Doctor of Philosophy, The Ohio State University, 2004, Chemistry

The processes of iron-sulfur cluster transfer from holo ISU and ISA to apo Fd were studied. Direct cluster transfer from holo ISU to apo Fd was demonstrated by Mossbauer spectroscopy. Experimental data shows that an intact [2Fe-2S] core is transferred to target proteins. Substitution of a key aspartate residue (Asp 37) of ISU is found to decrease the rate of cluster transfer by at least an order of magnitude. The rate constants of cluster transfer from native ISA to Fd are shown to be similar to those of cluster transfer from Cys derivatives of ISA. The factors that contribute to cluster transfer efficiency also appear to be similar, including the importance of cluster lability and solvent accessibility. Both ISA- and ISU-mediated Fe-S cluster transfer to apo Fd have been shown to be pH-dependent. The pKa of ISA is 7.8, slightly higher than the value of 6.9 found for ISU. This is consistent with deprotonation of a solvent accessible cysteine which would thereby provide a more effective nucleophilic center for facilitating cluster transfer to apo Fd. Viscosity studies show that diffusion is not the rate-limiting step, which is most likely the process of Fe-S cluster transfer between the intermediate complexes, ISU-Fd and ISA-Fd. The influences of the binding of DnaK to IscU were studied. DnaK can maintain a stable structure of ferric ion-bound IscU and avoids aggregation triggered by ferric ions. Also, DnaK-bound IscU has more stable iron sulfur clusters. Kinetic studies of Fe-S cluster transfer from holo IscU to apo Fd in the presence of DnaK reveal that the binding of DnaK to IscU slows down the rate of Fe-S cluster transfer from IscU, especially formation of IscU-Fd complexes. Binding of DnaK decreases the rates of forming IscU-Fd complexes (greater than 8-fold) and also causes a minor change on the rates of iron sulfur cluster transfer in IscU-Fd complexes. In other words, binding of DnaK to IscU does not facilitate the process of Fe-S cluster transfer from IscU.

Committee: James Cowan (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy, The Ohio State University, 2017, Biochemistry Program, Ohio State

Metals play diverse, yet essential roles in biological systems, especially the transition metals with their unique chemical properties. These versatile properties enable varied function as cofactors of biological macromolecules, but also require a high level of regulation, since perturbed concentrations of metal ions can result in toxic cellular consequences. Despite their essential nature, many of the processes of metallocofactor biosynthesis, trafficking, and incorporation are not well characterized; furthermore, misregulation of pathways responsible for metal ion incorporation often results in disease, and an in depth understanding of these pathways is key for treatment of disease states. This study focuses on a particular metal cofactor, iron-sulfur (Fe-S) clusters to elucidate mechanisms of cluster biogenesis, trafficking, and the molecular basis for disease. Fe-S cluster proteins constitute the largest class of metalloproteins and demonstrate varied functional roles, which include electron transport, transcriptional and translational regulation, substrate binding and catalysis, structural stabilization, and DNA-mediated charge transfer. The complex process of bringing together free iron and sulfide ions has been investigated in depth and is the first step in the de novo production of Fe-S clusters; however, the downstream maturation and delivery of Fe-S clusters to target proteins is not fully understood. The Fe-S cluster protein Nfu has been the central focus of this work, as its function in the process of cluster biogenesis is unclear. Furthermore, mutations on Nfu result in the condition of Multiple Mitochondrial Dysfunctions Syndrome (MMDS). A detailed characterization of this protein was conducted to establish cluster binding properties and elucidate potential partners in cluster trafficking via kinetic and spectroscopic means. This examination of the Nfu protein (also termed NFU1) was extended to demonstrate its role as an alternative scaffold for Fe (open full item for complete abstract)

Committee: James Cowan (Advisor); Ross Dalbey (Committee Member); Jane Jackman (Committee Member); Hannah Shafaat (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, The Ohio State University, 2011, Biochemistry Program, Ohio State

