Master of Science in Chemistry, Youngstown State University, 2007, Department of Chemistry

Metal catalyzed oxidation may result in structural damage to proteins and has been implicated in aging and disease, including neurological disorders like Alzheimer's. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect and may have dramatic consequences on physical and functional properties of the oxidized protein molecule. This thesis is about the investigation of the site specificity of metal catalyzed oxidation of the H15S+N77H double mutant of hen egg white lysozyme. An extracellular mutant was generated using the PCR overlap extension method. The mutant was cloned into the pCR® 4-TOPO® vector. Sequencing showed the successful generation of an H15S+N77H double mutant capable of extracellular expression into pPICZBα B vector.The double mutant gene insert was digested out of pCR® 4-TOPO® vector and ligated in the pPICZα B vector. Future work includes the linearization of pPICZα B and transformation into X-33 competent cells, followed by the extracellular expression of mg quantities of the mutant protein. The results of metal catalyzed oxidation experiments will be compared to those of the native enzyme to reach conclusions about the relationships between site-specific oxidation and protein structure.

Committee: Michael Serra (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy (PhD), Wright State University, 2007, Biomedical Sciences PhD

The role of subunit III (SIII) in cytochrome c oxidase structure and function was investigated using enzyme isolated from the bacterium Rhodobacter sphaeroides. Energy minimization calculations suggested that in the absence of SIII, subunit I (SI) adopted a more open conformation. This observation was tested through the use of limited proteolysis using, a-chymotrypsin. The results showed that in the absence of SIII the solution structures of wild-type and I/II oxidase were not significantly different, and that proteolysis occurred exclusively at the N and C-termini of SI. Upon inactivation of I/II oxidase by catalytic turnover, and subsequent digestion with the protease a-chymotrypsin it was concluded that SI underwent significant conformational changes. This change in conformation was probably due to CuB, located in the active site, dissociating from SI, upon turnover induced inactivation. A similar digestion pattern was observed in a mutant lacking CuB, which indicated that the large scale change in conformation was due to the loss of CuB from the active site. The functional role of SIII was also investigated by predicting and mutating interhelical hydrogen bonds within SIII. A cluster of amino acids consisting of Glu90, His212, and Tyr246 form a highly conserved triad of interhelical connectivity, perhaps linked by hydrogen bonds. Mutation of Glu90 to an alanine (E90A) resulted in a significant loss of SIII content, accompanied by a phenotype of turnover induced inactivation and reduced proton pumping efficiency, when reconstituted in phospholipid vesicles. Mutation of His212 to either an alanine or phenyalanine (H212A and H212F) resulted in wild-type expression of SIII and when reconstituted into a phospholipid vesicle, these mutants exhibited turnover induced inactivation as well as reduced proton pumping efficiency. Mutation of Tyr246 to a phenyalanine (Y246F) resulted in a mutant that was indistinguishable from wild-type enzyme. Therefore, it was concluded th (open full item for complete abstract)

Committee: Lawrence Prochaska (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy (PhD), Wright State University, 2007, Biomedical Sciences PhD

Telomeres are the structures that protect the ends of linear chromosomes from fusion and degradation. The telomere consists of tandem repeated DNA sequences that can range from hundreds of bases to kilo-bases depending on the organism. As the cells of an organism replicate their DNA, these repeats are lost due to the end replication problem, where the ends of linear DNA cannot be fully replicated. As the telomeres are shortened through each round of replication, they eventually reach a critical point. Once the telomeres are too short and the cell risks losing coding sequences, a signaling pathway is initiated that causes the cell to senesce. However, cells that require continuous replication (i.e., stem cells, germ cells, and cancer cells) require constant maintenance of their telomeres in order to not enter senescence. The majority of these cells use the multimeric protein telomerase and a host of other proteins to maintain the lengths of their chromosomes. Eukaryotic telomerase is a nucleo-protein complex consisting of the telomerase RNA (TR), telomere end reverse transcriptase (TERT), and telomerase associated protein 1 (TEP1). Furthermore, telomeric length is regulated by a host of telomeric binding proteins. This thesis focuses on two proteins important for human telomeric maintenance. The first is human TEP1 (hTEP1) which is a subunit of telomerase. This large protein contains the RNA binding domain that binds hTR. Though the RNA binding subunit of hTEP1 has been partially purified before, full-length hTEP1 has been refractory to biochemical analysis due to the inability to express and purify this large protein. Here we reveal the very first purification of full-length hTEP1. Furthermore, where the RNA binding domain of hTEP1 alone does not show specific interaction with hTR, we show that full-length hTEP1 binds hTR specifically. The second protein of interest in this thesis is the human telomeric repeat binding factor 1 (hTRF1). This protein is one of the tel (open full item for complete abstract)

