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Os calcógenos de európio (EuX, onde X representa O, S, Se ou Te) possuem propriedades magneto-ópticas únicas e interessantes, devido ao enorme magnetismo gerado dos elétrons na camada f do átomo pertencente a família dos terras raras, tornando estes materiais atraentes para aplicações na spintrônica (eletrônica baseada nos transporte de spins e não de carga). Neste trabalho investigamos em baixa temperatura o espectro de absorção utilizando luz circularmente polarizada na região próxima do limiar da banda para o telureto de európio EuTe e o seleneto de európio EuSe em alto campo magnético no ordenamento ferromagnético dos spins de Eu^{2+} da rede cristalina. As amostras crescidas por epitaxia por feixe molecular apresentaram um dicroísmo circular magnético intenso no espectro de absorção para a configuração de Faraday. O par de linhas estreitas observadas estão separadas de aproximadamente 200 meV para o EuTe e 300 meV para o EuSe. Em seguida, formulamos um modelo teórico para a interpretação deste espectro de absorção no arcabouço do modelo de transições eletrônicas entre o estado fundamental 4f^{7}({8}^S_{7/2}) e o estado excitado formado dos estados do caroço remanescente 4f^{6}({7}^F_{J=0...6}) mais o estado em que o elétron se encontra na banda de condução 5d(t_{2g}), resultando em uma excelente concordancia qualitativa e quantitativa com o experimento. / Europium chalcogenides (EuX, where X stands for O, S, Se or Te) have very interesting and unique magneto-optical properties, due to the huge magnetism that arises from the electrons in the f?shell of the rare earth element and which makes them attractive for spintronics applications ( spin transport electronics or spin basedelectronics) In this work we investigate the band-edge optical absorption in high magnetic fields in the Faraday geometry for EuTe and EuSe in the ferromagnetic order attained at low temperatures. In thin layers grown by molecular beam epitaxy, an intense magnetic circular dichroism were observed. The doublet of absorption lines showed a separation by about 200meV in EuTe and 300meV in EuSe. Next, we developed a theoretical model for the interpretation of the absorption spectrum, based in the framework of the model of an electronic transition from a localized ground state 4f^{7}({8}^S_{7/2)) to an excited state formed by the core states 4f^{6}({7}^F_{J=0...6}) and the electron extended state in the 5d(t_{2g}) conduction band, yielding an excellent qualitative and quantitavie agreement with experiment.

Understanding the local conformations of DNA at the level of individual nucleic acid bases is important for the study of the mechanism of DNA sequence-dependent behavior. Here we apply linear absorption, circular dichroism (CD), and fluorescence spectroscopy to study the DNA local base conformation using 6-methyl Isoxanthopterin (6-MI) labeled DNA. We interpret excitation–emission peak shift (EES) measurements of the 6-MI, both as a ribonucleotide monophosphate in solution and as a site-specific substituent for guanine in various DNA constructs, by implementing a simple two-state model. We show that the spectroscopic properties of the 6-MI probe in DNA can be used to obtain detailed information about local base conformations and conformational heterogeneity and fluctuations. Based on these findings, we apply a simple theoretical model to calculate CD of 6-MI substituted DNA constructs. We find that our model can be used to extract basesequence- dependent information about the local conformation of the 6-MI probe as modulated by the local base or base-pair environment. We next apply 6-MI to probe the ligand insertion of small molecules to duplex DNA, further extending the potential of 6- MI as a useful reporter of local nucleic acid base conformation. These studies served to establish a new level of sophistication in qualitatively analyzing 6-MI structural behavior in terms of local base stacking and unstacking conformations. v This dissertation contains previously published and unpublished co-authored material.