Fe/S cluster proteins are key players in diverse pathways, such as mitochondrial respiration, gene regulation, and DNA/RNA metabolism. It is generally believed that an Fe/S cluster is synthesized inside the mitochondria before it is exported out to the cytosol. But the details of this export pathway are still unclear; especially what is the substrate that is exported. This substance should be essentially small enough to go through the exporter Atm1p, stable enough to survive the transport process and labile enough to deliver the cluster to cytosolic proteins. It has been shown that glutathione (GSH) or its derivatives might be involved in this pathway. Several Glutaredoxin protein (Grx) structures revealed that Grx is able to coordinate a Fe/S cluster along with two molecules of GSH. The cluster transfer between Grx and scaffold protein ISU was found to be reversible, which indicates Grx may deliver the cluster to the export pathway and plays important role in Fe/S cluster biosynthesis. However, the role of this cluster is still not well understood. A complex of four GSH-coordinated Fe/S cluster was successfully synthesized and characterized and found to be stable under physiological conditions, but undergoes a reversible exchange with scaffold protein ISU. Considering the high cellular concentration of GSH and the stability of this complex under physiological conditions, this complex may contribute to a labile cellular Fe/S cluster pool. The GSH cluster complex is a very intriguing candidate for the substrate of mitochondrial Fe/S cluster exporter. A liposome system was constructed onto which transporter was successfully reconstituted. The stimulation of proteoliposome ATPase activity by GSH cluster complex indicates that the GSH cluster complex is very likely to be the exported substance. Evidence indicates frataxin to be the general iron donor protein in Fe/S cluster biosynthesis. However, a thorough search of the genomic database shows that based on sequence (open full item for complete abstract)

Committee: James Cowan PhD (Advisor); Ross Dalbey PhD (Committee Member); Karin Musier-Forsyth PhD (Committee Member); Richard Swenson PhD (Committee Member) Subjects: Biochemistry; Biophysics; Cellular Biology; Chemistry

Doctor of Philosophy, The Ohio State University, 2005, Chemistry

Frataxin is a nuclear-encoded mitochondrial protein involved in iron homeostasis in the mitochondrion. Mutations of frataxin, or low cellular levels of frataxin cause Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative disease. Although there are many data indicating a connection between frataxin and iron metabolism in the mitochondrion, the exact function of frataxin is still unknown. Therefore, a variety of biophysical techniques were used to investigate frataxin in order to gain insight into its role in iron metabolism. First, we directly monitored iron binding to frataxin. It was observed that frataxin directly binds 6 ~ 7 irons, with 10 ~ 55 microM dissociation constants (KD), by several independent methods, including fluorescence quenching and ITC (isothermal titration calorimetry). Iron-bound frataxin (holo frataxin) was further observed to form a complex with ISU-proteins with KD ~ 0.15 microM, and a 1:1 stoichiometry. Moreover, holo frataxin-mediated iron-sulfur cluster reconstitution was easily observed by UV-Vis and CD spectrometries with kobs ~ 0.075 min –1. Thus, we confirmed that frataxin delivers iron atoms for mitochondrial iron-sulfur clusters. Heme biosynthesis is the other major iron metabolic process found in the mitochondrion. We also observed complex formation between holo frataxin and ferrochelatase, a protein involved in the final step of heme biosynthesis. Interestingly, the complex formed with high binding affinity, KD ~ 17 nM and 1:1 stoichiometry between holo frataxin and a ferrochelatase dimer (the active form). Ferrochelatase activity was sharply optimized in the narrow range of 1 frataxin per ferrochelatase dimer. This is consistent with the formation of a tight complex, thus confirming that holo frataxin also delivers iron to ferrochelatase for heme biosynthesis. Herein we show that frataxin delivers iron to both ISU proteins and to ferrochelatase for iron-sulfur cluster and heme biosyntheses, respectively. Furthermore, (open full item for complete abstract)

Committee: James Cowan (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy, The Ohio State University, 2003, Ohio State Biochemistry Program

