Biochemistry Commons^™

Impact Of The Pre-A Motif On Truncated Hemoglobin N Activity, Alexander Shayne Drena

Theses and Dissertations

Tuberculosis (TB) remains the leading cause of death by an infectious agent and therefore a global health crisis, according to the most recent report by the World Health Organization. This is due, in part, to Mycobacterium tuberculosis’ impressive defensive mechanisms against immune response, as well as the rise of Multi-Drug Resistant strains that have recently developed. Towards the turn of the century, a small heme protein called truncated hemoglobin N (trHbN) was discovered to protect the bacteria against reactive nitrogen species by converting nitric oxide (NO) to nitrate at rates far exceeding those of myoglobin and closer to those of …

The Effects Of An R103q Mutation On The Chemical And Physical Properties Of The Enzyme Cytochrome C Nitrite Reductase (Ccnir), Shahama Alam

Theses and Dissertations

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic homodimeric decaheme enzyme that catalyzes the reduction of nitrite to ammonium in a process that involves six electrons and eight protons. The project described herein explored the properties of the Shewanella oneidensis R103Q ccNiR variant and compared them to the properties of the wild type enzyme. Under standard assay conditions, which use the strong reducing agent methyl viologen monocation radical (MVred) as an electron source, the R103Q variant still catalyzes reduction of nitrite to ammonium, albeit with an MVred turnover rate that is 20% of the wild type’s (780 ± 50 s-1 …

Development Of New Treatments For Asthma And Neuropathic Pain Based On Ɣ-Aminobutyric Acid A Receptor (Gabaar) Ligands, Nicolas Mark Zahn

Theses and Dissertations

The γ-aminobutyric acid A receptor (GABAAR) is a ligand-gated pentameric chloride channel consisting of several identified subunits: α1-6, β1-3, γ1-3, δ, ε, π, θ, ρ1-3.1-2 Typical arrangement of subunits consists of two α subunits, two β subunits, and one γ subunit.3 GABAARs have two binding sites for the endogenous ligand γ-aminobutyric acid (GABA), between the α and β subunits. GABAARs also have a binding site for positive allosteric modulators, such as benzodiazepines, between the α and γ subunits.4-5 Due to their ability to treat anxiety, epilepsy, insomnia, and muscle relaxation, benzodiazepines are widely prescribed pharmaceuticals.6-7 Still, adverse effects result from …

Protein-Ligand Binding As A Tool To Identify Antiviral Drugs., Rajdeep Virdi

Theses and Dissertations

Viruses are the most abundant biological entities on the planet. A virus is a collection of essential genetic material encapsulated in a protein coat that is incapable of replicating without a host. A virus must inject its genetic material into a suitable host cell to utilize host machinery to replicate. During the process of replication, a virus hijacks cellular functions, avoids or inhibits host antiviral defenses, and sometimes causes disease in the host organism. One effective way to fight viral infection is to identify molecules that inhibit the function of essential viral proteins. The studies described in this dissertation focus …

Part I: Development Of Small-Molecule-Based Probes For The Vitamin D Receptor; Part Ii: Development Of A Scalable Manufacturing Process For Orcein Dye, Tania Roseann Mutchie

Theses and Dissertations

PART I:The vitamin D receptor (VDR) is a ligand-dependent transcription factor and member of the nuclear hormone receptor superfamily. VDR is expressed in the epithelia of endocrine organs, digestive system, bronchi, kidneys, and thymus, as well as being present in leukocytes and bone cells. Cell proliferation, cell differentiation, and immunomodulation, along with calcium and phosphate homeostasis, are all processes regulated by the receptor. Within the cell, VDR can be membrane-bound or located in the nucleus. Nuclear localization of VDR transpires following the binding of vitamin D metabolites, the most active of which is 1α,25-dihydroxyvitamin D3 (calcitriol). Within the nucleus, interactions …

