One third of proteins in nature have been revealed as metalloproteins, whereas most of them remain uncharacterized, probably due to the lack of robust methods especially for tracking metalloproteins within the living context. Fluorescent labeling is capable to detect biomolecules with molecular resolution in living cells. Tracking metal-binding proteins in living cells by fluorescence could provide invaluable information in understanding their localization and potential functions in the native environment. A synthetic molecular probe NTA-AC was designed and synthesized to track metal-associated proteins in living cells upon chelation with metal ions. The fluorescent probe consists of a small molecular fluorophores, a metal-chelating moiety to direct the metal-chelated probe to the protein targets, and a photo-active crosslinker. Metal being chelated could help further explore potential binding targets and direct the fluorescent agent to the appropriate region, then subsequently covalent linkage to targets could be generated through photo-activation. NTA-AC was therefore chelated with different metals to examine its binding preference to different proteins. The Ni2+-chelating probe was applied to track Ni2+-binding proteins as an example to validate its applicability. Ni2+-NTA-AC preferentially binds to histidine-rich peptides and proteins thus verified its binding specificity. The Ni2+-chelated probe was further exploited to light up over-expressed histidine-rich proteins in Escherichia coli cells to validate its membrane permeability and binding specificity. In addition, the probe was applied to label His-tagged proteins expressed in tobacco plant cells to further evaluate its applicability in detecting and localizing the protein targets in eukaryotic cells. Afterwards, Ni2+-NTA-AC was exploited to track Ni2+-binding proteins in living Helicobacter pylori cells and incorporated with gel electrophoresis and mass spectrometry for protein identification. Many proteins identified are correlated to Ni2+-association and thus validating the applicability of the probe. Bi3+-chelated NTA-AC was therefore used to mine potential targets in H. pylori. Intense fluorescence was observed within H. pylori cells thus indicating the effectiveness of the fluorescent labeling. Protein separation and identification was therefore initiated to trace potential targets, while finding that some of the Bi 3+-coordinated proteins participate in various functioning pathways of the pathogens. The effects of colloidal bismuth subcitrate (CBS) on pH buffering and redox defense systems were therefore determined and verified, confirming that respective proteins could be potential therapeutic targets of the drug. Cr3+-NTA-AC was further applied to human Hep G2 cell line to determine Cr3+-binding targets in mammalian cells. Their localization on mitochondria was revealed, implying the potential effects of Cr3+ on mitochondria. Further confirmation of protein targets was performed through protein separation and identification. Proteins identified could be positively correlated to mitochondrial functions and thus revealing that Cr3+ might exert its effect at mitochondria. Addition of Cr3+ to Hep G2 could prevent mitochondrial fragmentation induced by hyperglycemia, which thus suggests the possible therapeutic function of Cr3+. The extensive application of NTA-AC in tracking Ni2+-, Bi3+- and Cr3+-associated proteins has validated the effectiveness of such strategy in detecting and localizing metalloproteins within the living context and thus could be extended to investigate other metalloproteomes. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

The direct (unmediated) electrochemistry of a variety of redox proteins has been studied at a range of electrode materials. Electrochemical studies using cytochromes, iron-sulphur proteins and copper proteins show a marked enhancement of the heterogeneous electron-transfer rate at the "edge" plane of pyrolytic graphite. Parallel ESCA studies have shown that mechanical polishing of edge graphite provides an electrode with a high surface coverage of oxidised functional groups. These results indicate the importance of oxidised functional groups in facilitating productive interactions with the proteins. The importance of multivalent cations has also been established. For proteins with negatively-charged physiological interaction domains, such as plastocyanin or 2[4Fe-4S] ferredoxin, elect reactivity at "edge"-oriented graphite is promoted and stabilised in the presence of multivalent cations such as Cr(NH,sub>3</sub>)₆³⁺. On the other hand, the electrochemistry of positively-charged cytochrome ̲c is inhibited and destabilised in the presence of such cations. Promotion and inhibition profiles for a range of proteins, together with various cations, indicate the participation of specific electrode-cation-protein interactions. Studies on plastocyanin, whose electrochemistry is troubled by a time-dependent deterioration of response, have demonstrated the importance of low protein concentration, low pH, low temperatures (3°C) and, in particular, Pt(NH₃)₆⁴⁺ at stabilising faradaic responses. Surface modification of edge pyrolytic graphite has been achieved electrochemically by exploiting the contrasting substitutional reactivity of chromium(III) and chromium(II). The modified-surface, incorporating chromium(III) complexes, promoted reversible direct electrochemistry of plastocyanin. The direct electrochemistry of plastocyanin, at pH 4, has provided an insight into the possible importance of a kinetically-inactive protonated form of the reduced protein. The ^t1/2 for deprotonation of Cu(I)-plastocyanin is estimated to be < 1 ms. Finally, the direct electrochemistry of azurin was exploited in the design of an electrocatalytic system. The electrochemical oxidation of p-cresol to p-hydroxybenzaldehyde was effected enzymically.

