Metal-RNA interactions are important to neutralize the negative charge and aid in correctly folding the RNA. Spectroscopically active metal ions, especially Mn2+, have been used to probe the type of interaction the metal has with RNA. In previous studies, the hammerhead ribozyme, an RNA motif that catalyzes a site-specific phosphodiester bond cleavage reaction, was determined by room temperature EPR (electron paramagnetic resonance) studies to have a set of tightly and weakly bound metal ions. Under high salt concentrations, the hammerhead was found to bind a single Mn2+ ion with high affinity and with a characteristic low temperature EPR signal. Using site specific 15N labeling of a guanine residue in conjunction with ESEEM (electron spin echo envelope modulation) spectroscopy, the high affinity Mn2+ ion was conclusively determined coordinated to G10.1 of the proposed A9/G10.1 site with four water molecules coordinated to the Mn2+ ion. EPR power saturation studies determined that under low salt conditions the hammerhead coordinates up to four Mn2+ ions in relatively close proximity compared to an RNA duplex. EXAFS (extended X-ray absorption fine structure) spectroscopy was used to determine that a Cd2+ ion coordinates to both the Rp and Sp sulfur atoms of a phosphorothioate modification at the A9 phosphate of the hammerhead. Previous EXAFS results for the Mn2+ substituted A9 phosphorothioate suggested that the Mn2+ ion coordinates to the oxygen atom for both isomers. Molecular modeling suggested that the A9/G10.1 metal site will twist the phosphate group in order to accommodate this coordination. A Mn-GMP and Mn-phosphate model complexes were prepared and characterized by EXAFS to assign the origin of the features observed for the hammerhead sample. A series of RNA sequences with internal loops containing the sheared G-A metal ion binding motif showed greater thermal stabilization of the RNA structure in the presence of Mn2+ ions compared to sequences without the motif. The EPR binding isotherms also showed a set of moderately tight metal ion interaction while circular dichroism spectroscopy was used to investigate structural differences between the sequences. These results suggest a mostly electrostatic, not structural role, for the Mn2+ ion interactions with these sequences.

Techn. Universiẗat, Diss., 2006--Darmstadt.

No description available.

No description available.

