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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
101

Multi-component protein films by layer-by-layer : assembly and electron transfer

Dronov, Roman January 2007 (has links)
Electron transfer phenomena in proteins represent one of the most common types of biochemical reactions. They play a central role in energy conversion pathways in living cells, and are crucial components in respiration and photosynthesis. These complex biochemical reaction cascades consist of a series of proteins and protein complexes that couple a charge transfer to different forms of chemical energy. The efficiency and sophisticated optimisation of signal transfer in these natural redox chains has inspired engineering of artificial architectures mimicking essential properties of their natural analogues. Implementation of direct electron transfer (DET) in protein assemblies was a breakthrough in bioelectronics, providing a simple and efficient way for coupling biological recognition events to a signal transducer. DET avoids the use of redox mediators, reducing potential interferences and side reactions, as well as being more compatible with in vivo conditions. However, only a few haem proteins, including the redox protein cytochrome c (cyt.c), and blue copper enzymes show efficient DET on different kinds of electrodes. Previous investigations with cyt.c have mainly focused on heterogeneous electron transfer of monolayers of this protein on gold. An important advance was the fabrication of cyt.c multilayers by electrostatic layer-by-layer self-assembly. The ease of fabrication, the stability, and the controllable permeability of polyelectrolyte multilayers have made them particularly attractive for electroanalytical applications. With cyt.c and sulfonated polyaniline it was for the first time possible that fully electro-active multilayers of the redox protein could be prepared. This approach was extended to design an analytical signal chain based on multilayers of cyt.c and xanthine oxidase (XOD). The system does not need an external mediator but relies on an in situ generation of a mediating radical and thus allows a signal transfer from hypoxanthine via the substrate converting enzyme and cyt.c to the electrode. Another kind of a signal chain is based on assembling proteins in complexes on electrodes in such a way that a direct protein-protein electron transfer becomes feasible. This design does not need a redox mediator in analogy to natural protein communication. For this purpose, cyt.c and the enzyme bilirubin oxidase (BOD, EC 1.3.3.5) are co-immobilized in a self-assembled polyelectrolyte multilayer on gold electrodes. Although these two proteins are not natural reaction partners, the protein architecture facilitates an electron transfer from the electrode via multiple protein layers to molecular oxygen resulting in a significant catalytic reduction current. Finally, we describe a novel strategy for multi-protein layer-by-layer self-assembly combining cyt.c with an enzyme sulfite oxidase (SOx) without use of any additional polymer. Electrostatic interactions between these two proteins with rather separated pI values during the assembly process from a low ionic strength buffer were found sufficient for the layer-by-layer deposition of the both biomolecules. It is anticipated that the concepts described in this work will stimulate further progress in multilayer design of even more complex biomimetic signal cascades taking advantage of direct communication between proteins. / Elektronentransferphänomene in Proteinen stellen den häufigsten Typ biochemischer Reaktionen dar. Sie spielen