The aim of this study is to further our understanding of the forces that contribute to protein stability and to investigate how site-directed mutagenesis might be used for increasing protein stability. Eleven proteins ranging from 36 to 370 residues have been studied here. A 36-residue VHP and a 337-residue VlsE were used as model systems for studying the contribution of the hydrophobic effect on protein stability. Mutations were made in both proteins which replaced bulky hydrophobic side chains with smaller ones. All variants were less stable than their wild-type proteins. For VHP, the destabilizing effects of mutations were smaller when compared with similar mutations reported in the literature. For VlsE, a similarity was observed. This different behavior was investigated and reconciled by the difference in hydrophobicity and cavity modeling for both proteins. Therefore, the stabilizing mechanism of the hydrophobic effect appears to be similar for both proteins. Eight proteins were used as model systems for studying the effects of mutating non-proline and non-glycine residues to statistically favored proline and glycine residues in ?-turns. The results suggest that proline mutations generally increase protein stability, provided that the replaced residues are solvent exposed. The glycine mutations, however, only have a stabilizing effect when the wild-type residues have ?, ? angles in the L? region of Ramachandran plot. Nevertheless, this strategy still proves to be a simple and efficient way for increasing protein stability. Finally, using a combination of eight previously identified stabilizing mutations; we successfully designed two RNase Sa variants (7S, 8S) that have both much higher Tms and conformational stabilities than wild-type protein over the entire pH range studied. Further studies of the heat capacity change upon unfolding (?Cps) for both proteins and their variants suggest that residual structure may exist in the denatured state of the 8S variant. An analysis of stability curves for both variants suggests that they achieve their stabilization through different mechanisms, partly attributed to the different role of their denatured states. The 7S variants may have a more rigid denatured state and the 8S variant may have a compact denatured state in comparison with that of wild-type RNase Sa.

The recognition of modified amino acids by reader proteins is governed by the competing interplay of weak, attractive, intermolecular forces and solvation effects. For the recognition of hydrophobic cations like methyl-lysines and methyl-arginines, native reader proteins utilize structural cages always containing multiple aromatic amino acids and sometimes an occasional acidic residue. Through the highly ordered arrangement of multiple aromatic surfaces, reader proteins can invoke the attractive forces of electrostatic, cation-pi, and in the case of arginine, pi-pi interactions. The hydrophobic effect can also significantly affect these binding events in aqueous environments. In this thesis, a number of small molecule, synthetic cages containing significant aromatic surface area have been synthesized. Variation in both total host hydrophobicity and degree of flexibility were explored to determine what effect they have on the overall binding of methylated amino acids in water. Significant flexibility in the first generation of highly aromatic hosts was shown to be detrimental to binding. However, strong binding was observed for guests with significant hydrophobic character despite this flexibility. The cause of the strong affinities in this family of synthetic cages was shown to be due to the hydrophobic effect, rather than any attraction due to cation-pi interactions. Synthetic efforts towards hosts with more rigid structures led to the use of Tröger’s base as a structural building block. Hosts incorporating Tröger’s bases into well-defined aromatic cavities were found to exhibit strong binding to both methyl-lysine and methyl-arginine derivatives in pure water. Differences in guest selectivity were due to the rigid altered host geometry introduced by the Tröger’s base cleft. / Graduate

This thesis research presents the synthesis and first application of bis-amino acid-based spiroligomers towards the development of organocatalysis, from small molecules to moderate size spiroligomers, and to macromolecules. By synthesizing a toolbox of cyclic monomers called "bis-amino acids", the Schafmeister group has developed an approach to construct both small and macromolecules named "Spiroligomers". These molecules arrange catalytic functional groups in a shape-persistent and programmable backbone. Unlike proteins and small peptides, spiroligomers do not fold; rather, their polycyclic backbone structures are controlled by the sequence and stereochemistry of the component monomers. Firstly, we demonstrated a structure/catalytic activity relationship together with computational modeling that suggests that a specific hydrophobic interaction between the modified pro4 catalyst and the aldehyde substrate is responsible for an observed rate enhancement in the aldol reaction. For the moderate size molecules, several spiroligomer libraries were prepared through solid phase or solution phase synthesis and screened for either the alcohol kinetic resolution reaction or the aldol reaction. The poor activity and selectivity suggest that the scaffolds involved cannot create the necessary chiral environment for asymmetric catalysis. Finally, a synthetic method of macromolecules using cross metathesis coupling was developed and a series of tetra-functionalized macrocyclic spiroligomers were synthesized. Three of these macromolecules were examined as asymmetric catalysts in the aldol reaction and gave moderate activity and selectivity. The NMR analysis of these macromolecules indicates their dynamic nature. As the first application of bis-amino acid based macromolecules in organocatalysis area, although these catalysts only generated moderate activity and selectivity, they provided evidence that changing the configuration of one stereocenter of the fourteen available within these macromolecules can alter the selectivity. This synthetic methodology also provides an effective way to create more complicated pocket like spiroligomer macromolecules for the future applications in catalysis and molecular recognition. / Chemistry