A investigação estrutural de materiais desordenados tem grande interesse devido ao rápido progresso de suas aplicações tecnológicas. A ressonância magnética, particularmente a ressonância paramagnética eletrônica (RPE) de onda contínua (CW) e pulsada, tem demonstrado ser um método altamente sensível para a análise das propriedades físicas e químicas de materiais vítreos e amorfos. Neste trabalho, aplicamos essas técnicas, em particular as técnicas de RPE pulsada, electron spin echo envelope modulation (ESEEM) com seqüências de dois e três pulsos e a de echo detected field sweep (EDFS) no estudo de três materiais distintos. No primeiro trabalho é apresentado um estudo das mudanças estruturais fotoinduzidas em filmes do sistema vítreo [Sb(PO3)3]nSb203 dopados com Cu2+.Esse material possui a propriedade de fotocontração quando exposto à luz ultravioleta. As medidas de RPE CW e de EDFS mostram que ocorre uma distorção na estrutura local quando o filme é irradiado por UV. Os espectros de ESEEM mostram a presença e uma linha de 31P que diminui drasticamente quando o filme é irradiado. O segundo trabalho descreve o estudo do xerogel pentóxido de vanádio, V2O3:nH2O com n = 1.8. Os espectros de RPE CW do V4+ na temperatura ambiente exibem uma estrutura hiperfina isotrópica tipicamente observada em líquidos. Em baixas temperaturas (65K) o espectro é anisotrópico e corresponde ao observado para amostras sólidas em pó. As simulações dos espectros CW estão em acordo com os resultados experimentais. Estudos de ESEEM em baixas temperaturas, efetuados para diversos valores do campo externo mostram que as modulações correspondentes aos núcleos 1 H existem somente para as componentes perpendiculares do espectro de ii EDFS. Os resultados obtidos são consistentes com o modelo estrutural no qual quatro radicais OH estão situados em posições equivalentes no plano equatorial da estrutura do V4+. Uma molécula de água pode estar localizada no sitio oposto da ligação axial V=0. No terceiro trabalho são estudados vidros foto-termo-refrativos (phototherino - refractive ou PTR). Vidros PTR do sistema O-Si-Na-Zn-Al-K-F-Br dopados com Ce, Ag, Sn, Sb exibem cristalização em nano-escala, induzidas após irradiação com luz ultravioleta e subseqüentes tratamentos térmicos. Neste trabalho, conjuntos de amostras irradiadas e/ou tratadas termicamente são analisados por RPE CW, EDFS e ESEEM. RPE CW não forneceu informações confiáveis sobre a estrutura do vidro. Mudanças significativas foram observadas nos espectros de EDFS, indicando mudanças estruturais na vizinhança dos centros paramagnéticos. Medidas sistemáticas de ESEEM em várias posições de campo magnético mostram claramente a modulação característica de 23Na, indicando que núcleos de Na são parte da estrutura ligante ao redor do centro observado. Para a amostra irradiada e tratada por 1 hora a 450°C e 2 horas a 520°C, o espectro de ESEEM obtido nas proximidades de g = 2 mostra a presença adicional de uma linha que pode ser atribuída a íons de 19F. Comparando-se com resultados de RMN, pode-se concluir que domínios de NaF são formados nas proximidades desse centro paramagnético. O quarto trabalho está relacionado com a simulação numérica de espectros de RPE CW e consiste em um estudo teórico a respeito da determinação dos autocampos de um Hamiltoniano de spin. O algoritmo proposto pode ser resolvido pelo \"Filter Diagonalization Method\", ou FDM, sugerindo uma nova metodologia capaz de resolver o problema de uma maneira mais direta do que a empregada pelos métodos usuais / The structural investigation of disordered materiais has been of great interest due to fast progress in technological applications. Magnetic resonance, particularly the pulsed and continuous wave electron paramagnetic resonance (EPR), has demonstrated to be a high sensibility method to analyze chemical and physical properties of disordered and vitreous materiais. In this work, we applied these techniques, particularly the electron spin echo envelope modulation (ESEEM) with two and three pulses and echo detected field sweep (EDFS) to investigate three different materiais. In the first investigation, we show a study of photo-induced structural changes in the [Sb(PO3)3]nSb203 vitreous system films doped with Cu2+ ions. This material has photo-contraction properties when exposed to UV light. EPR CW and EDFS measurements show that the local structure is distorted when the fim is UV irradiated. The ESEEM spectra show the presence of a 31P line that vanishes for irradiated films. The second work describes the study of vanadium pentoxide xerogel V205: nH2O with n = 1.8. The CW EPR spectra of V4+ at room temperature exhibts the isotropic hyperfine structure typical of liquid phases. At low temperatures (65 K) the spectrum is anysotropic, as expected for solid powder samples. Numerical simulations of the EPR spectra in the two temperature limits are in good agreement with the experimental data. An ESEEM study carried out at low temperatures and for different static field values show that the nuclear modulations associated to 1H nuclei are observable only for the perpendicular components of the EDFS spectrum. Results are consistent with the structural model in which the four OH radicals are placed in iv equivalent positions of the V4+ structure equatorial plane. A water molecule can be placed in the opposite site of the axial V=O bond. In the third work, CW and pulsed EPR spectroscopy techniques were applied to study the effects of the thermal treatments and UV irradiation on photo-thermorefractive (PTR) glasses. PTR glasses of the system 0-Si-Na-Zn-Al-K-F-Br doped with Ce, Ag, Sn, Sb exhibit NaF crystallization at nano-scale, induced after U.V. irradiation and subsequent thermal treatments. Sets of irradiated and/or thermal treated samples were analyzed at the X-band by CW and pulsed techniques, such as EDFS and ESEEM. The forth work is related to the numerical simulation of CW EPR spectra and reports a new technical approach to the resonance field problem associated with a given spin hamiltonian. The proposed alghoritm can be solved by the \"filter diagonalization method\" (PIM), suggesting a new methodology, which is capable to solve the problem in a more straightforward way when compared to existing methods

