Dissertations / Theses on the topic 'Iron proteins – Analysis'

Precisely how Hephaestin (Heph) facilitate iron release from cells is poorly understood. The work in this thesis tried to establish the role of different iron metabolic proteins, Mitoferrin (Mfrn), IRPs and Heph in iron homeostasis. Analysis of 18 tagSNPs in the Mfrn gene was carried out in an AsianCaucasian population to establish any correlation between the Mfrn tagSNPs, haemoglobin levels and birth weight in the presence of covariates such as sex of the fetus, gestational age and mother's booking weight. Two-way ANCOVA analysis was carried out to check if the covariates have any influence on the dependent variable in the presence of fixed factors. From the ANCOVA analysis of Mfrn tagSN Ps it can be concluded that neither the haemoglobin levels nor the birth weight are dependent on the genotype, fetal sex, nor on their interaction. Owing to the significance in identifying the interacting partners of IRPs and Heph to understand more about their role in iron metabolism, protein-protein interaction studies were also carried out. IRPs and Heph genes were successfully cloned with One-Strep tag. Full length clones were sequence confirmed for any variation after PCR. Before carrying out immunoprecipitation to identify the interacting partners, transfection efficiency, viability and the role of magnetic particles on K562 cells was performed by using IRPs and Heph cloned with One-Strep tag. Lipofectamine-L TX plus transfection had more viable cells and higher efficiency compared with magnetic-assisted transfection . Also, this study confirms that magnetic nanoparticles do not have any adverse or significant effect on IRPs during the transfection. An unsuccessful attempt was made to identify the interacting partners of IRPs and Heph by immunoprecipitation. The current thesis work also involved identification of a potential ferroxidase . Ceruloplasmin (Cp) was used as a postive control. Non-denaturing gel eletrophoresis of the K562, MDA-MB-231 and PNT2-C2 cell fractions confirmed the presence of the extra band establishing the ubiquitous nature of the band. Mass spectrometry analysis identified the excised band as Calreticulin (CALR). This is the first report of calreticulin having ferroxidase activity.

Iron is an essential nutrient for growth of photosynthetic microorganisms such as cyanobacteria and algae. Iron is required for proteins involved in the important processes of carbon and nitrogen assimilation. Low concentrations of iron in cultures or natural waters can lead to iron limitation which affects many aspects of algal metabolism. In natural waters, iron limitation can have effects on the patterns and rates of primary productivity. The cellular content of certain proteins can be affected by media iron concentrations. Methods have been used that assay components of the cell as an indirect measure of iron nutritional status. For example, spectroscopy can be performed to determine the cellular concentration of iron-containing proteins involved in photosynthesis. Organisms grown in media that imitate natural conditions, or organisms collected from their natural habitat are usually dilute. Methods that assay iron nutritional status such as spectroscopy and column chromatography require large sample sizes which are difficult to obtain from natural samples. In addition, methods that utilize techniques such as immunology or radioactive labelling are complex and time-consuming. These considerations led to the necessity of developing a technique that would be simple, rapid and effective on dilute samples. The method developed here utilized fast protein liquid chromatography (FPLC), which fulfilled these requirements. A complete analysis could be done within two to three hours with minimal sample treatment. The FPLC was simple to operate and was effective on a sample containing less than 100 μg of protein. Some photosynthetic organisms, when iron-depleted, can produce the flavin-containing protein flavodoxin (Flv). This protein substitutes for the iron-containing protein ferredoxin (Fd) in Fd-dependent reactions such as the light-induced reduction of NADP. The FPLC technique identified and quantified, in relative terms, Fd and Flv in the cell. Optical spectroscopy was used to verify FPLC retention time assignments. The results illustrated how the FPLC could be used to observe the changes in relative Fd and Flv content as a function of media iron concentration in cultures of the cyanobacterium Anabaena grown in the laboratory. It was found that Fd content decreased and Flv content increased with decreasing media iron concentration. In addition, samples of the cyanobacterium Trichodesmium collected from the ocean near Barbados were analyzed using FPLC to assay relative Fd and Flv content. By analogy with Anabaena, Fd and Flv retention times were identified. Using this technique conclusions could be drawn regarding the changing iron nutritional status of Trichodesmium in its natural habitat .