Investigation Of G Protein-Coupled Receptor Quaternary Structure Through Fluorescence Micro-Spectroscopy And Theoretical Modeling: Interdependence Between Receptor-Receptor And Receptor-Ligand Interactions, Joel David Paprocki

Theses and Dissertations

Proteins are of high interest in biophysics research due to the important roles they play within cells, such as sensing of chemical (ions and small molecules) and physical (e.g., light) stimuli, providing structure, transporting ions/molecules, signaling, and intercellular communication. The studies described in this dissertation focus on a particular type of membrane proteins known as G protein-coupled receptors (GPCR), which play a key role in cellular response to external stimuli. We used the sterile 2 α-factor mating pheromone receptor (Ste2), a prototypical class D GPCR present within Saccharomyces cerevisiae (baker’s yeast). Ste2 is responsible for initiating the second messenger signal …

Structure & Function Of Enzymes In Two Uncharacterized Gene Clusters From Pseudomonas Brassicacearum & Streptomyces Griseofuscus, Lamia Tabassum Badhon

Theses and Dissertations

Pyridoxal 5’-phosphate (PLP)-dependent enzymes harness this versatile cofactor to catalyze a variety of reactions including transamination, decarboxylation, racemization and various elemination/subsitution reactions. Several years ago, a new class of PLP-dependent enzymes was discovered that uses PLP and molecular oxygen to catalyze the 4-electron oxidation of L-arginine to 4-hydroxy-2-ketoarginine. Work with the prototypical enzyme of this class, MppP from Streptomyces wadayamensis (SwMppP), showed that the dioxygen consumed during the reaction is reduced to hydrogen peroxide, and that the hydroxyl group installed in the product derives from water. Thus, SwMppP is an L-arginine oxidase, and not an oxygenase. This was surprising given …

A Mechanistic Investigation Of Cytochrome C Nitrite Reductase Catalyzed Reduction Of Nitrite To Ammonia: The Search For Catalytic Intermediates, Shahid Shahid

Theses and Dissertations

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic homodimeric decaheme enzyme that catalyzes the reduction of nitrite to ammonium in a process that involves six electrons and eight protons. Under standard assay conditions, which use a strong reducing agent as an electron source, catalysis takes place rapidly without producing detectable intermediates. However, intermediates do accumulate when weaker reducing agents are employed, allowing the ccNiR mechanism to be studied. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated in isolation by using the weak reducing agents N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) and the 2-electron reduced form of indigo trisulfonate. Experiments were …

Structural And Functional Characterization Of L-Enduracididine Biosynthetic Enzymes, Nemanja Vuksanovic

Theses and Dissertations

The mannopeptimycins produced by Streptomyces hygroscopicus are non-ribosomal peptide antibiotics with activity against a number of Gram-positive pathogens, including drug-resistant strains like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Attempts to synthesize more potent mannopeptimycin analogues have so far not been successful. One reason this has proven difficult is that the mannopeptimycins contain the unusual non-proteinogenic amino acid L-enduracididine (L-End), or its hydroxylated derivative β-hydroxy enduracididine (βhEnd,). This amino acid is not commercially available and, while synthetic routes have been developed, they are multi-step processes starting from an advanced intermediate. Understanding enzymatic synthesis of L-End is an important goal, …

Quagga Mussel Induced Phosphorus Cycling Changes In Lake Michigan, Rae-Ann Maclellan-Hurd

Theses and Dissertations

Quagga mussels (Dreissena rostriformis bugensis) are an invasive ecosystem engineer that have successfully colonized both profundal and nearshore regions in Southern Lake Michigan. Quagga mussels directly altered the flow of nutrients by filtering particles, excretion of soluble forms of nutrients in the benthos, and through the production of biodeposits. Mussel excretion, egestion, and capture rates of P were compared between a 25 meter and a 55 meter deep site near Milwaukee Harbor to determine the retention of nutrients in these regions. The capture rate of particulate phosphorus at the 25m site (278 ± 388 μmol m-2) was similar to that …