A investigação estrutural de materiais desordenados tem grande interesse devido ao rápido progresso de suas aplicações tecnológicas. A ressonância magnética, particularmente a ressonância paramagnética eletrônica (RPE) de onda contínua (CW) e pulsada, tem demonstrado ser um método altamente sensível para a análise das propriedades físicas e químicas de materiais vítreos e amorfos. Neste trabalho, aplicamos essas técnicas, em particular as técnicas de RPE pulsada, electron spin echo envelope modulation (ESEEM) com seqüências de dois e três pulsos e a de echo detected field sweep (EDFS) no estudo de três materiais distintos. No primeiro trabalho é apresentado um estudo das mudanças estruturais fotoinduzidas em filmes do sistema vítreo [Sb(PO3)3]nSb203 dopados com Cu2+.Esse material possui a propriedade de fotocontração quando exposto à luz ultravioleta. As medidas de RPE CW e de EDFS mostram que ocorre uma distorção na estrutura local quando o filme é irradiado por UV. Os espectros de ESEEM mostram a presença e uma linha de 31P que diminui drasticamente quando o filme é irradiado. O segundo trabalho descreve o estudo do xerogel pentóxido de vanádio, V2O3:nH2O com n = 1.8. Os espectros de RPE CW do V4+ na temperatura ambiente exibem uma estrutura hiperfina isotrópica tipicamente observada em líquidos. Em baixas temperaturas (65K) o espectro é anisotrópico e corresponde ao observado para amostras sólidas em pó. As simulações dos espectros CW estão em acordo com os resultados experimentais. Estudos de ESEEM em baixas temperaturas, efetuados para diversos valores do campo externo mostram que as modulações correspondentes aos núcleos 1 H existem somente para as componentes perpendiculares do espectro de ii EDFS. Os resultados obtidos são consistentes com o modelo estrutural no qual quatro radicais OH estão situados em posições equivalentes no plano equatorial da estrutura do V4+. Uma molécula de água pode estar localizada no sitio oposto da ligação axial V=0. No terceiro trabalho são estudados vidros foto-termo-refrativos (phototherino - refractive ou PTR). Vidros PTR do sistema O-Si-Na-Zn-Al-K-F-Br dopados com Ce, Ag, Sn, Sb exibem cristalização em nano-escala, induzidas após irradiação com luz ultravioleta e subseqüentes tratamentos térmicos. Neste trabalho, conjuntos de amostras irradiadas e/ou tratadas termicamente são analisados por RPE CW, EDFS e ESEEM. RPE CW não forneceu informações confiáveis sobre a estrutura do vidro. Mudanças significativas foram observadas nos espectros de EDFS, indicando mudanças estruturais na vizinhança dos centros paramagnéticos. Medidas sistemáticas de ESEEM em várias posições de campo magnético mostram claramente a modulação característica de 23Na, indicando que núcleos de Na são parte da estrutura ligante ao redor do centro observado. Para a amostra irradiada e tratada por 1 hora a 450°C e 2 horas a 520°C, o espectro de ESEEM obtido nas proximidades de g = 2 mostra a presença adicional de uma linha que pode ser atribuída a íons de 19F. Comparando-se com resultados de RMN, pode-se concluir que domínios de NaF são formados nas proximidades desse centro paramagnético. O quarto trabalho está relacionado com a simulação numérica de espectros de RPE CW e consiste em um estudo teórico a respeito da determinação dos autocampos de um Hamiltoniano de spin. O algoritmo proposto pode ser resolvido pelo \"Filter Diagonalization Method\", ou FDM, sugerindo uma nova metodologia capaz de resolver o problema de uma maneira mais direta do que a empregada pelos métodos usuais / The structural investigation of disordered materiais has been of great interest due to fast progress in technological applications. Magnetic resonance, particularly the pulsed and continuous wave electron paramagnetic resonance (EPR), has demonstrated to be a high sensibility method to analyze chemical and physical properties of disordered and vitreous materiais. In this work, we applied these techniques, particularly the electron spin echo envelope modulation (ESEEM) with two and three pulses and echo detected field sweep (EDFS) to investigate three different materiais. In the first investigation, we show a study of photo-induced structural changes in the [Sb(PO3)3]nSb203 vitreous system films doped with Cu2+ ions. This material has photo-contraction properties when exposed to UV light. EPR CW and EDFS measurements show that the local structure is distorted when the fim is UV irradiated. The ESEEM spectra show the presence of a 31P line that vanishes for irradiated films. The second work describes the study of vanadium pentoxide xerogel V205: nH2O with n = 1.8. The CW EPR spectra of V4+ at room temperature exhibts the isotropic hyperfine structure typical of liquid phases. At low temperatures (65 K) the spectrum is anysotropic, as expected for solid powder samples. Numerical simulations of the EPR spectra in the two temperature limits are in good agreement with the experimental data. An ESEEM study carried out at low temperatures and for different static field values show that the nuclear modulations associated to 1H nuclei are observable only for the perpendicular components of the EDFS spectrum. Results are consistent with the structural model in which the four OH radicals are placed in iv equivalent positions of the V4+ structure equatorial plane. A water molecule can be placed in the opposite site of the axial V=O bond. In the third work, CW and pulsed EPR spectroscopy techniques were applied to study the effects of the thermal treatments and UV irradiation on photo-thermorefractive (PTR) glasses. PTR glasses of the system 0-Si-Na-Zn-Al-K-F-Br doped with Ce, Ag, Sn, Sb exhibit NaF crystallization at nano-scale, induced after U.V. irradiation and subsequent thermal treatments. Sets of irradiated and/or thermal treated samples were analyzed at the X-band by CW and pulsed techniques, such as EDFS and ESEEM. The forth work is related to the numerical simulation of CW EPR spectra and reports a new technical approach to the resonance field problem associated with a given spin hamiltonian. The proposed alghoritm can be solved by the \"filter diagonalization method\" (PIM), suggesting a new methodology, which is capable to solve the problem in a more straightforward way when compared to existing methods

