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CueO is a multicopper oxidase and catalyses the four-electron reduction of dioxygen to water and functions to protect Escherichia coli against copper-induced toxicity. The mechanism of oxygen reduction in multicopper oxidases has been well studied, but the key structures of the reaction intermediates are not known. A combination of kinetic measurements, mutagenesis and X-ray crystallographic studies were conducted to entrap and structurally characterize the reaction states in CueO. CueO has a methionine-rich insert and a labile copper binding site, two features found only in multicopper oxidases involved in copper detoxification. The role of these features in CueO activity has been investigated. In a separate study, a simple mathematical model based on infectivity amongst clustered receptors was developed to explain the chemotactic sensitivity, response range and other key features of chemotaxis.This study describes the successful entrapment of three out of four functional states in CueO. The crystal structures of these reaction states are presented. Using single-turnover oxygen reduction kinetics that were measured using a stopped-flow device, the optical absorption features of three different fully oxidized forms of CueO were captured: the native intermediate, the resting oxidized state and another intermediate lying between them. Stopped-flow studies combined with electron transfer kinetic measurements revealed a role of the conserved residue, E506, in either the protonation of the native intermediate or the release of water molecules formed as a product of the reaction.Cu(I) and Ag(I) bound crystal structures of CueO were determined revealing three binding sites along the methionine-rich helix used by both metal ions. The labile, regulatory copper site in CueO was shown to be a Cu(I) susbtrate oxidation site. Ag(I) was shown to be a potent inhibitor of all CueO activities in vitro and copper detoxification by the cue system in vivo. The cus system was discovered

This dissertation is a study in how heme facilitates biology using two heme proteins as examples. I write about my mechanistic studies on Cimex nitrophorin and preliminary studies on Ecdysone inducible protein 75, respectively. Nitrophorins are salivary heme proteins used by bloodfeeding insects to deliver NO to the victim, leading to vasodilation and antihemostasis. The bedbug nitrophorin cNP, a thiolate heme protein accomplishes this via an unusual heme-assisted S-nitrosation reaction, requiring proximal ligand cleavage. This dissertation explores this mechanism through mutational, crystallographic and transient kinetic approaches. I present the detailed investigation of the two NO binding events, one at the heme and the other at the proximal cysteine. The heme nitrosyl shows marked pH dependence arising out of the apparent protonation of the proximal cysteine ligand, a feature crucial to cNP function. The structures and spectroscopy of cNP mutant proteins reveal the SNO modification to be regulatory in nature. Laser flash photolysis measurements and the structures of mutant proteins reveal the negative influence of steric hindrance on SNO stability.Studies of insect embryogenesis and metamorphosis reveal the regulatory role of the hormone ecdysone via its target, the ecdysone receptor. Ecdysone triggers expression of several nuclear receptors in a time and tissue dependant fashion, which in turn carry out gene regulation. Ecdysone inducible protein 75 (E75), a nuclear receptor and an early ecdysone responsive gene product, regulates a subset of the developmental activities attributed to ecdysone. We are investigating E75 from Aedes aegypti to uncover its role in ecdysone signaling in mosquitoes. I have expressed and partially purified the full length protein using the baculovirus driven expression in SF9 cells, and purified to homogeneity the heme binding domain resolubilized from inclusion bodies obtained by expression in E. coli. Preliminary characterization of

The number of proteins shown to be S-nitrosated in vivo is increasing steadily, highlighting the importance of this redox based post translational modification. S-nitrosoglutathione reductase (GSNOR) reduces S-nitrosoglutathione (GSNO) to GSNHOH, removing GSNO from the cytosol. GSNOR has been linked to asthma in humans and has roles in thermotolerance and disease resistance in the model organism Arabidopsis thaliana. I have studied structure by x-ray crystallography and kinetics by steady state measurements and isothermal titration calorimetry of recombinant GSNOR from both these organisms. I present several structures, including the novel structure of Arabidopsis thaliana GSNOR to 1.4 Å resolution and a ternary complex of human GSNOR with GSNO and NAD+ to 1.5 Å resolution. GSNOR’s apparent ability to reduce GSNO in an environment where the high ratio of NAD⁺/NADH should prevent reductive chemistry from occurring had been unexplained. I present steady state and isothermal titration calorimetry data that demonstrate that GSNOR preferentially binds NADH, with several fold higher affinity than NAD⁺, allowing the enzyme to selectively bind NADH and reduce GSNO.

