Tesi sul tema "Proteins Oxidation"
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1
Osborn, Anna. "Measurements of Human Plasma Oxidation". Thesis, University of Canterbury. Biological Sciences, 2006. http://hdl.handle.net/10092/1426.
Testo completoAbstract (sommario):
The oxidation of lipids and antioxidants has been extensively studied in human plasma but little attention has been given to how plasma proteins are oxidised. Proteins make up the majority of biomolecules in cells and plasma and therefore are the most likely reactants with oxidants and free radicals. Previous studies in the laboratory had shown that peroxyl radicals generated by the thermolytic decay of 2-azobis (2-amdinopropane) dihydrochloride (AAPH) generated significant amounts of protein hydroperoxides, but only after a six hour lag period. In this study the existence of the six hour lag period was confirmed and shown by dialysis of the plasma to be due to the presence of low molecular weight antioxidants. The addition of both uric acid and ascorbic acid to the dialysed plasma restored the lag phase suggesting that in vivo these antioxidants act to prevent protein hydroperoxide formation. Lipid oxidation was also observed in the plasma but only after a two hour lag phase. This was the first time lipid oxidation has been observed in the absence of protein oxidation. The lipid lag phase was also abolished by dialysis of the plasma and restored by the addition of ascorbic acid and uric acid. The kinetics of tocopherol loss suggests that the tocopherol radicals act to inhibit lipid oxidation by transferring the electrons to the water-soluble ascorbate. The loss of ascorbate appears to cause the formation of a tocopherol radical mediate the lipid peroxidation process. Overall the data shows ascorbic acid scavenging the peroxyl radicals while uric acid acts to reduce the overall AAPH generated radical flux. In a separate investigation, the production of protein-bound DOPA (PB-DOPA) on albumin during X-ray radiolysis and copper mediate Fenton oxidation was investigated using a fluorescence based derivatisation method (ED-DOPA), which was compared with the more specific acid hydrolysis and HPLC analysis method. The ED-DOPA method consistently gave a much higher reading that the HPLC based methods, suggesting that the ED-DOPA method was measuring DOPA plus DOPA oxidation products. This was confirmed by oxidising X-ray radiolysis generated PB-DOPA with Cu++ to cause DOPA oxidation. The Cu++ treatment drastically increased the level of signal given by the ED-DOPA assay while HPLC analysis showed all the DOPA had been oxidised.
2
Du, Aiguo. "Prediction of oxidation states of cysteines and disulphide bridges in proteins". unrestricted, 2007. http://etd.gsu.edu/theses/available/etd-11272007-024411/.
Testo completoAbstract (sommario):
Thesis (Ph. D.)--Georgia State University, 2007.Title from file title page. Y. Pan, committee chair; G. Qin, A. Bourgeois, A. Zelikovski, committee members. Electronic text (124 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed June 3, 2008. Includes bibliographical references (p. 111-124).
3
Beilen, Jan Berthold van. "Alkane oxidation by Pseudomonas oleovorans: genes and proteins". [S.l. : [Groningen : s.n.] ; University Library Groningen] [Host], 1994. http://irs.ub.rug.nl/ppn/292892500.
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4
Fredriksson, Åsa. "On the role of protein oxidation and heat shock proteins in senescence and fitness /". Göteborg : Göteborg University, 2006. http://www.loc.gov/catdir/toc/fy0708/2006421399.html.
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5
Shutova, Tatiana. "Photosynthetic water oxidation : the function of two extrinsic proteins". Doctoral thesis, Umeå : Department of Plant Physiology, Umeå Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1476.
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6
Kapavarapu, Susmita. "Extracellular expression, oxidation and purification of hen egg white lysozyme double mutant (H15S+N77H) /". Connect to resource online, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1197658857.
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7
Wood, Geoffrey Paul Farra. "Theoretical Investigations of Radical-Mediated Protein Oxidation". Thesis, The University of Sydney, 2006. http://hdl.handle.net/2123/1413.
Testo completoAbstract (sommario):
This thesis primarily details the application of high-level ab initio quantum chemistry techniques in order to understand aspects of free-radical mediated protein oxidation. Traditionally, product analysis and electron paramagnetic resonance (EPR) spectroscopy are the primary means for elucidating the chemistry of protein oxidation. However, in experiments involving relatively small proteins reacting with a controlled radical-flux, a vast array of compounds can be produced, which are often difficult to analyse. Quantum chemical techniques on the other hand, can calculate the properties of any particular species directly, without suffering from the problems associated with experiment, such as side-reactions and chain processes. The results presented in this thesis are aimed at elucidating mechanistic details of protein oxidation, which might otherwise be difficult to probe experimentally. Chapter 1 gives an overview of the free-radical hypothesis of disease and ageing. Protein-derived radicals can undergo a variety of reactions, with the particular reaction that occurs depending on numerous aspects. Many types of reactions have been identified through radiolysis experiments of amino acids, and these are detailed in this chapter. In addition, the key reactive species are characterized and their different chemistries explained. Chapter 2 details the theoretical tools used throughout this thesis. Species with unpaired electrons (radicals) present unique problems for quantum chemistry to handle, thus an appropriate choice of theoretical technique is needed. The approach taken in this thesis is to use high-level compound methods, many of which have been directly formulated to give improved results for radical species, to provide benchmark quality results by which other less demanding techniques can be assessed. During the course of this study, it became apparent there was a void in the armoury of tools that could be used for the theoretical chemistry calculations. Chapter 3 details the formulation of a new tool in an attempt to fill this gap. Historically, the formulation of this new procedure came after much of the work in this thesis had been carried out. Thus, for the study of many of the reactions of this thesis the new method has not been used. However, it is most appropriate to place its formulation after summarizing the current status of techniques in common use today. Chapters 4 and 5 detail computations carried out on models of peptides containing backbone carbon- and nitrogen-centered radicals. A number of different theoretical techniques are used in these chapters, ranging from the highly accurate and computationally intensive to the less reliable and less demanding. The highly accurate techniques are used to gauge the accuracy of the other less demanding theoretical techniques so that the latter can be used with confidence in larger systems. Not only is the choice of theoretical technique important but also the judicious choice of model is essential. With this in mind, models are incrementally built until convergence of the particular property of interest is reached. Chapters 6 and 7 detail the calculations of β-scission reactions of alkoxyl radicals, which are a particular class of reaction known to occur on peptide backbones. Alkoxyl radicals are particularly difficult for theory to describe correctly. Therefore, Chapter 6 extensively assesses and then identifies the theoretical methods needed to portray them. Chapter 7 uses the techniques identified in the previous chapter in order to predict how the preference for a particular type of β-scission reaction changes.
8
Wood, Geoffrey Paul Farra. "Theoretical Investigations of Radical-Mediated Protein Oxidation". University of Sydney, 2006. http://hdl.handle.net/2123/1413.
Testo completoAbstract (sommario):
Doctor of Philosophy (PhD)This thesis primarily details the application of high-level ab initio quantum chemistry techniques in order to understand aspects of free-radical mediated protein oxidation. Traditionally, product analysis and electron paramagnetic resonance (EPR) spectroscopy are the primary means for elucidating the chemistry of protein oxidation. However, in experiments involving relatively small proteins reacting with a controlled radical-flux, a vast array of compounds can be produced, which are often difficult to analyse. Quantum chemical techniques on the other hand, can calculate the properties of any particular species directly, without suffering from the problems associated with experiment, such as side-reactions and chain processes. The results presented in this thesis are aimed at elucidating mechanistic details of protein oxidation, which might otherwise be difficult to probe experimentally. Chapter 1 gives an overview of the free-radical hypothesis of disease and ageing. Protein-derived radicals can undergo a variety of reactions, with the particular reaction that occurs depending on numerous aspects. Many types of reactions have been identified through radiolysis experiments of amino acids, and these are detailed in this chapter. In addition, the key reactive species are characterized and their different chemistries explained. Chapter 2 details the theoretical tools used throughout this thesis. Species with unpaired electrons (radicals) present unique problems for quantum chemistry to handle, thus an appropriate choice of theoretical technique is needed. The approach taken in this thesis is to use high-level compound methods, many of which have been directly formulated to give improved results for radical species, to provide benchmark quality results by which other less demanding techniques can be assessed. During the course of this study, it became apparent there was a void in the armoury of tools that could be used for the theoretical chemistry calculations. Chapter 3 details the formulation of a new tool in an attempt to fill this gap. Historically, the formulation of this new procedure came after much of the work in this thesis had been carried out. Thus, for the study of many of the reactions of this thesis the new method has not been used. However, it is most appropriate to place its formulation after summarizing the current status of techniques in common use today. Chapters 4 and 5 detail computations carried out on models of peptides containing backbone carbon- and nitrogen-centered radicals. A number of different theoretical techniques are used in these chapters, ranging from the highly accurate and computationally intensive to the less reliable and less demanding. The highly accurate techniques are used to gauge the accuracy of the other less demanding theoretical techniques so that the latter can be used with confidence in larger systems. Not only is the choice of theoretical technique important but also the judicious choice of model is essential. With this in mind, models are incrementally built until convergence of the particular property of interest is reached. Chapters 6 and 7 detail the calculations of β-scission reactions of alkoxyl radicals, which are a particular class of reaction known to occur on peptide backbones. Alkoxyl radicals are particularly difficult for theory to describe correctly. Therefore, Chapter 6 extensively assesses and then identifies the theoretical methods needed to portray them. Chapter 7 uses the techniques identified in the previous chapter in order to predict how the preference for a particular type of β-scission reaction changes.
9
Yi, Dong-Hui Chemistry Faculty of Science UNSW. "The Study of Biomarkers of Protein Oxidative Damage and Aging by Mass Spectrometry". Awarded by:University of New South Wales. School of Chemistry, 1999. http://handle.unsw.edu.au/1959.4/17636.