No description available.

No description available.

Tyrosine Hydroxylase (TyrH) is a pterin-dependent mononuclear non-heme iron oxygenase. TyrH catalyzes the hydroxylation reaction of tyrosine to dihydroxyphenylalanine (DOPA). This reaction is the first and the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. The active site iron in TyrH is coordinated by the common facial triad motif, 2-His-1-Glu. A combination of kinetic and spectroscopic techniques was applied in order to obtain insight into the catalytic mechanism of this physiologically important enzyme. Analysis of the TyrH reaction by rapid freeze-quench Mossbauer spectroscopy allowed the first direct characterization of an Fe(IV) intermediate in a mononuclear nonheme enzyme catalyzing aromatic hydroxylation. Further rapid kinetic studies established the kinetic competency of this intermediate to be the long-postulated hydroxylating species, Fe(IV)O. Spectroscopic investigations of wild-type (WT) and mutant TyrH complexes using magnetic circular dichroism (MCD) and X-ray absorption spectroscopy (XAS) showed that the active site iron is 6-coordinate in the resting form of the enzyme and that binding of either tyrosine or 6MPH4 alone does not change the coordination. However, when both tyrosine and 6MPH4 are bound, the active site becomes 5-coordinate, creating an open site for reaction with O2. Investigation of the kinetics of oxygen reactivity of TyrH complexes in the absence and presence of tyrosine and/or 6MPH4 indicated that there is a significant enhancement in reactivity in the 5-coordinate complex in comparison to the 6-coordinate form. Similar investigations with E332A TyrH showed that Glu332 residue plays a role in directing the protonation of the bridged complex that forms prior to the formation of Fe(IV)O. Rapid chemical quench analyses of DOPA formation showed a burst of product formation, suggesting a slow product release step. Steady-state viscosity experiments established a diffusional step as being significantly rate-limiting. Further studies with stopped-flow spectroscopy indicated that the rate of TyrH reaction is determined by a combination of a number of physical and chemical steps. Investigation of the NO complexes of TyrH by means of optical absorption, electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) techniques revealed the relative positions of the substrate and cofactor with respect to NO, an O2 mimic, and provided further insight into how the active site is tuned for catalytic reactivity upon substrate and cofactor binding.

Die Elektronen-Spin-Resonanz-Spektroskopie (ESR) in Verbindung mit ortsspezifischer Spinmarkierung stellt eine hervorragende Möglichkeit dar, um die Struktur und Dynamik von Proteinen aufzuklären. In dieser Dissertation wurden mit Hilfe der Hochfeld-ESR-Spektroskopie (W-Band, 95 GHz, T=160 K) für dreizehn spinmarkierte Colicin A Proben die Polarität und die Protizität der Umgebung der Spinlabelbindestelle bestimmt. Wasserzugänglichkeiten und Wasserstoffbrückenbindungen zum Spinlabel wurden mittels Puls-ESR Methoden (3-Puls-D-ESEEM und Hahn-Echozerfall) bestimmt und die Ergebnisse mit den Polaritäts- und Protizitätswerten korreliert. Raumtemperaturspektren dieser Proben im X-Band (9.5 GHz), Q-Band (34 GHz) und W-Band (95 GHz) liefern Informationen über die Spinlabelbewegung. Mit Hilfe von Molekulardynamiksimulationen (MD) der spinmarkierten kanalbildenden Domäne von Colicin A konnten die Konformationen (Rotameranalyse) und die Dynamik der Spinlabelseitenketten in den unterschiedlichen Umgebungen charakterisiert werden. Der Vergleich der experimentellen mit den aus MD-Trajektorien berechneten ESR-Spektren liefert die Beiträge der unterschiedlichen Rotamerübergänge, die für die beobachteten Spektrenformen charakteristisch sind.