We describe the first general approach for the DNA templated reassembly of proteins, which we term SEquence-Enabled Reassembly or SEER. SEER makes use of dissected signaling domains which are each attached to separate, sequence specific DNA-binding proteins. Described herein is an embodiment of SEER in which DNA catalyzes the reassembly of the green fluorescent protein which leads to a direct fluorescence readout of the corresponding DNA sequence. This strategy has also been extended to the first direct method for the site specific detection of DNA methylation. This mCpG-SEER system is capable of discriminating between methylated versus nonmethylated DNA with a 40-fold increase in fluorescence signal.In a separate undertaking we tested the efficiency of disulfide bond formation within the context of the ribosome display in vitro selection methodology. We established conditions for the enrichment of a cyclic peptide, which is specific for Neutravidin, by 2 x 10^6-fold. Using the knowledge gained from the above experiments, we combined the rapid protein expression and folding benefits of cell-free translation systems with a sensitive split-luciferase reassembly assay to yield the most rapid method to date for the detection of protein-nucleic acid and protein-protein interactions. Furthermore, we have shown that these split-luciferase cell-free reassembly systems can be compartmentalized, allowing for future molecular evolution studies.Lastly, we have applied this rapid cell-free split-luciferase assay system to the direct detection of clinically relevant proteins. We have engineered a system for the rapid characterization of HIV-1 clades utilizing single-chain antibody specificities. We also demonstrate that this platform can be used to determine the relative amounts of HER2 expression in human breast cancer cells, using a homogeneous assay format in which cells and reagents are mixed and luminescence is monitored directly.We envision that the assay platforms describe

Soluble guanylyl/guanylate cyclase (sGC), the primary receptor for nitric oxide (NO), is a heme containing heterodimeric enzyme involved in numerous physiological events in animals. The small molecule YC-1 stimulates sGC, but the mechanism behind and the location of binding are unknown. I have developed a prokaryotic expression system for insect ( Manduca sexta) sGC. The recombinant holoenzyme, like its mammalian counterpart, is responsive to NO, CO and YC-1, displaying a 175-fold increase in activity on binding. Truncated constructs containing the N-terminal two-thirds of both subunits (msGC-NT) were designed to facilitate expression. With the highly pure material, we investigated NO and CO binding, reaction kinetics and regulation. Binding of NO to msGC-NT heme forms a six-coordinate intermediate followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex. The conversion rate is insensitive to nucleotides, YC-1 and changes in NO concentration up to ~30 micromolar. In contrast, NO release from msGC-NT is biphasic in the absence of YC-1, while binding of YC-1 eliminates the fast phase but has little effect on the slower phase. CO binding to msGC-NT is also regulated by YC-1. The CO release rate is reduced by YC-1 while the on rate remains unchanged, which leads to an ~50-fold increase in binding affinity. YC-1 binding leads to a substantial geminate recombination of CO to msGC-NT upon photolysis. Our data are consistent with a model for allosteric activation in which (1) YC-1 binds away from the catalytic site and (2) sGC undergoes a conformational switch between two states of an open and a closed heme pocket. The final catalysis results from the integration of the influence of numerous allosteric effectors on the equilibrium between these two states.S -nitrosoglutathione (GSNO) exists in vivo and plays important roles in NO signaling. We have developed a model cell line, in which inducible NO synthase and human sGC genes were included.

Biometals such as copper, cobalt and zinc are essential to life. These transition metals are used as cofactors in many enzymes. Nonetheless, these metals cause deleterious effects if their intracellular concentration exceeds the cells' requirement. Prokaryotic organisms usually employ efflux systems to maintain metals in appropriate intracellular concentrations.The Cus system of Escherichia coli plays a crucial part in the copper homeostasis of the organism. This system is a tetrapartite efflux system, which includes an additional component compared to similar efflux systems. This fourth component is a small periplasmic protein, CusF. CusF is essential for full copper resistance, yet its role within the Cus system has not been characterized. It could potentially serve in the role of a metallochaperone or as a regulator to the Cus system.To gain insight into the molecular mechanism of resistance of this system, I have structurally and biochemically characterized CusF. Using X-ray crystallography I determined the CusF structure. CusF displays a novel fold for a copper binding protein. Through multiple sequence alignment and NMR chemical shift experiments, I proposed a metal binding site in CusF, which I confirmed through determination of the structure of CusF-Ag(I). CusF displays a novel coordination of Ag(I) and Cu(I) through a Met2His motif and a cation-pi interaction between the metal ion and a tryptophan sidechain. Furthermore, I have shown that CusF binds Cu(I) and Ag(I) specifically and tightly.I investigated the role of the tryptophan at the binding site to establish its effect on metal binding and function of CusF. I have shown through competitive binding assays, NMR studies and through collaborative EXAFS studies that the tryptophan plays an essential role in CusF metal handling. The affinity of CusF for Cu(I) is influenced by this residue. Moreover, the tryptophan also caps the binding site such that oxidation of the bound metal as well access to adventitiou