Neste trabalho, utilizando técnicas magneto-ópticas, estudamos tempos de relaxação spin-rede (T1) do estado fundamental de centros \'F\' e, cristais de halogenetos alcalinos (KCl-KBr). Descrevemos um sistema semi-automático para medidas ópticas de T1, capaz de medir tempos de relação curtos (~1mS), baseado na medida do Dicroísmo Circular magnético (DCM) que apresentam aqueles sais quando portadores de centros paramagnéticos. Obtivemos a dependência de T1 com o campo magnético H (até 65 Kgauss), bem como os espectros de DCM para diferentes concentrações nas matrizes mistas. Uma teoria desenvolvida por Panepucci e Mollenauer (1) para matrizes puras, foi adaptadas para explicar a relaxação spin-rede nos cristais mistos. Os resultados obtidos para o processo direto (T~2.0 K), confrontados com auqela teoria, mostram que o mecanismo de relaxação dominante até 25 KGauss continua sendo a modulação por fônons da interação hiperfins entre o elétron \'F\' e os núcleos vizinhos. / Using magneto-optic techniques we have studied the ground state spin- lattice relaxation times (T1) of \'F\' centers in mixed Alkali Halide cristals (KCl-KBr). We describe a computer assisted system to optically measure short relaxation times (~1mS). The technique is based on the measurement of the Magnetic Circular Dicroism (MCD) presented by F centers. We obtained the T1 magnetic field dependency at 2 K up to 65 kGauss), as well as the MCD spectra for different relative concentration at the mixed matrices. The theory developed by Panepucci and Mollenauer for F centers spin-lattice relaxation in pure matrices was modified to explain the behavior of T1 in mixed cristais. The Direct Process results (T~2.0 K) compared against that theory shows that the main relaxation mecanism, Up to 25 kGauss, continues to be phonon modulation of the hyperfine interaction between \'F\' electrons and surrounding nuclei.

Circular dichroism (CD) spectra were obtained for bacteriophage T4 lysozyme and three of its mutants in the presence and absence of colloidal silica nanoparticles. Mutant lysozymes were produced by substitution of the isoleucine at position 3 with tryptophan, cysteine and leucine. Each substitution resulted in an altered structural stability, quantified by a difference in free energy of unfolding from the wild type. CD spectra recorded in the absence of colloidal silica agreed with x-ray diffraction data in that the mutants and wild type showed similar secondary structures. CD spectra of protein-nanoparticle complexes recorded after contact for 90 minutes showed significant differences from those recorded in the absence of nanoparticles, and these differences varied among the proteins. The percentage of a-helix lost in these proteins upon adsorption to silica nanoparticles was also recorded as a function of time by CD. For a 1:2 protein to particle mixture, different kinetic behaviors were observed among the proteins. The more unstable the protein, the greater the rate and extent of secondary structure loss upon adsorption. For a 1:1 protein to particle mixture, only results recorded with the tryptophan mutant were significantly different from the other variants. The kinetic data recorded for the 1:2 protein to particle ratio was evaluated using two different protein adsorption models. Both models allow proteins at an interface to exist in two different states: state 1 molecules retain their native conformation, while state 2 molecules lose a certain amount of their native secondary structure and occupy more surface area than state 1 molecules. The main difference between these two models is that one allows state 2 molecules to be adsorbed directly from solution, while the other requires that state 2 molecules be generated by surface-induced conversion of state 1 molecules. The former model showed a better fit to the data than the latter from a least squares comparison. Both models indicated that proteins of lower thermal stability have a greater tendency to adopt state 2 on silica. / Graduation date: 1997

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 228-251).

To understand protein stability and the mechanism of protein folding, it is essential that we gain a better understanding of the ensemble of conformations that make up the denatured state of a protein. The primary goal of the research described here was to see what we might learn about the denatured state using fluorescence. To this end, tryptophan was introduced at five sites in Ribonuclease Sa (RNase Sa): D1W, Y52W, Y55W, T76W, and Y81W. The fluorescent properties of the denatured states of these five proteins were studied and compared to the fluorescent properties of eight model compounds: N-acetyl-tryptophan-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and five pentapeptides based on the sequence around the original tryptophan substitutions in RNase Sa. Regardless of the denaturant, λmax for the proteins and model compounds differed very little, 349.3 ± 1.2 nm. However, significant differences were observed in the fluorescence intensity at λmax (IF), suggesting that IF is more sensitive to the immediate environment than λmax. The differences in IF are due in part to quenching by neighboring side chains. More importantly, IF was always significantly greater in the protein than in its corresponding pentapeptide, indicating that the protein exerts an effect on the tryptophan, which cannot be mimicked by the pentapeptide models. Acrylamide and iodide quenching experiments were also performed on the model compounds and proteins. Significant differences in the Stern-Volmer quenching constant (KSV) were also observed between the proteins and between the proteins and their corresponding pentapeptides. Importantly, the KSV for the protein was always less than in its corresponding pentapeptide. These data along with the IF data show that non-local structure in the unfolded state influences tryptophan fluorescence and accessibility. In summary, these and our other studies show that fluorescence can be used to gain a better understanding of the denatured states of proteins.