Committee: John Turchi (Advisor) Subjects: Chemistry, Biochemistry

Master of Science (MS), Wright State University, 2005, Biochemistry and Molecular Biology

Forquer, Isaac Paul. M.S. Department of Biochemistry and Molecular Biology,WrightState University, 2005. Characterization of Photosynthetic Reaction Centers from Bradyrhizobium strain BTAi 1 Photosynthetic rhizobia have been studied for about 15 years now. They are now considered to be metabolically aligned with a relatively recently discovered group of bacteria, the anoxygenic aerobic phototrophs (AAP's).Rhizobia form symbiotic relationships with plants from the Fabaceae family. Photosynthetic rhizobia not only nodulate the roots, as most other rhizobia do, but they also form nodules on the stems of certain leguminous plants. The plant provides carbon to the bacteria and the bacteria provides the plant with soluble nitrogen fixed from the biologically inert but abundant atmospheric N2. A key question regarding photosynthetic rhizobia and other AAP's derives from the observation that photosynthesis in these organisms shuts down under anaerobic conditions. It has been proposed, and is the hypothesis of this thesis that the primary electron acceptor (QA) in the photosynthetic reaction center has a higher midpoint potential than in reaction centers found in the AAP's counterparts, the anaerobic purple bacteria. If QA had a higher midpoint potential, it would be more labile to overreduction under anoxic conditions, and if QA is reduced, then photosynthetic electron transport is blocked. A redox titration was done to measure the midpoint potential of Q in the reaction centers of BTAi 1. This was done by observing the level of P (primary electron donor) bleaching upon excitation with bright light at different ambient redox potentials. The level of P bleaching is proportional to the fraction of QA that is not reduced, since P cannot bleach and donate an electron if QA is already reduced. Reaction centers from BTAi 1 were purified using two techniques, both involving ion exchange chromatography and one involving ammonium sulfate precipitation. Reaction centers were characte (open full item for complete abstract)

Committee: Darrell Fleischman (Advisor) Subjects: Chemistry, Biochemistry

PhD, University of Cincinnati, 2001, Arts and Sciences : Chemistry

Penicillin-class antibiotics, known as Beta-lactams, are one of medicine's most valuable weapons against bacterial disease. These drugs infiltrate susceptible bacteria and interrupt normal growth via interactions with a class of cell wall-synthesizing enzymes known as penicillin-binding proteins (PBPs). Some pathogens, however, can escape such a fate. Beta-lactam antibiotics are not currently prescribed for the treatment of tuberculosis, although it has now been shown that commercially available lactams can bind to PBPs in membrane fractions from Mycobacterium tuberculosis (MTB), the causative agent. This lab has previously documented that PBPs form native protein complexes within H. influenzae and E. coli. If similar PBP complexes could be identified within mycobacteria, it would improve our understanding of the enzymology of cell wall biosynthesis in these organisms. Mycobacterial PBPs and the proteins with which they naturally interact are valuable targets for antibiotic research, especially in light of the emergence of multidrug-resistant tuberculosis (MDRTB) in certain populations worldwide. The approach outlined here allows covalent labeling of PBPs within intact cells before protein isolation; the advantage provided by this scheme is the opportunity to cross-link PBPs in their native protein-protein associations. Cross-linking can take place after interaction with (-lactam tags, but before disruption of cells. This lab has previous experience exploring the topography of bacterial PBPs using cyanogen (ethanedinitrile) as a cross-linking agent. It has been shown to permeate intact cells and to covalently link native PBP complexes. Dansylated penicillin has been used to covalently label purified serine protease enzymes, though to the best of our knowledge it has not been used to label mycobacterial proteins. When accompanied by Beta-lactamase inhibitors, Beta-lactams have been shown to interfere with the growth of mycobacteria in cultures and intracellularly (open full item for complete abstract)