Increased emergence of antibiotic resistance in bacterial pathogens has posed a serious threat to human health. Due to similar structural and functional characteristics of metal and antibiotic resistance systems in gram-negative bacteria, there is a growing concern that metal contamination functions as a selective agent in the proliferation of antibiotic resistance. The CusCFBA copper/silver resistance system of Escherichia coli forms a protein complex that spans the inner and outer membranes and functions in the efflux of metal from the periplasm to the extracellular space. In order to understand the molecular details of metal resistance by the Cus system and more specifically, to define the role of the periplasmic components in CBA type metal transporters, I characterized CusB and probed its interactions with CusF using various structural and biochemical tools. CusB was previously thought to play a relatively passive role as an adaptor protein that stabilized the association of the inner and outer membrane proteins. Through isothermal titration calorimetry (ITC), X-ray absorption spectroscopy (XAS), and mutagenesis, I have shown that CusB binds Cu(I)/Ag(I) with high affinity using three conserved methionines. Gel filtration chromatography experiments showed that upon binding Ag(I), CusB undergoes a substantial conformational change. Importantly, functional metal binding by CusB is essential for cell survival in environments with elevated copper concentrations. The small periplasmic metal binding protein CusF is a unique component of monovalent metal resistance systems serving an unknown function. To determine the nature and specificity of interaction between CusF and CusB, ITC and NMR were used to show that the interaction between CusF and CusB is metal-dependent and specific for the components of Cus system. From NMR chemical shift perturbations, the CusB interaction face on CusF was determined to overlap with the metal binding site. XAS experiments demonstrate m

Type II polyketide synthases (PKSs) are responsible for the generation of structurally diverse and clinically important aromatic polyketides. The bacteriostatic agent enterocin (enc) isolated from the marine microbe "Streptomyces maritimus" is derived from a rare benzoate primer unit and contains a unique nonaromatic caged core structure resulting from a Favorskii-like carbon skeleton rearrangement. The apparent diversion between enterocin biosynthesis and all other type II PKS pathways offered the opportunity to discover novel enzymatic strategies that may be exploited to diversify the chemical structures of polyketides. A comprehensive biochemical analysis was performed in order to characterize the key steps in enterocin biosynthesis and finally to reconstitute the whole pathway in vitro using purified recombinant enzymes.A nonribosomal peptide synthetase (NRPS)-like priming mechanism was discovered for the selective activation of a benzoic acid starter unit and its subsequent attachment to the enc PKS to initiate polyketide biosynthesis. This is the first example of a type II PKS that employs an NPRS-like priming mechanism to utilize alternative non- acetate starter units. Secondly, the minimal enc PKS was reconstituted in vitro to give three novel acetate-primed metabolites that had never been identified by heterologous in vivo expression of recombinant enc PKS gene sets. The minimal enc PKS was then merged with the NRPS-like chain initiation module and the resulting multienzyme complex catalyzed the formation of benzoate-primed natural products wailupemycin F and wailupemycin G. Favorskii-like rearrangement of the nascent polyketide chain was replicated in vitro and the flavin-dependent oxygenase EncM was confirmed to be solely responsible for catalyzing this unprecedented rearrangement. Other biosynthetic steps in the late stage of the enc pathway were also replicated in vitro, including the methylation of desmethyl-5-deoxyenterocin to 5-deoxyenterocin and the

