No description available.

No description available.

<p>Inhibitors belonging to the serpin (serine protease inhibitor) family control proteases involved in various physiological processes. All serpins have a common tertiary structure based on the dominant b-sheet A, but they have different inhibitory specificity. The specificity of a serpin is determined by the Pl-Pl’ peptide bond acting as a bait for the target protease which is made up of an exposed reactive centre loop (RCL). The serpin plasminogen activator inhibitor type-1 (PAI-1) is the main physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA, respectively). Elevated plasma levels of PAI-l have been correlated with a higher risk of deep venous thrombosis, and PAI-1 is a risk factor for recurrent myocardial infarction. Furthermore, PAI-1 has a role in cell migration and has been suggested to regulate tumor growth and angiogenesis. PAI-1 is unique among the serpins in that it can spontaneously and rapidly convert into its latent form. This involves full insertion of the RCL into b-sheet A. </p><p>There were two partially overlapping goals for this thesis. The first was to use latent PAI-1 as model for development of a fluorescence-based method, Donor-Donor Energy Migration for intramolecular distance measurements. The second goal was to use DDEM, together with other biochemical methods, to reveal the structure of the PAI-1/uPA complex, the conformation of the RCL in active PAI-1, and molecular determinants responsible for the conversion of PAI-1 from the active to the latent form.</p><p>The use of molecular genetics for introduction of fluorescent molecules enables the use of DDEM to determine intramolecular distances in a variety of proteins. This approach can be applied to examin the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this work, the accuracy of the DDEM method has been evaluated by experiments with the latent PAI-1 for which X-ray structure is known. Our data show that distances approximating the Förster radius (57±1 Å) obtained by DDEM are in good agreement (within 5.5 Å) with the distances obtained by X-ray crystallography.</p><p>The molecular details of the inhibitory mechanism of serpins and the structure of the serpin/protease complex have remained unclear. To obtain the structural insights required to discriminate between different models of serpin inhibition, we used fluorescence spectroscopy and cross-linking techniques to map sites of PAI-1/uPA interaction, and distance measurement by DDEM to triangulate the position of the uPA in the complex. The data have demonstrated clearly that in the covalent PAI-1/uPA complex, the uPA is located at the distal end of the PAI-1 molecule relative to the initial docking site. This indicates that serpin inhibition involves reactive center cleavage followed by full loop insertion, whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one. </p><p>To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by DDEM, the newly a developed method based on probe quenching and biochemical methods revealed that the RCL in PAI-1 is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants, and it can be partially inserted. This possibly explains for the ability of PAI-1 to convert spontaneously to its latent form. </p>

Within this study, the focus will be on oligoglycines. Numerous studies pertaining to the mobility and conformations of oligoglycines have been completed, as this is a driving force for the study. The oligopeptide is modeled using a “coarse-grained” model created in the Allison lab at Georgia State University [Xin,Y.,et. al, J. Phys. Chem. B 2006, 110, 1038-1045], which will be briefly explained within this paper. Oligoglycines will be studied in a few different systems, as the overall charge on the peptide and system will affect its mobility. The conclusion drawn is that the peptide adopts three different conformations based on the temperature of the system and length of the peptide; random conformation at high temperatures, and compact conformations at low temperature. Oligoglycines of length three to five amino acids adopts a cyclic conformation at low temperatures. [Allison, S., et al., J. Sep. Sci. 2010, 33, 2430- 2438.]

Die Endlagerung von radioaktivem Abfall ist eine der vordringlichsten Aufgaben auf dem Gebiet der Kerntechnik. Als Teil der Sicherheitsanforderungen steht dabei der Schutz von Mensch und Umwelt vor den Gefahren der radioaktiven Stoffe selbst im Falle einer Freisetzung dieser Stoffe aus dem Endlager im Vordergrund. Als Basis für Langzeitsicherheitsanalysen dienen Modellierungen. Für diese sind umfassende Kenntnisse der chemisch-physikalischen Effekte und Einflüsse, die eine Mobilisierung und den Transport der Actiniden bewirken können, erforderlich. Diese Arbeit war ein eigenständiger Teil eines Projektes, welches sich mit der Aufklärung des Einflusses von Mikroorganismen auf die Ausbreitung von Actiniden bei einer Freisetzung dieser aus dem Endlager beschäftigt. Dabei wurde der Einfluss von mikrobiell produzierten Substanzen auf die Mobilisierung ausgewählter Actiniden untersucht. Die in diesem Projekt untersuchten mikrobiell produzierten Substanzen, sogenannte Bioliganden, wurden von Bakterien des Genus Pseudomonas unter speziellen Bedingungen produziert. Die von den Pseudomonaden freigesetzten Bioliganden, hier Siderophore vom Pyoverdin-Typ, haben ein hohes Potential, Metalle, insbesondere Eisen(III), zu komplexieren und so zu transportieren. Es wurde untersucht, in welcher Weise und unter welchen Bedingungen diese Bioliganden in der Lage sind, auch radioaktive Schadstoffe zu komplexieren und damit zu mobilisieren. Für die Untersuchungen wurden die α-strahlenden Actiniden Uran, Curium und Neptunium ausgewählt, weil diese auf Grund ihrer Langlebigkeit und Radiotoxizität von besonderem