Biochemistry, Biophysics, and Structural Biology Commons^™

Enzymatic Characterization Of The Ammonia Tunnel In Helicobacter Pylori Asp-Trnaasn/Glu-Trnagln Amidotransferase, Liangjun Zhao

Wayne State University Dissertations

The Helicobacter pylori (H. pylori) Asp-tRNA^Asn/Glu-tRNA^Gln amidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity; however, its inter-domain communication and ammonia delivery mechanisms are not well understood. In the present study, we investigated the three activities of H. pylori AdT (glutaminase, kinase and transamidase) and used these reactions as probes to examine the inter-domain communication and ammonia delivery mechanisms between this enzyme's two isolated active sites. We adapted and optimized an assay to kinetically characterize a series of mutations at conserved positions throughout the putative AdT ammonia tunnel. The kinase assay enabled us …

Development Of Peptide Inhibitors Targeting Clostridium Difficile Toxins A/B And Characterizing The Regulatory Role Of A Putative Negative Regulator Tcdc In Clostridium Difficile Toxin Gene Expression, Sanofar Jainul Abdeen

Wayne State University Dissertations

Clostridium difficile infections cause one of the most common and vital hospitalacquired

diseases often associated with broad-spectrum antibiotic usage. TcdA and TcdB

are the key virulence factors involved in major patho-physiology. While standard

antibiotics provide some respite, due to the high relapse rates and the emergence of more

severe disease presentations, antibiotics alone have often proven to be suboptimal.

Therefore there is a desperate need to develop an effective non-antimicrobial

therapeutics. Part of this work focuses on identification and further characterization of

peptide therapeutic that target the major virulence factor TcdA/TcdB. Towards

development of mechanistic-based anti-toxin agent, phage display was …

Aβ Alters The Dna Methylation Status Of Cell-Fate Genes In An Alzheimer’S Disease Model, Gary D. Isaacs, Noor Taher, Courtney Mckenzie, Rebecca Garrett, Matthew Baker, Nena Fox

Faculty Publications and Presentations

Alzheimer’s disease (AD) is characterized by neurofibrillary tangles and extracellular amyloid-β plaques (Aβ). Despite ongoing research, some ambiguity remains surrounding the role of Aβ in the pathogenesis of this neurodegenerative disease. While several studies have focused on the mutations associated with AD, our understanding of the epigenetic contributions to the disease remains less clear. To that end, we determined the changes in DNA methylation in differentiated human neurons with and without Aβ treatment. We isolated the DNA from neurons treated with Aβ or vehicle, and digested the two samples with either a methylation-sensitive (HpaII) or a methylation-insensitive (MspI) restriction endonuclease. …

Development Of A Starch-Based Mussel-Mimetic Adhesive Polymer, Jeffrey Kazimir De Kozlowski

Open Access Theses

Mussel-mimetic adhesive polymers have gained lots of attention for their strong adhesive strength, moisture resistance, and unique ability to crosslink. These properties are mainly attributed to the high content of catecholic 3,4-dihydroxyphenylalanine (DOPA) in mussel adhesive proteins. While there has been success in creating mussel-mimetic synthetic polymers, less effort has been given to create a renewable, green, biocompatible counterpart. This thesis explores the possibilities of starch-based mussel-mimetic adhesives. Carboxymethyl starch of various molecular weights and degree of substitution was synthesized and subsequent conjugation of dopamine to these polymers by 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was investigated. The polymers suffered from very low substitution (DScatechol …

Interaction Between Maltose Binding Protein And Escherichia Coli Maltose Transporter, Yan Huang

Open Access Theses

The ATP-binding cassette (ABC) transporter superfamily is one of the largest families of transport proteins. The ABC transporters are responsible for selective permeability of solute across membranes energized by ATP hydrolysis, which occurs in all domains of life. Maltose transporter is an ABC importer that mediates maltose/maltodextrin uptake in bacteria and archaea. It is identified as an essential virulence factor in pathogenic species of Streptococcis pyogenes and Vibrio cholera (1, 2). Escherichia coli maltose transporter is a well-characterized system with crystal structures and exclusive biochemical studies available. Knowledge of the E. coli maltose transport mechanism will lead to a better …