Committee: R. Day (Advisor) Subjects: Chemistry, Biochemistry

PhD, University of Cincinnati, 2001, Arts and Sciences : Chemistry

Due to their ability to hydrolyze the lactams, the beta lactamases constitute the major mechanism by which bacteria become resistant to beta lactam antibiotics. The Class A beta lactamases (termed serine enzymes) are inhibited by clinically available compounds such as sulbactam, tazobactam and clavulanic acid. However, the metallo or class B enzymes are not affected by inhibitors of the Class A enzymes. Here we present the de novo synthesis and characterization of a dansylated monocyclic beta lactam (1-(5-dimethylamino-1-napthalenesulfonyl hydrazido)-3-acetamido-4-methoxy-2-azetidinone). The dansylated monocyclic beta lactam was characterized via infrared spectroscopy, mass spectrometry, NMR, bioassay, and purified by HPLC. This 2-azetidinone was found to be a covalent, irreversible inhibitor of the metallo beta lactamase CCrA from Bacteriodes fragilis . The CCrA metallo beta lactamase was characterized via HPLC, SDS-PAGE, densitometry, and activity assays. The HPLC elution profile of the beta lactamase showed a consistent retention time of 28.94 minutes with a 1-100% acetonitrile gradient. The enzyme was determined to be inactive (e.g. unable to hydrolyze nitrocefin) in the presence of EDTA. MALDI-TOF analysis of an in-gel tryptic digest of the CCrA beta lactamase gave eight peptides (MH+) that corresponded to the masses of theoretical peptides: 775.398, 1433.651, 1505.730, 1637.778, 1765.912, 2070.961, 2101.977, and 2483.065. The presence of metallo beta lactamases in clinically relevant bacteria has become an obvious threat to the efficacy of existing antibiotics. The ability of bacteria to escape the lethal action of beta lactam antibiotics highlights the importance of the discovery of new compounds that can either escape or inhibit the activity of the beta lactamases, particularly the clinically relevant metallo beta lactamases. There have been no previous reports of beta lactam inhibitors of the metallo beta lactamases.

Committee: Richard Day (Advisor) Subjects: Chemistry, Biochemistry

PhD, University of Cincinnati, 2007, Arts and Sciences : Chemistry

Two facets of nitroxides are examined in this thesis. First, the reproducibility of their experimental detection by EPR (Electron Paramagnetic Resonance) on two table top Bruker e-scans in view of their potential use for gene detection, and, second, their use for long range distance measurements by molecular modeling in complex biological systems. EPR signal heights as a function of microwave power showed non-linear behavior on organic radicals (5-and 6-membered nitroxides), and on an inorganic standard (Cr3+) under non-saturating conditions with two Bruker table top e-scans. h-1/ho ratio measurements for determining gene probe degradation gave values with a small standard deviation at 10-6 M [nitroxide], especially with the e-scan that features higher microwave power output and uses an internal standard to correct for magnetic field drifts. Therefore, EPR based gene detection will no longer require high-end EPR instrumentation. For nitroxide – nitroxide distance determinations various software and strategies were examined for bi-nitroxide-labeled DNA and nitroxide-labeled DNA – nitroxide-labeled reverse transcriptase (RT) complexes. Of particular interest was the effect of the tether property (rigid, semi-rigid, and flexible) on nitroxide – nitroxide distances. The distances determined by molecular modeling are discussed in the context of recent EPR measurements on bi-nitroxide-labeled DNAs and complexes consisting of reverse transcriptase (RT) and DNA with strategically placed nitroxides. WebLab ViewerPro, a simple visualization program, was used to calculate nitroxide – nitroxide distances with rigid and semi-flexible tethered nitroxides. This program approximated experimental distances within the experimental error limits of EPR data only with rigid tethered nitroxides known to perturb molecular structures. The biologically more relevant semi- or flexible tethered nitroxides that have been shown to cause minimal structural perturbation required more advanced pro (open full item for complete abstract)