Members of the IscU family of proteins are among the most conserved of all protein groups, extending across all three kingdoms of life. IscU is believed to be involved in iron-sulfur cluster delivery to apo iron-sulfur proteins. However, most of the evidence supporting the function of IscU stems from genetic and cellular biological studies. Therefore, we set out to biochemically characterize human, yeast, and prokaryotic IscU proteins. A variety of spectroscopic techniques were used to evaluate IscU including, UV-visible absorption, Mossbauer, near- and far- UV circular dichroism, mass spectrometry, atomic absorption, and nuclear magnetic resonance. Herein we demonstrate that IscU proteins coordinate reductively labile [2Fe-2S]2+ centers and are capable of mediating delivery of intact cluster to apo protein targets. Furthermore, extensive structural and dynamic data of a hyperthermophilic homologue, Thermotoga maritima IscU, revealed that IscU adopts a mobile molten globule-like state that is vastly different from the previously identified ferredoxin-like fold that has thus far been characterized for other metallochaperones. Such a dynamic molecule may allow for the flexibility that is necessary for the multiple roles of Fe-S cluster assembly, and recognition and delivery of that cluster to a target protein. Additionally, we utilized X-ray crystallography to elucidate a high resolution structure of an oxygen ligated [4Fe-4S] high potential iron protein.

Committee: James Cowan (Advisor) Subjects: Chemistry, Biochemistry

Bachelor of Arts, Miami University, 2011, College of Arts and Sciences - Microbiology

Acinetobacter baumannii is a gram-negative bacterium that causes severe infections in immunocompromised patients, such as newborns, burn patients, and the elderly. Because the bacteria are strongly resistant to antibiotics, there is a dire need to develop new therapeutics to treat A. baumannii infections. Potential targets are proteins involved in bacterial iron metabolism, since iron is an essential micro-nutrient. Accordingly, random insertion mutagenesis analysis showed that the NfuA protein is needed when cells were cultured in the presence of 2,2'-dipyridyl, a synthetic iron chelator that generates iron-limiting conditions, and hydrogen peroxide and cumene hydroperoxide, which were used to mimic oxidative conditions. The role of NfuA was further confirmed by the observation that the genetic complementation of an A. baumannii ATCC 19606T mutant with the parental allele was enough to restore the iron metabolism and oxidative stress phenotypes expressed by the wild-type strain. Electron paramagnetic resonance (EPR) analysis of overexpressed and purified NfuA demonstrated that this protein harbors an iron-sulfur cluster, which is a prosthetic group required in central metabolic processes. Interestingly, the inactivation of NfuA did not affect bacterial growth under non-oxidative and non-chelated conditions and did not impair the ability of the mutant to express the acinetobactin siderophore-mediated iron acquisition system. On the other hand, the ability of the A. baumannii ATCC 19606T nfuA mutant to replicate inside human epithelial cells was significantly impaired when compared with the parental strain. Taken together, these observations suggest that NfuA plays a defined and important role in iron metabolism, resistance to oxidation, and intracellular replication without affecting bacterial iron acquisition processes. By understanding the function of NfuA and its importance to the A. baumannii virulence properties, we will come closer to understanding basic metab (open full item for complete abstract)

Committee: Luis Actis PhD (Advisor); Daniel Zimbler (Committee Member); William Penwell (Committee Member) Subjects: Microbiology