Force Generation By Kinesin And Myosin Cytoskeletal Motor Proteins, F. Jon Kull, Sharyn A. Endow

Dartmouth Scholarship

Kinesins and myosins hydrolyze ATP, producing force that drives spindle assembly, vesicle transport and muscle contraction. How do motors do this? Here we discuss mechanisms of motor force transduction, based on their mechanochemical cycles and conformational changes observed in crystal structures. Distortion or twisting of the central β-sheet - proposed to trigger actin-induced Pi and ADP release by myosin, and microtubule-induced ADP release by kinesins - is shown in a movie depicting the transition between myosin ATP-like and nucleotide-free states. Structural changes in the switch I region form a tube that governs ATP hydrolysis and Pi release by the motors, …

Sir2 Is Required For Clr4 To Initiate Centromeric Heterochromatin Assembly In Fission Yeast, Benjamin J. Alper, Godwin Job, Rajesh K. Yadav, Sreenath Shanker, Brandon R. Lowe, Janet F. Partridge

Chemistry & Physics Faculty Publications

Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4-mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4-mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 …

Investigating The Metal Binding Affinity Of Bacterial Zinc Transporters: Zupt And Truncated Znta (Znta-Pina), Fei Nie

Wayne State University Theses

Zinc is essential trace elements for all living organisms and serves as cofactor and structural cofactors for more than 300 enzymes. ZupT is one of the members of ZIP super family of transporter proteins, which were identified as iron and zinc transporter in Eukaryotes at first. However, recent evidence showed that some of members can also transport other metal ions, such as manganese or cadmium, and ZIP proteins also exist in bacteria. ZupT is responsible for zinc uptake in E. coli; however, its full metal selectivity is not yet characterized. In addition, the mechanism of transport is also unknown. Therefore, …

Decoration Of Graphene With Metal Nanoparticles And Its Application For Bioanalysis, Keun Soo Kwon

Theses, Dissertations and Culminating Projects

Graphene is a two-dimensional monolayer of tightly packed sp² hybridized Carbon atoms. Formed from graphite, the theoretical surface area of the monolayer is about 2630 m²/g, and the initial formation of graphene oxide from graphite oxide introduces a variety o f functional groups, such as -COOH, -C=0, and -OH, on its surface. The high surface area and the rich presence of functional groups, along with the potential formation o f sp² bonding networks within graphene oxide, all serve as critical factors allowing for additional chemical modifications. There are extensive research attempts being performed to explore and …

The Genetic Engineering Of Motor Proteins, Rachael M. Hartz

Legacy Theses & Dissertations (2009 - 2024)

Molecular motors are a remarkable feature within living organisms that are responsible for directional mechanical motion, which is driven by adenosine triphosphate (ATP) hydrolysis. Actin-binding molecular motors are of specific interest in the field of nanotechnology as filamentous actin is capable of carrying cargo, such as quantum dots, while it is translocated along a motor coated surface. The binding regions of motor proteins, which are known to interact with actin, such as Myosin, have been thoroughly examined and identified. Rapid genetic engineering of the ATP-hydrolyzing enzyme, adenosine kinase, to incorporate these binding regions is possible through the use of site- …

Protein Structures Under Physiological Conditions, Karl Michael Bertrand

Legacy Theses & Dissertations (2009 - 2024)

My research focused on the evaluation of protein structures and protein dynamics inside eukaryotic cells under physiological conditions. The primary analyses of my research involved the use of in-cell Nuclear Magnetic Resonance spectroscopy using Heteronuclear Single Quantum Coherence experiments. This allowed me to visualize protein structures at an atomic resolution level, as well as, study the interactions of these proteins with small molecules.

