothesis of equiprobability of all jammed states holds only at the (un)jamming density, that is precisely the point of practical significance for many granular systems. Furthermore, two new recipes for the computation of high-dimensional volumes are presented, that improve on the established approach by either providing more statistically robust estimates of the volume or by exploiting the trajectories of the paths of steepest descent. Both methods also produce as a natural by-product unprecedented details on the structures of high-dimensional basins of attraction. Finally, we present a novel Monte Carlo algorithm to tackle problems with fluctuating weight functions. The method is shown to improve accuracy in the computation of the ‘volume’ of high dimensional ‘fluctuating’ basins of attraction and to be able to identify transition states along known reaction coordinates. We argue that the approach can be extended to the optimisation of the experimental conditions for observing certain phenomena, for which individual measurements are stochastic and provide little guidance.