Doctor of Philosophy, Miami University, 2013, Microbiology

Acinetobacter baumannii is an important opportunistic human pathogen that causes severe nosocomial infections. The bacterium must overcome iron starvation and oxidative stress conditions imposed by the host in order to propagate and cause disease. This work further investigates the transport and utilization of iron by A. baumannii, and their involvement in virulence. The transport of iron is an active process and requires energy; A. baumannii ATCC 19606T contains and expresses three gene loci encoding functions in the TonB energy-transducing complex to provide the energy needed for iron transport. Transformation of Escherichia coli KP1344 with plasmids harboring the TonB components of these A. baumannii TonB systems promoted cell growth under iron-chelated conditions, which shows that these systems provide the necessary energy needed for iron acquisition. Inactivation of tonB1 and tonB2 in A. baumannii resulted in growth restriction under iron-chelation, indicating these genes are involved in iron transport. The Galleria mellonella infection model showed that TonB1 and TonB2 are involved, but are not essential for bacterial virulence, indicating A. baumannii carries redundant functional TonBs. Furthermore, TonB2 plays an additional role in the interaction with A549 human alveolar cells. Inactivation of dppA1A2 and dppBC, components of an inner membrane ABC transporter, did not affect growth under iron-chelation, suggesting alternative transport functions. However, inactivation of cirA, which codes for an iron-regulated outer membrane receptor resulted in reduced growth under iron-chelated conditions, indicating CirA has a role in iron transport. Furthermore, A. baumannii expresses hemin utilization functions independent of production and transport of acinetobactin-siderophore. Following transport, iron must be integrated into the intracellular iron pool. NfuA, a [Fe-S] cluster carrier protein was found to be involved in the ability of cells to respond to (open full item for complete abstract)

Committee: Luis Actis PhD (Advisor); Kelly Abshire PhD (Committee Member); Rachael Morgan-Kiss PhD (Committee Member); Gary Janssen PhD (Committee Member); David Tierney PhD (Committee Member) Subjects: Microbiology

Doctor of Philosophy, The Ohio State University, 2023, Chemistry

In nature, metal containing proteins are found in all kingdoms of life and perform some of the most biologically important chemical reactions. One class of metallocofactors, the iron-sulfur cluster, is responsible for a wide array of biological processes from electron transfer to energy storage and conversion in biological metabolism. While homometallic iron-sulfur clusters are widely observed, there are fewer examples of heterometallic iron-sulfur clusters, which contain a unique heterometal coordinated within the cluster scaffold. These clusters typically have a singular function; to catalyze difficult chemical reactions such as nitrogen or carbon fixation. One such enzyme, carbon monoxide dehydrogenase (CODH), contains a heterometallic [NiFe4S4] cluster active site called the C-cluster. This enzyme is of great interest with respect to current energy challenges, as it performs the reduction of carbon dioxide (CO2) to the chemical feedstock carbon monoxide (CO) with perfect selectivity and negligible overpotential under ambient conditions, characteristics current anthropogenic catalysts have been unable to achieve. However, due to the complexity of the system, there are significant gaps in our knowledge of the mechanism. Moreover, previous attempts to model this system using synthetic analogous have not been shown to operate with any functionality. Alternatively, it is possible to model metalloenzymes by repurposing existing metalloproteins, thus providing accurate structural models within a biological scaffold. We have chosen a ferredoxin (Fd) from Pyrococcus furiosus which contains a site-differentiated [Fe4S4] cluster. This site-differentiation allows for facile substitution of the unique site with Ni2+ to structurally model the active site of the CODH. The [NiFe3S4] Fd (NiFd) cluster exhibits reversible electron transfer and the ability to bind both CO and cyanide (CN-), a substrate and inhibitor of CODH respectively. We have extensively characterized the el (open full item for complete abstract)

Committee: Hannah Shafaat (Advisor); Christine Thomas (Advisor) Subjects: Chemistry

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2018, Photochemical Sciences

This dissertation consists of two parts that together broadly aim to understand the relationship between the structure and function of protein. Part I is a study of the Fe-S cluster assembly system U-type scaffold protein, SufU, from the Gram positive bacterium Bacillus subtilis. The conformational changes of the protein, upon interaction with two different metals, Zn2+ and Fe3+, were investigated. The results indicate that purified SufU that had been stripped of its bound Zn2+ undergoes conformational changes upon reconstitution with Zn2+ ions, but there is no evidence of such changes upon the addition of Fe3+ ions. Thus, B. subtilis SufU discriminates between Zn2+ and Fe3+, and preferentially binds Zn2+ ions. Similar results have been reported for two other U-type proteins, namely, Escherichia coli IscU and Streptococcus mutans SufU, which suggests that these properties are widespread among U-type proteins. Part II is an investigation of the role of the post-translation acetylation modification of the Rhodobacter sphaeroides Puc1B protein. Together with PucA, PucB comprises the structural component of one of two light harvesting (LH) complexes that deliver photons to the photosynthesis reaction center. Rba. sphaeroides encodes two such sets of PucB and A proteins; Puc1B and Puc1A, and Puc2B and Puc2A. While the amino acid sequence of Puc1B differs by only 3 residues from Puc2B, mass spectrometry data indicate that only the former is acetylated. The findings presented here indicate that acetylation is important for efficient assembly of LH2. The specificity required to acetylate Puc1B, and not Puc2B, evokes an enzyme-catalyzed process, which raises the possibility that Puc1B acetylation is a regulated event, and the rate of acetylation of Puc1B might then govern the rate of LH2 assembly.