Thesis (Ph. D.)--Oregon Graduate Center, 1988.

Thesis (Ph.D.)--OGI School of Science & Engineering at OHSU, Oct. 2005. / Includes bibliographical references (leaves 115-134).

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, December 2003. / Includes bibliographical references. Also available on the Internet.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Iron is important for human health and serves as a co-factor in a variety of proteins and enzymes. Human serum transferrin (hTF) is an Fe(III) transporter in blood plasma which delivers metal to cells via a receptor-mediated endocytosis. In the first part, crystal structures of FeNFeC-hTF and BiNFeC-hTF have been characterized. The N-lobes of the two structures adopt “partially opened” conformations between holo-hTF’s “closed” and apo-hTF’s “fully-opened” states. The N-lobe of BiNFeC-hTF opens wider than FeNFeC-hTF. Their metal-bound C-lobes are totally closed. Rigid-body movement and different inter-lobal hydrogen bonds for the “partially opened” conformations are observed. The binding affinities of four putative binding residues are in the order: Tyr188>Tyr95>Asp63~His249. In the N-lobe of BiNFeC-hTF, Tyr188, bicarbonate and a nitrilotriacetate (NTA) ion bind to Bi(III), whilst Tyr95 and Asp63 interact with NTA ligand. One (BiNFeC-hTF) or two (FeNFeC-hTF) glycan molecules are identified on the surface area of C-lobe. In the second part, biocoordination chemistry of selected metal ions was investigated using hTF as a template. The Al(III), Fe(III), Ga(III), Dy(III) and Yb(III)-bound hTF exhibit closed conformations in the C-lobe and “fully-opened” conformations in the N-lobe. In these structures, malonate serves as an anion in the C-lobe and provides two tunable ligation sites that lead to a less distorted octahedral coordination geometry. As a result, the large lanthanide ions (Dy(III) and Yb(III)) turn from their favored high coordination numbers (8~12) and fit into the protein’s hexadental pocket. Unexpectedly, in the presence of malonate ion and the excess amount of Dy(III) ion, the Ga(III) can be partially replaced by Dy(III), although Ga(III) has a much higher affinity than Dy(III) towards the protein. The chaperone system in Helicobacter pylorithat helps protein refold is assembled with HspB and HspA. In the third part, preliminary crystallographic work is reported for HspB and HspA. The chaperone HspB has been crystallized under various conditions and currently the diffraction resolution is 6.8Å. The co-chaperone HspA, which binds Bi(III) tightly, although its crystals diffract to 1.6Å, still needs improvement for data collection due to radiation damage.The crystal structure of HspB revealed that HspB presents as a single-ring heptamer, though it is a mixture of dimer, tetramer and a higher oligomer in solution. The interactions between HspB monomers in crystal structure are significantly weaker than that of GroEL (counterpart in Escherichia coli) monomers which may makes the HspB heptamer easier to dissociate in solution. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Thesis (Ph.D.)--Michigan State University. Dept. of Biochemistry and Molecular Biology, 2008. / Title from PDF t.p. (viewed on Mar. 30, 2009) Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--University of Iowa, 2008. / Thesis supervisor: Sonya J. Franklin. Includes bibliographical references (leaves 129-134).

[NiFe] hydrogenases are found in a variety of microorganisms and catalyze the reversible oxidation of hydrogen gas to protons and electrons. This enzyme has generated intense interest due to its contribution to pathogenicity in certain organisms as well as its application in bioremediation and the production of hydrogen as an alternative fuel source. The biosynthesis of the dinuclear active site requires a number of accessory proteins to chaperone and insert the metal cofactors to the awaiting large subunit of hydrogenase. The proteins responsible for nickel delivery to Escherichia coli hydrogenase 3 are HypA, HypB, and SlyD, however the mechanism by which this is accomplished is unclear. The goal of this work was to analyze the metal-binding abilities and protein interactions of these nickel insertion proteins to enhance our understanding of their roles. Isolated N-terminal peptide of HypB has similar high-affinity metal-binding to the full-length protein. This peptide binds nickel in a square planar site with three cysteinyl and a fourth N-terminal amine ligand. Additionally, studies with SlyD and HypA reveal protein interactions that occur during hydrogenase maturation. Pull-down experiments of a tagged variant of hydrogenase revealed multi-protein complexes with HypA, HypB, and SlyD. A complex between SlyD and hydrogenase forms prior to both nickel and iron insertion, supporting chaperone activity of SlyD during hydrogenase maturation. HypA can interact with hydrogenase in the absence of HypB and SlyD, and a possible role as the bridging protein during the nickel insertion event is proposed. In addition, fluorescent imaging of E. coli cells using a fluorescently labeled streptavidin conjugate revealed localization of both Strep-tagged II hydrogenase and HypA at or near the cell membrane, suggesting that enzyme maturation occurs proximal to metal transporters. This work provided a deeper understanding of the role that each of these proteins play in [NiFe] hydrogenase assembly and is helpful for any future applications of this enzyme.