Testo completoAbstract (sommario):
The physiologically important free radicals, nitrogen monoxide and superoxide, can combine to form the reactive intermediate peroxynitrite. Peroxynitrite can react with proteins and their constituent amino acids, such as tyrosine, resulting in protein peroxidation, oxidation and nitration. The nitration of proteins, assessed by the analysis of 3-nitrotyrosine, is a proposed index of pathophysiological activity of peroxynitrite. The aim of the work was to investigate the reaction products between peroxynitrite and protein, develop an assay for 3-nitrotyrosine and measure its levels in biological samples. To study the amino acid products arising from the reaction of peroxynitrite and protein, both liquid chromatography (LC) and gas chromatography (GC) combined with mass spectrometry (MS) were adopted. Approaches to 3-nitrotyrosine assay development were first, to take advantage of the intrinsic sensitivity of electron capture negative ionization GC-MS. Secondly, to avoid possible artefactual problems associated with the derivatisation step in GC-MS, an assay for 3-nitrotyrosine based on combined LC-MS-MS was developed. When a selection of peptides was exposed to peroxynitrite under physiological conditions in vitro, the hydrolysis products showed that 3-nitrotyrosine was the major product. Detectable minor products were 3,5-dinitrotyrosine and DOPA. The GC-MS assay was found to be fraught with difficulty due to artefactual formation of 3-nitrotyrosine. In order to quantify and correct for artefact formation, this complication was approached by incorporating a second isotopomer. This method, however, was confounded by large errors that reduced the overall sensitivity. Either negative or zero levels of endogenous 3-nitrotyrosine were found in tested samples after correction for artefact formation. The LC-MS-MS assay was then used to analyse 3-nitrotyrosine levels in a range of biological samples, including human plasma from healthy volunteers, synovial fluid samples from arthritis patients and tissue extracts from a mouse model of amyotropic lateral sclerosis. In contrast to published data, 3-nitrotyrosine levels were found to be below the limit of detection (1 pg/????L, 10 pg o/c) for all samples - a result somewhat consistent with the negative GC-MS data. It is suggested that the high 3-nitrotyrosine levels previously reported in the literature might reflect artefactual generation of 3-nitrotyrosine and that other approaches to assessing pathophysiological nitration should be sought in future.
10
Dales, Simon Leslie. "The structure, function and biosynthesis of proteins involved in methanol oxidation". Thesis, University of Southampton, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296270.
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11
Wright, Adam. "Investigations of singlet oxygen-mediated amino acid, peptide and protein oxidation". Thesis, The University of Sydney, 2002. https://hdl.handle.net/2123/27830.
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12
Beard, Collen Alana. "Rubredoxin cobalt substitution and crystallization attempts". Thesis, Georgia Institute of Technology, 1989. http://hdl.handle.net/1853/29863.
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13
Sharar, Mona. "Molecular and Elemental Mass Spectrometric Approaches for Monitoring Oxidation Processes in Proteins". Doctoral thesis, Humboldt-Universität zu Berlin, 2017. http://dx.doi.org/10.18452/18520.
Testo completoAbstract (sommario):
Die oxidative Transformation der Thiol-Gruppe des Cysteins in verschiedene andere funktionelle Gruppen wird als sehr wichtige posttranslationale Modifikation (PTM) angesehen. Cysteinsulfensäure (SA) ist eine Zwischenstufe der Thiol-Oxidation: Sie kann entweder mit freien Thiolen reagieren, um Disulfide zu bilden oder durch reaktive Sauerstoffspezies (reactiveoxygenspecies, ROS) weiter oxidiert werden.
Jede Störung des zellulären Redox-Haushalts wird mit altersbedingten Erkrankungen , daher stellt die Überwachung des SA-Spiegels einen vielversprechenden Wegdar, den Status dieses Redox-Haushalts festzustellen. Da bereits kleinste Änderungen der Proteinmengen und PTMs tiefe Einblicke in den Zustand des biologischen Systems liefern können, ist eine quantitative Bestimmung von großer Bedeutung.Technologische Fortschritte im Bereich der Trennungsmethoden und Massenspektrometrie (MS) erlaubten die Entwicklung umfassender Möglichkeiten in der Protein-Analytik.
In dieser Arbeit wurde eine neue, hochsensitive und selektive Methode zur Detektion von SA entwickelt. Dafür wurde ein Alkin-β-Ketoester (KE) an einen Lanthanid-haltigen (Ln) Chelatkomplex. Zum Nachweis des Funktionsprinzips wurden, mittels H2O2, Sulfensäuren in verschiedenen Peptidsequenzen erzeugt, um die in biologischen Systemen durch ROS hervorgerufenen Oxidationen nachzustellen. Diese Sulfensäuren wurden anschließend durch den Ln-DOTA-KE-Komplex gebunden. Die Bildung dieser SA-Ln-DOTA-KE-Einheit wurde mittels (Elektronenspray-Ionisation/ ESI-MS) und (induktiv gekoppeltem Plasma/ICP-MS) nachverfolgt.
Die entwickelte Methode wurde weiterhin auf die Bestimmung von SA-Bildung in humanem Serum angewandt, humanes Serumalbumin wurde angereichert via Affinitätschromatographie. ICP-MS diente der Bestimmung der SA-Ln-DOTA-KE-Einheit, durch Kombination mit einer Isotopenverdünnungsanalyse (IDA) wurde eine absolute Quantifizierung durchgeführt. Die Ergebnisse zeigen oxidative Schäden bis zu 40 % des vorhandenen Albumins.Oxidative transformation of cysteine thiol group into different functional groups is considered a significant posttranslational modification (PTM) of great importance. Cysteine sulfenic acid (SA) is the transient state for thiol group oxidation; it can react with free thiols to form disulfide bonds or can be further oxidized with reactive oxygen species (ROS) to form sulfinic and sulfonic acids. As any disturbance in the cellular reduction-oxidation (redox) balance is correlated to age-related diseases, the detection of SA transient state formed a sensor for such redox-mediated events. Whereas only any small change in the quantity of proteins, as well as the formed PTMs, can provide deeper insights into the status of the biological system, quantitative analysis should be carried out to reveal the status of the system. On the other hand, the technological advances, in particular the separation techniques and mass spectrometry (MS), allowed the development of several approaches for the comprehensive assessment of proteome analysis. Herein, we provide a new strategy for the highly sensitive and specific detection of SA using alkyne β-ketoester (KE) previously linked to a lanthanide (Ln)-containing chelator (Ln-DOTA. SA was generated by hydrogen peroxide (H2O2) in different peptide sequences by ROS and was detected by the prepared compound Ln-DOTA-KE. Molecular mass spectrometry (electrospray/ ESI-MS) and (Inductively coupled plasma mass spectrometry /ICP-MS) have been used to monitor the formation of SA linked to Ln-DOTA-KE. The developed strategy has been further applied to the determination of SA-induced formation in human serum by using affinity chromatography for purification of albumin followed by ICP-MS to monitor the formed SA linked to Ln-DOTA-KE in combination with isotope dilution analysis (IDA) for the absolute quantification. Quantitative results showed levels of oxidative damage regarding SA formation in human serum up to 40% of the albumin present.
14
Sauvé, Véronique. "Biochemical and structural characterisation of proteins involved in the sulfur oxidation (sox) system". Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670036.
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15
Liu, Pei. "Development of redox proteomics methods and the identification of redox-sensitive proteins in arabidopsis". HKBU Institutional Repository, 2015. https://repository.hkbu.edu.hk/etd_oa/184.
Testo completoAbstract (sommario):
Cellular redox homeostasis mediates a wide range of physiological and developmental processes. Various stresses trigger over-production of reactive oxygen/nitrogen species which leads to oxidative modifications of redox-sensitive proteins. Identification and characterization of redox-sensitive proteins are important steps toward understanding molecular mechanisms of stress responses. In the study, a high-throughput quantitative proteomic approach termed OxiTRAQ was developed for identifying proteins whose thiols undergo reversible oxidative modifications in Arabidopsis cells subjected to oxidative stress. In this approach, a biotinylated thiol-reactive reagent is used for differential labeling of reduced and oxidized thiols, and the biotin-tagged peptides are affinity-purified and labeled with iTRAQ reagents for quantitation. This approach allows identification of the specific redox-regulated cysteine residues in proteins and offers an effective tool for elucidation of redox proteomes. With this approach, we identified 195 cysteine-containing peptides from 179 proteins whose thiols underwent oxidative modifications in Arabidopsis cells following the treatment with hydrogen peroxide. A majority of those redox-sensitive proteins, including several transcription factors, were not identified by previous redox proteomics studies. Besides, this method was also used to identify proteins that underwent oxidative modifications in Arabidopsis cells subjected to 15 minute treatment of salicylate (a key signaling molecule in the plant defense pathway) or flg22 (a peptide from bacterial flagellin that induces pathogen associated molecular patterns-triggered immunity). In total, 127 peptides from 111 distinct proteins were identified as salicylate- and/or flg22-responsive redox-sensitive proteins. Among the identified redox sensitive proteins are many regulatory proteins including those involved in chromatin remodeling, transcription, nucleocytoplasmic shutting, and posttranslational regulation. Furthermore, in vivo 15N metabolic labeling method combined with a cysteine-containing peptide enrichment technique was applied to identify proteins that undergo oxidative modifications in plants in response to pathogen attack. The identification of redox-sensitive proteins provides a foundation from which further study can be conducted toward understanding the biological significance of redox signaling in plant stress response.
16
Karimi, Maryam. "Characterization of Disulfide Bond Oxidation in Peptides and Proteins by Myeloperoxidase-Derived Oxidants". Thesis, The University of Sydney, 2017. https://hdl.handle.net/2123/22302.
