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Tapping mode analysis of lambda-DNA and carboplatin interactionsSabo, Michael J. 17 November 2015 (has links)
<p> The purpose of this research was to examine the complexation of carboplatin and λ-DNA via atomic force microscopy. This project had the challenge of getting the necessary resolution which lead to the need to examine and improve upon the experimental protocol. These resolution issues were fixed by eliminating contamination, and by developing more consistent means of DNA application. The carboplatin and DNA complexation was then able to be observed. Initial indications are consistent with expectations because the DNA appears to become more condensed over time but further examination is required.</p>
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Controlled oxygen activation in human oxygen sensor FIHSaban, Evren 01 January 2011 (has links)
One of the primary oxygen sensors in human cells, which controls gene expression by hydroxylating the hypoxia inducible transcription factor (HIFα) is the factor inhibiting HIF (FIH). As FIH is an alpha-ketoglutarate dependent non-heme iron dioxygenase, oxygen activation is thought to precede substrate hydroxylation. The coupling between oxygen activation and substrate hydroxylation was hypothesized to be very tight, in order for FIH to fulfill its function as a regulatory enzyme. Coupling was investigated by looking for reactive oxygen species production during turnover. Alkylsulfatase (AtsK), a metabolic bacterial enzyme with a related mechanism and similar turnover frequency, was used for comparison, and both FIH and AtsK were tested for H2O 2, O2- and OH· formation under steady and substrate-depleted conditions. Coupling ratios were determined by comparing the ratio of substrate consumed to product formed. AtsK reacted with O2 on the seconds timescale in the absence of prime substrate, and uncoupled during turnover to produce H2O2; neither O2- nor OH· were detected. In contrast, FIH was unreactive toward O2 on the minutes timescale in the absence of prime substrate, and tightly coupled during steady-state turnover; any reactive oxygen species produced by FIH was not available for detection. Inactivation mechanisms of these enzymes were also investigated. AtsK likely inactivated due to deoligomerization, whereas FIH inactivated by slow autohydroxylation. Autohydroxylated FIH could not be reactivated by dithiothreitol (DTT) nor is ascorbate, suggesting that autohydroxylation likely to be irreversible under physiological conditions. Iron in the FIH active site is coordinated by a (His2Asp) facial triad, αKG, and H2O. Hydrogen bonding between the facial triad, the HIF-Asn803 sidechain, and various second-sphere residues suggests a functional role for the second coordination sphere in tuning the chemistry of the Fe(II) center. Point mutants of FIH were prepared to test the functional role of the αKG-centered (Asn205, Asn294) or HIF-Asn803 centered (Arg238 , Gln239) second-sphere residues. The second sphere was tested for local effects on priming Fe(II) to react with O2, oxidative decarboxylation, and substrate positioning. Steady-state kinetics were used to test for overall catalytic effects, autohydroxylation rates were used to test for priming and positioning, and electronic spectroscopy was used to assess the primary coordination sphere and the electrophilicity of αKG. Asn205→Ala and Asn294→Ala exhibited diminished rates of steady-state turnover, while minimally affecting autohydroxylation, consistent with impaired oxidative decarboxylation. Blue shifted MLCT transitions for (Fe+αKG)FIH indicated that these point mutations destabilized the π* orbitals of αKG, further supporting a slowed rate of oxidative decarboxylation. The Arg238→Met mutant exhibited steady-state rates too low to measure and diminished product yields, suggesting impaired substrate positioning or priming; Arg238 →Met was capable of O2-activation for the autohydroxylation reaction. The Gln239→Asn mutant exhibited significantly slowed steady-state kinetics and diminished product yields, suggesting impaired substrate positioning or priming. As HIF binding to Gln239→Asn stimulated autohydroxylation, it is more likely that this point mutant simply mis-positions the HIF-Asn803 sidechain. By combining kinetics and spectroscopy, it was shown that these second sphere hydrogen bonds play roles in promoting oxidative decarboxylation, priming Fe(II) to bind O 2, and positioning HIF-Asn803.