Molecular Biochemical Study Of The Cataract-Associated Mutants In Human Gammad-Crystallin, Cindy Yeung

Legacy Theses & Dissertations (2009 - 2024)

Cataract is a vision impairment disease that leads to light scattering and opacity of the eye lens. It is recognized as the most prevalent cause of blindness. Most cataracts occur due to aging, but those that occur in childhood or in young adults are typically of genetic origin. In the young (developing) eye, post–surgical complications such as glaucoma are common after cataract surgery. Hence there is a great need to develop non-surgical intervention strategies for the treatment of cataract. Here, two cataract-associated mutants of human γD-crystallin (HGD): i) Arg76 to Ser (R76S) and ii) a truncation mutation at Trp156 (W156X …

Structural Biology Of The Receptor For Advanced Glycation End Products (Rage), Jing Xue

Legacy Theses & Dissertations (2009 - 2024)

The receptor for advanced glycated end products (RAGE), due to its location in the major histocompatibility complex class III (MHC III) region, suggests its involvement in immune responses. Because RAGE has been linked to complications of diabetes and chronic inflammation, the severity of Alzheimer's disease and cancer, it's significant to understanding the biological mechanism of signal transduction of RAGE.

New Tools To Study Amyloid Fibrils And Intrinsically Disordered Proteins In Vitro And In Vivo, Jacqueline D. Washington

Legacy Theses & Dissertations (2009 - 2024)

Amyloid fibrils are β-sheet-rich protein aggregates commonly found in the organs and tissues of patients with various amyloid-associated diseases. The structure of insulin fibrils was characterized by deep ultraviolet resonance Raman and Nuclear Magnetic Resonance spectroscopy combined with hydrogen-deuterium exchange. Our new approach of combining NMR and Raman spectroscopy with molecular dynamic simulations for characterizing amyloid fibrils provided exclusive knowledge about fibril structure at amino acid residue resolution.

Y-Family Dna Polymerases : Mechanism Of Single-Base Deletion And Effect Of Pcna Interaction, Yifeng Wu

Legacy Theses & Dissertations (2009 - 2024)

Translesional synthesis (TLS) by specialized Y-family DNA polymerases is a DNA damage tolerance pathway to bypass DNA lesions that have not been repaired by other DNA repair mechanisms. Despite their valuable lesion bypass ability, the Y-family DNA polymerases display a much lower fidelity upon replicating undamaged DNA. Their activity therefore needs to be well regulated in the cell. In our first project, we examined how an archaeal Y-family DNA polymerase, Dpo4, makes single-base deletions during replication. Dpo4 belongs to the DinB subfamily which is known to make single-base deletions. Although previous studies suggested that it uses a dNTP-stabilized misalignment mechanism, …

The Sxrna Platform : Biophysical Characterization Of Trans-Acting Rna Switches For Gene Expression Control, Christine Bazinet

Legacy Theses & Dissertations (2009 - 2024)

In the past decade, microRNAs (miRNAs) have become a hot topic in biochemical research. These very short ~ 22 nucleotide strands of RNA have been recognized as a functional RNA group and found to play a key role in mediating gene regulation along with RNA-binding proteins (RBPs). For example, by binding to the histone stem-loop (HSL) region of the target mRNA, normally in the 3'- untranslated region (UTR), gene expression can either be activated or deactivated for up or down regulation of the gene product. The majority of miRNAs have been found to down regulate gene expression upon binding to …

2'-O-Methyl Substitutions In The Yeast Telomerase Pseudoknot And Their Effects On Telomerase Activity, Katelyn Mae Jasper

Legacy Theses & Dissertations (2009 - 2024)

Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomeric repeats at the ends of linear chromosomes to form the DNA–protein complexes known as telomeres. Telomeres protect the ends of chromosomes from degradation during replication due to the end-replication problem. When replication occurs, gaps are created at the beginning of the lagging and leading strands that result in the loss of a small amount of DNA at every replication cycle. By adding thousands of copies of telomeric repeats, the repeats are lost in the replication process and not precious genetic information. The telomerase RNA varies drastically among different species both in …

(S)-Trifluoroselenomethionine : A New Non-Natural Amino Acid With Enhanced Methioninase-Induced Cytotoxicity Toward Human Colon Cancer Cells, Stephene Nathele Lodge

Legacy Theses & Dissertations (2009 - 2024)