Understanding The Role Of Protein Kinases Kin1 And Kin2 In The Protein Folding Pathways In The Yeast Saccharomyces Cerevisiae, Chandrima Ghosh

Theses and Dissertations

Eukaryotic protein kinases catalyze the transfer of the -phosphate of an ATP to a serine/threonine/tyrosine residue present in a protein substrate. The phosphorylation of proteins has profound effects on their activity and protein-protein interactions, thus regulating a plethora of cellular processes, including cell growth, differentiation and protein homeostasis (or proteostasis). Our lab is the first to demonstrate that protein kinases Kin1 and its paralog Kin2 in the budding yeast Saccharomyces cerevisiae, orthologs of human microtubule affinity-regulating kinase (MARK), contribute to protein-folding homeostasis inside the endoplasmic reticulum (ER), in addition to their canonical roles in cellular exocytosis. The main aim of …

Zinc Proteomics: Interactions Of Zn2+, Cd2+, And Metal-Binding Ligands With Zn-Binding Sites In The Proteome, Eric Daniel Lund

Theses and Dissertations

Cadmium ion causes toxicity in humans, most prominently in the kidney. This thesis focuses on mechanisms by which Cd2+ harms kidney proximal tubule cells. Previous experiments have shown the time-dependent distribution of Cd2+ and Zn2+ within supernatant of pig kidney, LLC-PK1 cells after exposure to Cd2+ plus the ionophore pyrithione1. The first part of this research describes the trafficking of cadmium within LLC-PK1 proximal tubule cells, including the time dependent distribution, speciation, and quantification of Cd2+ and Zn2+ in cells exposed to Cd2+ and Cd2+ plus pyrithione. The latter treatment introduces Cd2+ into cells at time zero and permits the …

The Curious Case Of Nad-Cleaving Nudix Hydrolases, Atreyei Ray

Theses and Dissertations

Nudix proteins are members of a large family of homologous enzymes that hydrolyze nucleoside diphosphates linked to other compounds (x). These enzymes have catalytic activity on a wide range of substrates such as dNTPs (both canonical and their oxidized forms), nucleotide sugars, alcohols, dinucleoside polyphosphates, dinucleotide cofactors, and nucleoside diphosphates linked to RNA. The bacterial genome encodes around 13, while the human genome encodes for 22 such nudix proteins.

The E. coli genome encodes for a mutT mutator gene, the progenitor of the family expressing the MutT pyrophosphohydrolase (NudA) protein (Treffers et al., 1954; Bhatnagar et al., 1988). The enzyme …

Targeting The Γ-Aminobutyric Acid A Receptor (Gabaar) To Alleviate Inflammation For Asthma And Neuropathic Pain, Amanda Nicole Nieman

Theses and Dissertations

The γ-Aminobutyric Acid A Receptor (GABAAR) is a ligand-gated, pentameric chloride channel composed of subunits that include α1-6, β1-3, γ1-3, δ, ε, π, θ, ρ1-3.1-2 The most common arrangement includes two α subunits, two β subunits, and a γ subunit.3 This receptor includes two binding sites for the endogenous ligand γ-aminobutyric acid (GABA) between the α and β subunits and a binding site between the α and γ subunit for benzodiazepines, a large family of positive allosteric modulators.4-5

Benzodiazepines are one of the most prescribed classes of pharmaceuticals to treat anxiety, insomnia, and epilepsy as well as for muscle relaxation.6-7 …

Design And Synthesis Of Achiral And Chiral Imidazodiazepine (Imdz) Gaba(A)R Subtype Selective Ligands For The Treatment Of Cns Disorders, As Well As Asthma, Guanguan Li

Theses and Dissertations

Gamma-aminobutyric acid type A receptors (GABAAR) are transmembrane pentameric ligand-gated chloride ion channels that respond to GABA, the major inhibitory neurotransmitter in the central nervous systems (CNS). The benzodiazepines (BZDs) bind at the extracellular interface of the α+γ2-subunits of GABAAR. The binding of ligands at different subunits of GABAA receptors specifically at α1-6β2/3 γ2 ion channels, can affect a wide variety of brain functions. The α1-subtype selective ion channels of GABAARs are involved in the sedative, ataxic, amnesic, anticonvulsant and addictive effects, which should be avoided, with the exception of the anticonvulsant effects, when designing ligands for this BZ allosteric …