<p>Molecular recognition is arguably the most elementary physical process essential for life that arises at the molecular scale. Molecular recognition drives events across virtually all length scales, from the folding of proteins and binding of ligands, to the organization of membranes and the function of muscles. Understanding such events at the molecular level is massively complicated by the unique medium in which life occurs: water. In contrast to recognition in non-aqueous solvents, which are driven largely by attractive interactions between binding partners, binding reactions in water are driven in large measure by the properties of the medium itself. Aqueous binding involves the loss of solute-solvent interactions (desolvation) and the concomitant formation of solute-solute interactions. Despite decades of research, aqueous binding remains poorly understood, a deficit that profoundly limits our ability to design effective pharmaceuticals and new enzymes. Particularly problematic is understanding the energetic consequences of aqueous desolvation, an area the Toone and Beratan groups have considered for many years.</p><p> In this dissertation, we embark on a quest to shed new light on aqueous desolvation from two perspectives. In one component of this research, we improve current computational tools to study aqueous desolvation, employing quantum mechanics (QM), molecular dynamics (MD) and Monte Carlo (MC) simulations to better understand the behavior of water near molecular surfaces. In the other, we use a synthetic host, cucurbit[7]uril (CB[7]), in conjunction with a de novo series of ligands to study the structure and thermodynamics of aqueous desolvation in the context of ligand binding with atomic precision, a feat hitherto impossible. A simple and rigid macrocycle, CB[7] alleviates the drawbacks of protein systems for the study of aqueous ligand binding, that arise from conformational heterogeneity and prohibitive computational costs to model.</p><p> </p><p> We first constructed a novel host-guest system that facilitates internalization of the trimethylammonium (methonium) group from bulk water to the hydrophobic cavity of CB[7] with precise (atomic-scale) control over the position of the ligand with respect to the cavity. The process of internalization was investigated energetically using isothermal titration microcalorimetry and structurally by nuclear magnetic resonance (NMR) spectroscopy. We show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by an unfavorable desolvation enthalpy of just 0.49±0.27 kcal*mol-1, a value significantly less endothermic than those values suggested from previous gas-phase model studies. Our results offer a rationale for the wide distribution of methonium in biology and demonstrate important limitations to computational estimates of binding affinities based on simple solvent-accessible surface area approaches.</p><p> To better understand our experimental results, we developed a two-dimensional lattice model of water based on random cluster structures that successfully reproduces the temperature-density anomaly of water with minimum computational cost. Using reported well-characterized ligands of CB[7], we probed water structure within the CB[7] cavity and identified an energetically perturbed cluster of water. We offer both experimental and computational evidence that this unstable water cluster provides a significant portion of the driving force for encapsulation of hydrophobic guests.</p><p> The studies reported herein shed important light on the thermodynamic and structural nature of aqueous desolvation, and bring our previous understanding of the hydrophobic effect based on ordered water and buried surface area into question. Our approach provides new tools to quantify the thermodynamics of functional group desolvation in the context of ligand binding, which will be of tremendous value for future research on ligand/drug design.</p> / Dissertation