Metallic flavors are of concern for many industries including food, health, and water. Metallic off-flavor, induced by ferrous sulfate solution (10mg/L), and its remediation using pre- and post-rinse treatments of water (control) or metal chelators, were studied. Metal chelators included lactoferrin (1 ?M), a natural metal-binding protein in milk and saliva, and EDTA (36 ?M), a synthetic chelator. Time-intensity (TI) evaluation (n=6, 4 female; age 40-70) of lingering metallic flavor indicated that metallic flavor decreased with a post-rinse adjuvant treatment of lactoferrin as indicated by a reduced maximum intensity and area under the curve compared to a pre-rinse treatment; EDTA and water post-rinses were equally effective for three of the TI parameters. Alterations in salivary components were studied in saliva collected (n=8; 5 female, age 40-70) after sipping a lactoferrin solution (1?M) followed with a ferrous sulfate sample (10 mg/ml) to stimulate metallic flavor, as compared to unstimulated whole saliva. Protein concentration, oral lipid oxidation as indicated by thiobarbituric acid reactive substances assay, and iron concentration were determined on individual saliva samples, with no significant differences found between treatments (p>0.05). Protein patterns were qualitatively characterized for each pre-rinse and metallic stimuli from four panelists by two-dimensional gel electrophoresis. A consistent pattern of regions containing major salivary components was observed. This research has shown that lactoferrin protein is a potential natural alternative to synthetic EDTA for reducing iron-induced metallic off-flavors. This study provides a foundation of method development to better understand salivary protein interaction with metals and flavor perception.
Master of Science in Life Sciences

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.

Sample preparation as an essential step in mass spectrometry-based analysis, plays a critical role in proteomics studies. Magnetic nanoparticles (MNPs) have been widely used in protein and peptide sample preparation due to their magnetic properties, biocompatibility, easy synthesis and surface functionalization. MNPs loaded with analyte or analyte modification reagent can be easily separated from the reaction medium by an externally applied magnetic field. The small size of MNPs provides high analyte loading and extraction capacity. Additionally, MNP can be decorated with different functional groups to achieve selective modification or extraction of analyte. In this study we have utilized silica coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs) for protein and peptide sample preparation. Fluorescence-based methods were utilized for quantitative and qualitative characterization of N-hydrosucccinimidyl (NHS) ester groups on the surface of Fe3O4@SiO2 MNPs. Fluorophore Dansylcadaverine was conjugated to NHS ester functional groups. Fluorometric measurement of cleaved dansylcadaveine was employed to determine the number of NHS ester groups per MNPs that was found to be 2.6 × 102 and 3.4 × 103for 20 nm and 100 nm Fe3O4@SiO2 MNPrespectively. The efficiency of labeling native bovine serum albumin (BSA) by NHS ester coated Fe3O4@SiO2 MNPs was also explored in terms of maximizing the number of MNPs conjugated per BSA molecule or maximizing the number of BSA molecules conjugated per each MNP. Lysine residues of apolipoprotein B-100 (apoB-100) on the surface of intact human low density lipoprotein (LDL) were labeled by NHS ester modified Fe3O4@SiO2 MNPs in aqueous solvents at room temperature. The MNP labeledapoB-100 was treated by SDS to remove lipids and then digested using trypsin. Tryptic peptides were eluted from MNPs by cleaving disulfide linkage between labeled peptides and MNPs. LC-MS/MS analysis found 28 peptides containing labeled lysine residues. These lysine residues should be on the solvent exposed surface of LDL since the large size of MNPs prevents contact of the labeling reagent to those lysines embedded inside the structure of LDL. TCEP- immobilized Fe3O4@SiO2MNPs were fabricated and utilized for reduction of disulfide bonds in bovine pancreas insulin and two different cyclic peptides. Disulfide bonds were efficiently cleaved at room temperature in both organic and aqueous solvents confirmed by LC-MS/MS analysis of reduced/alkylated protein and peptides. Disulfide reduction and alkylation reactions was performed in one step and the reducing agent was simply separated from peptide and protein solution by magnetic separation.