Testo completoAbstract (sommario):
Activated neutrophils play an important role in the immune response to invading pathogens and bacteria. These cells generate large fluxes of reactive oxidants via an oxidative burst to kill pathogens. Inappropriate or misdirected stimulation of this mechanism, such as occurs during inflammation, can damage the host tissue. This inflammation-induced damage has been linked to the development and/or progression of multiple diseases such as atherosclerosis, arthritis, asthma and some cancers. One of the major sources of these reactive oxidants at sites of inflammation is the heme enzyme myeloperoxidase (MPO) that is released by activated leukocytes. Hydrogen peroxide (H2O2) is generated from multiple sources, including the NADPH oxidase system of activated leukocytes. MPO uses H2O2 in the presence of halide (Cl ͞ ,Br ͞ )and pseudo-halide (SCN ͞ ) to generate highly reactive oxidants hypohalous acids (HOX) including hypochlorous acid (HOCl, the major component of household bleach), hypothiocyanous acid (HOSCN) and at lower quantity hypobromous acid (HOBr). HOCl is a strong oxidant with antibacterial properties but excessive or misplaced production of HOCl have been reported to be involved in several diseases. HOCl has many biological targets, but proteins are major targets due to their abundance and their high reactivity. Previous studies have shown that the most reactive protein residues with hypohalous acids are the sulfur- and selenium-containing amino acids cysteine (Cys), methionine (Met) and selenocysteine (Sec). Lysine (Lys) side-chains, disulfide bonds and the aromatic side-chains of tryptophan (Trp) and histidine (His) also prone to damage. The order of second-order rate constant for the various side-chains present in proteins and peptides was determined to be: Cys > Met >> Cystine ~ His ~ α-amino > Trp >Lys >> Tyr ~ Arg > Gln ~ Asn. These data show that disulfides (e.g. cystine) also react, but the values of k2 and the effect of structure and environment on these values have not been examined in detail. Disulfides are among the most important bonds in proteins and peptides, which are conventionally known as elements to stabilize proteins structure but many studies have categorized disulfides in two subproteomes. One group provides structural stabilizing and a second redox-active group which provides catalytic activity. Insight into disulfide oxidation e.g. the kinetics, specificity and mechanism of this oxidation by hypohalous acids is of pivotal importance, for example in understanding protein stability. The studies reported in Chapter 3 show that the second-order rate constant for the reaction of a variety of model disulfides with HOCl determined using stopped-flow instrumentation and competition kinetic methodology are highly variable. The k2 values determined for the reaction of HOCl with acyclic (linear) disulfides vary from 6×103 to 5×105 M– s–1. Further studies confirmed this high reactivity but showed that for cyclic disulfides the variation is ~ 10,000-fold. The second-order rate constants for reaction of HOCl with 5-membered ring species (e.g., lipoic acid, lipoamide) were determined as ~ 1×108 M–1 s–1. These data suggest that a conformation of a disulfide bond has a dramatic effect on its oxidation kinetics. The higher rate constant of the 5-membered ring species compared with 6-membered rings and acyclic species is particularly notable. α-Lipoic acid and α-lipoamide may, therefore, be significant targets for these oxidants. These suggest that if proteins contain strained disulfide bonds then these may be more susceptible to oxidation than thermodynamically stable disulfides. The high rate constant of these compounds makes them potential protective agents against damage to proteins with which they are associated. The kinetic values of two protein model compound with several disulfide bonds (insulin and α-lactalbumin) are significantly higher than for any of the model compounds with the exception of the 5-membered ring structures. The wide spectrum of products was reported to be generated from Cys reaction with hypohalous acids including disulfides (RSSR'); sulfenic (RSOH), sulfinic (RSO2H) or sulfonic (RSO3H) acids, sulfinamides (RS(O)NR'), sulfonamides (RS(O)2NR') and RS-NO species. In contrast to the abundance of data on Cys and Met, little is known about the oxidation products of cystine residues and particularly those in proteins. The studies in Chapter 4 assessed and identified disulfide bond oxidation products and kinetics of a fluorescently tagged model peptide (Naph-SS) on reaction with HOCl. Oxidation of Naph-SS with HOCl demonstrated the formation of disulfide-S-oxides, sulfenic, sulfinic and sulfonic acids as well as peptide cleavage between Trp and Arg. The reaction of the oxidized products with thiols (GSH, N-Ac-Cys) did not result in a repair but generated mixed disulfide products with Naph-SS and the cleaved peptide which may exacerbate the damage. This study was subsequently extended to two other compounds; a synthetic dimer of the tripeptide (GSH) with a disulfide bond between the Cys residues and the α-amino groups blocked with N-acetyl groups ((N-Ac)2GSSG) and the biologically relevant protein insulin. The primary oxidation products detected on reaction with HOCl and HOBr were the RS(O)SR and, the disulfide-S-dioxide, which have been characterized by mass spectrometry. Higher oxidation state products including sulfinic and sulfonic acids were characterized by LC-MS/MS. The studies reported in Chapter 6 investigated whether there is a possibility to visualize disulfide bond oxidation products by Raman spectroscopy. In a wide range of inflammatory diseases, MPO-derived oxidants are implicated. 3-chlorotyrosine is commonly used as a marker of HOCl formation which is a relatively minor product, but very specific. Analysis of HOCl-treated model peptides containing sulfur and amine groups by Raman spectroscopy showed dose-dependent changes in the wavelength and intensity of characteristic Raman bands assigned to disulfides (500–550 cm-1), and the formation of a new Raman absorption at ~ 2255 cm-1 which has been tentatively assigned to bond stretching of nitrile groups. The latter peak was detected in a dose-dependent manner on the HOCl treatment of Lys-containing protein and peptide models, N-acetyl-histidine and oxidized glutathione (GSSG). Thus HOCl-mediated oxidation of proteins and peptides modifies protein and peptide side-chains in a manner that can be readily detected by Raman spectroscopy in isolated systems. The absorption band attributed to C≡N stretching at ~ 2255 cm-1 is well removed from other features in the Raman spectra, making it a potential marker of HOCl-mediated protein oxidation, chloramine formation and decomposition. Overall the studies presented in this thesis are consistent with selective damage to the specific disulfides, such as in proteins, with this resulting in structural perturbation. Oxidation of protein disulfides by HOCl may alter the stability of protein structures and enhance unfolding by incorporation of oxygen atoms and, disulfide or backbone cleavage. This may be of particular relevance for proteins such as integrins and extracellular proteins, where cystine residues are abundant, and Cys and Met levels low.
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Little, Laura Grace. "Response of a NEIL1 deficient murine epithelial cell line to chromate". CONNECT TO THIS TITLE ONLINE, 2008. http://etd.lib.umt.edu/theses/available/etd-04172008-090537/.
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Liu, Chia-chi. "Oxidation of ascorbate by protein radicals in simple systems and in cells". Phd thesis, Australia : Macquarie University, 2007. http://hdl.handle.net/1959.14/16746.
Testo completoAbstract (sommario):
Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007.Bibliography: leaves 295-322.
Generation of peroxide groups in proteins exposed to a wide variety of reactive oxygen species (ROS) requires an initial formation of protein carbon-centred or peroxyl free radicals, which can be reduced to hydroperoxides. Both protein radicals and protein hydroperoxides are capable of oxidizing important biomolecules and thus initiate biological damage. In this study, we investigated the inhibition of protein hydroperoxide formation by ascorbate and GSH in gamma-irradiated HL-60 cells.--We used HL-60 cells as a model for general protection of living organisms by ascorbate (Asc) and glutathione (GSH) from the deleterious effects of protein hydroperoxides generated by radicals produced by gamma radiation. Measurement by HPLC indicated that incubation of HL-60 cells with Asc in the presence of ascorbate oxidase resulted in the accumulation of intracellular Asc. The intracellular Asc levels were lowered by irradiation, demonstrating intracellular consumption of Asc by the radiation-generated radicals. Exposure of HL-60 cells to increasing gamma irradiation doses resulted in increasing accumulation of protein peroxides in the cells. This was measured by the FOX assay. A significant decrease in intracellular protein hydroperoxides was noted when the cells were treated with ascorbic acid before irradiation. A dose-dependent protective effect of Asc was observed. Asc loading also provided strong protection from radiation-generated protein hydroperoxides independently of the composition of the external medium, showing that only the radicals formed within the cells were effective in oxidizing the cell proteins. Similarly, protein peroxidation was inhibited in cells with enhanced levels of GSH and increased when the intracellular GSH concentration was reduced. These findings indicate that ascorbate and GSH are important antioxidants in protecting cells from oxidative stress associated with the generation of protein hydroperoxide.
Mode of access: World Wide Web.
xxix, 322 leaves ill
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Geier, George Richard. "The preparation of a metalloporphyrin-peptide conjugate artificial protein for the catalytic oxidation of alkenes /". Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/8546.
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Kwolek, Kathleen A. "Investigation of Site-Specific Metal-Catalyzed Oxidation of Proteins Using Dipeptides as Model Compounds". Youngstown State University / OhioLINK, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=ysu997720041.
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Love, Dominic Thomas. "The Role of HOSCN in the Oxidation of Proteins and Cellular Damage in Atherosclerosis". Thesis, The University of Sydney, 2015. http://hdl.handle.net/2123/14705.
Testo completoAbstract (sommario):
Myeloperoxidase forms the reactive oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). While HOCl is an extremely reactive oxidant that causes extensive damage to all manner of biomolecules, HOSCN reacts preferentially with protein thiols. This targeting of thiols can lead to protein inactivation and inhibition, and has led to HOSCN being implicated in the alteration of numerous cellular redox pathways. This Thesis compares the ability of HOCl and HOSCN to form reversibly oxidised cysteine products in murine J774A.1 macrophages, examining changes to the protein thiol levels and the manner of oxidised cysteine products formed in the cells upon oxidation, with changes to the cellular chemistry observed via WB analysis, FTIR and Raman microscopy. Further studies employ real-time analysis into the functional changes in metabolism of J774A.1 cells after HOSCN treatment. Results show that HOSCN is able to affect macrophage glucose metabolism via the oxidation of glycolytic proteins. The oxidation of glycolytic proteins causes a reduction in the glycolytic end product, pyruvate, and extend to mitochondrial oxidative phosphorylation, mitochondrial permeability transition pore function and ATP production. In summary, the studies of this Thesis may be significant in understanding the inflammatory process of atherosclerosis, especially in cigarette smokers, who have elevated levels of thiocyanate, the parent ion of HOSCN in their plasma.
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Bhattacharyya, Anjan Kumar. "Intramolecular and intracomplex electron transfer in water soluble redox proteins". Diss., The University of Arizona, 1988. http://hdl.handle.net/10150/184339.
Testo completoAbstract (sommario):
Electron transfer to and between the redox centers of milk xanthine oxidase was investigated by laser flash-photolysis. Evidence is presented for slow equilibration of electrons (k < 38 s⁻¹) between the various redox centers of the enzyme. The enzyme-bound flavin and the heme moieties of the flavoprotein and cytochrome subunits of p-cresol methyl hydroxylase from Pseudomonas putida are both reduced rapidly in a second order manner by 5-dRF generated by the laser flash, followed by slower first order intramolecular electron transfer (k = 220 s⁻¹) from the protein-bound neutral flavin radical to the oxidized cytochrome. Complex formation between spinach ferredoxin:NADP⁺-reductase (FNRₒᵪ), spinach ferredoxin (Fdₒᵪ), rubredoxin (Rdₒᵪ) from Clostridium pasteurianum, two homologous HIPIP's from Ectothiorhodospira halophila (iso-1 and iso-2) and two homologous cytochromes (cytochromes-c₂ from Paracoccus denitrificans and Rhodospirrilum rubrum) have been investigated. Evidence is presented supporting the formation of 1:1 complexes that are stabilized by attractive electrostatic interactions at low ionic strength. Kinetic studies of the above-mentioned complexes provide evidence for extremely rapid to relatively slower intracomplex electron transfer rates (k 7000 s⁻¹ to 4 s⁻¹). In addition the effect of complexation on the degree of accessibility of the various redox centers of the respective complexes to reduction by small reductants such as 5-dRF· and LfH· generated by the laser flash has been evaluated. The effect of both pH and ionic strength on the second order rate of reduction and the intracomplex rates in the respective complexes have also been investigated. The results have been interpreted in terms of redox potential differences, electrostatic and structural features that influence the electron transfer rates in these systems.
23
Elliott, Nathan Andrew. "Prevention of Oxidative Damage by Yeast and Human OXR1: A Dissertation". eScholarship@UMMS, 2004. https://escholarship.umassmed.edu/gsbs_diss/96.
Testo completo
24
Thirumangalathu, Renuka. "Understanding physical and chemical stability of proteins in solution : relevance to therapeutic protein and monoclonal antibody formulations /". Connect to abstract via ProQuest. Full text is not available online, 2007.