Abstract

Voltage-Controlled Enzyme-Catalyzed Glucose–Gluconolactone Conversion Using A Field-Effect Enzymatic Detector, Siu Tung Yau, Yan Xu, Yang Song, Ye Feng, Jiapeng Wang

Chemistry Faculty Publications

The field-effect enzymatic detection (FEED) technique was used to control the kinetics of the enzymatic conversion of glucose to gluconolactone. The glucose–gluconolactone conversion occurring at an enzyme-immobilized electrode, a well-studied process, was confirmed using mass spectrometry. Electrochemical studies showed that the glucose oxidation current depends on the gating voltage VG and the ion concentration of the sample solution. Additionally, the depletion of glucose in the sample also showed a dependence on VG. FEED was used to detect H2O2 on the zepto-molar level in order to show the ultrasensitive detection capability of the technique. These results, while providing evidence for the …

Molecular Considerations In The Design Of Novel Alpha/Beta Hydrolase Inhibitors, Shana Victoria Stoddard

Electronic Theses and Dissertations

Alpha/beta hydrolases (ABHs) are a superfamily of hydrolytic enzymes that process a wide variety of substrates. A subfamily of ABHs called carboxylesterases (CEs) are important enzymes that catalyze biological detoxification, hydrolysis of certain pesticides, and metabolism of many esterified drugs. The chemotherapy drug irinotecan used for treatment of colorectal cancer is metabolized to SN-38, the active drug metabolite, by two CE isozymes CES1 (localized in the liver) and CES2 (localized in the small intestines). CES2's ability to activate irinotecan at a faster rate than CES1 creates a localization of activated SN-38 in the gut epithelium, resulting in the dose limiting …

Spliceosomal Prp24 Unwinds A Minimal U2/U6 Complex From Yeast, Chandani Manoja Warnasooriya

Wayne State University Theses

Splicing plays a major role in eukaryotic gene expression by processing pre-mRNA to form mature mRNA. Pre-mRNAs undergo splicing to remove introns, non–protein coding regions, and religate exons, protein coding regions. This process is catalyzed by the spliceosome, which consists of five small nuclear ribonucleoprotein particles (snRNPs: U1, U2, U4, U5 and U6) and numerous protein factors. Proper assembly of spliceosomal components is critical for function, and thus, defects in assembly can be lethal. Several spliceosomal proteins facilitate structural rearrangements important for spliceosomal assembly and function. Prp24 is an essential factor in U6 snRNP assembly, and it has been proposed …

Investigation Of Bacterial Rna-Directed Dna Methylation Via Dcm And Hfq, Dandan Li

Wayne State University Theses

Bacterial small RNAs and the RNA chaperone Hfq play crucial roles in post-transcriptional gene regulation, often as parts of stress-response pathways, but little is known about their roles in regulation of gene transcription. A recent report showed that changes in methylation patterns caused by DNA cytosine methyltransferase (Dcm) were linked to gene regulation occurring during the transition to stationary phase. Here, we show that Dcm involves in the stress responses under nutrient starvation and cold stress. Dcm and Hfq together mediate gene expression under cold stress. Hfq promotes Dcm-catalyzed cytosine methylation at specific sites near the rpoS promoter, which is …

Lc3i And Lc3ii As Autophagy Markers For The Development And Improvement Of Products And Techniques Used In Research, Caitlin Jane Williams

Wayne State University Theses

Abstract

LC3I and LC3II as Autphagy Markers for the Development and Improvement of Products and Techniques used in Research

By

Caitlin J. Williams

May 2013

Advisor: Dr. Robert Akins

Major: Biochemistry & Molecular Biology

Degree: Master of Science

Autophagy is an intracellular process that functions to maintain homeostasis in the cell by degrading misfolded proteins, old or nonfunctioning organelles, and outside invaders such as bacteria or viruses. This process can be split into three different types, microautophagy, chaperone mediated autophagy and macroautophagy. Macroautophagy is the most commonly studied form and is believed to be regulated by Atg proteins, as well …