La necesidad de polímeros bioestables para fabricación de implantes protésicos queda patente, entre otros indicadores, por la proliferación de dispositivos actualmente comercializados. La caracterización físico-química así como la respuesta biológica de un conjunto de materiales poliméricos bioestables es el objetivo último de esta tesis. En este trabajo se han sintetizado diferentes materiales poliméricos de la familia de los acrilatos y metacrilatos variando sutilmente sus características superficiales, como el grado de hidrofilia o la distribución de cargas eléctricas. El procedimiento consistió en la copolimerización via radical de acrilato de etilo, EA, acrilato de 2-hidroxietilo, HEA, y ácido metacrílico, MAAc. Se ha caracterizado los materiales en estado seco y en presencia de diferentes contenidos de agua mediante calorimetría diferencial de barrido, DSC, análisis dinámico-mecánico, DMA, microscopía de fuerza atómica, AFM, análisis dieléctrico, DRS, contenido de agua en equilibrio, EWC, y energía superficial, SE, persiguiendo el objetivo de dilucidar si el agua es capaz de inducir cambios conformacionales en las cadenas poliméricas que den lugar a una separación de fases. Sobre los materiales en forma de scaffold poroso con poros esféricos interconectados se ha cultivado fibroblastos y endoteliales. La compatibilidad de las células endoteliales se midió en términos de viabilidad celular y la adecuada diferenciación endotelial y su funcionamiento. Se han realizado cultivos de células endoteliales humanas primarias, HUVEC, y se ha determinado si su morfología y función se vio afectada por el material. Se examinó la adhesión y proliferación de las mismas, así como un marcador importante de activación endotelial, la E-selectina. Se evaluó si se mantuvieron los fenotipos endoteliales normales y sus funciones observadas in vivo mediante análisis de los contactos célula-célula y la regulación de la expresión génica del marcador de activación E-selectina cuando se añadió un estímulo (LPS). Además, como posible aplicación de estos materiales en una prótesis de córnea artificial, y dado que los fibroblastos del estroma de la córnea (es decir, los queratocitos) son de relevancia en la cicatrización de la córnea se determinó cómo afectaba la hidrofilicidad del substrato a la adhesión celular de la línea de fibroblastos humanos MRC-5, como modelo celular para estudiar la disposición del citoesqueleto tras la adhesión a los diferentes soportes mediante la detección de F-actina. Asimismo, se ha sembrado células epiteliales evaluando su comportamiento/funcionamiento celular ya que uno de los requisitos esenciales para que un implante de queratoprótesis tenga éxito es que se cree y mantenga una capa de células epiteliales que impidan entrar a las bacterias al interior del ojo y permita la difusión la capa lagrimal de manera estable en el tiempo. Así, se han analizado parámetros celulares como adhesión, proliferación y viabilidad de una línea de células epiteliales de conjuntiva humana, NHC, cultivada sobre substratos poliméricos con diferentes grados de hidrofilia y cargas eléctricas superficiales buscando qué grado de hidrofilicidad permite la epitelización del substrato y podría darle al material flexibilidad y la hidrofilicidad necesaria para un mejor contacto con los párpados y lágrima. Los resultados obtenidos se han correlacionado con la adsorción y conformación de una proteína de la matriz extracelular, la fibronectina. / Campillo Fernández, AJ. (2014). Polímeros bioestables para fabricación de implantes protésicos: caracterización físico-química y respuesta biológica in vitro [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/44232 / TESIS / Premios Extraordinarios de tesis doctorales

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第20481号 / エネ博第350号 / 新制||エネ||70(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 木下 正弘, 教授 森井 孝, 教授 片平 正人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

Comprehension of reactive chemical dynamics in the gas phase and at the gas/organic-surface interface and non-reactive dynamics at the interface between hydrophobic surfaces and water requires an understanding of the fundamental atomic and molecular interactions that undergird these important phenomena. In an effort to study these regimes of chemical interaction, we have performed computational simulations that probe the dynamics of chemical systems that exemplify each of these domains. To study gas-phase chemical dynamics, we reparametrized semiempirical Hamiltonians so that they can accurately describe the potential energy surfaces for two distinct atom+alkane reactions. In addition to their demonstrated accuracy, these methods possess the attractive quality of being computationally inexpensive enough to afford extensive direct-dynamics trajectory studies. Our results on the dynamics of atom+alkane hydrogen-abstraction reactions have shown good agreement with experimental metrics that are as diverse as product velocity distributions, excitation functions, angular distributions and rovibrational state distributions for diatomic products of the abstraction. We have demonstrated that our reparametrized Hamiltonians are suitable for investigating gas-phase reactions with up to 15 (5 heavy) atoms and that they are appropriate for studying reactions beyond the gas phase, especially gas/surface reactions. By employing our semiempirical methods within a quantum-mechanics/molecular-mechanics hybrid scheme we are able to examine hydrogen-abstraction reactions of fluorine atoms with alkanethiolate self-assembled monolayers. Our simulations reproduce the general trends of experimental results for the cousin F+squalane reaction. Our simulations also probe the role that secondary collisions play in determining the final internal and translational energy of the product HF molecules. For instance, we determined that very few interactions with the SAM surface were required to cool rotational and translational modes of the HF product, while its vibrational energy remains unchanged on the time scale that HF molecules trap on the SAM surface. Moving beyond the gas/organic surface interface, we have also performed molecular-dynamics simulations of thin water films confined between hydrophobic SAM surfaces. These simulations illuminated the structural and dynamics behavior induced in the water films by confinement in hydrophobic environments. While most effects of the surface do not penetrate deep into the water layers we have noted that enhanced lateral diffusion of water molecules can persist in these films with > 1 nm length scales. We have elucidated a possible mechanistic precursor for the attractive forces seen in experimental measurement of the hydrophobic effect. / Ph. D.