Interesse sind. Diese Arbeit beschäftigte sich mit der Wechselwirkung der Actiniden U(VI), Np(V) und Cm(III) mit Modellliganden, die die Funktionalitäten der Pyoverdine simulieren. Für die Metallbindung der Pyoverdine sind die Katecholgruppe des Chromophors und die funktionellen Gruppen der Peptidkette (Hydroxamsäuregruppen und α-Hydroxysäurereste) verantwortlich. Für die Simulation der Hydroxamsäuregruppen kamen dabei die Monohydroxamate Salicylhydroxamsäure (SHA) und Benzohydroxamsäure (BHA) und das natürliche Trihydroxamat Desferrioxamin B (DFO) zum Einsatz und für die Katecholgruppe das 6-Hydroxychinolin (6HQ) und 2,3-Dihydroxynaphthalin (NAP). Als Vergleichsligand wurde außerdem Benzoesäure (BA) untersucht. Für die Bestimmung der Stabilitätskonstanten zur Einschätzung der Stärke der gebildeten Komplexe, die Aufklärung der Struktur der Actinid-Ligand-Verbindungen und die Verfolgung der Änderung der Speziation der Actiniden vor und nach der Wechselwirkung mit den Modellliganden kamen verschiedene spektroskopische Verfahren wie Absorptionsspektroskopie, Laserfluoreszenzspektroskopie, Röntgenabsorptionsspektroskopie und Schwingungsspektroskopie zum Einsatz. Außerdem wurden erstmals theoretische Modellierungen zur Aufklärung der Struktur der Actinid-Modellligand-Komplexe durchgeführt. Die Ziele dieser Arbeit waren also die spektroskopische Charakterisierung und Bestimmung der Speziation und Komplexbildungskonstanten sowohl der ausgewählten Modellliganden als auch der gebildeten Actinid-Modellligand-Komplexe, die Aufklärung möglicher Strukturen der Komplexe sowie ein Vergleich der Ergebnisse mit denen der Pyoverdine. Der Vergleich der Stabilitätskonstanten der untersuchten Liganden mit den drei Actiniden U(VI), Cm(III) und Np(V) ergab im Wesentlichen folgende Reihenfolge der Komplexstärke: PYO ≥ DFO &amp;gt; NAP &amp;gt; 6HQ &amp;gt; SHA ≥ BHA &amp;gt; BA. Benzoesäure (hier wurde nur die Komplexbildung mit U(VI) untersucht) besitzt als einziger Ligand eine Carboxylfunktionalität und weist mit 103 die geringste Stabilitätskonstante auf. Die beiden Monohydroxamate SHA und BHA bilden mit allen drei Actiniden ähnlich starke 1:1-Komplexe. Bei den 1:2-Komplexen besitzt SHA mit Cm(III) und Np(V) etwas höhere Stabilitätskonstanten als BHA, wahrscheinlich verursacht durch einen stabilisierenden Einfluss der zusätzlichen phenolischen OH-Gruppe. Dieser Trend wurde auch in den theoretischen Modellierungen für die U(VI)-Komplexe beobachtet. Die natürlichen Siderophore DFO und PYO bilden die stärksten Komplexe mit den Actiniden (Stabilitätskonstanten von 1012 bis 1034). Dies liegt in der Struktur und der hohen Anzahl an funktionellen Gruppen begründet; DFO besitzt drei Hydroxamatgruppen, das Pyoverdinmolekül neben den Hydroxamatgruppen noch die Katecholgruppen der Chromophorfunktionalität. Die Modellliganden für die Chromophorfunktionalität, NAP und 6HQ, bilden stärkere Komplexe als die Monohydroxamate SHA und BHA, aber schwächere Komplexe als DFO und PYO. Daraus lässt sich schlussfolgern, dass die Chromophorfunktionalität eine wichtige Rolle bei der Anbindung der Actiniden an die Pyoverdine spielt. Der Vergleich der Stabilitätskonstanten der Komplexe der Liganden SHA, BHA und 6HQ mit den drei untersuchten Actiniden U(VI), Cm(III) und Np(V) untereinander zeigte, dass die Stärke der Komplexe von U(VI) über Cm(III) zu Np(V) abnimmt. Der Grund dafür liegt in den unterschiedlichen Ladungsdichten der Actinidionen. Während das UO2 2+-Ion mit einer Koordinationszahl von 5 und einem Ionenradius von ~ 0.6 eine effektive Ladung von + 3.3 besitzt, hat das Cm3+-Ion eine effektive Ladung von + 2.6 und das NpO2+-Ion von + 2.3. Damit besitzt das NpO2+-Ion die geringste Ladungsdichte der untersuchten Actinidionen und bildet damit auch die schwächsten Komplexe mit den niedrigsten Stabilitätskonstanten. Die Stärke der Komplexe der Liganden NAP, DFO und PYO nimmt von Cm(III) über U(VI) zu Np(V) ab. Obwohl Cm(III) eine geringere effektive Ladung als U(VI) hat, bildet es stärkere Komplexe als U(VI). Eventuell sind dafür strukturelle Behinderungen der Koordination durch die lineare O=U=O Einheit verantwortlich. Die Struktur der wässrigen U(VI)-Komplexe wurde mittels EXAFS-Spektroskopie und ATRFTIR-Spektroskopie untersucht. Aus den EXAFS-Spektren ließ sich schließen, dass die Koordination des Uranylions an die Hydroxamsäuregruppen der Liganden SHA, BHA und DFO eine Verkürzung des Abstandes der äquatorialen Sauerstoffatome zur Folge hat. Im Gegensatz dazu resultiert eine Koordination des Uranylions an die Carboxylgruppe des Liganden BA in einer Verlängerung des U-Oäq Abstandes. Die Ergebnisse des NAP als Modellligand für die Chromophorfunktionalität des Pyoverdins und die Ergebnisse des Pyoverdins selbst zeigten, dass das Uranylion mit großer Wahrscheinlichkeit an die katecholischen OH-Gruppen der Chromophorfunktionalität des Pyoverdinmoleküls gebunden ist. In den Spektren der ATR-FTIR-Spektroskopie ist besonders der Bereich um die Schwingungsbande des Uranylions (961 cm-1) für die Beobachtung der Komplexbildung interessant. Dabei zeigte sich im U(VI)-BHA- und U(VI)-SHA-System eine Mischung aus zwei Komplexen mit 1:1- und 1:2-Stöchiometrie, die auch durch Speziationsrechnungen nachgewiesen werden konnten. Außerdem ließ sich anhand der Schwingungsbanden des Liganden feststellen, dass die Hydroxamsäuregruppe von SHA und BHA während der Komplexierung deprotoniert und direkt an der Komplexbildung beteiligt ist. Im Falle von SHA konnte weiterhin nachgewiesen werden, dass die phenolische OH-Gruppe bei den untersuchten pH-Werten nicht deprotoniert ist. Die pH-abhängigen Spektren des U(VI)-DFOSystems zeigten bei pH 3 die Bildung eines 1:1-Komplexes ähnlich dem der Monohydroxamate, bei Erhöhung des pH-Wertes bis pH 4 dann die Bildung eines 1:1- Komplexes, bei dem das Uranylion an