Committee: Dr. Albert Bobst (Advisor) Subjects: Chemistry, Biochemistry

MS, University of Cincinnati, 2006, Arts and Sciences : Chemistry

There continues to be a great demand for synthetic hosts that recognize targeted guests as efficiently as proteins recognize endogenous ligands. Artificial protein mimetics could act as chemical sensors, catalysts, or agents that transport drugs across cell membranes. We have created new protein mimetics with amino acid recognition elements that converge to a hydrophobic pocket in order to provide maximum binding free energy with a guest. The dynamic component of these host-[2]rotaxanes allows them to adjust to their environment, whether aqueous or non-aqueous, and does not detract significantly from the binding free energy. The host-[2]rotaxanes bind a variety of biomolecules like oligopeptides and some of them efficiently transport fluorescein and some fluoresceinated peptides into eukaryotic cells.

Committee: Dr. David Smithrud (Advisor) Subjects: Chemistry, Biochemistry

PhD, University of Cincinnati, 2005, Arts and Sciences : Chemistry

Alpha-1 acid glycoprotein (OMD) is an acute phase serum protein known to bind to various ligands. As a member of the lipocalin family, OMD has a tertiary structure of a beta-barrel core with alpha-helices external to the barrel core and two disulfide bonds. The structure of OMD is influenced by the environment which dictates its stability and ligand binding ability. OMD can have the disulfides reduced and the tertiary structure and binding site is affected by the oxidative state of the two disulfides. Reduction of the disulfides decreases stability and modifies the binding site of the protein to be more relaxed, perhaps even in a molten globular form. The reduced OMD is unable to bind to ligands such as warfarin in this state. Within the life of the protein, OMD is often crowded by other macromolecule. Cellular concentrations of macromolecules are between 30-40% and plasma concentrations are about 8%. Crowding with 10% (w/v) PEG leads to a decrease in the solvent accessible surface area of the protein, which increases the stability of OMD, since the unfolded state is disfavored. Crowding also changes the binding site of OMD and appears to be less accessible for the three tested ligands. The environment of the protein greatly influences the activity and shape of OMD. The different environments of the body can have a profound effect on OMD changing the pharmacological impact of this protein.

Committee: Brian Halsall (Advisor) Subjects: Chemistry, Biochemistry

PhD, University of Cincinnati, 2004, Medicine : Molecular Genetics, Biochemistry, and Microbiology

Hedgehog (Hh) deficient flies, mice and humans reveal a conserved developmental requirement for Hh signaling, while aberrant Hh signaling causes basal cell carcinoma, medulloblastoma, and is implicated in other tumors including breast, lung and pancreas. We address the poorly understood mechanism of Hh signal transduction from the plasma membrane to the nucleus, taking advantage of the extensive genetic characterization of the Hh pathway in Drosophila melanogaster. The secreted protein Hh binds its receptor Patched (Ptc), relieving Ptc inhibition of the GPCR-like protein, Smoothened (Smo). Downstream of Smo is the microtubule associated Hedgehog Signaling Complex (HSC). The HSC consists of the Kinesin related protein (KRP) Costal2 (Cos2), the serine/threonine protein kinase Fused (Fu) and Cubitus interruptus (Ci), the zinc finger transcription factor that regulates Hh target genes. Cos2 and Fu are required to produce transcriptional repressor and activator forms of Ci. Drosophila genetics suggest that fu, cos2, ci and Suppressor of fused [Su(fu)], which suppresses fu phenotypes, interact in a non linear fashion, and phenotypes of the Drosophila wing suggest two complexes could be involved. However, it is not known how Smo signals to the HSC, nor how this complex that tethers Ci from the nucleus, also serves to activate Ci. We demonstrate that Su(fu) associates with the HSC, however, Su(fu) does not appear to bind the microtubule associated complex, and the HSC is stable in the absence of Su(fu). While Ci accumulates in the nucleus of cells exposed to Hh, we show that other HSC members and Su(fu) do not. Cos2 tethers the HSC to vesicular membranes, and releases membranes in response to Hh. Cos2 also binds Smo, although with lower affinity than membranes and with no Hh sensitivity. Additionally, the bulk of Cos2 membrane association is Smo independent. We have thus provided biochemical evidence for two HSC complexes, targeted by Cos2 to Smo or membranes. Differential (open full item for complete abstract)