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Structure and function in a nickel metallochaperone, HypA and nickel dependent superoxide dismutaseHerbst, Robert Walter 01 January 2010 (has links)
Nickel enzymes are critical for the survival of many different organisms; Urease and NiFe-hydrogenase are essential to acid viability in Helicobacter pylori, and Nickel dependent superoxide dismutases (NiSOD) provide defense against oxidative damage in Streptomyces species. The work herein focuses on understanding structure/function relationships in a metallochaperone involved in nickel trafficking and the unique role played by nickel in redox active enzymes. HypA is a metallochaperone in H. pylori that is able to sense both nickel loading and pH changes, which are critical to its proper function. XAS studies show that the structural zinc site is able to change ligand coordination (from a distorted tetrathiolate site to a tetrahedral site) upon Ni binding. Upon lowering the pH, the zinc site undergoes ligand substitution and forms a mixed N/S-donor site. Mutagenesis of the two conserved CxxC motifs, as well as two flanking histidine residues shows how changes at the zinc site are able to affect the nickel binding properties and conformation of HypA, and help sense fluctuations in pH. Superoxide dismutases rely on structural elements to adjust the redox potential of the active site to an optimum value, ∼300 mV (vs NHE), to provide a source of protons for catalysis, and to control the access of anions to the active site. These aspects of the catalytic mechanism are examined herein for NiSOD and a series of mutants that affect a key tyrosine residue, Tyr9. Structural studies show that second sphere mutations do not affect the first coordination sphere of NiSOD. Kinetic investigations show that the mutant proteins have impaired but measurable activity. In the case of Y9F-NiSOD, the enzyme exhibits saturation behavior that is not observed in WT-NiSOD and suggests that release of peroxide is inhibited. The crystal structure of Y9F-NiSOD reveals an anion binding site that is occupied by either Cl- or Br- and is located close, but not within bonding distance of the nickel center. The structure of D3A-NiSOD reveals that in addition to affecting the interaction between subunits, this mutation repositions Tyr9 and leads to altered chemistry with peroxide. Proper positioning of second-sphere residues is critical in maintaining the optimal efficiency of NiSOD.
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Metal specificity in nickel responsive transcriptional regulatorsLeitch, Sharon Virginia LaMont 01 January 2008 (has links)
Nickel is a required nutrient for bacteria. In E. coli, the regulation of nickel levels is achieved by NikR and RcnR, two nickel-responsive transcriptional regulatory proteins. Work herein illustrates connections between metal-site structure and protein function, proposing a mechanism for nickel-specific response. The high-affinity site of NikR is substituted with Co(II), Ni(II), Cu(II), Cu(I), and Zn(II) and the coordination environments characterized with XAS. The cognate nickel binds in a four-coordinate planar His3Cys1 coordination environment. All other metals exhibit significant differences in coordination environment. Cu(II) and Zn(II) bind with the same ligands as Ni(II), however Cu(II) is distorted and Zn(II) is tetrahedral. Cu(I) is three coordinate, with a loss of an N/O-donor. Co(II) differs most compared to Ni(II). It is octahedral and is the only metal to lose the Cys S-donor. Concurrent results showed that different metals elicit different protein dynamics and function. These results implicate coordination geometry and ligand selection as a mechanism for metal-ion selectivity. Further structural studies of NikR are carried out on the low-affinity nickel site. The low-affinity site is six-coordinate and solvent accessible. RcnR is a recently characterized nickel-responsive regulatory protein. Herein, structure-function studies are used to characterize the metal binding sites. Ni-RcnR and Co-RcnR share many of the same ligands, but have distinct coordination spheres. Both metals have pseudooctaheral geometries, with a nickel coordination sphere of (NH2)NHis3NHis64SCys35(N/O) 2 and a cobalt coordination sphere of (NH2)NHis3N His60NHis64SCys35(N/O). The possible role in metal recognition of the invariant cysteine ligand is examined for both NikR and RcnR. In NikR, the absence of C95 drastically reduces the binding affinity for nickel. In RcnR, C35 is required for cobalt function, but not nickel function, implicating it as a structural residue in Ni-RcnR and a functional residue, perhaps as an electronic sensor and point of allosteric communication, in Co-RcnR. The data herein supports a two part mechanism, cognate metal recognition through coordination environment elicits an allosteric response in the regulator, which alters DNA binding affinity.