Cerca il testo completoAbstract (sommario):
Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 133-143). Online version available via ProQuest Digital Dissertations.
25
Mensah, Eric. "Creation of a Site-Directed Mutant of Hen Egg White Lysozyme Working Toward Site-Specific Oxidation as it Relates to Protein Structure". Connect to resource online, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1251756763.
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26
Fernström, Maria. "Effects of endurance exercise on mitochondrial efficiency, uncoupling and lipid oxidation in human skeletal muscle /". Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-059-6/.
Testo completo
27
Traore, Souleymane. "Caractérisation biochimique de muscles de porc riches en glycogène : relation avec les phénomènes d'oxydation". Thesis, Clermont-Ferrand 2, 2011. http://www.theses.fr/2011CLF22207.
Testo completoAbstract (sommario):
L’objectif de notre étude est de comprendre les mécanismes impliqués dans la variation des qualités sensorielles et technologiques de la viande de porc. Les différentes expérimentations nous ont permis de mettre en évidence le rôle de l’oxydation des protéines dans la variation de qualités des viandes. Les modifications structurales qui ont en découlé participent également à l’élucidation des mécanismes à l’origine de la diminution du pouvoir en rétention d’eau. L’application des traitements thermiques sur la viande accentue les phénomènes oxydatifs dans lesquels la myosine et de l’actine sont des cibles privilégiées, impliquées dans la formation d’agrégats protéiques. Enfin, le rôle du glycogène comme potentialisateur de l’oxydation protéique a été démontréWe aimed to better understand the mechanisms underlying sensorial and technological meat qualities. The experimental design put into relief the important role of protein oxidation in meat quality. As a consequence of oxidation, structural changes of proteins demonstrated also their implication in water holding capacity. Heating enhanced the oxidative process in which myosin and actin can be considered as favoured protein target. Moreover these proteins are implicated in aggregation /polymerization. Finally, glycogen as a catalyst of protein oxidation was demonstrated
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Heine, George F. "Functional analysis of P1, a model R2R3 MYB domain transcription factor". Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1148487881.
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Kuldyushev, Nikita [Verfasser], Stefan H. [Gutachter] Heinemann, Lars-Oliver [Gutachter] Klotz e Jochen [Gutachter] Balbach. "Monitoring of methionine oxidation with fluorescent proteins / Nikita Kuldyushev ; Gutachter: Stefan H. Heinemann, Lars-Oliver Klotz, Jochen Balbach". Jena : Friedrich-Schiller-Universität Jena, 2021. http://d-nb.info/1238142265/34.
Testo completo
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Niland, Hannah. "Detection and analysis of proteins in the solid phase using extrinsic and intrinsic fluorescence". Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28771.
Testo completoAbstract (sommario):
Over the past two decades a body of evidence concerning residual biological contamination on cleaned surgical instruments has accumulated. This is substantiated by the number of yearly surgery cancellations due to visible residue on instruments in surgical packs and incidences of iatrogenic Creutzfeldt-Jakob disease (iCJD). It is therefore imperative to develop a method of protein detection for use in clinical sterile services departments (SSDs) for monitoring of decontamination quality. This Thesis describes the development and use of an epifluorescence surface scanner (EFScan) technology in the assessment of proteinaceous residue on surgical instruments, by detecting protein pre-labelled with fluorescein isothiocyanate (FITC), and exploratory studies on the feasibility of label-free detection, using intrinsic protein fluorescence. Measurements using FITC labelling showed that residual protein on the order of micrograms can be found on new, single-use instruments (i.e. prior to use). This is comparable to the amount of residual protein found on retired, reusable instruments. To confirm the suitability of fluorescence techniques in the detection and quantification of proteinaceous residue, a blind, pilot study was carried out in conjunction with groups from Queen Mary University and the University of Southampton. Each University used a different labelling and detection method, and results showed good agreement between these methods. This showed that fluorescence is a suitable technique for the detection and quantification of proteinaceous contamination on surgical instruments. The next step in the project focussed on detection of contamination via intrinsic protein fluorescence from tryptophan residues, with a view to elimination of the labelling step. Intrinsic fluorescence of proteins in solution is widely characterised; however, fluorescence characteristics of solid or surface-bound protein have been little studied. Therefore, the characterisation of solid protein fluorescence and the emission characteristics of protein adsorbed onto stainless steel was undertaken. Analysis of the commonly used protein standard bovine serum albumin (BSA) showed that the two tryptophan residues it contains are highly susceptible to photo-oxidation in the solid state, resulting in conversion to the fluorescent photoproducts n-formylkynurenine (NFK) and kynurenine. Therefore, BSA is not suitable for use as a standard in the development of intrinsic fluorescence detection of surface-bound protein. The 72-tryptophan protein fibrinogen, as well as a series of other multi-tryptophan proteins, were assessed and it was found that photo-oxidation of the tryptophan residues did not occur on the irradiation timescale of 1 hour utilized. Therefore, it was concluded that lysozyme or gamma-globulins, a prominent group of serum proteins, would be more suitable candidates as a standard in subsequent research into the intrinsic detection and quantification of proteinaceous contamination. A third study explored the potential use of fluorescence in the early diagnosis of cataract. This involved the fluorescence characterisation of healthy porcine lenses and the use of UV irradiation of the lens to attempt to create cataract in vitro. There was found to be a large variation in fluorescence characteristics from lens to lens, suggesting that fluorophore concentrations can vary significantly. This implies that identification of a suitable standard for the early detection of cataract may be problematic. Attempts to create cataract resulted in the photo-oxidation pathway which had been observed in BSA, and although NFK and kynurenine play a role in cataractogenesis, the accumulation of these photoproducts is not analogous to a natural cataract. It was found that these products could be destroyed by irradiation of the lens at appropriate photo-bleaching wavelengths. However, this also destroyed intrinsic, protective fluorophores within the lens, suggesting that a light-based method of cataract treatment may not be achievable.
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Taskinen, J. (Jukka). "Protein crystallographic studies of CoA-dependent proteins: new insight into the binding mode and exchange mechanism of acyl-CoA". Doctoral thesis, University of Oulu, 2006. http://urn.fi/urn:isbn:9514280377.
Testo completoAbstract (sommario):
Abstract Multifunctional enzyme type 1 (MFE-1) is a monomeric member of the hydratase/isomerase superfamily (H/I) involved in the β-oxidation of fatty acids. MFE-1 has 2-enoyl-CoA hydratase-1, Δ3-Δ2-enoyl-CoA isomerase, and several other enoyl-CoA isomerase activities at the N-terminus. The C-terminus has (3S)-hydroxyacyl-CoA dehydrogenase activity. MFE-1 can also convert certain hydroxylated C27 bile acid synthesis intermediates. In these studies, a domain assignment of MFE-1 by sequence alignment with the H/I family (domains A and B in MFE-1) and mitochondrial monofunctional 3-hydroxyacyl-CoA dehydrogenases (HAD, domains C, D and E) was proposed. This was further improved with the structural information obtained from the crystal structure of the construct containing domains B, C, D and E (MFE1-DH). The structure of MFE1-DH resembles the bilobal structure of the α-subunit of the bacterial fatty acid metabolising complex and the mammalian HAD enzyme. The N-terminal linker helix of MFE1-DH (domain B) corresponds to helix-10 of the hydratase/isomerase enzymes having residues important for substrate contacts. Domain C adopts the classical Rossmann fold and forms the first lobe of the MFE1-DH structure. The C-terminal domains D and E form the second lobe and have local symmetry between each other. This local symmetry corresponds to the D domain-mediated dimerisation of the HAD dimer. The domain deletion studies showed that the presence of domains D and E, but not domain C, was essential to obtain a functional hydratase 1 enzyme; this can be understood from stabilising contacts from domain E to the linker helix, as seen in the MFE1-DH structure. The structure of human ACBP from liver was determined with and without a physiological ligand. This structure adopts the classical four-helix bundle of the ACBP family. The ligand binding mode seen in the presence of myristoyl-CoA shows that one ligand molecule is bound jointly by the two protein molecules of the asymmetric unit such that the fatty acid tail is bound by one protein molecule, and the 3'-phosphate AMP moiety of the CoA is bound by the other protein molecule, essentially as in known complexed ACBP structures in the monomeric binding mode. The observed ligand binding mode suggests a new model for the ACBP-mediated ligand transfer observed in biochemical in vitro studiesTiivistelmä Tyypin 1 monitoiminen entsyymi (MFE-1) on hydrataasi/isomeraasiperheen (H/I) jäsen ja se osallistuu rasvahappojen β-oksidaatioon. MFE-1:n N-päädyssä on 2-enoyyli-CoA-hydrataasi 1- ja Δ3-Δ2-enoyyli-CoA-isomeraasiaktiivisuus sekä useita muita enoyyli-CoA-isomeraasiaktiivisuuksia. C-päädyssä on (3S)-hydroksiasyyli-CoA-dehydrogenaasiaktiivisuus. MFE-1 voi myös katalysoida tiettyjen hydroksyloitujen C27-sappihapposynteesin välituotteiden reaktioita. Tässä tutkimuksessa määritettiin MFE-1:n domeenirakenne H/I-perheen (MFE-1:n domeenit A ja B) ja 3-hydroksiasyyli-CoA-dehydrogenaasiperheen (HAD, domeenit C, D ja E) sekvenssilinjausten perusteella. Rakennetta tarkennettiin domeenit B, C, D ja E sisältävän konstruktin (MFE1-DH) kiderakenteesta saadun tiedon avulla. MFE1-DH:n kiderakenne muistuttaa bakteerien rasvahappoja hajottavan kompleksin α-alayksikön sekä nisäkkäiden HAD-entsyymin kahdesta alayksiköstä muodostuvaa rakennetta. MFE1-DH:n N-päädyn α-kierre vastaa H/I-entsyymien kierre-10:tä, jossa sijaitsee substraattikontaktien kannalta tärkeitä aminohappotähteitä. C-domeeni muodostaa Rossmann-laskoksen ja se on MFE1-DH:n rakenteen ensimmäinen alayksikkö. C-päädyssä sijaitsevat D- ja E-domeenit muodostavat yhdessä toisen alayksikön ja niiden välillä on symmetria, joka vastaa D-domeenien välittämää HAD-entsyymien dimerisaatiota. Domeenitutkimukset osoittivat, että D- ja E-domeenien läsnä olo oli välttämätöntä hydrataasi 1:n toiminnalle, mutta C-domeeni voitiin poistaa ja säilyttää hydrataasi 1 -aktiivisuus. Havainto voitiin selittää MFE1-DH:n rakenteen avulla, jossa nähdään stabiloivia vuorovaikutuksia E-domeenin ja N-päädyn α-kierteen välillä. Ihmisen maksan ACBP:n kiderakenne määritettiin fysiologisen ligandin kanssa sekä ilman ligandia. Tämä rakenne laskostuu ACBP-perheelle tyypilliseksi neljän kierteen nipuksi. Myristoyyli-CoA:n läsnä ollessa havaitussa ligandin sitoutumistavassa yksi ligandimolekyyli on sitoutunut kahden proteiinimolekyylin välille siten, että rasvahappo-osa sitoutuu toiseen proteiinimolekyyliin ja CoA:n 3'-fosfaatti-AMP-osa sitoutuu toiseen proteiinimolekyyliin kuten tunnetuissa monomeerisissä ACBP:n sitomistavoissa. Havaitun sitoutumisen avulla voidaan ehdottaa uutta mallia ACBP:n välittämälle ligandinsiirrolle, joka on havaittu aikaisemmin biokemiallisissa in vitro -tutkimuksissa
32
Harrington, Jane Colleen. "Regulation of Reactive Nitrogen Species (RNS) Metabolism and Resistance Mechanisms in Haemophilus influenzae: A Dissertation". eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/401.