Committee: Dr. David Robbins (Advisor) Subjects: Chemistry, Biochemistry

PhD, University of Cincinnati, 2003, Medicine : Molecular Genetics, Biochemistry and Microbiology

Bloom's syndrome (BS) is a rare autosomal recessive disorder that greatly predisposes affected individuals to cancer. Such individuals also are small in size, sensitive to the sun, have immune dysfunction and gross genomic instability. The cytogenetics of BS cells have been extensively studied and have shown increased levels of homologous recombination, quadriradial formations, telomeric associations and chromosome breakage. The gene responsible for BS has been positionally cloned and and encodes a RecQ helicase family member with strand displacement activity that is dependent on ATP and Mg2+. In order to have a greater understanding of BLM helicase function in the cell in regards to DNA replication, recombination and repair, we identified protein-partners of BLM. The C-terminus of BLM identified the DNA mismatch repair protein MLH1 from a yeast two-hybrid screen. In vitro and in vivo immunoprecipitations confirmed the interaction between these two proteins. Using an in vitro mismatch repair assay, BS cell extracts were tested for their ability to correct a single nucleotide mismatch. The BS cell extracts were able to remove the single nucleotide mismatch from the plasmid DNA, demonstrating that the BLM-MLH1 interaction is not necessary to correct a single nucleotide mismatch substrates. Helicase assays then were performed which demonstrated that MLH1 or the mutL heterodimer modulates the enzymatic activity of BLM by stimulating BLM's strand displacement activity on the double-overhang (DO) substrate. Finally, we performed experiments with the supF20 mutagenesis system and demonstrated that extracts from BS cells are unable to utilize micro-homology elements within the supF20 gene to restore supF function following the induction of a double strand break (DSB). Additional experiments with the pUC18 mutagenesis system demonstrate that although the efficiency and fidelity of DSB repair by BS extracts are comparable to those of normal extracts when ligatable ends are pr (open full item for complete abstract)

Committee: Dr. Joanna Groden (Advisor) Subjects: Chemistry, Biochemistry

PhD, University of Cincinnati, 2002, Arts and Sciences : Chemistry

Salt bridges between self-associating hen egg-white (HEW) lysozyme and bovine insulin molecules were converted to covalent links by ethanedinitrile (cyanogen) and identified using mass spectrometry. Peptides resulting from cyanogen-mediated intermolecular cross-linking of HEW lysozyme were detected using in-gel digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sequence data from electrospray ionization quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF MS) revealed that one of the peptides has a covalent bond between Asp 66 and Arg 14. Human lysozyme is one of twenty known proteins that form amyloid fibrils, a characteristic feature of systemic amyloidosis. The reported cases of lysozyme amyloidosis result from single point mutations in the lysozyme gene. One of the variant proteins contains a mutation at Asp67His (corresponding to Asp66His in HEW lysozyme), the same amino acid residue identified cross-linked in native HEW lysozyme associations using cyanogen. The self-assembly of another amyloid fibril protein, bovine insulin, was also investigated using cyanogen. Using high performance liquid chromatography (HPLC) coupled with ESI-Q-TOF MS, an intermolecular salt bridge association was identified by covalently linking the B chain C-terminal carboxyl group of Ala 30 and the charged imidazole of His 5 (B chain). These two residues are not known to associate closely in native insulin self-assembly. However a recent model of insulin fibril formation, proposed by Brange and colleagues, places these two residues in close intermolecular proximity. A method was developed incorporating enzymatic digestion, 1D gel electrophoresis, MALDI-TOF MS, and HPLC ESI-Q-TOF MS to identify amino acid residues participating in salt bridge formations at protein-protein interfaces. The same methodology was applied to protein-protein associations in Bacillus subtilis . Computer based searching and HPLC ESI-Q-TOF MS sequence data (open full item for complete abstract)