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Metal selectivity in the E. coli Nickel(II)- and Cobalt(II)-responsive transcriptional regulator, RcnRHiggins, Khadine Athena 01 January 2012 (has links)
Cellular trafficking systems for transition metals require importers, exporters, chaperones and regulators that respond to specific metals. Understanding how different metals are recognized by proteins in order to generate specific biological responses is a goal of this research. E. coli RcnR is a 40.4 kDa tetrameric transcriptional repressor that responds to the binding of Ni(II) or Co(II) at micromolar concentrations to allow the expression of the exporter, RcnA. E. coli RcnR has significant amino acid sequence homology with M. tuberculosis CsoR, a Cu(I) responsive regulatory protein. RcnR and CsoR are members of a new structural class of transcriptional regulators characterized by an &agr;-helical structure consisting of a four-helix bundle. RcnR binds to a variety of metals in vitro, and it is thought that the metal ion-selective biological response derives from both preferences in ligand selection and coordination number. This model is similar to that seen for E. coli NikR, the Ni-responsive transcription regulator responsible for the repression of the nickel importer, NikABCDE. Prior data obtained from XAS show that RcnR forms six-coordinate complexes with its cognate metal ions (Ni(II) and Co(II)) that have M(N/O)5S ligand donor atom sets. Mutagenesis and lacZ data suggests that these ligands are derived from the coordination of the N-terminal amine and the side chains of Cys35, His3, His60 and His64. The largest structural difference between the Co(II) and Ni(II) complexes was found to be the M-Scys35 distance (2.31 Å and 2.62 Å for Co(II) and Ni(II), respectively). Metal substitutes of both wild-type and mutant RcnR proteins (A2*, H3L, H3C, H3E, H60C, H64C and H67C) have been prepared and characterized. The metal complexes were assayed for RcnA de-repression using LacZ reporter assays. The data from the wild-type metal complexes with RcnR show that metal responsiveness in RcnR is linked to the coordination number and geometry of the metal ions; the cognate metal ions Ni(II) and Co(II) are six-coordinate while the non-cognate metals have low coordination numbers. All the metals bind at the same locus, involving C35, the only cysteine residue in the protein. However, only binding the six-coordinate metal ions, Ni(II) and Co(II), results in de-repression of RcnA. The role of specific ligands in creating the metal binding site was addressed by mutagenesis and XAS. These studies show that the binding of the N-terminal amine is important in discriminating cognate from non-cognate metals. Further, Ni(II) and Co(II) are recognized differently; the complexes formed have distinct interactions with His3 and very different M-SCys35 distances. Mutation of His3 to aspartate resulted in a protein that now responds to the binding of Zn(II) ions, to our knowledge the first example of changing the metal-specificity of a transcriptional regulator.
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Chemically directed assembly of nanoparticles for material and biological applicationsPark, Myoung-Hwan 01 January 2012 (has links)
The unique electronic, magnetic, and optical properties of nanoparticles (NPs) make them useful building blocks for nanodevices and biofabrication. Site-selective immobilization/deposition of NPs on surfaces at desired positions is an important fabrication step in realizing the potential of nanomaterials in these applications. In this thesis, my research has focused on developing new strategies for mono- and multilayered-NP deposition on surfaces, increasing the stability of NP-assembles upon various surfaces for practical use of NP-based devices. Chemically directed dithiocarbamate binding of amine groups to NPs in the presence of CS2 was used for enhancing the robustness of NP assembles. Such patterning methodologies have allowed me to use site-directed NP immobilization in applications as diverse as microcontact printing, nanomolding in capillaries, nanoimprint lithography, and photolithography. Also, I have developed a simple and reliable one-step technique to form robust dendrimer-NP nanocomposites using dithiocarbamate-based chemistry. These composites are able to encapsulate and release various therapeutics, providing controllable sustained release and to separate small molecules and biomacromolecules.
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Mutational analysis of geopilin function in Geobacter sulfurreducensRichter, Lubna V 01 January 2011 (has links)
Geobacter sulfurreducens possesses type IV pili that are considered to be conductive nanowires and a crucial structural element in biofilm formation, enabling electron transfer to insoluble metal oxides in anaerobic sediments and to graphite anodes in microbial fuel cells. The molecular mechanism by which electrons are transferred through the nanowires to the electron acceptor is not fully understood. Prior to the work described in this thesis, the gene (pilA) encoding the structural pilus subunit had been identified, but little was known about the functional translation start codon, the length of the mature secreted protein, or what renders the pili conductive. Using mass spectrometry, I found that a tyrosine residue (Y32) near the carboxyl terminus of the mature PilA protein is posttranslationally modified by attachment of glycerophosphate. I studied the significance of Y32 for biofilm formation on various surfaces and for growth of G. sulfurreducens with insoluble electron acceptors. A mutant in which Y32 was replaced by phenylalanine lacked the glycerophosphate; biofilm formation on graphite surfaces was severely diminished and current production in microbial fuel cells was initiated only after a long lag phase. Moreover, cells with Y32F mutation in the pilA gene exhibited growth deficiency when Fe(III) oxide was the sole electron acceptor. My data confirm the role of G. sulfurreducens pili in biofilm formation and electron transfer to Fe(III) oxide and identify an amino acid in the PilA protein that is essential for these two processes. I also confirmed the existence of two functional translation start codons for the pilA gene and identified two isoforms (short and long) of the PilA preprotein by series of genetic complementation experiments. The short PilA isoform is found predominantly in an intracellular fraction, and seems to stabilize the long isoform and influence the secretion of several outer surface c-type cytochromes. The long PilA isoform, on the other hand, is required for secretion of PilA to the outer surface of the cell, a process that requires co-expression of pilA and the nine genes on its 3' side. The long isoform is essential for biofilm formation on various surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxide. This study provides new insight concerning the function and biogenesis of Geobacter type IV PilA, as well as a foundation for further research that will be conducted on microbial nanowires.