eine zentrale Rolle bei der Energieumwandlung in der Zelle und sind entscheidende Komponenten in der Atmung und Photosynthese. Diese komplexen Kaskaden biochemischer Reaktionen setzen sich aus einer Reihe von Proteinen und Proteinkomplexen zusammen, die den Energietransfer an verschiedene Formen chemischer Energie koppeln. Die große Effektivität und Selektivität des Signaltransfers in diesen natürlichen Redoxketten war Vorbild für die Entwicklung künstlicher Architekturen, die die wesentlichen Eigenschaften ihrer natürlichen Analoga nachahmen. Die Implementierung des direkten Elektronentransfers (DET) von Proteinen mit Elektroden war ein Durchbruch im Bereich der Bioelektronik. Sie lieferte einen einfachen und effizienten Weg für das Koppeln biologischer Erkennungsereignisse an einen Signalumwandler. Durch den DET können Redoxmediatoren vermieden und damit potentielle Grenzflächen und Nebenreaktionen reduziert werden. Ebenso wird damit die Kompatibilität für in vivo Bedingungen erhöht. Jedoch zeigen nur einige Hämproteine wie das Redoxprotein Cytochrom c (Cyt c) und blaue Kupferproteine einen effizienten DET auf verschiedenen Elektrodentypen. Bisherige Untersuchungen mit Cyt c konzentrierten sich hauptsächlich auf den heterogenen Elektronentransfer von Monoschichten dieses Proteins auf Gold. Ein wichtiger Fortschritt war die Herstellung von Cyt c Multischichten durch die elektrostatische Layer-by-Layer-Technik. Die einfache Herstellung, die Stabilität sowie die kontrollierbaren Permeationseigenschaften von Polyelektrolyt-Multischichten machte sie besonders attraktiv für elektroanalytische Anwendungen. So gelang es auch zum ersten Mal vollständig elektroaktive Multischichten aus Cyt c und Polyanilinsulfonsäure zu präparieren. Dieser Ansatz wurde hier erweitert, um eine analytische Signalkette auf der Basis von Multischichten aus Cyt c und Xanthinoxidase zu entwerfen. Das System bedarf keinen externen Mediator, es hängt jedoch von der in situ Generierung eines vermittelnden Radikals ab und erlaubt daher einen Signaltransfer von Hypoxanthin über ein substratumwandelndes Enzym und Cyt c zur Elektrode. Eine andere Art von Signalketten basiert auf der Assemblierung von Proteinen in Komplexen auf Elektroden in solcher Art und Weise, daß ein direkter Protein-Protein-Elektronentransfer möglich wird. Dieser Ansatz benötigt keinen Redoxmediator in Analogie zu Beispielen aus dem biologischen Signaltransfer. Zu diesem Zweck werden Cyt c und das Enzym Bilirubinoxidase mit einem selbst-assemblierenden Polyelektrolyten auf einer Goldelektrode koimmobilisiert. Obwohl diese zwei Proteine keine natürlichen Reaktionspartner sind, unterstützt die Protein-Architektur einen Elektronentransfer von der Elektrode über mehrere Proteinschichten zu molekularem Sauerstoff und ergibt einen signifikanten katalytischen Reduktionsstrom. Schließlich wird eine neue Strategie beschrieben für eine Selbstassemblierung von Proteinen ohne zusätzlichen Polyelektrolyten - am Beispiel der Kombination von Cyt c mit Sulfitoxidase. Es stellte sich heraus, daß die elektrostatische Wechselwirkung zwischen diesen zwei Proteinen mit ziemlich weit voneinander entfernt liegenden pI-Werten während des Assemblierungsprozesses durch einen Puffer mit geringer Ionenstärke ausreicht um die beiden Biomoleküle nach dem Layer-by-Layer-Prinzip auf einer Elektrode abzuscheiden. Es wird erwartet, daß das entwickelte Konzept von Multiprotein-Assemblaten auf Elektroden weitere Fortschritte bei dem Entwurf von Multischichten und sogar noch komplexeren biomimetischen Signalkaskaden anregen wird und dabei der Vorteil der direkten Kommunikation zwischen Proteinen genutzt wird.
102