Committee: Dr. Richard A. Day (Advisor) Subjects: Chemistry, Biochemistry

MS, University of Cincinnati, 2001, Arts and Sciences : Biological Sciences

The American chestnut tree Castanea dentata (Marsh.) Borkh was one of the largest and most valued trees in the Eastern United States until the accidental introduction of the blight fungus Cryphonectria parasitica (Murrill). The blight virtually eradicated the American chestnut from its former range. It was imported on Asian chestnut trees, in the early part of the 20th century. We have used a combination of molecular and biochemical techniques in an attempt to identify the compound(s) in the Chinese chestnut, Castanea mollissima (Blume), that give it resistance to the chestnut blight. A broad survey of components of Chinese chestnut bark was evaluated for anti-fungal activity. This survey identified that the 938 molecular weight compound chestanin consistently encouraged fungal growth. A separated fraction containing at least two compounds with apparent molecular weight around 1300 Daltons was also found. Preliminary data shows the compounds in this fraction decrease the rate of mycelial growth and sporulation. As a result of experiments using different media, an improved recipe for callus production from immature green leaves or embryo tissues of American chestnut was identified.

Committee: Dr. G. Douglas Winget (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy, University of Toledo, 2007, Chemistry

The processes of DNA replication, repair and recombination have been studied for many years using model systems such as E. coli. One key aspect of DNA replication is the role of proteins at the replication fork. It is important to understand the interactions of these proteins with the other proteins involved in the process or with a DNA substrate. In PART I of this dissertation, studies of the Archaeal Aeropyrum pernix and Archaeoglobus fulgidus single-stranded DNA binding protein are presented. Archaea are more closely related to eukaryotes than the commonly studied E. coli model system. The SSB proteins have an involvement in Okazaki fragment processing. The FEN-1 enzyme removes DNA flaps that are 3 to 5 nucleotides long. If the length of the flap is more than 5 to 7 nucleotides, two other proteins are then involved in the removal of these flaps, the SSB protein that binds to the flap, and dna2 needed to shorten the flap. Using PCR and molecular cloning techniques and protein chemistry, milligram quantities of the purified Ape SSB protein were obtained. Preliminary attempts to crystallize the full length protein failed, most likely due to highly flexible C-terminal region. To overcome this problem, a truncated version of the protein was prepared. The truncated protein crystallized in two different crystallization conditions. One produced beautiful large 1mm hexagonal crystals that appeared twinned on diffraction. The other crystal form, rod shaped small crystals, diffracted to almost 1.6 A. The structure of the truncated protein was solved using molecular replacement with an already known SSB protein from the species Sulfolobus sulfataricus. Biophysical studies, Dynamic Light Scattering (DLS) and Fluorescence Anisotropy, have been carried out to characterize the Ape SSB protein. In PART II, work done on DNA helicases (dnaB) from different organisms is presented. Expression and purification protocols for Vibrio cholerae dnaB were successfully established and crysta (open full item for complete abstract)

Committee: Timothy Mueser (Advisor) Subjects: Biology, Microbiology; Biology, Molecular; Biophysics, General; Chemistry, Biochemistry; Chemistry, General

Master of Science, University of Toledo, 2006, College of Arts and Sciences

The lipoxygenase enzymes are non-heme, non-sulfur iron dioxygenases that are widely distributed throughout plants, animals, fungi and some bacteria. This family of enzymes plays a major role in polyunsaturated fatty acid metabolism because they catalyze the incorporation of molecular oxygen into fatty acids containing a 1,4-pentadiene moiety producing hydroperoxide products. In mammals, the products of the dioxygenation catalyzed by lipoxygenases, hydroperoxyeicosatetraenoic acid (HPETEs), are intermediates in the formation of bioregulators including leukotrienes, hepoxilins and HETEs, that are implicated in a variety of human conditions such as inflammation, fever, arthritis and cancer. 12-Lipoxygenases are mammalian lipoxygenases that catalyze the conversion of arachidonic acid into 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE). Actually, 12-HPETE is a precursor to the formation of the hydroxy fatty acid 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and also epoxy fatty acids called hepoxylins. There are three isoforms of 12-lipoxygenase named after the cells where they are found: leukocyte, platelet and epidermis type-enzymes. Even if the leukocyte and the platelet 12-lipoxygenase catalyze the same reaction, they differ by their tissue distribution, their substrate specificity, their primary structure and size. They are also involved in different kinds of diseases as inflammation, hypertension and diabetes. The goal of this research was to investigate the human platelet and porcine leukocyte 12-lipoxygenases. In order to study both 12-lipoxygenases, they need to be expressed, purified and in the best case crystallized to get the 3D structure. This thesis presents the expression and purification protocols that lead to the preparation of large amounts of pure porcine leukocyte 12-lipoxygenase. Several biophysical analyses were performed to characterize the protein in solution such as dynamic light scattering (DLS), differential scanning calorimet (open full item for complete abstract)