Testo completoAbstract (sommario):
Haemophilus influenzae encounters niches within the human host that are predicted to differ in availability of oxygen and reactive nitrogen species (RNS: nitrite and nitric oxide), which influence the environmental redox state. Previously reported data has indicated that an altered redox condition could serve as a signal recognized by H. influenzae to optimize its survival within host microenvironments. To elucidate the role of redox signaling in virulence, we examined regulation by the FNR homolog of H. influenzae, whose counterpart in E. coli has been reported to be a direct oxygen sensor and a regulator of genes responsible for RNS metabolism and resistance. Many members of the FNR regulon are subject to coordinated transcriptional control by NarP, a regulator in E. coli that is activated by cognate sensor NarQ in response to environmental nitrite. To study the regulatory activities of FNR and NarQ-NarP in H. influenzae, I targeted a gene predicted to be FNR-regulated, nrfA, which encodes nitrite reductase, a periplasmic cytochrome-c involved in anaerobic respiration. The fnr, narP and nrfA mutants were assayed for nitrite reduction, which implicated the roles of FNR, NarP and NrfA in RNS metabolism. Using Western blot detection of an epitope-tagged reporter protein fused to the endogenous nrf promoter (Pnrf-HA), I demonstrate that FNR and NarP, but not NarQ, are required for full activation of the nrf promoter. Additionally, Pnrf-HA expression increases as oxygen becomes depleted and decreases when exposed to high concentrations of nitrite, implying that the nrfpromoter is modulated by environmental redox signals.
FNR of E. coli has been implicated in regulation of resistance mechanisms to a reactive nitrogen species, nitric oxide (NO), which is produced by innate immune cells during infection as a host defense mechanism. A mutant lacking FNR is more sensitive to NO exposure and killing by activated macrophages than wild type H. influenzae after anaerobic pre-growth. Mutants of nrfA and narP have been tested and initial experiments have shown both mutants have a lesser NO sensitivity phenotype as compared to the fnr mutant, suggesting that other factors could be involved in FNR-mediated NO resistance in H. influenzae. Upon examination of potential factors that might be involved to this phenotype, we discovered FNR-regulated gene, ytfE, which contributes to defense against nitrosative stress. The fnr and ytfE mutants are more susceptible to killing by activated macrophages indicating that FNR regulation of ytfE might be important for in vivo infection.
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Wang, Xu. "CONTROLLED OXIDATIVE MODIFICATION WITH GLUCOSE OXIDASE TO ENHANCE THE RHEOLOGICAL AND GELLING PROPERTIES OF MYOFIBRILLAR PROTEINS". UKnowledge, 2017. http://uknowledge.uky.edu/animalsci_etds/74.
Testo completoAbstract (sommario):
This study investigated the feasibility of oxidative modification with glucose oxidase (GluOx) to enhance the rheological and gelling properties of myofibrillar protein. Differential oxidative modifications of myofibrillar protein (MP) by hydroxyl radicals generated in an enzymatic system with glucose oxidase (GluOx) in the presence of glucose/FeSO4 compared to a Fenton system (H2O2/FeSO4) were investigated. Firmer and more elastic MP gels were produced by the GluOx-oxidizing system than by the Fenton system at comparable H2O2 levels due to an altered radical reaction pathway.
The study further explored the effect of GluOx-mediated oxidation on the efficacy of transglutaminase (TGase) cross-linking of MP in 0.6 M NaCl and the rheological properties of GluOx oxidation/TGase treated MP in MP–lipid emulsion composite gels. The GluOx-mediated oxidation promoted the formation of both soluble and insoluble protein aggregates via disulfide bonds and occlusions of hydrophobic groups. The subsequent TGase treatment converted protein aggregates into highly cross-linked polymers. MP–lipid emulsion composite gels formed with such polymers exhibited markedly enhanced gelling capacity: up to a 4.4-fold increase in gel firmness and a 3.5-fold increase in gel elasticity over untreated protein. Microstructural examination showed small oil droplets dispersed in a densely packed gel matrix when MP was oxidatively modified, and the TGase treatment further contributed to such packing.
Comparison of the modification of MP via GluOx oxidation/TGase cross-linking pathway under different salt concentrations (0.3 and 0.6 M NaCl) showed different patterns of MP cross-linking, resulting in different extents of aggregation. Under low-salt condition (0.3 M NaCl), the GluOx/TGase treatment increased the gel strength to the same level as those treated with TGase in 0.6 M NaCl, suggesting a potential application of GluOx/TGase for improving gel strength in low ionic strength conditions.
Finally, the application of GluOx oxidation in the development of emulsion-type sausages was studied. The GluOx oxidation/TGase cross-linking improved the textural properties (firmness, chewiness, and rupture force) of emulsion-type sausages under both salt levels (P < 0.05). Under low-salt condition (1.5% NaCl), GluOx/TGase treatment can increase the sausage binding strength to the same level as the control sample under high-salt condition (3% NaCl). The GluOx oxidation/TGase treatment shows promise to improve the textural properties of emulsified meat products. However, the significant decrease of a* value and increase of b* value indicated GluOx-induced color deterioration.
34
Chou, Danny Kochen. "Mechanistic insights into physical and chemical stability of albumin fusion proteins in aqueous solution /". Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2008.
Cerca il testo completoAbstract (sommario):
Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2008.Typescript. Includes bibliographical references (leaves 219-242). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
35
López, Martínez Montserrat. "Electrochemical tunneling microscopy and spectroscopy of electron transfer proteins". Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/462883.
Testo completoAbstract (sommario):
Electron Transfer (ET) plays essential roles in crucial biological processes such as cell respiration and photosynthesis. It takes place between redox proteins and in protein complexes that display an outstanding efficiency and environmental adaptability. Although the fundamental aspects of ET processes are well understood, more experimental methods are needed to determine electronic pathways. Understanding how ET works is important not only for fundamental reasons, but also for the potential technological applications of these redox‐active nanoscale systems.
The general objective of this thesis is to investigate electron transfer in redox proteins at the single molecule level. To that end, we use Electrochemical Scanning Tunneling Microscopy (ECSTM) and conductive Atomic Force Microscopy (cAFM), excellent tools to study electronic materials and redox molecules including proteins. In this thesis, we focused on two redox protein systems: azurin, a small electron carrier protein and photosystem I, a light‐sensitive oxidoreductase protein complex.
In azurin, we studied the protein conductance as a function of its redox state and location on the protein surface, and the effect of technical parameters such as the contact properties between azurin and the metal electrodes, and the mechanical force applied in such contact. For that we adapted our ECSTM setup for an alternating current method often used in ultrahigh vacuum (UHV) STMs. We also worked in the development of a methodology that combines AFM‐based single‐molecule force measurements with single‐molecule electrical measurements, while working in an electrochemically controlled environment. These techniques can lead to a more detailed description of the ET pathways, and to a deeper understanding of the complex relation between the structure of redox proteins and their electronic properties.
In photosystem I, developed a method to immobilize complexes on a substrate suitable for ECSTM imaging and spectroscopy, atomically flat gold. In these conditions, we characterized photosystem I by imaging and spectroscopy, and evaluated its conductance and distance‐decay properties in a wide range of biologically relevant electrochemical potentials.
The characterization of conduction pathways in redox proteins at the nanoscale would enable important advances in biochemistry and would cause a high impact in the field of nanotechnology.La transferencia de electrones (ET) desempeña papeles esenciales en procesos biológicos cruciales como la respiración celular y la fotosíntesis. Tiene lugar inter‐ e intra‐ proteínas redox y en complejos de proteínas que muestran una eficiencia excepcional y gran capacidad de adaptación ambiental. Aunque los aspectos fundamentales de los procesos de ET se han estudiado en profundidad, se necesitan más métodos experimentales para determinar las vías electrónicas de ET. La comprensión de cómo funciona la ET es importante no sólo por razones fundamentales, sino también por las potenciales aplicaciones tecnológicas de estos sistemas redox nanoscópicos. El objetivo general de esta tesis es investigar la transferencia de electrones en las proteínas redox a nivel de molécula individual. Para ello utilizamos la Microscopía de Túnel Electroquímico (ECSTM) y la Microscopía de Fuerza Atómica Conductor (cAFM), que son excelentes herramientas para estudiar materiales electrónicos y moléculas redox, incluyendo proteínas. En esta tesis, nos centramos en dos sistemas de proteínas redox: azurina, una pequeña proteína portadora de electrones y el fotosistema I, un complejo de proteína oxidorreductasa sensible a la luz. En el estudio de la azurina, estudiamos la conductancia de las proteínas en función de su estado redox y el efecto de parámetros técnicos como las propiedades de contacto entre la azurina y los electrodos metálicos, y la fuerza mecánica aplicada en dicho contacto. Para ello hemos adaptado nuestra configuración de ECSTM para un método de corriente alterna a menudo utilizado en Microscopía de Túnel de ultra alto vacío (UHV‐STM). También trabajamos en el desarrollo de una metodología que combina medidas de fuerza de una sola molécula basadas en AFM con medidas eléctricas, mientras trabajamos en un ambiente controlado electroquímicamente. Estas técnicas pueden conducir a una comprensión más profunda de las vías de ET y de la compleja relación entre la estructura de las proteínas redox y sus propiedades electrónicas. En el estudio del fotosistema I, desarrollamos un método para inmovilizar complejos sobre un sustrato adecuado para la obtención de imágenes y espectroscopía con ECSTM, oro atómicamente plano. En estas condiciones, caracterizamos el fotosistema I mediante imágenes y espectroscopia, y evaluamos sus propiedades de conductancia y sus parámetros de decaimiento de la corriente con la distancia, en una amplia gama de potenciales electroquímicos biológicamente relevantes. La caracterización de las vías de conducción en las proteínas redox a escala nanométrica puede permitir importantes avances en bioquímica y causar un alto impacto en el campo de la nanotecnología.