Engineered human cytochrome c : investigation of superoxide and protein-protein interaction and application in bioelectronic systems

Wegerich, Franziska January 2010 (has links)
The aim of this thesis is the design, expression and purification of human cytochrome c mutants and their characterization with regard to electrochemical and structural properties as well as with respect to the reaction with the superoxide radical and the selected proteins sulfite oxidase from human and fungi bilirubin oxidase. All three interaction partners are studied here for the first time with human cyt c and with mutant forms of cyt c. A further aim is the incorporation of the different cyt c forms in two bioelectronic systems: an electrochemical superoxide biosensor with an enhanced sensitivity and a protein multilayer assembly with and without bilirubin oxidase on electrodes. The first part of the thesis is dedicated to the design, expression and characterization of the mutants. A focus is here the electrochemical characterization of the protein in solution and immobilized on electrodes. Further the reaction of these mutants with superoxide was investigated and the possible reaction mechanisms are discussed. In the second part of the work an amperometric superoxide biosensor with selected human cytochrome c mutants was constructed and the performance of the sensor electrodes was studied. The human wild-type and four of the five mutant electrodes could be applied successfully for the detection of the superoxide radical. In the third part of the thesis the reaction of horse heart cyt c, the human wild-type and seven human cyt c mutants with the two proteins sulfite oxidase and bilirubin oxidase was studied electrochemically and the influence of the mutations on the electron transfer reactions was discussed. Finally protein multilayer electrodes with different cyt form including the mutant forms G77K and N70K which exhibit different reaction rates towards BOD were investigated and BOD together with the wild-type and engineered cyt c was embedded in the multilayer assembly. The relevant electron transfer steps and the kinetic behavior of the multilayer electrodes are investigated since the functionality of electroactive multilayer assemblies with incorporated redox proteins is often limited by the electron transfer abilities of the proteins within the multilayer. The formation via the layer-by-layer technique and the kinetic behavior of the mono and bi-protein multilayer system are studied by SPR and cyclic voltammetry. In conclusion this thesis shows that protein engineering is a helpful instrument to study protein reactions as well as electron transfer mechanisms of complex bioelectronic systems (such as bi-protein multilayers). Furthermore, the possibility to design tailored recognition elements for the construction of biosensors with an improved performance is demonstrated. / Ziel dieser Arbeit ist es genetisch veränderte Formen von humanem Cytochrom c herzustellen und diese einerseits hinsichtlich der Reaktion mit dem Sauerstoff-Radikal Superoxid aber auch mit anderen Proteinen zu untersuchen. Zusätzlich sollen die verschiedenen Protein-Mutanten in neuartige bioelektronische Systeme eingebracht werden. Es wurden insgesamt 20 Cytochrome c Mutanten designt, rekombinant exprimiert und aufgereinigt. Es konnte in dieser Arbeit gezeigt werden, dass sich die Reaktion von Cytochrom c mit dem negativ geladenen Superoxid durch gezielte Mutationen, die zusätzliche positive Ladungen in das Molekül bringen, um bis zu 30 % erhöhen lässt. Es wurde aber auch deutlich, dass andere Eigenschaften des Proteins sowie dessen Struktur durch die Mutationen geändert werden können. Cytochrom c Mutanten mit einer erhöhten Reaktionsrate mit Superoxid konnten erfolgreich in einen Superoxid-Biosensor mit erhöhter Sensitivität eingebracht werden. Weiterhin wurde einige Mutanten hinsichtlich Ihrer Interaktion mit den zwei Enzymen Sulfitoxidase und Bilirubinoxidase untersucht. Hier konnten ebenfalls unterschiedliche Reaktivitäten festgestellt werden. Schließlich wurden ausgewählte Protein-Varianten mit und ohne den zuvor untersuchten Enzymen in ein Multischicht-Elektroden-System eingebettet und dessen kinetisches Verhalten untersucht. Es wurde gefunden, dass die Schnelligkeit mit der Cytochrom c mit sich selbst Elektronen austauschen kann, eine Limitierung der Größenordnung der katalytischen Ströme darstellt. Diese Selbstaustausschrate wurde durch die eingeführten Mutationen verändert. So verdeutlicht diese Arbeit, dass „Protein-Engineering“ ein gutes Hilfsmittel sein kann, um einerseits Proteinreaktionen und komplexe Elektronentransferreaktionen in Multischichten zu untersuchen, aber auch ein potentes Werkzeug darstellt mit dem zugeschnittene Biokomponenten für Sensoren mit erhöhter Leistungsfähigkeit generiert werden können.
103

Validation of docking performance in the context of a structural water molecule using model system