Committee: Max O. Funk (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy, University of Toledo, 2005, Chemistry

The duplication of genomic information is central to the survival of organisms in all kingdoms of life. DNA replication and repair processes are essential for maintaining the fidelity and genomic stability required for life. Many proteins are involved directly in a number of coordinated interactions to ensure the accurate and efficient replication and repair of DNA. However, a number of these coordinated interactions during the replication and repair of DNA remain uncharacterized. Therefore, studying the nature of the various protein protein and protein substrate interactions can provide a more comprehensive understanding of both DNA replication and repair in all forms of life. Specifically, the fidelity of DNA replication is highly dependent on the function of the flap endonuclease (FEN 1) family of enzymes. The FEN-1 family of DNA replication associated DNA repair enzymes are structure specific 5' to 3' endonucleases that are members of the RAD2/RAD27 family of eukaryotic nucleases. The FEN 1 family of enzymes are also functionally related to both the bacteriophage and prokaryotic 5' to 3' exonucleases. Many of the enzymes in the RAD2/RAD27 family of nucleases are involved in the processing of Okazaki fragment primers during lagging-strand DNA synthesis and in processing strands displaced during DNA synthesis associated with repair. However, a comprehensive structural characterization of the structure specific substrate recognition of the FEN 1 family of enzymes has not yet been completed. This work was focused primarily on structural studies of the archaeal Aeropyrum pernix (Ape) FEN-1 enzyme and the T4 RNase H, a FEN 1 homologue in the bacteriophage T4. A number of X ray crystallographic studies were focused on understanding the molecular basis of nucleic acid substrate recognition and the role of divalent metal ions in the catalytic mechanism of these enzymes. These structural studies have provided a more complete understanding of how catalysis is facilitated b (open full item for complete abstract)

Committee: Timothy Mueser (Advisor) Subjects: Chemistry, Biochemistry

Master of Science, University of Toledo, 2005, Chemistry

Understanding the mechanisms of DNA replication has been one of the main research topics in biochemistry for decades, due to the tremendous potential applications involved, such as medical treatments and better knowledge of the biological evolution of organisms. In this perspective, macromolecular X ray crystallography provides a very powerful tool. By solving the 3 dimensional structure of the proteins involved in replication mechanisms, one can get a good insight at complicated biological systems at the atomic level. It is also important to study the protein-protein and protein-substrate interactions when they form biological complexes. This work focused on studying several proteins which are associated with the assembly of the replication restart primosome in Escherichia coli, during the repair of damaged DNA. The crystallization of PriA, the most important of the restart primosome proteins, was investigated. Two truncations of PriA (the PriA N and PriA NI domains) were expressed, purified and characterized to be prepared for crystallization. Three additional replication restart proteins: PriB, PriC and DnaT, were expressed and purified. The solubility of each protein was optimized by identifying the best solution conditions. The proteins were characterized, alone and in complexes, using several biophysical techniques. DnaT was successfully crystallized and screened for X-ray diffraction. Finally, using ten standard test proteins, the correlation between the optimization of protein solubility (and solution conditions) and the success of crystal screening was studied.

Committee: Timothy Mueser (Advisor) Subjects: Chemistry, Biochemistry

Master of Science, University of Toledo, 2004, Chemistry

In the Mueser laboratory, we study how DNA replication and repair proteins recognize DNA in a structure-specific manner. Bacteriophage T4 is used as a model system to study DNA replication as it encodes all ten proteins required for DNA replication. Much is known about how the individual proteins function in replication but not much is known about the structural aspects of the protein-protein or protein-DNA interactions at the replication fork. The goal of our research is to study how these replication proteins interact with each other and with DNA. We work towards achieving this goal by crystallizing the protein-protein and protein-DNA complexes and then solving their structures, using macromolecular crystallography techniques. We then use the structural information gathered to analyze the interactions. The overall goal of this master's thesis project was to learn many of the techniques involved in protein chemistry and protein crystallization. My research was tailored to protein expression, purification and crystallization so I could learn an array of techniques and become familiar with various pieces of instrumentation. I wanted to be able to use this knowledge in future research positions. My work was focused on two of the replication proteins from Bacteriophage T4: T4 gene 59 helicase assembly protein and T4 gene 32 single-stranded binding protein. These two proteins interact in the absence of DNA and form a complex at the replication fork. I was responsible for expressing mutated and truncated forms of the native proteins on a large scale and developing purification protocols in order to prepare pure protein for crystal screening. After my research with the T4 helicase assembly protein began, I also started working on a similar helicase assembly protein from a related system – bacteriophage KVP40 59 protein. I was also responsible for developing a purification protocol for single-stranded DNA substrates that were used to prepare forked substrates for the cryst (open full item for complete abstract)