The transferrin iron acquisition system of Neisseria consists of two dissimilar proteins, transferrin binding protein A and B (TbpA and TbpB). TbpA and TbpB both specifically and independently bind human transferrin (Tf). TbpA is a TonB-dependent transporter, expression of which is necessary for Tf iron acquisition. In contrast, the lipoprotein TbpB is not necessary for iron internalization; however it makes this process more efficient. The role of TbpB in the transferrin iron acquisition system has not been completely elucidated. It has been suggested that TbpB is entirely surface exposed and tethered to the outer membrane by its lipid moiety. We inserted the hemagluttinin antigen (HA) epitope into TbpB in an effort to examine surface accessible and functional domains of the lipoprotein. We determined that TbpB was entirely surface exposed from just beyond the mature N-terminus. It was previously reported that the N- and C-terminus of TbpB independently bind Tf. HA epitope analysis defined both the N-terminal and C-terminal binding domains. TbpB was previously reported to play an important role in the release of Tf from the receptor. We established that TbpB exhibited a biphasic dissociation pattern; a C-terminal rapid release followed by a slower N-terminal release. These results suggested that the C-terminus plays a role in ligand turnover of the wild-type receptor. Little is known about the transport of TbpB to the outer membrane. In an attempt to identify the signals/mechanisms required for TbpB localization, the signal sequence of the protein was altered. In the absence of lipid modification, TbpB remained associated with the cell, localized to the periplasm. We also noted that internal cysteine residues were not critical for TbpB localization. Our results suggested that TbpB was transported by a lipoprotein-specific mechanism. Additionally, we demonstrated the major outer membrane secretin, PilQ, was not necessary for proper localization of TbpB. The mechanism responsible for this process remains elusive. This body of work represents the first comprehensive study of TbpB topology and function, utilizing the lipoprotein expressed in its native membrane. These results may translate to other, similar lipoprotein receptors of the pathogenic Neisseria, helping to shed light on these poorly understood proteins.

One of the most challenging goals in nanoparticle research is to develop successful protocols for the large-scale, simple and possibly low-cost preparation of morphologically pure nanoparticles with enhanced properties. The work presented in this thesis was focused on the synthesis, characterisation and testing of magnetic nanoparticles and their potential applications. There are a number of magnetic nano-materials prepared for specific applications such as metal oxide nanoparticles encapsulated with various porous materials including Fe₃O₄/Fe₂O₃ coated with soft bio-organic materials such as glycol chitosan and bovine serum albumin and hard materials such as silica (SiO₂) and zinc sulphide (ZnS). The preparation of these materials was achieved principally by bottom-up methods with different approaches including micro-emulsion, precipitation, electrostatic and thermolysis processes. The thesis also presents the uses of various analytical techniques for characterising different types of nano-materials including Attenuated Total Reflection Fourier Transformer Infrared Vibrational Spectroscopy (ATR-FTIR), Ultraviolet Visible- Near Infrared (UV-Vis-NIR) Spectroscopy, Zeta Potentiometric Surface Charge Analysis, Superconducting Quantum Interference Device (SQUID) and Vibration Sample Magnetometry (VSM) for magnetic analysis and powder X-Ray Diffraction (XRD) for crystallographic pattern analysis. There are many applications of magnetic nanoparticles, including nano-carriers for biological and catalytic reagents. The magnetic nanoparticles can facilitate separation in order to isolate the carriers from solution mixtures as compared to many inefficient and expensive classic methods, which include dialysis membrane, electrophoresis, ultracentrifugation, precipitation and column separation methods. There are six key chapters in this thesis: the first chapter introduces the up-to-date literature regarding magnetic nano-materials. The uses of magnetic nano-materials in drug binding and for protein separation are discussed in the second and third chapters. The fourth chapter presents the use of magnetic nanoparticle in conjunction with a photo-catalytic porous overlayer for the photo-catalytic reduction of organic molecules. The fifth chapter describes different analytical techniques used for the characterisation of nanoparticles and the underlying principles and the experimental details are also given. The sixth chapter summarises the results and provides an overview of the work in a wider context of future applications of magnetic nanoparticles.