Structure-specific recognition of nucleic acids is a promising method to reduce the size of the recognition unit required to achieve the necessary selectivity and binding affinity for small molecules. It has been demonstrated recently that G-quadruplex DNA structures can be targeted by organic cations in a structure-specific manner. Structural targets of quadruplexes include the planar end surfaces of the G-tetrad stacked columns as well as four grooves. The significant structural differences between quadruplex DNA and duplex DNA make quadruplex DNA a very attractive target for highly selective, structure-specific drug design. We have used a variety of biophysical techniques including circular dichroism, surface plasmon resonance, thermal melting and absorbance spectroscopy to investigate small molecules that can selectively bind to the ends of human telomeric DNA as well as the ends of the G-quadruplex structure formed by the purine-rich promoter region of the c-MYC oncogene. We have also screened a library of heterocyclic diamidines, and identified one that binds selectively in the grooves of human telomeric quadruplex DNA. This compound is an excellent starting point for the design of new anti-cancer and anti-parasitic compounds with high affinity and selectivity for human telomeric DNA.

It has been shown that heterocyclic diamidines, a class of minor groove binders, are promising antimicrobial agents. These compounds bind none covalently to the minor groove of A/T rich regions of the kinetoplast DNA and kill the parasite. The mechanism of action of these compounds is not well understood, yet many hypotheses have been proposed. One of the methods that improve the specificity is cooperative binding. Since there are many binding sites available in k-DNA thus the cooperativity in adjacent binding sites is desirable. A library of compounds has been scanned and few of those compounds identified that are able to bind to two adjacent A/T binding sites separated by a single G. Many biophysical methods such as isothermal titration calorimetry, surface Plasmon resonance, circular dichroism and thermal melting have been used to explore the thermodynamic profiles and binding mode of these compounds. The pulsed field gradient NMR was used to investigate the structural changes to the DNA sequence upon binding of the minor groove binders and find a correlation between their biological difference and structural changes. The molecular dynamics was applied to look at the interaction of some of the heterocyclic diamidines to the DNA with more details and predict the unknown structures.

The rotavirus NSP4 protein is the first described viral enterotoxin. Abundant data from our laboratory reveals that NSP4 binds both the N- and C-termini of caveolin- 1 (aa2-31 and 161-178, respectively). Yeast two-hybrid and peptide binding analysis mapped the caveolin-1 binding site to three hydrophobic residues within the amphipathic a-helix, enterotoxic peptide domain (aa114-135). The research studies herein utilized peptides to investigate the interaction between NSP4 and caveolin-1. Peptides were synthesized corresponding to the amphipathic a-helix and caveolin-1 binding domain of NSP4 (aa112-140) and to the N- (aa2-20 and 19-40) and C- (161-178) termini of caveolin-1, and were utilized in structural and functional studies. Fluorescence binding assays revealed that NSP4 (aa112-140) binds to the N-terminus (aa19-40) of caveolin-1 with a stronger affinity than the C-terminus (aa161-178). In addition, this assay further delineated the NSP4 binding domain on caveolin-1 to aa19-40. Secondary structural changes following NSP4-caveolin-1 peptide-peptide interactions were investigated by circular dichroism analysis. Changes in a-helix formation were observed only upon interaction of the NSP4112-140 peptide with the C-terminal caveolin-1 peptide (C-Cav161- 178). The NSP4112-140 peptide contains a potential cholesterol recognition amino acid consensus (CRAC) sequence. Therefore this peptide was examined for cholesterol binding. Results of the binding assay revealed NSP4 binds cholesterol with a Kd of 7.67 +/- 1.49nM and this interaction occurs via aa112-140. Mutation of amino acid residues within the CRAC motif resulted in weaker binding affinities between each of the corresponding mutant peptides and cholesterol. NSP4 peptides containing mutations within the hydrophobic and charged faces of the amphipathic a-helix, enterotoxic peptide and caveolin-1 binding domain of NSP4 were examined for changes in secondary structure as well as diarrhea induction in mouse pups. Circular dichroism analysis revealed that mutation of hydrophobic residues resulted in a decrease in a-helix formation, whereas mutation of acidic and basic charged residues caused little to no change in a-helical content. When tested for diarrhea induction in mouse pups, the peptides containing mutations of either the hydrophobic or basic charged residues did not cause diarrhea. Taken together, the results of this research suggest a complex interplay between NSP4 secondary structure, caveolin-1 and cholesterol binding and diarrheagenic function.