zwei Hydroxamsäuregruppen gebunden ist. Dies stützt die Annahme einer 112-Stöchiometrie des Komplexes, die bei den anderen verwendeten experimentellen Methoden getätigt wurde. Durch Ausfällung aus wässrigen U(VI)-SHA- und U(VI)-BHA-Lösungen wurden Feststoffe der U(VI)-Komplexe hergestellt. Die Struktur dieser ausgefällten, pulverförmigen Feststoffe wurde mittels EXAFS, XRD und FTIR untersucht. Die Untersuchung der ausgefällten Feststoffe ergab, dass die Feststoffkomplexe mit sehr hoher Wahrscheinlichkeit den in Lösung gefundenen Komplexen mit 1:2-Stöchiometrie entsprechen. Der Vergleich der Uran und Kohlenstoffgehalte der Feststoffe mit den in der Literatur beschriebenen Uranverbindungen (zur gravimetrischen Bestimmung von Urangehalten) zeigte übereinstimmende Werte. In den FTIR-Messungen wurden Banden bei 916 cm-1 beobachtet, die denen in der Lösung dem 1:2-Komplex zugeordneten Banden entsprechen. Die Ergebnisse der EXAFS-Messungen deuten auf eine unterschiedliche Nahordnung des U(VI) im Feststoff und in der Lösung hin. So ergab der Vergleich der Strukturparameter der Hydroxamat- Feststoffe mit den U(VI)-Hydroxamat-Komplexen in Lösung deutliche Unterschiede zwischen den Feststoffkomplexen und denen in Lösung. So ist in wässriger Lösung der Abstand der äquatorialen Sauerstoffatome mit 2.41 Å signifikant kürzer als der der Feststoffkomplexe mit 2.47 Å (SHA) und 2.44 Å (BHA). Die röntgendiffraktogrammischen Messungen der Festphasen ergaben reflexreiche Spektren mit signifikanten Peaks, die sich allerdings keinen bekannten U(VI)-Festphasen zuordnen ließen. In einer Kooperation mit dem Institut für Theoretische Chemie der Universität zu Köln wurden für die 1:1- und 1:2-Komplexe der wässrigen U(VI)-SHA-, U(VI)-BHA- und U(VI)-BA-Systeme erstmals theoretische Modellierungen durchgeführt. Dabei wurden die Strukturen der Komplexe sowohl in der Gasphase als auch unter Berücksichtigung der Solvatation optimiert und die relativen Stabilitäten und Anregungsspektren berechnet. Die mit DFT berechneten Bindungsenergien bestätigen die experimentell anhand der Stabilitätskonstanten log β ermittelte Reihenfolge der Komplexstabilitäten (SHA ≥ BHA &amp;gt; BA). Außerdem zeigen die höheren Bindungsenergien der 1:2-Komplexe, dass diese stabiler sind als die 1:1-Komplexe. Dies lässt sich auch anhand der experimentell ermittelten Stabilitätskonstanten nachweisen. Die Maxima der mit TD-DFT berechneten Anregungsspektren weichen um 0.4 ± 0.2 eV von den experimentellen UV-Vis Spektren ab. Dies zeigt die gute Übereinstimmung der berechneten Anregungsspektren mit den gemessenen UV-Vis Spektren. Für den 1:1-Komplex des U(VI)-SHA-Systems konnte mit Hilfe der theoretischen Modellierung die strukturelle Anbindung des Uranylions an die Hydroxamsäuregruppe aufgeklärt werden. Der Vergleich der berechneten Strukturen, Bindungsenergien, Bindungslängen und Anregungsspektren der beiden möglichen Anbindungsmodi [O,O] und [N,O’] zeigte deutlich, dass das Uranylion bevorzugt über die beiden Sauerstoffatome der Hydroxamsäuregruppe, also den [O,O]-Modus, gebunden wird. Die Methode der DFT konnte also dazu beitragen, Defizite in der experimentellen Aufklärung der Komplexstruktur im Fall des U(VI)-SHA-Systems zu beheben. Die Modellliganden und deren Komplexe mit U(VI), Cm(III) und Np(V) wurden zum größten Teil erstmals spektroskopisch charakterisiert sowie deren bisher weitgehend unbekannten Stabilitätskonstanten bestimmt. Außerdem konnte die Struktur der U(VI)-Hydroxamat- Komplexe mit Hilfe der ATR-FTIR-Spektroskopie und der theoretischen Modellierung aufgeklärt werden. Im Vergleich der Ergebnisse der Modellliganden mit denen der Pyoverdine konnte festgestellt werden, dass die Katecholfunktionalität der Pyoverdine eine große Rolle bei der Komplexierung mit den Actiniden spielen wird. Weiterhin ließen sich aus den Ergebnissen Schlussfolgerungen zur Stärke der gebildeten Actinid-Modellligand- und Actinid-Pyoverdin-Komplexe ziehen. Die Pyoverdine bildeten mit U(VI) Komplexe mit Stabilitätskonstanten bis 1030, mit Cm(III) bis 1032 und mit Np(V) bis 1020. Die wichtigsten, in höheren Konzentrationen vorkommenden anorganischen Komplexbildner in natürlichen Wässern sind das Hydroxidion OH- sowie das Carbonation CO32-. Diese besitzen eine hohe Komplexierungsfähigkeit und bilden mit den drei Actiniden U(VI), Cm(III) und Np(V) Komplexe mit Stabilitätskonstanten von 102 bis 1020. Der Vergleich der Konstanten von OH und CO32- mit denen der organischen, mikrobiellen Pyoverdin-Liganden zeigt, dass die Pyoverdine ähnlich starke bzw. teilweise stärkere Komplexe mit den Actiniden bilden als die anorganischen Komplexbildner. Daraus lässt sich ableiten, dass die Pyoverdine selbst in niedrigeren Konzentrationen ein hohes Potential besitzen, Actiniden in natürlichen Wässern zu binden und damit zu transportieren. Die untersuchten Bioliganden sind also in der Lage, bei Anwesenheit in der Natur in bestimmten Konzentrationen im Grundwasser Actiniden, z.B. durch Herauslösen aus Festphasen, zu mobilisieren. Damit können solche Bioliganden das Verhalten der Actiniden in der Umwelt entscheidend beeinflussen. Die Ergebnisse dieser Arbeit tragen dazu bei, den Einfluss der mikrobiellen Liganden auf die Mobilisierung und Ausbreitung der Actiniden besser einschätzen zu können. Damit können die Ergebnisse zur Quantifizierung des Mobilisierungseffekts der Actiniden durch freigesetzte Bioliganden im Nahfeld genutzt werden. / One of the urgent tasks in the field of nuclear technology is the final storage of radioactive substances. As a part of the safety requirements the protection of humans and the environment from the danger of radioactive substances in case of the release from the final storage is essential. For performing long-term safety calculations the detailed understanding of the physico-chemical effects and