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New insights into enzymatic CO₂ reduction using protein film electrochemistryWang, Vincent Cho-Chien January 2013 (has links)
Carbon monoxide dehydrogenase (CODH) is known to catalyze CO oxidation and CO₂ reduction reversibly with the minimal overpotential. A great advantage of protein film electrochemistry (PFE) is its ability to probe catalysis over a wide range of potentials, especially in the low potential region required for CO₂ reduction. CODH I and CODH II from Carboxydothermus hydrogenoformans(Ch) and the composite enzyme acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) from Moorella thermoacetica(Mt) are intensively studied throughout this thesis. The different catalytic redox-states in CODH, C<sub>ox</sub> (inactive state), C<sub>red1</sub> (for CO oxidation) and C<sub>red2</sub> (for CO₂ reduction) as characterized by spectroscopy, are studied by PFE in the presence of substrate-mimic inhibitors. Cyanide, isoelectronic with CO, mainly inhibits CO oxidation, whereas cyanate, isoelectronic with CO₂, mainly targets CO₂ reduction. Sulfide inhibits CODH rapidly when the potential is more positive than −50 mV, which suggests that sulfide reacts to form a state at the oxidation level of C<sub>ox</sub> in CODH and is not an activator for CODH catalysis as suggested before. Thiocyanate only partially inhibits CO oxidation. No inhibition of CODH by azide is detected, which is in contrast with previous studies with ACS/CODH<sub>Mt</sub>. The main differences between CODH I<sub>Ch</sub> and CODH II<sub>Ch</sub> are the stronger CO product inhibition and inhibition of CODH II<sub>Ch</sub> by cyanide. These discoveries might shed light on the possible role of CODH II<sub>Ch,/sub> in biological systems. In comparison with bidirectional (reversible) electrocatalysis by CODH I<sub>Ch</sub> and CODH II<sub>Ch</sub>, only unidirectional electrocatalysis for CO oxidation by ACS/CODH<sub>Mt</sub> is observed with an overpotential of 0.1 V and the electrocatalytic current is much smaller. In order to identify whether ACS influences the performance of CODH, several chemical reagents, such as sodium dodecyl sulfate (which separates CODH and ACS partially), 1, 10-phenanthroline, (which inhibits the active site in ACS) and acetyl-CoA (the product of the reaction carried out by ACS/CODH<sub>Mt</sub>) are added. However, we have yet to observe any electrocatalytic current from CO₂ reduction. Inhibition of ACS/CODH<sub>Mt</sub> by cyanide, cyanate and azide is consistent with previous studies by spectroscopy. Oxygen attack toward the active site in CODH is proved by cyanide protection. The inactive state, C<sub>ox</sub> can prevent oxygen attack and reductive reactivation restores CODH activity. In contrast, oxygen damages the active site irreversibly when CODH is in the C<sub>red1</sub> state. The new substrate, nitrous oxide (N₂O), isoelectronic with CO₂, is reduced by CODH and acts as the suicide substrate. Finally, hydrogen formation in the direction of CO oxidation and formate formation in the direction of CO₂ reduction by CODH are detected. The small solvent kinetic isotope effect is observed in CO oxidation. These findings suggest metal-hydride should play a role in CODH catalysis, which might provide a new direction to design better catalysts for CO₂ reduction.
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Characterizing Metallopeptide-Based Antimicrobials and Artificial Glycosidases: Progress in Artificial Metalloenzymes and TherapeuticsThompson, Zechariah January 2021 (has links)
No description available.
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