Thesis advisor: Udayan Mohanty / In chapter one, two simple models are used to estimate the electrostatic contributions to the stiffness of short DNA fragments. The first model views DNA as two strands that are appropriately parameterized and are wrapped helically around a straight cylinder radius equal to the radius of the DNA molecule. The potential energy of the DNA due to phosphate-phosphate electrostatic interactions is evaluated assuming that the charges interact through Debye-Hückle potentials. This potential energy is compared with the potential energy as computed using our second model in which DNA is viewed as two helical strands wrapping around a curved tube whose cross-section is a disk of radius equal to the radius of the DNA. The results are compared with counterion condensation models and experimental data (Guo et al. J. Phys. Chem. B, 2008, 112, 16163-16169). In chapter two, the fidelity of translation selection begins with the base pairing of codon-anticodon complex between the mRNA and tRNAs. Binding of cognate and near-cognate tRNAs induces 30S subunit of the ribosome to wrap around the ternary complex, EF-Tu(GTP)aa-tRNA. We have proposed that large thermal fluctuations play a crucial role in the selection process. The binding energies of over a dozen unique site-bound magnesium structural motifs are investigated and provide insights into the nature of interaction of divalent metal ions with the ribosome (Guo et al. Proc. Nat. Acad. Sci. 2011, 108, 3947-3951). In chapter three, we use extensive molecular dynamics simulations to study a series of stapled alpha helical peptides over a range of temperatures in solution. The peptides are found to exhibit substantial variations in predicted helicities that are in good agreement with the experimental value. In addition, we find significant variation in local structural flexibility of the peptides with the position of the linker, which appears to be more closely related to the observed differences in activity than the absolute alpha helical stability (Guo et al. Chem. Biol. Drug. Des. 2010, 75, 348-359.). In chapter four, the alpha helical conformation and structural stability of single and double stapled all-hydrocarbon cross-linked p53 peptides in solution and when bound to MDM2 is investigated. We determined the effects of the peptide sequence, the stereochemistry of the cross-linker, the conformation of the double bond in the alkene bridge, the length of the bridge, on the relative stability of the alpha helix structure. The conformation population distribution indicates a fully helical state and several partially folded states. The distribution of dihedral pairs of the stapled peptides in the bound state indicates a significant population around the alpha helical region. Sequences over which the linker spans tend to have the highest helical occupancy. Significant helical content is observed for a double stapled p53 peptide at 575 K. The probability to form native contacts is increased when the stapled peptides are bound to MDM2. The distribution of the end-to-end distance of the peptides is bimodal. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Cell adhesion plays an important role in inflammation and immunological responses. Adhesion molecules (e.g., selectins and integrins) are key modulators in mediating these cellular responses, such as leukocyte trafficking under shear stress. In this thesis, we use a bio-membrane force probe (BFP) to study force regulation on kinetics and conformations of selectin and LFA-1 integrin. A new BFP was built up, and a new assay, using thermal fluctuation of the BFP, was developed and used to monitoring selectins and their ligands association and dissociations. The new BFP was also used to investigate the force and force history dependence of selectin-ligand interactions. We found tri-phasic transition of force-dependent off-rates and force-history dependence of selectin/ligaind interactions. The BFP was also used to characterize force-dependent lifetimes of the LFA-1-ICAM-1 interaction. We found that LFA-1/ICAM-1 bonds behaved as catch bond and that LFA-1-ICAM-1's catch bonds were abolished blocking the downward movement of αA domain α7 helix. Finally, the BFP was applied to dynamically probe the global conformational changes of LFA-1 and to characterize force-regulated transitions among different conformational states on a living cell. We observed dynamic transitions of LFA-1 between extended and bent conformations on living cells. The observed average distance change of LFA-1's extensions was about 18nm, while that of the bending was only about 14nm. We also found that forces could facilitate extension but they slow down the bending of LFA-1. The observed transition time of extension was less than 0.1s, while that of contraction was longer than 0.2s. Our observations here are the first in-situ evidence to demonstrate how integrins dynamically transit different conformations and how force regulates these transitions.