influences which cause the mobilisation and transport of actinides are necessary. The presented work was a discrete part of a project, which was focused on the clarification of the influence of microorganisms on the migration of actinides in case of the release of actinides from a final storage. The influence of microbial produced substances on the mobilisation of selected actinides was studied thereby. The microbial produced substances studied in this project were synthesized by bacteria from the Pseudomonas genus under special conditions. Fluorescent Pseudomonads secrete bacterial pyoverdin-type siderophores with a high potential to complex and transport metals, especially iron(III). The aim of the project was to determine how and under which conditions the bioligands are able to complex also radioactive substances and therefore to transport them. For this work the alpha-emitting actinides uranium, curium and neptunium were chosen because their long-life cycle and their radiotoxicity are a matter of particular interest. This work dealed with the interaction of the actinides U(VI), Np(V) and Cm(III) with model ligands simulating the functionality of the pyoverdins. The functional groups that participate in the metal binding of the pyoverdins are the catechol group of the chromophore and the ligand sites in the peptide chain, i.e. the hydroxamate groups and the α-hydroxy acid moieties. For the simulation of the hydroxamate functionality the monohydroxamates salicylhydroxamic acid (SHA) and benzohydroxamic acid (BHA) and the natural trihydroxamate desferrioxamine B (DFO) and for the simulation of the catechol groups 6-hydroxyquinoline (6HQ) and 2,3-dihydroxynaphthalene (NAP) were used. A further ligand with carboxyl functionality, benzoic acid (BA), was used as a comparison. Absorption spectroscopy, laser fluorescence spectroscopy, X-ray absorption spectroscopy and vibrational spectroscopy were applied for the determination of the stability constants to assess the strength of the formed actinide-model ligand-complexes, for the clarification of the structures of the formed complexes and to observe the variation of the speciation of the actinides during the interaction with the ligands. Furthermore, for the first time density functional theory (DFT) calculations were performed to determine the molecular structure of the actinide-modelligand-complexes. Thus, the objectives of this work were the determination of the spectroscopic properties, speciation and stability constants of the model ligands and the formed actinide-model ligand-complexes, the clarification of the complex structures and a comparison of the results with those of the pyoverdins. The comparison of the stability constants of the studied ligands with the three actinides U(VI), Cm(III) and Np(V) systems results mainly in the following order of complex strength: PYO ≥ DFO &amp;gt; NAP &amp;gt; 6HQ &amp;gt; SHA ≥ BHA &amp;gt; BA. Benzoic acid, the ligand with the carboxyl functionality, has the lowest stability constant of 103. Both monohydroxamates, SHA and BHA, form 1:1 complexes with similar stability. The stability constants of the 1:2 complexes of SHA with Cm(III) and Np(V) are slightly higher than those of BHA, which is probably caused by a stabilizing effect of the additional phenolic OH-group of SHA. This behaviour was also found in the theoretical calculations of the U(VI)-complexes. The natural siderophores DFO and PYO have the highest stability constants with U(VI) and form the strongest complexes (constants from 1012 to 1034). The reason therefore is the structure and high number of functional groups of these ligands; DFO has three hydroxamate groups, the pyoverdin molecule has the catechol groups of the chromophore functionality in addition to the hydroxamate groups. The model ligands for the chromophore functionality, NAP and 6HQ, form stronger complexes than SHA and BHA, but weaker complexes than DFO and PYO. From this it can be reasoned that the chromophore functionality probably plays an important role for the coordination of the actinides to the pyoverdins. The comparison of the stability constants of the complexes of the ligands SHA, BHA and 6HQ with the studied actinides U(VI), Cm(III) and Np(V) shows that the strength of the complex formation decreases from U(VI) via Cm(III) to Np(V). The reason therefore is the different charge density of the actinide ions. The UO22+-ion has an effective charge of + 3.3 (with a coordination number of 5 and an ionic radius of ~ 0.6), the Cm3+-ion of + 2.6 and the NpO2+-ion of + 2.3. Therefore, the neptunyl ion has the lowest charge density of the studied actinide ions and on account of this it forms the weakest complexes with the lowest stability constants. The strength of the complex formation of the ligands NAP, DFO and PYO decreases from Cm(III) via U(VI) to Np(V). Cm(III) forms stronger complexes than U(VI) although Cm(III) has a lower effective charge. The reason therefore could be a possible structural hampering of the coordination through the linear O=U=O unit. The structure of the aqueous U(VI)-complexes was studied using EXAFS spectroscopy and FTIR spectroscopy. From the results of the EXAFS spectra one can conclude that the coordination of the uranyl ion to the hydroxamic acid groups of the SHA, BHA and DFO ligands results in a shortening of the distance of the equatorial oxygen atoms. In contrast to this the coordination of the uranyl ion to the carboxyl group of BA yields in a longer U-Oeq bond length. From the findings of the EXAFS studies with NAP and pyoverdin one can conclude a strong affinity of U(VI) to the catechol functionality of the pyoverdin molecule. For the observation of the complexation in the ATR-FTIR spectra the region around the vibration band of the uranyl ion (916 cm-1) is interesting to observe. In