Multivalent interactions are common in nature, such as influenza virus infecting epithelial cells, clearance of pathogens by antibody-mediated attachment to macrophages, etc. To mimic nature, we utilized a bottom-up approach to develop various multivalent self-assembling systems based on leucine-zipper peptides. We tethered several pairs of leucine-zipper peptides to PAMAM dendrimers to form leucine-zipper dendrimers (LZDs). We conjugated Fos/Jun to the dendrimer to make D0Fos4 and D0Jun4, and studied the interactions between these LZDs and their cognate peptide target, either Jun or Fos. Our experiments showed that the D0Fos4 can non-covalently assemble four copies of Jun, and this approach can be further used for the rapid non-covalently assembling of multimeric ligands. We also pursued the multivalent target of GPCRs with a Fos/Jun assembly, and found the complex can potentially be used as a molecular switch to target GPCRs with controlled ligand activity. In a related project for bio-material design based on self-assembly of LZDs, we synthesized a different pair of LZDs, D-Ez4 and D-Kz4, and established that they can assemble at neutral pH to form helical fibrils which display higher order self-organized structures, providing a new methodology for bio-material design. In another effort for studying multivalent interactions, we conjugated three copies of the F23, mini-protein that binds the HIV-1 capsid protein, to a trimesic acid and obtained a trivalent inhibitor, Tri-F23. Tri-F23 showed enhanced binding in ELISA against gp120, but was not significantly more effective preventing HIV entry. This methodology provides a new strategy for developing multivalent inhibitors for preventing HIV-1 infection at the entry level. In a related area, we are developing imaging agents based on quantum dots that can detect GPCRs on whole cells and at the single molecule level. To this end, a new method was developed for biocompatible amphphilic polymers to coat quantum dots. Thi

Nitrophorins are nitric oxide transport proteins that aid in an insect obtaining a blood meal. The nitrophorins from the saliva of Rhodnius prolixus, a blood-sucking insect, consist of seven closely related proteins (rNP1-7). These proteins have the classic hallmarks that define the lipocalin protein family: an eight-stranded beta-barrel, binds a hydrophobic molecule, contains a flexible omega loop that lines the entrance to the binding cavity, contains a N-terminal helix and a conserved Trp-Arg/Lys interaction, and a disulfide bridge pattern. This dissertation explores the functional implications of the rNP4 structure. An apo structure of rNP4 to 1.11 angstrom resolution revealed the beta-barrel of rNP4 remains a rigid structure in the absence of its heme cofactor and is similar to that of holo rNP4. An X-ray crystallographic study of alterations to protein and heme cofactor was used to explore how heme distortion arises. In all altered proteins, local changes were seen at the point of alteration. The protein has evolved so as to have all surrounding contacts, as well as electronic interactions between ligand and heme, contribute to distortion. An omega loop triple alanine mutant of rNP4 that was unable to collapse over the binding cavity was used to investigate ligand (nitric oxide) protection crystallographically and kinetically. A 1.00 angstrom resolution structure confirmed the mutant protein was unable to collapse. The loop mutant, as well as wild-type protein, display NO binding to heme that was unaffected by oxygen, indicating that NO binds to heme before reacting with surrounding oxygen. NO-protein complex decay was very slow (hours) under conditions where NO was allowed to rebind to heme, highlighting the protection properties of rNP4. In the presence of oxygen and/or glutathione, which can react with NO, decay was accelerated but still slow (minutes to hours). Taken together, these data suggest that NO binds to the heme iron and then dissociates and assoc

Motivated by the urgent need for new molecular targets and novel agents to treat pancreatic cancer, a target-based approach to drug discovery was implemented that led to the identification, validation and targeting of the Aurora family of kinases. The Aurora kinases (A, B and C) are mitotic serine/threonine kinases involved in various aspects of mitosis, including centrosome separation, bipolar spindle assembly, chromosome alignment and cytokinesis. In this dissertation, the potential use of the Aurora kinases as therapeutic targets to treat pancreatic cancer was investigated. It was found that both Aurora A and Aurora B are overexpressed in pancreatic adenocarcinomas, suggesting that some cancer cells are dependent upon their activity for continued proliferation and survival. To validate this hypothesis, antisense oligonucleotides were used in cell-based assays to evaluate the biological consequences of Aurora A and/or Aurora B inhibition. It was found that perturbations in Aurora kinase signaling result in cell cycle arrest and apoptosis. The biological fingerprints of Aurora A and Aurora B inhibition were compared and contrasted in an effort to identify the superior therapeutic target. It was concluded that an Aurora A-targeted therapy may have some beneficial consequences; however, a therapeutic approach discriminating between Aurora A and Aurora B is not straightforward. A fragment-based approach relying heavily on computer modeling was used to design and identify a nanomolar inhibitor of the Aurora kinases; however, it showed activity only at high micromolar concentrations in cell-based evaluations suggesting the compound possessed unfavorable characteristics that limited its biological activity. The preclinical development of analogues of the compound discovered by the work presented in this dissertation is ongoing. Finally, the pancreas-specific overexpression of Aurora A kinase was shown to be insufficient to induced pancreatic tumorigenesis in a mouse tran