Desenvolvemos dois modelos estatísticos para água, nos quais diferentes aspectos de sua estrutura são considerados. O modelo geométrico permite diferentes números de coordenação. Em uma aproximação de campo médio mostramos que sob pressão o modelo apresenta linha de coexistência entre fases de baixa e alta densidade, e ponto crítico. A entropia das ligações de hidrogênio tem papel fundamental na definição da inclinação da linha de coexistência. O comportamento do modelo pode estar realcionado como segundo ponto crítico da água super resfriada e com transições líquido-líquido em geral.O modelo da água quadrada é uma versão térmica do modelo do gelo, no qual considera-se a direcionalidade das ligações de hidrogênio. O modelo foi estudado na rede de Bethe e através de simulações de Monte Carlo em três situações diferentes: para a água pura e na presença de solutos polares e apolares. A água quadrada pura não apresenta transição de fase. No modelo para solvente com solutos apolares, medidas de frequência relativa de ligações e do tempo de correlação mostram que o modelo apresenta estruturação da camada de hidratação. Medidas de correlação temporal no modelo de Ising mostram comportamento oposto. Em um estudo preliminar para uma solução com solutos que realizam ligações de hidrogênio não conseguimos encontrar diagramas de coexistência com círculo fechado, para o conjunto de parâmetros utilizados, possivelmente devido à ausência de buracos. / We have developed two statistical models for water in which different features of water structure are considered. In the geometrical model different coordination numbers are present and the model allows for translational disorder. A mean-field treatment shows that under pressure the model exhibits phases of different densities and a coexistence line ending in a critical point. Entropy of the hydrogen network plays an essential role in defining the slope of the coexistence line. The model behavior might be related with the second critical point in supercooled water and to liquid-liquid transitions in general. The square water model is a thermal version of the ice model, and takes into account the directionality of the hydrogen bonds. The model was studied on a Bethe lattice and through Monte Carlo simulations, for three different situations: as pure water and in the presence of polar and apolar solutes. Pure square water does not present a phase transition. In the presence of apolar solutes, first shell square water presents ordering, as shown from comparison of relative frequency of bonds, as well as from study of time correlations. The latter was shown to present opposite behavior in case of an Ising system. In a preliminary study for a solution of hydrogen bonding solutes we were unable to find a closed loop for the sets of parameters chosen. Vacancies might need to be included.

It has long been observed that salts affect proteins in a variety of ways, yet comprehensive explanations for different salt effects are still lacking. In the work presented here, the effect of salts on proteins has been investigated through three different effects: the hydrophobic effect; their conformational stability; the hydrogen bonding network of water in a protein’s hydration shell. UV-vis absorbance and fluorescence spectroscopy were used to monitor changes in two model systems, the phenol-acetate contact pair and the model enzyme ribonuclease t1. It was shown that salts affect the hydrophobicity of the contact pair according to their charge density, induced image charges play an important role in the observed salt-induced increase of ribonuclease t1 stability, and that salts affect ribonuclease t1 activity through modulation of the hydrogen bonds of water in the enzyme’s hydration shell. This work contributes a greater understanding of the effect of salts on proteins.

It has long been observed that salts affect proteins in a variety of ways, yet comprehensive explanations for different salt effects are still lacking. In the work presented here, the effect of salts on proteins has been investigated through three different effects: the hydrophobic effect; their conformational stability; the hydrogen bonding network of water in a protein’s hydration shell. UV-vis absorbance and fluorescence spectroscopy were used to monitor changes in two model systems, the phenol-acetate contact pair and the model enzyme ribonuclease t1. It was shown that salts affect the hydrophobicity of the contact pair according to their charge density, induced image charges play an important role in the observed salt-induced increase of ribonuclease t1 stability, and that salts affect ribonuclease t1 activity through modulation of the hydrogen bonds of water in the enzyme’s hydration shell. This work contributes a greater understanding of the effect of salts on proteins.