the spectra of the U(VI)-BHA- and U(VI)-SHA-system a mixture of two complexes with 1:1 and 1:2 stoichiometry was observed, which was also existing in the speciation. Furthermore, on the basis of the vibration bands of the ligands it could be ascertained that the hydroxamate groups of SHA and BHA are deprotonated and directly involved in the complexation. Also, in case of SHA it could be verified that the phenolic OH-group is protonated at the investigated pH values. At pH 3 the pH dependent spectra of the U(VI)-DFO-system showed the formation of a 1:1 complex similar to those of the monohydroxamates. With increasing pH up to 4 the formation of a 1:1 complex was observed, in which the uranyl ion is bound to two hydroxamic acid groups. This underlines the assumption that the complex had a 112-stoichiometry, which was concluded on the basis of the other used experimental methods. Solid phases of U(VI) complexes were assembled by precipitation from the aqueous U(VI)-SHA and U(VI)-BHA solutions. The structure of these powder solids was analyzed using EXAFS, XRD and FTIR. The analysis of the solid phases showed that the solid complexes are most likely consistent with the complexes in aqueous solution with 1:2 stoichiometrie. The comparison of the uranium and carbon percentage of the solids with those of the uranium compounds described in the literature (for the gravimetric estimation of uranium contents) results in analogue values. In the FTIR spectra of the solids vibration bands at 916 cm-1 were observed according to the bands of the 1:2 complexes in aqueous solution. The results of the EXAFS measurements indicated a different short-range order of the U(VI) in solid phases and solutions. The comparison of the structural parameters of the solid phases with those of the aqueous U(VI)-hydroxamate complex species points to strong differences. Thus, in aqueous solution the distance of the equatorial oxygen atoms of 2.41 Å is significant shorter than those of the solid complexes with 2.47 Å (SHA) and 2.44 Å (BHA). The XRD measurements showed spectra high in reflexes and with significant peaks which could not be assigned to known U(VI) solid phases. In a cooperation with the Institute of Theoretical Chemistry at the University of Cologne density functional theory (DFT) calculations were performed to determine the molecular structure of 1:1 and 1:2 U(VI)-complexes with SHA, BHA and BA. The precise molecular structures of the complexes in gas phase have been calculated as well as the relative stabilities and the time-dependent DFT excitation spectra with consideration of the solvation effects. The relative stabilities calculated with DFT confirm the order of strength of the complexes determined using the stability constants log β (SHA ≥ BHA &amp;gt; BA). Furthermore, the higher binding energies of the 1:2 complexes point to a higher complex stability of these complexes in comparison to the corresponding 1:1 complexes. This could be also demonstrated by means of the stability constants determined by the experimental studies. The peak maxima of the TD-DFT excitation spectra deviate at 0.4 ± 0.2 eV from the absorption maxima of the experimental UV-vis spectra. Thus, calculated and experimental spectra show a good qualitative agreement. For the 1:1 complex of the U(VI)-SHA-system the structurally coordination of the uranium ion to the hydroxamate group could be clarified with the help of the theoretical modelling. The comparison of the calculated structures, binding energies, bond lengths and excitation spectra of the two possible coordination modes [O,O] and [N,O’] showed clearly that the uranyl ion is bound preferable to the two oxygen atoms of the hydroxamate group ([O,O]-mode). Therefore, the method of DFT could contribute to eliminate shortcomings in the experimental determination of the complex structure in case of the U(VI)-SHA-system. The model ligands and their complexes with U(VI), Cm(III) and Np(V) were characterized spectroscopically and their widely unknown stability constants were determined for the first time. Furthermore, the structures of the U(VI)-hydroxamate-complexes were clarified using ATR-FTIR spectroscopy and theoretical calculations. The comparison of the results of the model ligands with those of the pyoverdins showed that the chromophore functionality of the pyoverdins probably plays an important role for the coordination of the actinides to the pyoverdins. Furthermore, conclusions to the strength of the formed actinide-model ligandand actinide-pyoverdin-complexes could be drawn from those results. The pyoverdins formed U(VI)-complexes with stability constants up to 1030, Cm(III)-complexes with constants up to 1032 and Np(V)-complexes with values up to 1020. The hydroxide ion OH- and the carbonato ion CO32- are the most important inorganic complexing agents in natural aquatic systems. They are highly concentrated and have great complexing ability. With the three studied actinides U(VI), Cm(III) and Np(V) complexes with stability constants from 102 to 1020 were formed. The comparison of the constants of OH- and CO3 2- with those of the organic microbial ligands showed that the pyoverdins complexes the actinides with similar and particularly higher strength than the inorganic complexing agents. Thus, it appears that the pyoverdins have a high potential to bind actinides and transport them in natural aquatic systems even though the pyoverdins exist in lower concentrations. Therefore, the studied bioligands are able to mobilize the actinides in natural aquatic systems, for example through dissolving them from solid phases, if they are present in the nature in specific concentrations. So, such bioligands can essentially influence the behaviour of actinides in the environment. The results of this work contribute to a better understanding and assessment of the influence of the microbial ligands to the mobilisation and migration of the radionuclides. The outcomes could be used to quantify the actinide-mobilising effect of the bioligands, which are released, for example, in the vicinity of a nuclear waste disposal site.