Part I: An Investigation Of Calcitroic Acid And Its Phase Ii Conjugates. Part Ii: Toward The Development Of A Novel Orally-Available Asthma Treatment Targeting Gabaa Receptors In The Lungs, Olivia B. Yu

Theses and Dissertations

PART I

Calcitroic acid (CTA) was isolated and characterized more than four decades ago.5 Radiolabeled calcitriol (1,25-dihydroxyvitamin D3) was used at that time to enable the identification of radioactive CTA formed in vivo, which was subsequently extracted and characterized by derivatization.7 CTA was found to be predominantly formed in the liver and secreted into the gut through the bile duct via enterohepatic circulation, leading to fairly high concentrations of this metabolite of vitamin D in the intestine.8 However, assuming it was only a catabolic product of calcitriol, it was ignored thereafter.

Recently, novel experiments showed that CTA can bind the …

Part I: An Investigation Of Calcitroic Acid And Its Phase Ii Conjugates. Part Ii: Toward The Development Of A Novel Orally-Available Asthma Treatment Targeting Gabaa Receptors In The Lungs, Olivia B. Yu

Theses and Dissertations

PART I

Calcitroic acid (CTA) was isolated and characterized more than four decades ago.5 Radiolabeled calcitriol (1,25-dihydroxyvitamin D3) was used at that time to enable the identification of radioactive CTA formed in vivo, which was subsequently extracted and characterized by derivatization.7 CTA was found to be predominantly formed in the liver and secreted into the gut through the bile duct via enterohepatic circulation, leading to fairly high concentrations of this metabolite of vitamin D in the intestine.8 However, assuming it was only a catabolic product of calcitriol, it was ignored thereafter.

Recently, novel experiments showed that CTA can bind the …

Renalase As An Intracellular Metabolite Repair Enzyme, Matthew Robert Hoag

Theses and Dissertations

The human enzyme renalase was discovered in 2005 by nephrologist Gary Desir, who claimed the enzyme is secreted by the kidney into the blood where it was said to catabolize catecholamines in order to modulate blood pressure and heart rate. It has since been shown that the enzyme is expressed in all tissues and does not react with catecholamines. The research detailed in this dissertation led to the discovery that renalase oxidizes two highly toxic isomers of NAD(P)H to form innocuous NAD(P)+. We surmised that such an important cellular function would be pervasive in nature, and our lab was the …

Studies In Molecular Recognition: Non-Proteogenic Amino Acids For Antibiotic Studies And Chemosensors For Recognition And Reporting Of Metal-Ions, Sarah Anne Oehm

Theses and Dissertations

The field of molecular recognition focuses on the selective and reversible binding of small guest molecules to larger host molecules. This dissertation describes synthesis of small molecules as binding guests for enzymatic substrates as well as molecules as host chemosensors to detect and identify metal ions. Two approaches to new antibiotic drugs have been explored, and an array of sensors for the quantitation of aqueous metals is being commercialized.

As strains of deadly bacteria emerge with evolved resistance to known antibiotics, new drugs are needed with novel mechanisms of action. Natural product antibiotics containing enduracididine, a non-canonical amino acid derived …

Interactions Of Viral And Cellular Helicases, Megan Josephine Corby

Theses and Dissertations

The innate immune system is a part of the first line of defense against virus infection. An important subset of the innate immune system consists of a group of intracellular pattern recognition receptors (PRRs) which recognize conserved features of bacteria and viruses and initiate an interferon response. The RIG-I like receptors (RLRs) are PRRs that bind to RNA viruses (such as hepatitis c virus) and signal through the adaptor mitochondrial anti-viral signaling protein (MAVS).