36
Parker, Nicole Renee. "The role of kynurenine and UV light in lens protein modification". Access electronically, 2005. http://www.library.uow.edu.au/adt-NWU/public/adt-NWU20060720.111305/index.html.
Testo completoAbstract (sommario):
Thesis (Ph.D.)--University of Wollongong, 2005.Typescript. EMBARGOED - This thesis is subject to a 12 month embargo (07/03/06 to 07/03/07) and may only be viewed and copied with the permission of the author. For further information please Contact the Archivist. Includes bibliographical references: leaf 236-266.
37
Wilson, Cameron. "Mediation of Osteoblast Responses to Titanium Roughness by Adsorbed Proteins". Queensland University of Technology, 2005. http://eprints.qut.edu.au/16096/.
Testo completoAbstract (sommario):
Stable fixation of implants such as artificial teeth depends on the direct apposition of bone to the implanted material. While endosseous implants were traditionally allowed to "osseointegrate" over several months without carrying load, clinical and experimental data show that prostheses with roughened surfaces allow successful integration when subject to earlier loading and more challenging implant sites. However, to design implant surfaces for an optimal biological response requires an understanding of the mechanism by which roughened surfaces promote osseointegration. Research into this mechanism has, to date, focussed primarily on the response of osteoblastic cells to surface topography in vitro. While these have demonstrated some consistent trends in cell behaviour, the fundamental means by which cells sense and respond to roughness remain unclear. It has been suggested that cell responses to changes in topography may relate to differences in the proteins adsorbed from serum (in vitro). While experimental evidence indirectly suggests that physical features can affect protein adsorption, few studies have examined this with respect to surface roughness, particularly as a mediator of cell responses. To address this issue, cell culture and protein adsorption experiments were conducted on a limited range of surface textures. Titanium samples were ground to produce morphologically similar surfaces with three grades of roughness. A duplicate set of specimens were heated at 600°C for one hour, with the aim of masking potential variations in physicochemical properties with differing degrees of grinding. Osteoblast attachment and proliferation studies were conducted over a short time-frame of 48 hours or less, to highlight the effects of proteins adsorbed from serum rather than secreted by adherent cells. Gel electrophoresis provided a profile of the proteins adsorbed to each surface after 15 minutes, corresponding to the time by which the cells had settled onto the surface. Finally, confocal microscopy was used to examine cell morphology on each surface, and to visualize specific interactions between cellular structures and adsorbed adhesion-mediating proteins. Although the effects were inconsistent, attachment assays showed some indications that fewer cells attached in the first 90 minutes as roughness increased. This inverse cell number-roughness trend was significant at 48 hours; however, the variability in attachment assays prevented reliable separation of attachment and proliferation rate effects. While the reduction in cell number with increasing roughness is consistent with previous reports, it is typically observed at later time points, and thus may be increasingly confounded by contact inhibition and differentiation. Thermal oxidation of the titanium did not impact on osteoblast responses to roughness, although it significantly slowed cell proliferation. The latter result was unexpected on the basis of previous reports. One-dimensional gel electrophoresis revealed no significant differences in the composition of adsorbed layers with variations in roughness. However, as expected on account of wettability changes, the heat-treatment did correspond to significant changes in the adsorption profile. While this was not a highly sensitive analysis, it suggests that the cell responses to roughness changes were not governed by broadscale differences in the proteins initially available to adhering cells. In addition to the composition of the adsorbed layer, the distribution of proteins may also vary with topography. The immunofluorescence methods were not sufficiently sensitive to reveal the distribution of adsorbed adhesion proteins (vitronectin and fibronectin). However, the lack of clear labelling does suggest an absence of large accumulations due to specific topographic features. Further work is required to address this issue conclusively. Observations of cell morphology were consistent with widely-reported contact guidance phenomena on grooved surfaces, with elongation and alignment (with topography) increasing with groove depth. Cell elongation was also enhanced on the more hydrophilic, heat-treated titanium, but this effect diminished over time. Although increased elongation at 90 minutes corresponded to lower cell numbers at 48 hours, no causal relationship has yet been established.
38
Wilson, Cameron John. "Mediation of Osteoblast Responses to Titanium Roughness by Adsorbed Proteins". Thesis, Queensland University of Technology, 2005. https://eprints.qut.edu.au/16096/1/Cameron_Wilson_Thesis.pdf.
Testo completoAbstract (sommario):
Stable fixation of implants such as artificial teeth depends on the direct apposition of bone to the implanted material. While endosseous implants were traditionally allowed to "osseointegrate" over several months without carrying load, clinical and experimental data show that prostheses with roughened surfaces allow successful integration when subject to earlier loading and more challenging implant sites. However, to design implant surfaces for an optimal biological response requires an understanding of the mechanism by which roughened surfaces promote osseointegration. Research into this mechanism has, to date, focussed primarily on the response of osteoblastic cells to surface topography in vitro. While these have demonstrated some consistent trends in cell behaviour, the fundamental means by which cells sense and respond to roughness remain unclear. It has been suggested that cell responses to changes in topography may relate to differences in the proteins adsorbed from serum (in vitro). While experimental evidence indirectly suggests that physical features can affect protein adsorption, few studies have examined this with respect to surface roughness, particularly as a mediator of cell responses. To address this issue, cell culture and protein adsorption experiments were
conducted on a limited range of surface textures. Titanium samples were ground to
produce morphologically similar surfaces with three grades of roughness. A duplicate set of specimens were heated at 600°C for one hour, with the aim of masking potential variations in physicochemical properties with differing degrees of grinding. Osteoblast attachment and proliferation studies were conducted over a short time-frame of 48 hours or less, to highlight the effects of proteins adsorbed from serum rather than secreted by adherent cells. Gel electrophoresis provided a profile of the proteins adsorbed to each surface after 15 minutes, corresponding to the time by which the cells had settled onto the surface. Finally, confocal microscopy was used to examine cell morphology on each surface, and to visualize specific interactions between cellular structures and adsorbed adhesion-mediating proteins. Although the effects were inconsistent, attachment assays showed some indications that fewer cells attached in the first 90 minutes as roughness increased. This inverse cell number-roughness trend was significant at 48 hours; however, the variability in attachment assays prevented reliable separation of attachment and proliferation rate effects. While the reduction in cell number with increasing roughness is consistent with previous reports, it is typically observed at later time points, and thus may be increasingly confounded by contact inhibition and differentiation. Thermal oxidation of the titanium did not impact on osteoblast responses to roughness, although it significantly slowed cell proliferation. The latter result was unexpected on the basis of previous reports. One-dimensional gel electrophoresis revealed no significant differences in the composition of adsorbed layers with variations in roughness. However, as expected on account of wettability changes, the heat-treatment did correspond to significant changes in the adsorption profile. While this was not a highly sensitive analysis, it suggests that the cell responses to roughness changes were not governed by broadscale differences in the proteins initially available to adhering cells. In addition to the composition of the adsorbed layer, the distribution of proteins may also vary with topography. The immunofluorescence methods were not sufficiently sensitive to reveal the distribution of adsorbed adhesion proteins (vitronectin and fibronectin). However, the lack of clear labelling does suggest an absence of large accumulations due to specific topographic features. Further work is required to address this issue conclusively. Observations of cell morphology were consistent with widely-reported contact guidance phenomena on grooved surfaces, with elongation and alignment (with topography) increasing with groove depth. Cell elongation was also enhanced on the more hydrophilic, heat-treated titanium, but this effect diminished over time. Although increased elongation at 90 minutes corresponded to lower cell numbers at 48 hours, no causal relationship has yet been established.
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Grabarczyk, Daniel Ben. "Molecular characterisation of bacterial proteins that interact with sulfur or nitrogen compounds". Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:b6b3e3fd-620f-467d-b063-01311fa7a9a2.
Testo completoAbstract (sommario):
Many bacteria use inorganic nitrogen and sulfur compounds for energy metabolism. These compounds are often toxic and so bacteria must adapt to survive their deleterious effects. Bacteria use specific proteins in order to metabolise, sense and detoxify these compounds. In this thesis protein interactions with inorganic nitrogen and sulfur compounds are examined at the mechanistic level. Intermediates in the Sox sulfur oxidation pathway are covalently attached to a cysteine on the swinging arm of the substrate carrier protein SoxYZ. An interaction between the Sox pathway enzyme SoxB and the carrier protein SoxYZ is demonstrated. A crystal structure of a trapped SoxB-SoxYZ complex at 3.3 Å resolution identifies two sites of interaction, one between the SoxYZ carrier arm and the SoxB active site channel and the other at a patch distal to the active site. The presence of a distal interaction site suggests a mechanism for promiscuous specificity in the protein-protein interactions of the Sox pathway. Using biophysical methods it is shown that SoxB distinguishes between the substrate and product forms of the carrier protein through differences in interaction kinetics and that the carrier arm-bound substrate group is able to out-compete the adjacent C-terminal carboxylate for binding to the SoxB active site. The thiosulfate dehydrogenase TsdA has an unusual His/Cys coordinated heme. TsdA catalyses oxidative conjugation of two thiosulfate molecules to form tetrathionate. Mass spectrometry and UV/visible spectroscopy are used to identify an S-thiosulfonate reaction intermediate which is covalently attached to the cysteine heme ligand. A catalytic mechanism for TsdA is proposed using a crystal structure of TsdA at 1.3 Å resolution alongside site-directed mutagenesis of active site residues. Nitric oxide is produced by the mammalian immune response to kill bacterial pathogens. Part of the killing mechanism occurs through the reaction of nitric oxide with protein-bound iron-sulfur clusters. However, the same type of reaction is also exploited by nitric oxide-sensing bacterial proteins. An infrared spectroscopy approach is developed to detect the products of iron-sulfur protein nitrosylation. Using this methodology it is shown that the presence of trace O2 strongly impacts which products are formed in these nitrosylation reactions. These observations are of physiological relevance because bacteria are often exposed to NO under aerobic conditions during an immune response.
40
Raimondi, Daniele. "The effect of genome variation on human proteins: understanding variants and improving their deleteriousness prediction through extensive contextualisation". Doctoral thesis, Universite Libre de Bruxelles, 2017. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/251313.