La realisation de mesures d'aimantation a saturation est, dans le cas des proteines a site actif binucleaire a fer non heminique ou a manganese, source d'informations structurales. En effet, de telles mesures permettent de determiner l'interaction d'echange j entre les ions du site actif, parametre qui est correle avec la nature des ligands pontant le site. Au cours de ce travail, nous decrivons tout d'abord le protocole a suivre pour realiser de telles mesures. Nous appliquons ensuite ce protocole a deux proteines a site actif binucleaire a fer non heminique: la ribonucleotide reductase de souris et la methane monoxygenase de methyloccocus capsulatus et a une proteine a site actif binucleaire a manganese: la catalase de thermus thermophilus. Comme attendu, la nature des ligands pontant le site actif de ces proteines est precisee grace a la determination de l'interaction d'echange j entre les ions du site. Mais, dans le cas des sites a fe#2#+ et surtout a mn#3#+, l'evaluation du parametre d'ecart en champ nul axial d associe a chaque ion du site permet d'obtenir des informations structurales supplementaires. Des mesures d'aimantation a saturation sont en outre utilisees pour caracteriser, en complement avec la rmn, les proprietes magnetiques d'analogues synthetiques a valence mixte des proteines a site actif binucleaire a fer non heminique

Face au besoin croissant de nouvelles molécules bioactives d’origine naturelle, les coproduits de l’industrie alimentaire et de transformation d’agroressources constituent une ressource stratégique à exploiter. En effet, l’hydrolyse enzymatique de protéines végétales ou animales permet de générer une grande variété de séquences peptidiques avec des propriétés biologiques potentielles : antihypertensive, antithrombotique, anticancéreuse, opioïde, antimicrobienne. Malgré le potentiel bioactif de certains peptides, leur présence incertaine et leur faible concentration au sein d’un hydrolysat protéique (mélange complexe constitué d’une dizaine à parfois plus d’une centaine de peptides) limitent leur purification et leur exploitation. Aussi, les peptides bioactifs pourraient être criblés préalablement à toute phase séparative afin de n’engager l’étape de séparation qu’en cas d’activité avérée. Le pouvoir antioxydant est un terme générique qui regroupe divers mécanismes chimiques tels que l’activité anti-radicalaire, l’inhibition de la peroxydation des lipides, ou encore la chélation de métaux. En chélatant les métaux de transition naturellement présents in vivo (fer, cuivre), les peptides chélateurs pourraient être utilisés comme antioxydants indirects et agir ainsi contre le stress oxydant. L’objectif principal de cette thèse est de développer des méthodes originales de criblage à haut débit des peptides chélateurs de fer présents dans des hydrolysats peptidiques. A terme, ces méthodes pourraient être appliquées à tous types de mélanges peptidiques complexes. La première approche développée met en œuvre la chromatographie d'affinité pour ions métalliques immobilisés (IMAC). Cette technique est incontournable pour purifier des peptides chélateurs de métaux au sein des hydrolysats. Grâce à la spécificité d’interaction entre un métal donné – immobilisé sur la phase stationnaire IMAC – et des motifs complexants déterminés, il est possible d’identifier sélectivement les composés chélateurs présents dans des mélanges complexes. Notre objectif étant de parvenir à une détection rapide de ces molécules d’intérêt, nous avons réalisé un couplage en ligne avec la spectrométrie de masse (MS). La deuxième stratégie consiste à évaluer la formation des complexes fer-peptide en solution. Dans ce cas, tous les sites accepteurs d’électrons du métal sont accessibles (au contraire de la technique IMAC qui présente un biais potentiel de ce point de vue) et, d’autre part, les conditions de solubilisation peuvent simuler le milieu visé (i.e. le milieu intracellulaire). Par ailleurs, l’observation de la forme complexée avec le fer (FeII ou FeIII) fournit une preuve directe et irréfutable de la capacité de chélation d’un peptide. Ainsi, la mise en évidence d’un peptide chélateur peut être réalisée par détection concomitante de sa forme libre (peptide seul) et de sa forme complexée (fer-peptide). Dans cette approche, la spectrométrie de masse – de par sa sensibilité et sa spécificité – est une technique de choix pour réaliser le criblage souhaité. Après avoir été testés sur des peptides des synthèse (sous forme pure et en mélange), les deux protocoles ont été appliqués à un hydrolysat protéique réel. Les résultats préliminaires obtenus sont prometteurs et permettent d'envisager, à court terme, le criblage automatisé de divers hydrolysats réels, pour la recherche de peptides chélateurs du fer(II) et du fer(III)
Faced with the growing need for new bioactive compounds of natural origin, by-products from the agro-food industry and the processing of agro-resources constitute a strategic resource to be exploited. In fact, the enzymatic hydrolysis of plant or animal proteins makes it possible to generate a wide variety of peptide sequences with potential biological properties: antihypertensive, antithrombotic, anticancer, opioid, antimicrobial. Despite the bioactive potential of certain peptides, their uncertain presence and their low concentration in a protein hydrolysate (a complex mixture sometimes made up of more than a hundred peptides) limit their purification and use. Also, bioactive peptides could be screened before their purification in order to initiate the separation step only if activity is proven. Antioxidant power is a generic term which groups together various chemical mechanisms such as anti-free radical activity, inhibition of lipid peroxidation, or even metal chelation. By chelating the transition metals naturally present in vivo (iron, copper), the chelating peptides could be used as indirect antioxidants and thus act against oxidative stress. The main objective of this PhD thesis is to develop original methods for high throughput screening of iron-chelating peptides present in protein hydrolysates. Ultimately, these methods could be applied to all types of complex peptide mixtures. The first approach is based on immobilized metal affinity chromatography (IMAC). IMAC is a reference technique for purifying metal-chelating peptides in hydrolysates. Thanks to the specificity of interaction between a given metal – immobilized on the stationary phase IMAC – and determined complexing groups, it is possible to selectively identify the chelators present in complex mixtures. Our objective being to achieve a rapid detection of these molecules of interest, we carried out an on-line coupling with mass spectrometry (MS). The second strategy consists of evaluating the formation of iron-peptide complexes in solution. In this case, all the electron acceptor sites of the metal are accessible (unlike the IMAC technique which presents a potential bias from this point of view) and, on the other hand, the solubilization conditions can simulate the target medium (i.e. the intracellular medium). In addition, the observation of the peptidic form complexed with iron (FeII or FeIII) provides direct and irrefutable proof of the chelating capacity of a peptide. Thus, the identification of a chelating peptide can be carried out by the concomitant detection of its free form (peptide) and of its complexed form (iron-peptide). In this approach, mass spectrometry – thanks to its sensitivity and its specificity - is a technique of choice for carrying out the desired screening. After having been tested on synthetic peptides (pure solutions and mixture), the two protocols were applied to a real protein hydrolysate. The preliminary results are promising and make it possible to envisage, in the short term, the automated screening of various real hydrolysates for the search for iron(II)- and iron(III)-chelating peptides