The aim of this thesis is to study the energy fluctuations (leading to thermal fluctuations) during thermal and enzymatic denaturation of biological molecules and to study the variation in fluctuations between simple molecules like the DNA (which have only a secondary structure) to molecules with higher order structures and packaging. We have developed a new technique - Thermal Fluctuation Spectroscopy (TFS) to study these fluctuations. The technique of Thermal Fluctuation Spectroscopy (TFS) is a combination of microcalorimetry and noise measurement techniques. The combination of these two powerful techniques has never been exploited before. In this technique any energy exchange between sample and the substrate is reflected as a thermal fluctuation of the substrate. The system resolution is few parts per billion (ppb) and fluctuations in energy ~ 100nJ (which correspond to temperature fluctuations ~ K) can be measured. Chromatin is the basic building block of chromosome and this thesis focuses on the constituents this fundamental building block - DNA, histones and nucleosomes. Heteropolymeric dsDNA shows extremely large non-Gaussian fluctuation around its melting temperature. For homopolymeric DNA the fluctuations during denaturation are smaller. The thermal fluctuation during denaturation of a heteropolymer in buffer is several orders larger than when the DNA is on a substrate while that for a homopolymer is comparable in both cases. Our measurements established that heteropolymeric dsDNA denaturation occurs in two stages. Initially, at around 330 K, bubbles are formed in the AT rich regions. At higher temperatures, the GC rich regions binding them denature in a cooperative transition causing extremely large fluctuations. TFS on histone monomers showed that H1 monomer shows an increase in thermal fluctuation in the temperature range studied, while the core histones did not. We infer that this is due to the fact that the core histones may not be properly folded when they exist as monomers. It was seen that H1 crosses an energy barrier of 17 kcal/mol to go from its native to denatured state. The transition was kinetically driven with a fixed barrier till 352 K. At 352K, the barrier softened by ~ 1 kcal/mol leading to faster denaturation. The core histones when assembled as dimers/oligomers showed an increase in fluctuation at temperatures below 350 K. The assembling of these histones and DNA into a mononucleosome causes a very large increase in fluctuation over the entire temperature range studied. TFS showed that the fluctuation during mononucleosome denaturation was much larger than a simple sum of the fluctuations of its constituents. From the data we were able to identify that the denaturation starts with dissociation and unfolding of the core histones and the denaturation of AT rich regions of the DNA which leads to the breaking of some of the histone-DNA contacts. At higher temperatures the linker histone H1 and the GC rich regions of the DNA denature, leading to a collapse of the entire nucleosome structure. The broadness of the transition region (the fact that the fluctuation is large over the entire temperature range) was attributed to the presence of different types of contacts and interactions (with different energies) stabilizing the nucleosome structure. The nucleosome was found to favour large energy jumps over smaller ones indicating that the denaturation has an element of cooperativity involved. Using TFS we have been able to determine the fluctuations involved in the denaturation of biomolecules like DNA, histones and nucleosomes. The energy barriers to denaturation have been determined. We have also been able to give models for the denaturation of these biomolecules. We have also shown that it is possible to study enzymatic digestion using TFS. Thus, the technique of TFS is a viable tool for the study of fluctuations in reactions, in biomolecules, during transitions and in any process where there is an energy exchange involved.

No description available.

O problema do folding de proteínas tem sido investigado intensamente há mais de sessenta anos. Entretanto ainda não é encontrado na literatura um modelo que seja capaz de explicar plenamente qual é o mecanismo responsável pelo processo de folding. Neste contexto, a presente tese de doutorado é uma proposta minimalista para investigar o papel de um código estereoquímico, que é centrado no efeito hidrofóbico e nos vínculos estéricos dos aminoácidos (o modelo estereoquímico), no processo em pauta. Esse modelo quando combinado com um método para incluir a flutuação térmica local no sistema cadeia protéica-solvente, possibilita a investigação de um dos aspectos mais extraordinários do problema, a saber, a rapidez do processo de folding, considerado aqui por meio da correlação entre a complexidade da estrutura nativa (alvo) e a taxa de folding. Esse método é motivado por argumentos físico-químicos e biológicos, e é fundamentado na Mecânica Estatística Não Extensiva, via o uso do peso de Tsallis em simulações Monte Carlo (MC). O tempo característico de folding (obtido daquelas simulações e utilizado aqui como um parâmetro analítico do problema), cobre várias ordens de grandeza para cadeias com o mesmo tamanho. Dois conjuntos principais de simulações foram considerados com a finalidade de análise: (i) alguns alvos foram especialmente selecionados e submetidos a simulações MC a várias temperaturas T do reservatório térmico (o meio solvente); e (ii) um total de duzentos alvos com topologias diversas foram submetidos a simulações à mesma temperatura T = 1 (unidades arbitrárias). Com essas simulações foi possível verificar comparativamente o efeito dos dois pesos estatísticos, o de Boltzmann e o de Tsallis, sobre a cinética do processo de folding, e assim revelar comportamentos intrigantes, porém consistentes com o fenômeno focado, como a robustez do processo de folding e , este último emergindo como uma grandeza que depende da complexidade da estrutura nativa. Para estruturas distintas, cobre quatro ordens de grandeza. Os resultados da investigação da correlação entre e parâmetros globais destinados a avaliar a complexidade da topologia da estrutura nativa, como a ordem de contato e a cooperatividade estrutural, corroboram com a noção de que a rapidez do processo de folding é determinada fundamentalmente pela complexidade da topologia da estrutura nativa. / The protein folding problem has been investigated for more than sixty years. However is not yet found in literature a model that is able to fully explain the mechanism behind the folding process. In this context, the present doctoral thesis is a speculative and minimalist proposal to investigate the role of a stereochemical code -grounded in the hydrophobic effect and steric constraints of amino acids (the stereochemical model), in the discussed process. This model, when combined with a method to include local thermal fluctuations in the protein chain-solvent system, enables the investigation of one of the most extraordinary aspects of the problem, namely, the fastness of the folding process, which is studied here by means of the correlation between the complexity of the native structure (target) and the folding process rate. This method is motivated by physical chemistry and biological arguments, and is based on the Nonextensive Statistical Mechanics, by the use of the Tsallis weight in Monte Carlo simulations (MC), where the entropic index q is adjusted at each new conformation of the protein chain. For chains with the same length, the characteristic folding time (estimated from those simulations and used here as an analytical parameter of the problem) span several order of magnitude. Two main sets of simulations were performed for analysis purposes. First, some targets were specially selected and submitted to MC simulations with several temperatures of the thermal reservoir (the solvent), and then a total of two hundred targets with diverse topologies were submitted to simulations with the same reservoir temperature T = 1 (arbitrary units). With such set of simulations, we could compare the effect of two statistical weights, namely the Boltzmann and the Tsallis weight, on the kinetics of the folding process. Intriguing but consistent behavior with respect to the folding phenomenon was reveled, such as the robustness of the process, and about the characteristic folding time , which emerges as an amount that depends on the complexity of the native structure. For distinct structures, covers four orders of magnitude. Our results about the correlation between and global parameters to assess the complexity of the topology of the native structure, such as contact order and structural cooperativity, support the notion that the fastness of the folding process is essentially determined by the complexity of the topology of native structure.