Testo completoAbstract (sommario):
Rapid technological advances are providing unprecedented insights in the biologicalsciences, with massive amounts of data generated on genomic and protein sequences.These data continue to grow exponentially, and they are extremely valuable for com-putational tools where the effect of genomic variants on human health is predicted.State of the art tools in this field give varying results and only tend to agree in thecase of single variants that are strongly correlated to disease. The aim of this workis to increase the reliability of these methods, as well as our understanding of theunderlying biological mechanisms that lead to disease. We first developed machinelearning (ML) based structural bioinformatics predictors that are able to predictmolecular features of proteins from the sequence alone. We then used these tools forin silico analysis of the molecular effects of known variants on the affected proteins,and integrated these data with other sources heterogenous sources of information,such as the essentiality of a gene, that put the variants into their broader biologicalcontext. With this information we created DEOGEN, a novel predictor in this field,which is able to deal with the two most common forms of genomic variation, namelySingle Nucleotide Variants (SNVs) and short Insertions and DELetions (INDELs).DEOGEN performs at least on par with other state of the art methods in this fieldon different datasets. The method was then extended with additional contextualdata and is now available as DEOGEN2 via a web server, which visualizes the pre-dicted results for all variants in most human proteins through an interactive interfacetargeted to both bioinformaticians and clinicians.Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Otten, Michael P. "Nitrogen in the Environment: Blue Copper Proteins Involved in Ammonia Oxidation and A Novel Smartphone-based Strategy for Colorimetric Water Quality Measurements". Bowling Green State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1467989136.
Testo completo
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Sousa, Roberta Regina Ruela de. "Estudo por oxido-redução de uma proteina tirosina fosfatase (CD45) purificada de membrana de linfocitos humanos". [s.n.], 2005. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314761.
Testo completoAbstract (sommario):
Orientador: Hiroshi AoyamaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-06T06:33:37Z (GMT). No. of bitstreams: 1 Sousa_RobertaReginaRuelade_M.pdf: 3257089 bytes, checksum: 29f24a432f8c0bfd7dd71af48cc7608a (MD5) Previous issue date: 2005
Resumo: As proteínas tirosina fosfatases (PTP) (EC 3.1.3.48) são enzimas regulatórias chaves que participam na transdução de sinal e são essenciais na regulação do crescimento, diferenciação, ciclos celulares, na transcrição gênica, resposta imune e outros processos. Esta classe de enzimas, que contém cisteína no sítio ativo, pode ser inativada por agentes oxidantes. Neste trabalho, estudamos os efeitos de peróxido de hidrogênio e t-butil hidroperóxido, compostos que induzem estresse oxidativo, na atividade de uma PTP purificada de membranas de linfócitos humanos, indicativamente a CD45. A PTP foi purificada de membranas de linfócitos humanos através de cromatografias de troca iônica (DEAE Sepharose) e exclusão molecular (Sephacryl S-200). A purificação enzimática foi acompanhada por SDS-PAGE e eletroforese bidimensional. A atividade enzimática foi determinada através de incubação a 37°C por 30 min em pH 5,0 em presença de 5 mM de p-nitrofenil fosfato (pNPP) como substrato. A enzima obtida da cromatografia de exclusão molecular apresentou uma massa molecular relativa de aproximadamente 200 kDa, reconheceu mais eficientemente tirosina fosfato (cerca de 3,2 vezes) como substrato quando comparado ao pNPP, e foi inibida por inibidores específicos de PTP, tais como vanadato (40%), pervanadato (100%), p-cloromercuribenzoato (20%) and Cu2+ (95%). Ácido okadáico, um inibidor específico de serina e treonina proteína fosfatases, não afetou a atividade da PTP de membranas de linfócitos. Estes resultados de caracterização sugerem fortemente que a PTP purificada de membranas de linfócitos humanos é a CD45. Peróxido de hidrogênio e t-butil hidroperóxido inibiram a atividade dessa proteína com valores de IC50 (concentração do composto que produz 50% de inibição enzimática) de 50 µM e 16 mM, respectivamente. Glutationa reduzida (GSH) protegeu parcialmente a enzima contra estes oxidantes, porém proteções totais foram obtidas quando se adicionava 250 mM de desferoxamina ao meio de ensaio. Nossos resultados sugerem que essa proteína pode ser regulada por alteração do estado de oxidação dos grupos funcionais do sítio ativo e que esta regulação poderia fornecer um mecanismo de controle celular através de espécies reativas de oxigênio
Abstract: Protein phosphatases, that dephosphorylate proteins in residues of threonine, serine and tyrosine, are a class of enzymes that can be stressed by compounds present in oxidant or reductant environments. In particular, the protein tyrosine phosphatases (PTP) (EC 3.1.3.48) are key regulatory enzymes that participate in signal transduction and are essential for regulating cellular growth, differentiation, cell cycle, gene transcription, immune response and other processes. This class of enzymes, which contain cysteine in the active site, can be inactivated by oxidant reagents. In this work we have studied the effect of hydrogen peroxide and t-butyl hydroperoxide, compounds that induce oxidative stress, on a purified PTP (CD45) from membrane human lymphocytes. PTP was purified from human lymphocyte membranes through ion exchange (DEAE Sepharose) and molecular exclusion (Sephacryl S-200) chromatographies. The enzyme purification was followed by SDS-PAGE and 2D electrophoresis. The enzyme activity was determined by incubation at 37oC for 30 minutes at pH 5.0 in presence of 5 mM p-nitrophenylphosphate (pNPP) as substrate. The enzyme obtained from molecular exclusion chromatography had a relative molecular mass of approximately 200 kDa, recognized more efficiently tyrosine phosphate (about 3.2-fold) as substrate when compared with p-NPP, and was inhibited by specific PTP inhibitors, such as, vanadate (40%), pervanadate (100%), p-chloromercuribenzoate (20%) and Cu2+ (95%). Okadaic acid, a specific serine and threonine protein phosphatases inhibitor, did not significantly affect the lymphocyte membrane PTP activity. These characterization results strongly suggest that the membrane PTP purified from human lymphocytes was the CD45. Hydrogen peroxide and t-butyl hydroperoxide inhibited the CD45 activities with IC50 (concentration of compound that produces 50% enzyme inhibition) values of 50 µM and 16 mM, respectively. Reduced glutathione (GSH) partially protected the enzyme against these oxidations, but full protections were observed when 250 mM deferoxamine were added to the assay medium. Our results suggest that CD45 can be regulated by altering the oxidation state of active site functional groups, and that this regulation could provide a mechanism of cell control by reactive oxygen species
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular
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Pozuelo, Ruiz Marta. "Bioengineering single-protein wires". Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/462906.
Testo completoAbstract (sommario):
Electron Transfer (ET) is undoubtedly one of the most important processes in life. Molecularly well-defined ET pathways in complex protein ensembles play a vital role in biological processes such as cell respiration or photosynthesis. The fundamental understanding of ET processes in biology is important not only to understand such key natural processes but also to advance in the design of biomolecule/electrode interfaces for Bioelectronic applications. The development of new techniques such as scanning probe microscopies (SPM) played a key role. In particular, the electrochemical scanning tunnelling microscopy (EC-STM) has been exploited to in situ monitor the ET rate as a function of the applied potential of individual metalloproteins immobilized on an Au electrode thanks to the single-molecule spatial resolution and the electrochemical gate capabilities.
Azurin from Pseudomonas aeruginosa is a widely studied redox protein model both in bulk and at the single molecule level. Its globular structure contains a coordinated copper ion, which makes the protein capable of exchanging electrons by switching its redox state (Cu I/II) and supports its role as a soluble electron carrier in the respiratory chain of bacteria.
In this thesis, we will show our advances on the design and characterization of single-protein devices using a Cu-Azurin metalloprotein model. We have demonstrated transistor like-behaviour in an electrochemically-gated single-protein wire that operates at very low voltages thanks to the Cu-Azurin redox properties. It was demonstrated that the conductance varies depending on the redox state of the Cu centre, having its maximum value at the redox-midpoint. We have also analysed the spontaneous formation of single-Azurin electrical contacts through the monitored current when the two ECSTM electrodes were placed at a fixed distance. Discrete switching events for the conductance were observed, whose frequency depends on the applied electrochemical conditions and, therefore, they were univocally ascribed to discrete changes in the redox state of the trapped protein.
In order to tailor the charge transport behaviour of the single-protein wire, we have synthesized several mutants of the protein by exploiting point-site bioengineering schemes at different positions of the protein second coordination sphere. Our results show that we can rationally change the transport mechanism of the single-protein device by studying the effect of the specific residue modification on the particular ET pathways in the protein backbone.La transferencia de electrones (ET) es uno de los procesos más importantes de la vida. La comprensión fundamental de los procesos de ET en biología es importante no sólo para comprender tales procesos naturales claves, sino también para avanzar en el diseño de interfaces biomolécula / electrodo para aplicaciones bioelectrónicas. En particular, se ha explotado la microscopía de efecto túnel con control electroquímico (EC-STM) para monitorizar in situ la constante de ET en función del potencial aplicado de las metaloproteínas. La Azurina de Pseudomonas aeruginosa es un modelo de proteína redox ampliamente estudiado, tanto en ‘bulk’ como a nivel de una sola proteina. Su estructura globular contiene un ion de cobre coordinado, que hace que la proteína sea capaz de intercambiar electrones cambiando su estado redox (Cu I/II). Este ion es el responsable de su rol como portador de electrones en la cadena respiratoria de las bacterias. En esta tesis, mostraremos nuestros avances en el diseño y caracterización de dispositivos de una sola proteína utilizando un modelo de metaloproteína Cu-Azurin. Hemos demostrado un comportamiento similar a un transistor en un hilo electroquímico de una sola proteína que funciona a muy bajos voltajes gracias a las propiedades redox de Cu-Azurin. Se demostró que la conductancia varía dependiendo del estado redox del centro de Cu, teniendo su valor máximo en el punto medio redox. También hemos analizado la formación espontánea de los contactos eléctricos de Azurin única a través de la corriente monitorizada cuando los dos electrodos ECSTM se colocaron a una distancia fija. Se observaron eventos discretos de conmutación para la conductancia, cuya frecuencia depende de las condiciones electroquímicas aplicadas y, por lo tanto, se atribuyeron unívocamente cambios discretos en el estado redox de la proteína atrapada. Con el fin de adaptar el comportamiento de transporte de carga de la unión uniproteica, hemos sintetizado varios mutantes de la misma proteína mediante bioingeniería en diferentes posiciones de la proteína. Nuestros resultados muestran que podemos cambiar racionalmente el mecanismo de transporte del dispositivo de una sola proteína mediante el estudio del efecto de la modificación de residuos específicos en las vías ET particular en el esqueleto de la proteína.
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Gao, Ming. "IDENTIFICATION AND ANALYSIS OF PROTEINS AND GENES RESPONSIBLE FOR MICROBIAL ANAEROBIC NITRATE-DEPENDENT IRON OXIDATION AND OVEREXPRESSION IN E. coli OF PERCHLORATE REDUCTASE". OpenSIUC, 2011. https://opensiuc.lib.siu.edu/dissertations/378.