The modeling of transport behavior of charged particles carried out in our laboratory is based on classical continuum electro kinetic theory. It is applied to a variety of systems from small electrolyte ions to macromolecules including peptides, DNA and nanoparticles. Systems range from weakly charged particles to highly charged ones. Transport properties studied include conductance, electrophoresis, and diffusion. In this dissertation, the conductance of polyvalent electrolytes ions is studied both by a “small ion” model [R.M. Fuoss, L. Onsager, J. Phys. Chem. 61 (1957) 668] and “large ion” model [R.W. O’Brien, L.R. White, J. Chem. Soc. Faraday Trans. 2 (74) (1978) 1607)]. Also, the coarse-grained continuum primitive model is developed and used to characterize the titration and electrical conductance behavior of aqueous solutions of fullerene hexa-malonic acid, which is a highly charged electrolyte with an absolute valence charge as high as 12. Free solution electrophoresis is closely related to conductance and a coarse-grained bead modeling methodology, BMM, developed in the Allison’s laboratory starting in 2006, is generalized to characterize peptide systems with respect to the charge, conformation, and possibly specific interactions with components of the BGE. For weakly charged peptides, the electrostatic potential is treated at the level of linear Poisson-Boltzmann equation, which predicts the electrophoretic mobility with considerable accuracy [S. Allison, H. Pei, U. Twahir, H. Wu, J. Sep. Sci., 2010, 33(16):2430-2438], but fails for highly charged systems. A new nonlinear Poisson-Boltzmann, NLPB-BM procedure is developed and applied to the free solution electrophoretic mobility of low molecular mass oligolysines. The difficulty of highly charged systems is twofold: more complex handeling of electrostatics and accounting for the relaxation effect. Both issues are addressed in this dissertation. A related problem we investigated deals with the retarding influence of a gel on the rotational motion of a macromolecule. This is investigated within the framework of the Effective Medium (EM) model and is applied to examine the electric birefringence decay of a 622 base pair DNA fragment in an agarose gel. Modeling is also compared with experiment.

Les chromènes photochromiques sont largement utilisés dans les technologies modernes en raison de leur capacité à changer les propriétés sous irradiation UV. Les chromènes présentés dans ce travail possèdent en outre des fragments pouvant participer à la coordination avec des cations métalliques, acides aminés, ou de l'ADN. Pour ce type de molécule, l'interdépendance éventuelle entre les propriétés photochromiques et complexantes permet d'envisager soit le photo-contrôle de la complexation, soit la modulation du photochromisme par le biais de coordination.Ce travail est divisé en deux parties : la première est consacrée à la préparation des molécules cibles et la seconde à l'étude de la complexation. Les approches synthétiques élaborées ont permis d'obtenir une série de nouveaux chromènes annelés par des éthers couronnes de taille et de composition hétéroatomique différentes. Le processus de complexation de certains dérivés a été étudié en détail par spectroscopie RMN et absorptionUV-Visible. Il a été établi que la nature des cations métalliques détermine la stoechiométrie du complexe formé ainsi que la structure spatiale. Pour tous les composés étudiés, la complexation affecte les paramètres photochromiques notamment la vitesse de décoloration. En ce qui concerne la complexation des chromènes synthétisés avec les acides aminés protonés, il a été établi qu'en fonction de la longueur de chaine de ces acides, la formation de complexe mono- ou ditopique est favorisée.De plus, l'interaction d'un nouveau chromène cationique avec l'ADN a été étudiée. Il a été constaté que contrairement à la forme initiale, la forme colorée de ce composé générée sous irradiation UV permet l'intercalation au sein de l'ADN / Photochromic chromenes are widely used in modern technologies due to their abilityto change their properties upon UV irradiation. The chromenes presented in this work alsopossess fragments able to participate to the coordination with metal cations, amino acids, orDNA. These properties may sustain mutual influence on each other resulting in either photocontrolof complexing ability or photochromism tunable by complex formation.This work is divided in two parts, one devoted to the synthesis of the targetcompounds and the second to study on the complexing ability of the substances,respectively. Thus, the synthetic approaches to photochromic benzo- and naphthopyrans,annelated to the crown ether moieties of different size and heteroatomic composition, weredeveloped. The complex formation of several chromenes with metal cations wasinvestigated by means of UV-Vis absorption and NMR spectroscopies. The metal cationnature was found to determine the stoichiometry of the complexes as well as their spatialstructure. The complex formation was found to affect the photochromic properties of thecompounds, especially the bleaching rate. Investiga.on of complexation of the chromeneswith protonated amino acids revealed that, depending on the length of the carbon chain ofthe acid used, mono- or ditopic complexes may be formed.The interaction of the new chromene, possessing a positively charged group, with DNAwas also studied. In contrast to the initial form, the photo-induced colored form was foundto intercalate with DNA.