Committee: Jill Zeilstra-Ryalls (Advisor); Jong Kwan Lee (Other); H. Peter Lu (Committee Member); R. Marshall Wilson (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry; Molecular Biology

Master of Science, The Ohio State University, 2017, Biochemistry

Iron-sulfur clusters are essential cofactors found across all domains of life. Their assembly and transfer are accomplished by highly conserved protein complexes and partners. In eukaryotes a [2Fe-2S] cluster is first assembled in the mitochondria on the scaffold protein IscU in tandem with iron, sulfide, and electron donors. The current model posits that a chaperone pair interacts with a cluster-bound IscU to facilitate cluster transfer to a monothiol glutaredoxin. In humans this protein is Grx5. Once the [2Fe-2S] cluster is transferred to Grx5, its cluster may be transferred to a variety of target apoproteins. My work has focused primarily on Grx5 and monitoring cluster transfers to and from this protein. To accomplish this, circular dichroism (CD) spectroscopy was used to great effect, owing to the unique chiral active site that is characteristic to many Fe-S proteins when bound to cluster. By monitoring the change in CD spectra from donor to acceptor Fe-S protein cluster transfer pathways can be mapped out with kinetic rates for these transfers. In this regard, we showed that the prokaryotic model, whereby the chaperones HscA and HscB are required for successful, efficient transfer of a [2Fe-2S] cluster from the IscU scaffold to a monothiol glutaredoxin, does not extend to the human system. The human chaperone homologues, HSPA9 and Hsc20, are not necessary for human IscU to transfer a cluster to Grx5. Interestingly, the presence of these chaperones seemed to reverse the possible transfer; with HSPA9 and Hsc20 no transfer was seen from IscU to Grx5, but instead a transfer from Grx5 to IscU occurred. This is also at odds with the current model of cluster assembly and transfer, highlighting significant differences between human systems with other organisms. Once cluster-bound, Grx5 was also able to donate cluster to Ferredoxins 1 and 2 as well as Nfu. The ferredoxins have been suggested to be terminal cluster acceptors that serve differing functions, while Nfu has (open full item for complete abstract)

Committee: James Cowan Dr. (Advisor); Ross Dalbey Dr. (Committee Member) Subjects: Biochemistry

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2016, Photochemical Sciences

Iron-sulfur (Fe-S) clusters are ancient cofactors that participate in numerous cellular processes, such as electron transfer, enzyme catalysis, redox sensing, DNA repair and gene regulation. To achieve high degree of regulation and control of free sulfide and iron ion three biosynthetic machineries were identified to assemble Fe-S clusters: ISC, SUF, and NIF. Scaffold protein represents a centerpiece of the biosynthetic pathway with two important roles. It must support the assembly of Fe-S clusters from constituent iron and sulfide species donated by other proteins followed by transfer of the nascent Fe-S clusters to other partners requiring clusters for their function. These two roles present an interesting conundrum, namely that scaffold proteins must initially exhibit robust affinity for Fe-S clusters as required for their assembly, and yet transfer of the Fe-S clusters to recipient proteins indicates lower affinity. A model explaining this apparent paradox is based on the ability of IscU, a well-known scaffold protein, to adopt two different conformational states with different affinities for Fe-S clusters and biosynthesis protein binding partners. During sequential steps of the Fe-S cluster biosynthesis cycle, IscU alternates its conformational state and affinity for Fe-S clusters, in order to accomplish Fe-S cluster assembly and transfer. Our interest is focused on the U-type protein from pathogenic Gram-positive bacteria Streptococcus mutans that belongs to a truncated version of typical SUF system represented by sufCDSUB operon. Even though, the SufU protein shares high sequence similarity with above mentioned IscU, it exhibits important distinguishing feature of an extra 19 amino acids that is denoted as the Gram-Positive Region (GPR). This dissertation is focused on the effect of conformational change of the scaffold protein SufU on the kinetic activity of its binding partner cysteine desulfurase SufS from Streptococcus mutans. Our aim is to charac (open full item for complete abstract)