The existence of multiple hexameric conformations of insulin has been well defined by x-ray crystallography. Insulin conformation within the hexamer has been shown to be dependent upon solution conditions, particularly, the presence of metal ions, protein concentration, pH and the presence of ligands, (Brange, 1987). Of particular interest is the induced structural change in residues Phe^(B1)-Gly^(B8) in the T₆ → R₆ hexameric transition. Until recently NMR studies have been of limited value in probing the structure of the various insulin hexamers due to the aggregation affinity of the protein. The determination of conditions under which the monomeric unit can be maintained (Kline, 1990, Weiss, 1989) is a vital first step in this process. Although the hexameric unit is too large for the NMR experiment, a method has been developed by which the dynamics of the T₆ and R₆ hexamers can be probed with the limitation that the NMR experiment be conducted on the monomer. Comparative analysis of the ¹H NMR spectra of human insulin shows that in the presence of phenol, the tertiary structuring of the protein is altered as evidenced by the decreased rate of amide hydrogen-deuterium exchange. In particular, exchange of amide protons in residues of the B chain helix (Ser^(B9) – Gly^(B20)) appear to be most significantly affected suggesting a stabilization of this helix. The amide exchange rates for amide resonances which exchange on a medium to slow time scale, particularly residues Leu^(A16), Ala^(B14), Leu^(B15), Tyr^(B16), Leu^(B17), Val^(B18), Cys^(B19), Gly^(B20) and Phe^(B24) are reported herein. Efforts to produce an insulin mutant with decreased aggregation affinity and hence rapid pharmacologic activity have been moderately successful (Brange, 1988, 1990, Brems, 1992, Bryant, 1992). One such mutant was produced by transposing two amino acid residues in the B chain, Lys^(B28)Pro^(B29) insulin. The NMR spectral assignment of this mutant has been completed and the dynamics o

The lambdoid coliphage HK022 can grow in either a lytic or lysogenic mode following infection. In λ phage the switch to either mode is controlled by the cI and Cro repressors which bind to operators in the λ chromosome and regulate gene expression of adjacent promoters. HK022 is organized similar to λ, but has its own unique repressors and operators which are different from λ. The major goal of this research was to characterize the properties of the CI repressor from HK022 to understand better how this protein regulates gene expression. Seven sites in HK022 with similar nucleotide sequences were identified by DNase I footprinting analyses as CI binding sites. Six were clustered in two groups of three, O(L)1-O(L)3 (operator left) and O(R)1-O(R)3 (operator right), while the remaining site O(FR) (operator far right), was alone. The positions of these sites on the HK022 chromosome from left to right was O(L)1-O(L)3, cI, O(R)3-O(R)1, cro, and O(FR). CI bound to adjacent operators, O(R)1 and O(R)2, with a high degree of cooperativity (cooperativity parameter, ω, of about 2000). When only a single site was present (as with O(FR)), the repressor also increased the affinity for adjacent non-specific DNA sites resulting in a "phased" pattern of binding. CI binding sites were defined as operators by analyses of virulent phage mutants, which were able to escape CI repression and grow on a lysogen. The vast majority of virulent mutants contained 2 mutations in O(R), with single mutations in O(R)1 and O(R)2, suggesting that mutations in both sites are required to overcome the high degree of cooperativity. However, one mutant contained only a single mutation in O(R) and a second mutation in the distant site, O(FR). When the single O(FR) mutation was recombined with a wild-type O(R), the resulting phage was not virulent, but virulent mutants arose from it at a high frequency by acquiring a single mutation in either O(R)1 or O(R)2. The mechanism of action by O(FR) is not understood.