Thesis (Ph.D.)--State University of New York at Buffalo, 2005.
Title from PDF title page (viewed on Mar. 21, 2006) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Kosman, Daniel J. Includes bibliographical references.

碩士
國立臺灣師範大學
化學系
98
Conductive-mode and magnetic-mode atomic force microscopic techniques (C-AFM and M-AFM) are potential tools for protein image mapping, and iron-storage protein, such as ferritin (FT), is an ideal model for such a study. As FT was subjected to C-AFM analysis, it showed 10 nm for its diameter and five layers, ~20 Å each in segregation, symmetrically distributed around the iron core, matching well with its 3D model. We also conducted M-AFM for structural comparison. Experimental results revealed that FT showed only vague image at lower lift height (~1 nm). Nevertheless, as it was subjected to electric bias, the image was greatly enhanced; the phase shift increased linearly with the amplitude of the applied bias. Noticeably, the resulting image was ~40 nm larger than that from the C-AFM counterpart. We attributed the discrepancy to the long range interaction between the magnetic moments of the probe and the substrate. Despite this, the interaction could in turn promote the phase shift of FT on ITO. We also characterized the electron tunneling in ferritin. The energy barrier for electrons to travel in the protein was about 2 eV, and the speed was 10-3~10-2 the speed of free electrons.