Committee: Andrew T. Torelli Dr. (Advisor); Alexander Izzo (Other); George S. Bullerjahn Dr. (Committee Member); H. Peter Lu Dr. (Committee Member) Subjects: Biochemistry; Chemistry; Inorganic Chemistry

Master of Science (MS), Bowling Green State University, 2015, Chemistry

IscU and SufU are proteins that play critical roles in iron-sulfur (Fe-S) cluster biosynthesis in Gram-negative and Gram-positive bacteria, respectively. Though the function of IscU has been well characterized, there is ongoing debate about the role of SufU in Fe-S cluster biosynthesis in Gram positive Firmicutes, a bacterial classification that includes several notable human pathogens. Furthermore, metal-dependent conformational changes that are believed to be important in the Fe-S cluster biosynthesis cycle have been observed for IscU, but have not been well studied in SufU. Here, we present a comparison of the effects of different transition metals on the solution structures of SufU from Streptococcus mutans with IscU from Escherichia coli and SufU from Bacillus subtilis using three complementary techniques. In spite of the proposed differences in their functional roles, our results show a striking similarity between the responses of the three proteins to the titrated metal ions, both in terms of the enthalpies of the interactions and the resulting structural changes. Interestingly, the three proteins also discriminate between different transition metal ions, wherein they exhibit affinity for Zn2+ and Co2+, but not Fe3+. The three correlated techniques employed in this study provide new information relevant to understanding the role of SufU in Fe-S biosynthesis, and also serve as a foundation for future studies into whether altering the structures of key Fe-S biosynthesis proteins is a possible mechanism for toxic transition metal stress.

Committee: Andrew Torelli Ph.D. (Advisor); Ksenija Glusac Ph.D. (Committee Member); Alexis Ostrowski Ph.D. (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, The Ohio State University, 2009, Chemistry

Iron-sulfur clusters are small inorganic cofactors found in all kingdoms of life. The iron-sulfur cluster biogenesis is a complex system which involves a surprisingly large group of proteins. In human cells, frataxin is believed to recruit ferrous iron from the labile iron pool and subsequently deliver it to the scaffold protein human ISU where the iron-sulfur clusters will form. Deficiency in cellular frataxin production results in a human disease, Friedreich's ataxia which affects 1 in 50,000 humans. By using mutagenesis, the activities of frataxin mutants were investigated. We found that frataxin may have a pool of potential sites that can stably bind an iron center when bridged to a variety of physiological targets; there may not be unique binding loci, but rather a number of locations that provide flexibility in the binding of physiological partner proteins. Frataxin has been found to repair the damaged cluster on mitochondrial aconitase. We herein investigated its functional role towards cytosolic aconitase, IRP1, which can register the iron level and is important for the iron homeostasis of human cells. Frataxin can bind to cytosolic aconitase and repair the damaged clusters ([3Fe-4S]) into [4Fe-4S] clusters. We reported the cloning and overexpression of human mitochondrial HscB, a J-type co-chaperone, and demonstrated an interaction between human frataxin and human HscB by cross-linking experiments and ITC. HscB was also shown to promote cleavage of the N-terminal domain of full-length human frataxin. Human ISU protein contains three highly conserved cysteine residues (C44, C70, and C113) that mutagenesis studies suggested to be directly coordinated to the cluster. The fourth ligand is still unclear. H112 was mutated into alanine or aspartate, and the effects of the mutation were evaluated. We found that H112 might be the fourth ligand that helps to stabilize the cluster, however, it was not absolutely needed for the formation of cluster; it might be the (open full item for complete abstract)