Hepatitis C virus (HCV) is a small enveloped RNA virus that belongs to the flaviviridae family of viruses. HCV infects hepatocytes and can cause a persistent …

The Heart Of The Madder: An Important Prehistoric Pigment And Its Botanical And Cultural Roots, Michelle Laberge

Theses and Dissertations

In recent years, an interest in natural botanical dye sources has prompted new research into the cultivation and processing of prehistoric dye plants. Advances in chemical analyses of ancient European textiles have provided more information about dye plants such as woad (Isatis tinctoria) weld (Reseda luteola) and madder (Rubia tinctorum), which were important sources of color in early textile production. Evidence of madder dye has been reported in the archaeological record of the European Bronze and Iron Ages in textiles preserved in the Hallstatt salt mines, Scandinavian bog sites and other elite European burials but the picture of madder usage …

New Direct Acting Anti-Virals Inhibiting Hepatitis C Virus Helicase And Insights Into How Atp Fuels Helicase Action, Mark Yerukhimovich

Theses and Dissertations

According to the World Health Organization, Hepatitis C Virus (HCV) has infected 130-150 million people worldwide. Approximately 700,000 of those die each year from chronic HCV related causes such as cirrhosis or cancer. Currently, there are numerous HCV drugs on the market; they target the protease, polymerase and NS5A proteins encoded by of HCV. These drugs are expensive and HCV can become resistant, thus there is constant need for new DAAs. The first part of this thesis examines the search for additional drugs that function by inhibiting the NS3 helicase, which have been challenging to develop.

Part of the reason …

Structure-Function Relationships In Bacterial Regulatory Proteins And An Enzyme Involved In Antibiotic Biosynthesis, Lanlan Han

Theses and Dissertations

The first part of my thesis is focused on a new family of two-component response regulator proteins: Aspartate-Less Regulators (ALR). They lack the catalytic aspartate residue required for the phosphorylation mechanism of typical two component response regulators. We are using biophysical tools to characterize two proteins with redox-sensitive ALR domains: repressor of iron transport regulator (RitR) from Streptococcus pneumonia R6 and diguanylate cyclase Q15Z91 from Pseudoalteromonas atalantica. The structure of inactive RitRC128S monomer showed that the ALR domain and the DNA-binding domain are linked by an α-helix that runs the length of the entire protein, with C128 near the C-terminal …

Interaction With Nitric Oxide Of The Nitrosomonas Europaea Tetraheme Protein Cytochrome C554, And Two Of Its Variants, In Increasingly Reducing Environments, Jennifer M. Mcgarry

Theses and Dissertations

A re-investigation of the interaction with NO of the small tetraheme protein cytochrome c554 (C554) from Nitrosomonas europaea has shown that the 5-coordinate heme II of the 2-electron or 4-electron reduced protein will nitrosylate reversibly. The nitrosylation process was found to be first order in C554, first-order in NO, and second-order overall. The rate constant for NO binding to the heme was determined to be 3000 ± 140 M-1s-1, while the rate constant for dissociation was 0.034 ± 0.009 s-1; the degree of protein reduction does not appear to significantly influence the nitrosylation rate. In contrast to a previous report, …

Part – I: Development Of A Two-Step Regiospecific Synthetic Route For Multigram Scale Synthesis Of Β-Carboline Analogs For Studies In Primates As Anti-Alcohol Agents,Part – Ii: Design And Synthesis Of Novel Antimicrobials For The Treatment Of Drug Resistant Bacterial Infections Part – Iii: A Novel Synthetic Method For The Synthesis Of The Key Quinine Metabolite (3s)-3-Hydroxyquinine, Veera Venkata Naga Phani Babu Tiruveedhula

Theses and Dissertations

PART – I

Development of a Two-Step Regiospecifc Synthetic Route for Multigram-Scale Synthesis of β-Carboline Analogs for Studies in Primates as Anti-Alcohol Agents