Iron-sulfur clusters, an important class of redox active cofactors, are ligated by protein-based Cys ligands in a variety of nuclearities. Traditionally, these clusters serve as one-electron transfer units, though many clusters are capable of catalytic activity and sensing functions. Recently, a greater number of iron-sulfur clusters with non-Cys ligation have been identified, wherein one or more of the Cys ligands are replaced by an alternative amino acid residue such as His or Asp. In most cases the role of this ligand substitution is unknown. Some hypotheses are that non-Cys ligation may modify reduction potential, allow for proton-coupled electron transfer, or modulate cluster stability. The human mitoNEET protein contains a 1-His, 3-Cys ligated [2Fe-2S] cluster, identified by the presence of a CDGSH peptide motif. MitoNEET is a binding target for the type II-diabetes drug, pioglitazone, and is implicated in controlling mitochondrial iron levels. How exactly mitoNEET functions in the cell is unknown, as is the role its uniquely ligated FeS cluster may play. This thesis uses mitoNEET as a model for the study of non-Cys ligated FeS clusters and their biological function. Protein film voltammetry was used to examine the pH-dependent electrochemical properties of the mitoNEET cluster, indicating that multiple as yet unidentified protonations control redox potential and that drug binding impacts cluster reduction and protonation. Additionally, the effect of reduction and protonation on cluster and protein structure instability was examined through absorbance and circular dichroism measurements, suggesting an important role for cluster lability in protein function. The CDGSH-motif family of [2Fe-2S] cluster-binding proteins was examined using protein similarity networks. This technique highlights the evolutionary relationship among these proteins, and has led to further work examining the DUF1271 domain containing proteins E. coli YjdI and A. vinosum Alvin0680 (a CDGSH-DUF1271 fusion). This work furthers the scientific knowledge of non-Cys ligated Fe-S clusters by improving our understanding of how the mitoNEET His-ligand contributes to proton-coupled electron transfer and cluster instability, and how the broader class of CDGSH-motif proteins is organized.

Iron is one of the most important micronutrients used by living organisms. Iron is frequently a limiting nutrient for plant growth, and plants are a major source of iron for human nutrition. The most prominent symptom of iron deficiency in plants is interveinal chlorosis, or yellowing between the veins, which appears first in the youngest leaves. Iron deficiency anemia (IDA) is the number one human nutritional deficiency worldwide. In order to solve the problem of iron deficiency, it is desirable to breed plants that have increased iron in those parts that are consumed by humans. To do this, we must first understand the molecular basis of Fe uptake, transport, and storage in plants. In soil, iron is quickly oxidized to Fe(III), and Fe(III) is relatively insoluble, thus difficult for plants to obtain. Our lab has been working on metal ion homeostasis mechanisms in plants and the ultimate goal of our research is to understand the mechanisms by which plants maintain the correct levels of iron, zinc and copper in each cell and tissue. The Yellow Stripe-like (YSL) family of proteins has been identified based on sequence similarity to maize Yellow stripe 1 (YS1). YS1 transports Fe(III) that is complexed by phytosiderophores (PS), strong Fe(III) chelators of the mugineic acid family of compounds. Non-grass species of plants neither make nor use PS, yet YSL family members are found in non-grass species including Arabidopsis thaliana. YSLs in non-grasses have been hypothesized to transport metals that are complexed by nicotianamine (NA), an iron chelator that is structurally similar to PS and which is found in all higher plants. In this dissertation, Arabidopsis YSL1 and YSL3 are demonstrated to be important in iron transport and also responsible for loading Fe, Cu, and Zn from leaves into seeds. Arabidopsis YSL4 and YSL6 are demonstrated to be involved in iron transport and metal mobilization into seeds. The transport function of Arabidopsis YSL1 and YSL2 are shown be partially overlapping to the function of Arabidopsis YSL3 in vegetative structures, but distinct in reproductive organs. Arabidopsis YSL3 and YSL6 are shown to have distinct functions in planta.

The thesis "The proteomic analysis in hematology: Identification of alfa2- macroglobulin as a specific carrier for the hormone hepcidin and proteomic analysis of the leukemic K562 cell differentiation induced by sodium butyrate" describes proteomic approaches, used for the identification and functional characterisation of proteins, which are binding and transporting the iron metabolism regulating hormone hepcidin. Proteomic techniques are also exploited for the identification of proteins, participating in erythroid differentiation of the model cell line K562. In the first section of the thesis, non-denaturing, native techniques, such as chromatography and native electrophoresis are used, in the second section, the control and butyrate - induced K562 cell proteomes are compared using the classical 2D - SDS polyacrylamide gel electrophoresis approach. The methods, described in the thesis are broadening the spectrum of available techniques in experimental hematology. The results, described in this thesis together with the accompanying published manuscripts broaden our knowledge in the function of proteins of iron metabolism and proteins, functioning in erythroid differentiation. Key words: proteomic analysis, hepcidin, alfa2-macroglobulin, iron metabolism, CML, K562, sodium butyrate