Wahlström, Rickard January 2009 (has links)
In silico ligand docking is a versatile and common technique when predicting ligands and inhibitors for protein binding sites. The various docking programmes aim to calculate binding energies and to predict interactions, thus identifying potential ligands.The currently available programmes lack satisfying means by which to account for structural water molecules which can either mediate protein-ligand contacts or be displaced upon ligand binding. The present project aims to generate data to facilitate the global work of developing scoring functions in docking programmes to account for structural water molecules contribution to ligand binding to fill the said void. This is done by validating the performance of docking using a simple model system (cytochrome C peroxidase (CCP) W191G) containing four well ordered, deeply buried structural water molecules which are known to either interact with a ligand or to be displaced upon ligand binding.Known ligands were docked into eight (crystallographically determined) receptor set-ups comprising the receptor and no, one or two of the water molecules. The performance was validated by comparison of the binding modes of the docked ligands and the crystal structures, comparison of docking scores of the ligands in the different set-ups, enrichment of the ligands from a database of decoys and finally by predicting new ligands from the decoy database. In addition a high resolution crystal structure of CCP W191G in complex with 3-aminopyridine (3AP) was determined in order to resolve ambiguities in the binding mode of this ligand.
104

Regulation of Apoptosis Following Mitochondrial Cytochrome c Release

Parrish, Amanda Baumann January 2010 (has links)
<p>Many pro-apoptotic signals trigger mitochondrial cytochrome c release, leading to caspase activation and ultimate cellular breakdown. Cell survival pathways, including the mitogen-activated protein kinase (MAPK) cascade, promote cell viability both by impeding mitochondrial cytochrome c release and by inhibiting subsequent activation of caspases. Cytosolic cytochrome c is directly responsible for initiating formation of the caspase-activating apoptosome, which, in many cell types, plays a crucial role in the apoptotic process. Given the important role of cytochrome c in dismantling the dying cell, we wanted to investigate the process of cytochrome c-induced apoptosis with the goal of understanding how this mechanism is altered in certain malignant conditions. </p> <p> First, we examined cytochrome c-induced caspase activation in normal and tumorigenic mammary epithelial cells. Although most tumor types have developed mechanisms for evading apoptosis, we surprisingly discovered that breast cancer cells were hypersensitive to cytochrome c when compared with their normal counterpart. Specifically, breast cancer cells show increased binding of caspase-9 to the Apaf-1 caspase recruitment domain. This altered apoptosome formation is mediated by overexpression of the protein PHAPI in the malignant mammary epithelial cells. Immunoblot analysis demonstrated that protein levels of PHAPI are also elevated in human breast tumors. These results suggest a novel paradigm where breast cancer cells are refractory to cytochrome c release in response to certain stimuli, but they are quite sensitive to apoptosis downstream of the mitochondria. </p> <p> Secondly, we describe a mechanism for the inhibition of cytochrome c-induced caspase activation by MAPK signaling, identifying a novel mode of apoptotic regulation exerted through Apaf-1 phosphorylation by the 90-kDa ribosomal S6 kinase (Rsk). This Apaf-1 phosphorylation results in impaired apoptosome formation, thereby inhibiting caspase activation. The Rsk effect on Apaf-1 is antagonized by protein phosphatase 1 (PP1), which promotes Apaf-1 dephosphorylation. High endogenous levels of Rsk in PC3 prostate cancer cells leads to Apaf-1 phosphorylation and renders them relatively insensitive to cytochrome c, suggesting a role for Rsk signaling in the apoptotic resistance of certain cancers. These results identify a novel locus of apoptosomal regulation wherein MAPK signaling promotes direct Rsk-catalyzed phosphorylation of Apaf-1, resulting in decreased cellular responsiveness to cytochrome c. Collectively, this work provides insight into novel mechanisms of regulation for cytochrome c-induced apoptosis.</p> / Dissertation
105

Cytochrome c maturation and redox homeostasis in uranium-reducing bacterium Shewanella putrefaciens