β-Carboline and their derivatives are important structural motifs in synthetic organic and medicinal chemistry because of their novel biological activity, especially in regard to the reduction of alcohol self-administration [binge drinking (BD)], a major problem increasing day by day in modern society. This anti-alcohol effect is proposed to be due to the activity of ligands at the benzodiazepine site of the GABAA receptor in the central nervous system acting as antagonists at the α1 subunit. The past …

The Role Of Proteome In Cellular Zn2+ Trafficking And In The Ability Of The Fluorescent Zinc Sensors To Image Intracellular Zn2+, Mohammad Rezaul Karim

Theses and Dissertations

Zinc is an essential biological trace metal used in as many as 3000 Zn-proteins, about 10% of the eukaryotic proteome, as either a structural constituent or a catalytic cofactor. These proteins include the zinc fingers, the most prevalent transcription factors that bind a wide range of gene promoters and thus regulate gene expression. A eukaryotic cell contains several hundred micromolar of Zn2+- almost all of it is bound to specific Zn-proteins. Recently, Zn2+ has been reported to serve as a regulatory signal and a neurotransmitter, suggesting that there also exists a dynamic Zn2+ pool in cells. These findings led to …

Structural And Functional Characterization Of Acetoacetate Decarboxylase-Like Enzymes, Lisa Mueller

Theses and Dissertations

The acetatoacetate decarboxylase-like superfamily (ADCSF) is a largely unexplored group of enzymes that may be a potential source of new biocatalysts. Bioinformatic analysis has grouped these approximately 2000 enzymes into seven different families based on comparison of predicted active site residues. To date, only the prototypical ADCs (Family I) that catalyze the decarboxylation of acetoacetate have been studied. Analysis of gene context suggests that Family V contains predominantly enzymes predicted to be involved in secondary metabolism. On average, these share about 20% sequence identity to the true ADCs. To learn more about the diversity of chemistries performed by members of …

The Function Of Renalase, Brett Allen Beaupre

Theses and Dissertations

Renalase was originally reported to be an enzyme secreted into the blood by the kidney to lower blood pressure and slow heart rate. Despite multiple reports claiming to confirm this activity in vivo there has been considerable discord in regards to the reaction catalyzed by renalase. The structural topology of renalase resembles that of known flavoprotein oxidases, monooxygenases and demethylases, but the conserved active site residues are unique to renalase. It has been reported that the catalytic function of renalase is to oxidize circulating catecholamines, however in vitro studies have failed to demonstrate a catalytic activity in the presence of …

Reaction Of Zinc Proteome With Biologically Important Metal Binding Ligands, Kaniz Fatema

Theses and Dissertations

Fluorescent sensors have been widely used as microscopic tools to image Zn2+ on a cellular level. Recently, it has been established that the sensors TSQ and Zinquin form adducts with Zn-proteins and image fractions of the Zn-proteome.1 Since TSQ and Zinquin bind specifically to many Zn-proteins, it is hypothesized that other metal binding ligands, both synthetic and natural, may also bind to the Zn-proteome. Biologically active 1,10-phenanthroline (Phen) and related molecules were investigated for their ability to bind to Zn-proteome. Similarly, the cellular tripeptide, glutathione was investigated. It was observed that Phen and some other metal binding, bidentate ligands, were …

Arylboronates As H2o2 Or Photo-Inducible Dna Cross-Linking Agents: Design, Synthesis, Mechanism, And Anticancer Activity, Yibin Wang

Theses and Dissertations

Interest in the development of cancer therapies with improved selectivity and reduced host toxicity has been growing. In this thesis, we designed and synthesized a series of novel non-toxic arylboronic ester and biarylboronic ester derivatives that can be activated by hydrogen peroxide (H2O2) to induce DNA interstrand cross-link formation. The mechanism of DNA cross-linking induced by these arylboronates involves generation of phenol intermediates 1 followed by departure of leaving group (L) leading to quinone methides (QMs) 2, which directly cross-link DNA via alkylation. The QM formation is the rate-determining step for DNA cross-linking. The activity and selectivity of these compounds …