Committee: Timothy Mueser (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy, The Ohio State University, 2008, Ohio State Biochemistry Program

The study presented herein provides a better understanding of the global sulfur cycle at the molecular level by exploring the enzymatic process whereby DMS is generated from from dimethylsulfoxide (DMSO) by examining the pathway that leads to the generation of this gas, the DMSO reductase pathway. Resonance Raman (rRaman) spectroscopic studies have also been undertaken in order to determine the roles of two active site residues, W116 and Y114, in the catalytic cycle of substrate turnover in DMSO reductase. We have found that whereas Y114F mutant forms a complex with DMSO substrate and W116F does not form the complex using multiple component analysis (MCA) and rRaman spectroscopy. The reaction mechanism of the properly redox cycled W116F form of the enzyme was determined and is reported. The physiological reductant of DMSO reductase is a pentaheme, membrane-bound c-type cytochrome protein known as DorC. The purification procedure for wild-type DorC has been successfully developed as part of this study. The limiting rate of electron transfer from DorC to DMSO reductase has been determined to be 2.66 s-1 using stopped-flow spectrophotometry and pseudo-first order reaction conditions. This experiment also yielded a Kd of 13.2 μM for binding of DorC to DMSOR. Electron paramagnetic resonance (EPR) spectroscopy has been used to analyze the possible generation of a Mo (V) intermediate and it does not appear as though a Mo (V) state is generated in the course of the reaction. Surface plasmon resonance (SPR or BIAcore) experiments have been undertaken to determine the dissociation constant (Kd) of the complex independent of electron transfer. The Kd was determined to be approximately 30 μM. This study has also produced a computational model of DorC using the computer program Rosetta. From this model, protein docking simulations have been calculated using Hex 4.5 and have produced a compelling working model for the structure of the protein complex with a specific route of elec (open full item for complete abstract)

Committee: Ross Dalbey (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy, The Ohio State University, 2007, Chemistry

One-bead one-compound (OBOC) peptide libraries have been useful tools in the biomedical sciences. However, OBOC peptide libraries usually display the N-termini of peptides on the surface as conventional solid phase peptide synthesis proceeds in the C to N direction. While large combinatorial libraries of cyclic peptides can be synthesized by the split-and-pool synthesis method, the sequence determination has been a challenge. Also, peptide libraries with free C-termini face the same problem as well as the difficulty of synthesis in the N to C direction. We report here the development of cyclic peptide libraries and C-terminal peptide libraries for high-throughput screening and sequencing. TentaGel microbeads (90 μm) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear encoding peptide. After screening of the cyclic peptide library, the identity of hit peptides was determined by sequencing the linear encoding peptides using a partial Edman degradation/mass spectrometry method. Using the same spatial segregation approach peptides were synthesized in the conventional C to N direction, with their C-termini attached to the support through an ester linkage on the bead surface but through an amide bond in the inner layer. The surface peptides were cyclized between N-terminal amine and a carboxyl group installed at a C-terminal linker sequence, while the internal peptides stayed in the linear form. Base hydrolysis of the ester linkage in the cyclic peptides exposed a free α-carboxyl group at the C-termini of the peptides attached to the resin via the N-termini. An inverted peptide library containing five random residues was synthesized and screened for binding to PDZ domains. The identity of the binding peptides was determined from the encoding peptides. Consensus recognition motifs were identified for the PDZ domains and representative peptides were (open full item for complete abstract)

Committee: Dehua Pei (Advisor) Subjects: Chemistry, Biochemistry