Dale, Jason Robert 11 October 2007 (has links)
Microbial metal reduction contributes to biogeochemical cycling, and reductive precipitation provides the basis for bioremediation strategies designed to immobilize radionuclide contaminants present in the subsurface. Facultatively anaerobic ×-proteobacteria of the genus Shewanella are present in many aquatic and terrestrial environments and are capable of respiration on a wide range of compounds as terminal electron acceptor including transition metals, uranium and transuranics. S. putrefaciens is readily cultivated in the laboratory and a genetic system was recently developed to study U(VI) reduction in this organism. U(VI) reduction-deficient S. putrefaciens point mutant Urr14 (hereafter referred to as CCMB1) was found to retain the ability to respire several alternate electron acceptors. In the present study, CCMB1 was tested on a suite of electron acceptors and found to retain growth on electron acceptors with high reduction potential (E¡¬0) [O2, Fe(III)-citrate, Mn(IV), Mn(III)-pyrophosphate, NO3-] but was impaired for anaerobic growth on electron acceptors with low E¡¬0 [NO2-, U(VI), dimethyl sulfoxide, trimethylamine N-oxide, fumarate, ×-FeOOH, SO32-, S2O32-]. Genetic complementation and sequencing analysis revealed that CCMB1 contained a point mutation (H108Y) in a CcmB homolog, an ABC transporter permease subunit required for c-type cytochrome maturation in E. coli. The periplasmic space of CCMB1 contained low levels of cytochrome c and elevated levels of free thiol equivalents (-SH), an indication that redox homeostasis was disrupted. Anaerobic growth ability, but not cytochrome c maturation activity, was restored to CCMB1 by adding exogenous disulfide bond-containing compounds (e.g., cystine) to the growth medium. To test the possibility that CcmB transports heme from the cytoplasm to the periplasm in S. putrefaciens, H108 was replaced with alanine, leucine, methionine and lysine residues via site-directed mutagenesis. Anaerobic growth, cytochrome c biosynthesis or redox homeostasis was disrupted in each of the site-directed mutants except H108M. The results of this study demonstrate, for the first time, that S. putrefaciens requires CcmB to produce c-type cytochromes under U(VI)-reducing conditions and maintain redox homeostasis during growth on electron acceptors with low E¡¬0. The present study is the first to examine CcmB activity during growth on electron acceptors with widely-ranging E¡¬0, and the results suggest that cytochrome c or free heme maintains periplasmic redox poise during growth on electron acceptors with E¡¬0 < 0.36V such as in the subsurface engineered for rapid U(VI) reduction or anoxic environments dominated by sulfate-reducing bacteria. A mechanism for CcmB heme translocation across the S. putrefaciens cytoplasmic membrane via heme coordination by H108 is proposed.
106

Trigonal based copper sites - a natural situation?

Coyle, Joanne Lyssa. January 1999 (has links)
Thesis (Ph. D.)--Open University. BLDSC no. DXN033657.
107

Nuclear magnetic resonance studies of modified eukaryotic cytochrome C

Boswell, Andrew Philip January 1981 (has links)
The central theme of this thesis is a study of the structural changes accompanying chemical modification and denaturation of eukaryotic cytochrome c as characterised by <sup>1</sup>II nuclear magnetic resonance (n.m.r.) spectroscopy. First, however, it was necessary to obtain and confirm assignments for individual resonances; this was achieved by a novel method of cross assignment between ferricytochrome c and ferrocytochrome c and by double resonance techniques. A variety of perturbations were caused to native cytochromes c, which ranged in degree from the elevation of temperature for ferrocytochrome c to the complete denaturation of the protein with urea or methanol. Modification at single sites both on the surface (e.g. Met 65, Tyr 74) and in the core ( e.g. Tyr 67) of the molecule were found to cause only small local effects to the structure, although the dynamic features of the molecules were altered. One single site modification, the breaking of the iron - sulphur cross linking bond, caused considerable disruption to one side of the molecule, although hydrophobic domains in the other side were preserved; this state of the molecule is analogous to the penultimate state in the refolding pathway. Modification of all the charged lysine residues caused small changes to the surface structure of the molecule, though the complete reversal of the charges in maleyl cytochrome c produced a species which unfolded reversibly from a native configuration with the increase of temperature. The unfolding of the protein is virtually identical with both methanol and urea, but the pathways are shown to differ for the oxidised and reduced proteins.
108

Connecting genotype to phenotype: drosophila simulans mitochondria as a model.