Committee: James Cowan PhD (Advisor); Karin Musier-Forsyth PhD (Committee Member); Ross Dalbey PhD (Committee Member) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 2007, Ohio State Biochemistry Program

How iron-sulfur clusters are syntheisized in vivo remains an interesting topic to explore. In this dissertation, studies were focused on human NFU, an important protein that participates in iron-sulfur cluster biosynthesis. Studies were first carried out on NFU's functional characterization. Due to the presence of the conserved CXXC motif in NFU, this protein was proposed and demonstrated to cleave the persulfide bond on NifS. In the presence of L-cysteine, a catalytic amount of NifS, ferrous iron and NFU, apo proteins lacking iron-sulfur clusters can be successfully reconstituted. The function of human NFU led to the hypothesis that this protein can interact with NifS. The binding of NFU to NifS was quantified by ITC (isothermal titration calorimetry). The temperature-dependence of binding was also examined to determine the underlying thermodynamics. A positive change in enthalpy with increased temperature suggested dominance of polar charged residues in the binding. The interaction of human NFU and the chaperone system was also studied in vitro. Both NFU and its C-terminal domain can stimulate the ATPase activity of the chaperone HscA. The ADP form of HscA binds to both NFU and the C-terminal domain more tightly than the ATP form, providing additional evidence for NFU interacting with the chaperone system. The binding of NFU to the HscA/HscB complex is tighter than binding to HscA alone, which is consistent with previous ATPase results. Besides functional characterization, structural characterization was also carried out. The near UV CD data for NFU were almost negligible, suggesting a possible molten globule property of human NFU. Results from ANS binding and tryptic digestion experiments were consistent with molten globule type proteins. Tryptic digestion suggested the rigid tertiary structure of the N-terminal domain. It was confirmed to possess a tertiary structure by the ANS experiment and the tryptic digestion experiment.

Committee: James Cowan (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2004, Chemistry

In this research, we primarily focus on the structure and function of both the nucleotide binding domain, and the full length membrane-spanning transporter. First, the soluble nucleotide binding domain of Atm1 (Atm1-C), an ABC transporter in yeast mitochondria, that has previously been implicated in the maturation of cytosolic iron-sulfur cluster proteins, has been overexpressed in E. coli, purified, and characterized. The full length version of Atm1 from Saccharomyces cerevisiae has been cloned, over-expressed, purified from a yeast expression system, and characterized. A fluorescent assay of liposome-loaded reconstituted Atm1p suggested that Atm1p only allowed small molecules and/or metal complexes to cross the channel. Both pH gradient and fluorescent assays also indicated that ADP-bound Atm1p existed in an open state that is different from the closed state for ATP-bound Atm1p. The further discovery of an iron carrying peptide, hepcidin, provides the first step toward understanding iron trafficking in living cells. With eight, well-conserved cysteine residues in the sequence, hepcidin may not only be a signal peptide, but could potentially serve as an iron carrier. The iron binding properties have been determined by UV-vis spectroscopy, mass spectroscopy, and isothermal titration calorimetry (ITC). The iron binding affinity has been determined in the micromolar range. Studies by circular dichroism (CD) reveal varying degrees of secondary structure within an apparent dynamic tertiary fold. Taken together, hepcidin clearly binds iron, and the secondary structure change induced by iron binding may be required for the full function of the peptide in iron homeostasis and antimicrobial activity. In Part II, a novel fluorescent assay has been developed for monitoring the cleavage of a target RNA by cooper kanamycin in vivo. However, demonstration of the efficacy of such reagents in vitro is only a first step. To demonstrate in vivo cleavage chemistry we have designed a (open full item for complete abstract)

Committee: James Cowan (Advisor) Subjects: Chemistry, Biochemistry