O problema do folding de proteínas tem sido investigado intensamente há mais de sessenta anos. Entretanto ainda não é encontrado na literatura um modelo que seja capaz de explicar plenamente qual é o mecanismo responsável pelo processo de folding. Neste contexto, a presente tese de doutorado é uma proposta minimalista para investigar o papel de um código estereoquímico, que é centrado no efeito hidrofóbico e nos vínculos estéricos dos aminoácidos (o modelo estereoquímico), no processo em pauta. Esse modelo quando combinado com um método para incluir a flutuação térmica local no sistema cadeia protéica-solvente, possibilita a investigação de um dos aspectos mais extraordinários do problema, a saber, a rapidez do processo de folding, considerado aqui por meio da correlação entre a complexidade da estrutura nativa (alvo) e a taxa de folding. Esse método é motivado por argumentos físico-químicos e biológicos, e é fundamentado na Mecânica Estatística Não Extensiva, via o uso do peso de Tsallis em simulações Monte Carlo (MC). O tempo característico de folding (obtido daquelas simulações e utilizado aqui como um parâmetro analítico do problema), cobre várias ordens de grandeza para cadeias com o mesmo tamanho. Dois conjuntos principais de simulações foram considerados com a finalidade de análise: (i) alguns alvos foram especialmente selecionados e submetidos a simulações MC a várias temperaturas T do reservatório térmico (o meio solvente); e (ii) um total de duzentos alvos com topologias diversas foram submetidos a simulações à mesma temperatura T = 1 (unidades arbitrárias). Com essas simulações foi possível verificar comparativamente o efeito dos dois pesos estatísticos, o de Boltzmann e o de Tsallis, sobre a cinética do processo de folding, e assim revelar comportamentos intrigantes, porém consistentes com o fenômeno focado, como a robustez do processo de folding e , este último emergindo como uma grandeza que depende da complexidade da estrutura nativa. Para estruturas distintas, cobre quatro ordens de grandeza. Os resultados da investigação da correlação entre e parâmetros globais destinados a avaliar a complexidade da topologia da estrutura nativa, como a ordem de contato e a cooperatividade estrutural, corroboram com a noção de que a rapidez do processo de folding é determinada fundamentalmente pela complexidade da topologia da estrutura nativa. / The protein folding problem has been investigated for more than sixty years. However is not yet found in literature a model that is able to fully explain the mechanism behind the folding process. In this context, the present doctoral thesis is a speculative and minimalist proposal to investigate the role of a stereochemical code -grounded in the hydrophobic effect and steric constraints of amino acids (the stereochemical model), in the discussed process. This model, when combined with a method to include local thermal fluctuations in the protein chain-solvent system, enables the investigation of one of the most extraordinary aspects of the problem, namely, the fastness of the folding process, which is studied here by means of the correlation between the complexity of the native structure (target) and the folding process rate. This method is motivated by physical chemistry and biological arguments, and is based on the Nonextensive Statistical Mechanics, by the use of the Tsallis weight in Monte Carlo simulations (MC), where the entropic index q is adjusted at each new conformation of the protein chain. For chains with the same length, the characteristic folding time (estimated from those simulations and used here as an analytical parameter of the problem) span several order of magnitude. Two main sets of simulations were performed for analysis purposes. First, some targets were specially selected and submitted to MC simulations with several temperatures of the thermal reservoir (the solvent), and then a total of two hundred targets with diverse topologies were submitted to simulations with the same reservoir temperature T = 1 (arbitrary units). With such set of simulations, we could compare the effect of two statistical weights, namely the Boltzmann and the Tsallis weight, on the kinetics of the folding process. Intriguing but consistent behavior with respect to the folding phenomenon was reveled, such as the robustness of the process, and about the characteristic folding time , which emerges as an amount that depends on the complexity of the native structure. For distinct structures, covers four orders of magnitude. Our results about the correlation between and global parameters to assess the complexity of the topology of the native structure, such as contact order and structural cooperativity, support the notion that the fastness of the folding process is essentially determined by the complexity of the topology of native structure.