Melvin, Richard G, Biotechnology & Biomolecular Science, UNSW January 2008 (has links)
The influence of genotype variation on phenotype has been a longstanding question in biology but it is now one of the greatest challenges of the post-genomics era. Discovering the link between common gene variants that affect phenotypes within and between populations is likely to provide insight into the molecular physiology of phenotypic traits and the mechanisms by which they evolve. The overall goal of this thesis is to link naturally occurring genotypic variation with the organism??s phenotype. The specific goal of this thesis is to connect natural variation in the mitochondrial genotype with the organismal phenotype using the model organism Drosophila simulans. Mitochondria are intracellular organelles found in most eukaryotes and produce over 90% of the energy needed by cells. Determining the connection of mitochondrial genotype to whole organism phenotype is of particular interest because of the broad use of mitochondrial (mt) DNA as a molecular marker in evolutionary biology and population genetics, the organelle??s central role in cellular energy production, the potential for the mitochondria to influence organismal distribution particularly in the face of climate change and in human degenerative disease. I use the model organism D. simulans because it has high genetic variability, can be easily sampled from the wild and manipulated in the lab, and the energy producing reactions that take place in its mitochondria are highly conserved among metazoa. I studied naturally occurring mutations to understand the influence of these changes in natural populations. The four studies in this thesis have employed a Genotype-Biochemistry-Phenotype (GBP) model to link naturally occurring variation in the mitochondrial genotype with organism phenotype in D. simulans mitochondria. Three major conclusions can be drawn from the thesis that follow the genotype to biochemistry to phenotype model. Firstly, a subset of the mutations in genes that comprise the mitochondrial genotype is functionally significant. Secondly, the biochemical efficiency of OXPHOS is regulated by mitochondrial homeostasis. Thirdly, key organismal life history traits influenced by the mitochondrial genotype and this is mediated through the biochemistry of OXPHOS.
109

Biochemical pathways in apoptosis

Nijhawan, Deepak. January 2003 (has links) (PDF)
Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 142-166.
110

The effect of photobiomodulation on cerebral blood flow

Iennaco, Maria 15 May 2021 (has links)
Photobiomodulation (PBM) therapy involves the irradiation of tissues with red to near- infrared (NIR) light at low power densities to stimulate healing, reduce inflammation, and promote optimal cellular functioning. These beneficial effects are thought to occur due to the absorption of NIR light by the chromophore, and terminal enzyme in the mitochondrial electron transport chain, cytochrome c oxidase (CCO). It is hypothesized that increased oxygen consumption due to the photostimulation of CCO, as well as photodissociation of the vasodilator nitric oxide from its binding site in the binuclear center of CCO, contribute to improved tissue healing by increasing blood flow to the irradiated region. Applied to the brain, PBM therapy has the potential to improve many neurological injuries and diseases for which reduced cerebral blood flow (CBF) is a common finding. This study examines whether cortical irradiation with NIR light has an impact on CBF in mice. Mice were administered brain PBM via 810nm, 190mW LED for 18 minutes. CBF was measured before, during, and after treatment using Doppler Optical Coherence Tomography. Results from 16 trials demonstrated a significant, 40% increase in CBF during NIR treatment. This CBF increase was not observed during control trials. Additionally, irradiation with a 730nm LED did not increase CBF, indicating that the blood flow increase observed with 810nm irradiation was not simply due to tissue heating. These findings provide support for the value of PBM therapy for the treatment of neurological conditions. / 2023-05-14T00:00:00Z

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