Cysteine Desulfurase And Isd11: A Drosophila Model, Rahul Ravindran Nair

Wayne State University Theses

ABSTRACT

Cysteine desulfurase and Isd11: A Drosophila model

Iron-sulfur clusters are cofactors with evolutionary origins that date back to the pre-biotic world. Ever since life originated, these cofactors have intermingled with proteins to play vital roles in sustaining life. My research focuses on one such protein, the cysteine desulfurase (Nfs) that has the PLP cofactor incorporated in its active site and avails of the catalytic property of PLP to provide sulphur for Iron-sulfur cluster biogenesis and assembly in a cell. Interestingly, in a eukaryotic cell, despite the versatility of PLP, cysteine desulfurase's role as a "sulphur-extractor" is incomplete without another …

Optimization Of Labeling Techniques; Determination Of Best Parameter For Olfactory Mucosal Progenitor Cell Delivery And Study Of Effects Of Methylene Blue And Polyethylene Glycol In An Animal Model Of Spinal Cord Injury, Kiran Kumar Rokkappanavar

Wayne State University Theses

In the United States, the incidence of TSCI is about 40 per million persons per year, with approximately 250,000 living survivors of TSCI in the United States in July 2005. A number of human clinical trials (85) are ongoing using stem cells to evaluate the methods to reduce the injury following TBI. Among the stem cells, olfactory mucosal progenitor cells have several advantages with respect to ease of obtaining, fate of cells, etc. Our study was intended to optimize the labeling technique and to determine the best parameter to deliver the stem cells to the site of injury. We found …

Characterizing Cyclin J By Identifying Conserved Protein-Protein Interactions, Phillip Jacob Selman

Wayne State University Theses

Cyclins are proteins that bind to Cyclin-dependent kinases, or Cdks, through a conserved domain called the Cyclin Box. Many Cyclins regulate the cell cycle. A few Cyclins impact cellular processes outside of the cell cycle. Also, a few Cyclins have poorly understood functions.

Cyclin J is a member of the Cyclin superfamily of proteins. Cyclin J is conserved among all metazoans, but is presently not well understood. All the research done on Cyclin J has been done in Drosophila.

Its mRNA is present in the early embryo, then disappears, only to reappear in adult females. When probing protein extracts with …

Spectroscopic Analysis Of Polymerization And Exonuclease Proofreading By A High-Fidelity Dna Polymerase During Translesion Dna Synthesis, Babho Devadoss, Irene Lee, Anthony J. Berdis

Chemistry Faculty Publications

High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2′-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient …

Towards Elucidation Of A Viral Dna Packaging Motor, Chad T. Schwartz

Theses and Dissertations--Pharmacy

Previously, gp16, the ATPase protein of phi29 DNA packaging motor, was an enigma due to its tendency to form multiple oligomeric states. Recently we employed new methodologies to decipher both its stoichiometry and also the mechanism in which the protein functions to hydrolyze ATP and provide the driving force for DNA packaging. The oligomeric states were determined by biochemical and biophysical approaches. Contrary to many reported intriguing models of viral DNA packaging, it was found that phi29 DNA packaging motor permits the translocation of DNA unidirectionally and driven cooperatively by three rings of defined shape. The mechanism for the generation …

Amalgamation Of Nucleosides And Amino Acids In Antibiotic Biosynthesis, Sandra H. Barnard

Theses and Dissertations--Pharmacy

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading …

Regulation Of 7-Dehydrocholesterol Reductase By Vitamin D3, Ling Zou

Theses and Dissertations--Pharmacy

7-Dehydrocholesterol (7-DHC) is the substrate of 7-dehydrocholesterol reductase (DHCR7) in the cholesterol synthesis pathway. Keratinocytes in human skin possess the enzymes necessary for cholesterol synthesis but are also responsible for vitamin D₃ synthesis from 7-DHC by exposure to UVB irradiation. It has been well established that DHCR7 is regulated by the SREBP pathway in the regulation of cholesterol synthesis, but little is known about the regulation of DHCR7 by the vitamin D pathway. In this study, the regulation of DHCR7 activity by vitamin D was explored. Treatment of adult human epidermal keratinocyte (HEKa) cells with vitamin D₃ resulted …