Testo completoAbstract (sommario):
SECTION 1 Iron is the fourth most abundant element on the earth crust as well as an essential nutrient for all living organisms. The cycling of iron between the environment and biological systems and the microbial-mediated transformation between Fe2+ and Fe3+ has a significant impact on the biogeochemistry of the environment. The recently discovered microbially-mediated anaerobic nitrate-dependent oxidation of Fe2+ has been shown to play an important role in global iron biogeochemical cycling. Furthermore, the formation of iron oxide from anaerobic nitrate-dependent Fe2+ oxidation results in the adsorption and precipitation of soluble toxic heavy metals and radionuclides from surrounding environment. Therefore, this metabolism has been attracting more and more attention because this process could serve as a cost-effective way to co-remediate nitrate, heavy metals and radionuclides at contaminated sites. Little is known about the molecular genetics of the anaerobic nitrate-dependent Fe2+ oxidation pathway so far. Previous studies in our lab using a microarray approach on Dechloromonas aromatica RCB uncovered the likely involvement of lipoproteins, transmembrane proteins in major operons, cytochromes and signal transduction enzymes in this metabolism. In an effort to further elucidate the metabolic process, a recently isolated bacterium strain Acidovorax ebreus strain TPSY capable of anaerobic nitrate-dependent Fe2+ oxidation was selected as a model organism in this study. By utilizing a 2-dimensional electrophoresis method, a list of candidate proteins which exhibited elevated levels of expression were identified by the comparison of whole cell protein profile between Fe2+-oxidizing strain TPSY cells and control cells. Conserved domain analysis of the protein candidates along with the locus analysis of their corresponding genes revealed two operons (Dtpsy_1460-1463 and Dtpsy_3433-3438) that could encode key components in the anaerobic nitrate-dependent Fe2+ oxidation pathway. An outer membrane efflux pump protein complex encoded by the Dtpsy_1460-1463 operon could play a role in the exportation of periplasmic-accumulated Fe3+ as a detoxification procedure. In addition, a putative ferric reductase protein Dtpsy_3433 and cytochrome reductase-like protein Dtpsy_3436 are likely critical electron transport chain components in this metabolism. Quantitative reverse transcription PCR provided further evidence for the involvement of this operon by demonstrating the transcriptional level up-regulation of the genes in the Dtpsy_3433-3438 operon. This study serves as the first attempt to identify the proteins and genes responsible for anaerobic nitrate-dependent iron oxidation in Acidovorax ebreus strain TPSY. This work has led to the successful identification of a few key proteins and genes responsible for anaerobic nitrate-dependent Fe2+ oxidation, thus providing information important for the elucidation of other components in this electron transport pathway. SECTION 2 Perchlorate is a wide-spread contaminant detected in drinking water and ground water systems in the United States. The current development of a highly sensitive enzymatic bioassay for in situ perchlorate concentration quantification created a need for high-quality and low-cost perchlorate reductase. Perchlorate reductase, originally isolated from DPRB (dissimilatory perchlorate reducing bacteria), is encoded by an operon containing four genes, pcrABCD. Enzymatically active perchlorate reductase purified by traditional methods is composed of two structural subunits, PcrA and PcrB, encoded by the pcrA and pcrB genes, respectively. The lengthy traditional protein purification process and the slow growth rate of DPRB hinder the industrial mass production of this enzyme. In this study, we report an attempt to use E. coli host to overexpress perchlorate reductase and use a polyhistidine tag to enable ease of the subsequent purification. The pcrAB genes encoding the structural subunits of perchlorate reductase were cloned into an expression vector in E. coli. The purification of the recombinant perchlorate reductase was performed under strict anaerobic and denaturing conditions and a highly purified form of the enzyme was obtained. Possible solutions to avoid the formation of inclusion bodies while still maintaining the enzyme activity were discussed. This work proved the feasibility of recombinant perchlorate reductase expression using an E.coli host and the usefulness of the histidine tag in the purification process. In addition, this work provided insights into factors that need to be taken into future consideration in order to obtain the recombinant enzyme with full enzymatic activity. As a final goal, this study will contribute to the development of enzyme-based bioassay for the detection of perchlorate in the environment by lowering the production and purification cost of its key component, the perchlorate reductase.
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Sharar, Mona [Verfasser], Maria [Gutachter] Montes-Bayon, Michael G. [Gutachter] Weller e Michael W. [Gutachter] Linscheid. "Molecular and Elemental Mass Spectrometric Approaches for Monitoring Oxidation Processes in Proteins / Mona Sharar ; Gutachter: Maria Montes-Bayon, Michael G. Weller, Michael W. Linscheid". Berlin : Humboldt-Universität zu Berlin, 2017. http://d-nb.info/1189326604/34.
Testo completo
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Šiurkus, Juozas [Verfasser], e Peter [Akademischer Betreuer] Neubauer. "Approach for production of sensitive to oxidation and aggregating proteins in E. coli at the example of a heterologous ribonuclease inhibitor / Juozas Šiurkus. Betreuer: Peter Neubauer". Berlin : Universitätsbibliothek der Technischen Universität Berlin, 2013. http://d-nb.info/1031075208/34.
Testo completo
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Liu, Jing. "EFFECT OF AMYLOSE AND PROTEIN OXIDATION ON THE THERMAL, RHEOLOGICAL, STRUCTURAL, AND DIGESTIVE PROPERTIES OF WAXY AND COMMON RICE FLOURS AND STARCHES". UKnowledge, 2013. http://uknowledge.uky.edu/animalsci_etds/23.
Testo completoAbstract (sommario):
The effects of oxidation by sodium hypochlorite (0, 0.8, 2, and 5%, NaOCl), the presence of endogenous proteins, and amylose content on waxy and common rice flours (WF, CF) and starches (WS, CS) were investigated in terms of in vitro starch digestibility, morphology and surface properties, and thermal and rheological characteristics.
The concentration of NaOCl had an effect on all the samples including WF, CF, WS, and CS. The carbonyl and carboxyl group contents increased up to 25 and 10 folds (P < 0.05) of oxidized starches (WS, CS), respectively. Only mild oxidation (P < 0.05) occurred in flours (WF, WS). In addition, endogenous proteins were oxidized according to amino acid analysis and SDS–PAGE results. Glu+Gln, Gly, His, Arg, Tyr, and Lys were more sensitive to NaOCl oxidation. Disulfide bonds, hydrophobic force, and hydrogen bonds were involved in protein polymerization after NaOCl oxidative modification. In granular state, the in vitro starch digestibility of WF, WS, and CS decreased by 5% NaOCl oxidation. After gelatinization, only 2 and 5% oxidized WS had lower digestibility.
Scanning electron microscopy and confocal laser scanning microscopy further demonstrated that protein existed on the surface of starch granules and had aggregation by oxidation. X-ray diffraction patterns showed the crystallinity of 5% oxidized flours and starches was reduced compared with all their non-oxidized samples.
Thermal and rheological properties were analyzed by differential scanning calorimetry and rheometer, respectively. Starch gelatinization peak temperature of flours (WF, RF) was increased by 3 °C, but starches (WS, CS) had a significantly decrease by 8 °C. Viscoelastic patterns were dramatically changed by oxidation. Oxidized WF and CF had increased in both viscosity and elasticity by oxidation, whereas both WS and CS had significantly lower viscoelasticity after oxidative modification.
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Polander, Brandon C. "The hydrogen-bonded water network in the oxygen-evolving complex of photosystem II". Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/50222.
Testo completoAbstract (sommario):
Protein dynamics play a key role in enzyme-catalyzed reactions. Vibrational spectroscopy provides a method to follow these structural changes and thereby describe the reaction coordinate as a function of space and time. A vibrational spectroscopic technique, reaction-induced FTIR spectroscopy, has been applied to the study of the oxygen-evolving complex (OEC) of photosystem II (PSII). In plant photosynthesis, PSII evolves oxygen from the substrate, water, by the accumulation of photo-oxidizing equivalents at the OEC. Molecular oxygen and protons are the products of this reaction, which is responsible for the maintenance of an aerobic atmosphere on earth. The OEC is a Mn4CaO5 cluster with nearby bound chloride ions. Sequentially oxidized states of the OEC are termed the S states. The dark-stable state is S1, and oxygen is released on the transition from S3 to S0. Using short laser flashes, individual S states are generated, allowing vibrational spectroscopy to be used to study these different oxidation states of the OEC. In current X-ray crystal structures, hydrogen bonds to water molecules are predicted to form an extensive network around the Mn4CaO5 cluster. In the OEC, four peptide carbonyl groups are linked to the water network, which extends to two Mn-bound and two Ca-bound water molecules. This dissertation discusses a vibrational spectroscopic method that uses these peptide carbonyl frequencies as reporters of solvatochromic changes in the OEC. This technique provides a new, high-resolution method with which to study water and protein dynamics in PSII and other enzymes.
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Wang, Wenzhong. "Mechanistic studies of flavoenzymes in fatty acid oxidation and oxidative protein folding". Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 233 p, 2007. http://proquest.umi.com/pqdweb?did=1362529911&sid=3&Fmt=2&clientId=8331&RQT=309&VName=PQD.
Testo completo
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Johansson, Patrik. "Structural Studies of a Xyloglucan Endotransglycosylase from Populus tremula x tremuloides and Three Conserved Hypothetical Proteins from Mycobacterium tuberculosis". Doctoral thesis, Uppsala universitet, Institutionen för cell- och molekylärbiologi, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6738.
Testo completoAbstract (sommario):
This thesis describes the structural studies of four different proteins from two organisms. Xyloglucan endotransglycosylases, XETs, are involved in plant cell wall expansion and remodeling by splitting and reconnecting xyloglucan-cellulose crosslinks. The first crystal structure of a XET enzyme has been determined to 1.8 Å. The structure provides insights into how XETs are able to bind a heavily branched xyloglucan sugar, as well as hints about the XET-transglycosylation mechanism. Mycobacterium tuberculosis (Mtb) is the cause of enormous human mortality each year. Despite the sequencing of the complete Mtb-genome, the biological function of a large fraction of the M. tuberculosis proteins is still unknown. We here report the crystal structures of three such proteins, Rv2740, Rv0216 and Rv0130. Rv2740 forms a Cystatin α+b fold with a deep active site pocket similar to a limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis. However, in contrast to the small limonene-based substrate of the Rhodococcus enzyme, Rv2740 is able to degrade large fatty acid and sterol epoxides, giving suggestions for the physiological substrates of this enzyme. The structure of M. tuberculosis Rv0216 exhibits a so-called double hotdog fold. Rv0216 shows similarity to a number of enzymes using thiol esters as substrates, including several R-enoyl hydratases and β-hydroxyacyl dehydratases. However, only parts of the hydratase / dehydratase catalytic site are conserved in Rv0216. Rv0130 in contrast, contains a highly conserved R-hydratase motif, housed in a dimer of two single hotdog folded molecules. This active site is situated in a long tunnel, formed by a sharp kink in the Rv0130 central helix. A number of previously predicted single / double hotdog folded proteins from M. tuberculosis seem to feature a similar substrate-binding tunnel, indicating that Rv0130 as well as some of these proteins, might act on long fatty enoyl chains.