Inhibitors belonging to the serpin (serine protease inhibitor) family control proteases involved in various physiological processes. All serpins have a common tertiary structure based on the dominant b-sheet A, but they have different inhibitory specificity. The specificity of a serpin is determined by the Pl-Pl’ peptide bond acting as a bait for the target protease which is made up of an exposed reactive centre loop (RCL). The serpin plasminogen activator inhibitor type-1 (PAI-1) is the main physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA, respectively). Elevated plasma levels of PAI-l have been correlated with a higher risk of deep venous thrombosis, and PAI-1 is a risk factor for recurrent myocardial infarction. Furthermore, PAI-1 has a role in cell migration and has been suggested to regulate tumor growth and angiogenesis. PAI-1 is unique among the serpins in that it can spontaneously and rapidly convert into its latent form. This involves full insertion of the RCL into b-sheet A. There were two partially overlapping goals for this thesis. The first was to use latent PAI-1 as model for development of a fluorescence-based method, Donor-Donor Energy Migration for intramolecular distance measurements. The second goal was to use DDEM, together with other biochemical methods, to reveal the structure of the PAI-1/uPA complex, the conformation of the RCL in active PAI-1, and molecular determinants responsible for the conversion of PAI-1 from the active to the latent form. The use of molecular genetics for introduction of fluorescent molecules enables the use of DDEM to determine intramolecular distances in a variety of proteins. This approach can be applied to examin the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this work, the accuracy of the DDEM method has been evaluated by experiments with the latent PAI-1 for which X-ray structure is known. Our data show that distances approximating the Förster radius (57±1 Å) obtained by DDEM are in good agreement (within 5.5 Å) with the distances obtained by X-ray crystallography. The molecular details of the inhibitory mechanism of serpins and the structure of the serpin/protease complex have remained unclear. To obtain the structural insights required to discriminate between different models of serpin inhibition, we used fluorescence spectroscopy and cross-linking techniques to map sites of PAI-1/uPA interaction, and distance measurement by DDEM to triangulate the position of the uPA in the complex. The data have demonstrated clearly that in the covalent PAI-1/uPA complex, the uPA is located at the distal end of the PAI-1 molecule relative to the initial docking site. This indicates that serpin inhibition involves reactive center cleavage followed by full loop insertion, whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by DDEM, the newly a developed method based on probe quenching and biochemical methods revealed that the RCL in PAI-1 is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants, and it can be partially inserted. This possibly explains for the ability of PAI-1 to convert spontaneously to its latent form.

Na parte inicial deste trabalho faz-se estudo monográfico sobre o íon azoteto e sua estrutura. Também nesta parte encontram-se reunidas referências sobre o íon uranilo em meio aquoso e os possíveis produtos de hidrólise, propostos na literatura, bem como, em presença de azoteto. Algumas considerações sobre a formação de complexos mistos polinucleares são também apresentadas. Sendo o ácido azotídrico um eletrólito fraco, as mudanças de pH nos tampões N-3/HN3 podem ser acompanhadas após a introdução de íons U2O2+5, potenciometricamente, com eletrodo de vidro. No intuito de obtenção de medidas mais precisas, determinou-se a resposta Nernstiana do eletrodo utilizado. O tratamento de dados aplicado à obtenção das constantes de formação, em meio azoteto, através do numero médio de ligantes e do grau de complexação, a diferentes concentrações de ligante, força iônica 2,00 M e temperatura de (25,0 ± 0,l) ºC, permitiu a avaliação de três constantes de equilíbrio: &#946;1 = 2,136 x 104 (M-1), &#946;2 = 6,090 x 105 (M-2), &#946;3 = 1,421 x 108 (M-3). Tais constantes de formação, nas condições estudadas, bem como o crescimento da estabilidade especialmente do primeiro complexo, sugerem a existência dos seguintes equilíbrios: (Ver arquivo PDF). É proposto um método alternativo , para a determinação de acidez livre das soluções padrões de câtions de metais hidrolisáveis, por medidas de pH condicional, à força iônica constante, por adição de padrão ácido. Correção de pH para respostas não Nernstianas do eletrodo de vidro recomenda o processo para resultados analíticos mais precisos. Estudos espectrofotométricos em meio aquoso complementam os estudos de hidrólise para o íon uranilo. Estudos das características espectrais dos sistemas U(VI), Fe(III) e Cu(II) em meio azotídrico, sugerem apresentação de um método analítico, para a determinação simultânea destes íons, baseado nas altas absortividades molares dos complexos formados: (Ver arquivo PDF). Em decorrência do estudo de interferentes, após extenso estudo sobre a oxidação e estabilidade dos sistemas cobalto (II) e níquel(II), em meio azotídrico, baseado nas trocas espectrais de cobalto(II) à cobalto(III), é apresentado um método alternativo para a detecção de traços de cobalto em diferentes sais de níquel (perclorato, nitrato, sulfato e cloreto), após estudo monográfico apresentando os clássicos para tal finalidade. Em meio 2,9 M de &#8204;N-3&#8204; e 0,10 M &#8204;HN3&#8204; pela ação de 10 mM de H2O2 à 365 - 353 nm marcantes diferenças espectrais devidas à oxidação de íons de cobalto recomendam o método da adição de padrão para limites entre 0,59 - 1,7 mg de Co(II) com interferências de níquel(II) em concentrações superiores à 250 mg . Medidas potenciométricas permitiram uma estimativa da constante global de formação da espécie &#8204;Co(N3)6&#8204;3- por oxidação anódica de íons de cobalto(II) em meio não complexante, a baixas temperaturas e pela ação de oxidante químico, PbO2, em meio complexante, como sendo da ordem de 1016 (M-6) em força iônica 2,00 M (NaClO4) à (25,0 ± 0,1) °C. / The study starts with a monographic study about the azide ion and its structure. In this part we can also meet bibliographic references about the uranyl ion, in aqueous medium, and its possible hydrolysis products is proposed in the literature. Some considerations of the mixed polinuclear complex formation are also presented. Being hydrazoic acida weak electrolyte, the change of pH in N-3/HN3 buffers can be followed after introduction of U2O2+5 ions, potentiometrically, with glass electrode. In order to obtain accurate measurements the Nernstian response of this electrode was determined. Data treatment was applied to obtain the hydrolysis formation constants , in azide medium, through the average ligand number and complexity function, at several ligand concentrations, ionic strenght 2.00 M and temperature of (25.0 ± 0.l) ºC. The following constants are avaliable: &#946;1 = 2,136 x 104 (M-1), &#946;2 = 6,090 x 105 (M-2), &#946;3 = 1,421 x 108 (M-3). Referred to the equilibria (3) - (5): (See file PDF). It has been proposed an alternative method for the determination of free strong acid in standard solutions of hydrolysable cations, can be achieved by measurements of the conditional pH, at constant ionic strenght, with standard acid addition. Corrections of the measured pH for non Nernstian response of the glass electrode is a recomended procedure for more accurate analytical results. Spectrophotometric studies, in aqueous medium, are completing the hydrolysis studies of uranyl ion. Spectral studies of the U(VI), Cu(II) and Fe(III) systems, in azide medium, to render possible a new analytical method, for simultaneous determination of these ions, based in high molar absortivities of the complex formed (See file PDF). Running allway of the foreign ins study, after the great study about the oxidation and stability of cobalt(II) and nickel(II) systems, in azide medium, based in spectral changes of cobalt(II) to cobalt(III), it has been formed an alternative method for determination of traces of cobalt in different nickel salts (perchlorate, nitrate, sulphate, and chloride). A monographic study, giving the different methods of the literature, has been completed. In 2.9 M &#8204;N-3&#8204; and 0.10 M &#8204;HN3&#8204; by the action of 10 mM of H2O2 at 365 - 353 nm a marked spectral differences arosen from oxidation of cobalt(II). A standard addition method leved to determination of 0.59 - 1.7 mg Co of nickel interferes when in concentrations higher than 250 mg, i.e., higher than 0.010 M. Potentiometric measurements lead to an estimation of overall formation constant of &#8204;Co(N3)6&#8204;3- specie by anodic oxidation of cobalt(II) ions, in non complexing medium, at low temperatures and by chemical oxidant action, PbO2, in complexing medium, as being 1016 (M-6) order in ionic strenght 2.00 M (NaClO4) at (25.0 ± 0.1) ºC.

On the bead modeling methodology, or BMM, a macromolecule is modeled as a rigid, non-overlapping bead array with arbitrary radii. The BMM approach was pioneered by Kirkwood and coworkers (Kirkwood, J.G., Macromolecules, E.P. Auer (Ed.), Gordon and Breach, New York, 1967; Kirkwood, J.G., Riseman, J., J. Chem. Phys., 1948, 16, 565) and applied to such transport properties as diffusion, sedimentation, and viscosity. With the availability of computers, a number of investigators extended the work to account for the detailed shape of biomolecules in the 1970s. A principle objective of my research has been to apply the BMM approach to more complex transport phenomena such as transport in a gel, electrophoresis (free solution and in a gel), and also transport in more complex media (such as the viscosity of alkanes and benzene). Variables considered by the BMM include the number of beads (N), the radii of the beads, net charge and charge distribution, conformations, salt type, and salt concentration. The BMM has been extended to: (1) account for the existence of a gel; (2) characterize the charge and secondary structure of macromolecules; (3) account more accurately for hydrodynamic interaction (remove the orientationnal preaveraging approximation of hydrodynamic interaction); (4) study the effect of ion relaxation for particles in arbitrary size, shape, and charge; (5) consider the salt dependence of electrokinetic properties; (6) account for the formation of possible complex between guest ions and BGE ions. We also did diffusion constant measurement by NMR for amino acids and short peptides in 10%D2O-90% H2O at room temperature and applied to our modeling study by BMM.