Biochemistry Commons^™

Investigating Hfq-Mrna Interactions In Bacteria, Martha Audra Faner

Wayne State University Dissertations

Regulatory RNAs (sRNAs) are essential for bacteria to thrive in diverse environments and they also play a key role in virulence [11]. Trans-sRNAs affect the stability and/or translation of their target mRNAs through complementary base-pairing. The base-pairing interaction is not perfect and requires the action of an RNA binding protein, Hfq. Hfq facilitates these RNA-RNA interactions by stabilizing duplex formation, aiding in structural rearrangements, increasing the rate of structural opening, and/or by increasing the rate of annealing [18-21]. Hfq has two well characterized binding surfaces: the proximal surface, which binds AU rich stretches typical of sRNAs, and the distal surface, …

Single-Molecule Studies Of Local And Global Nucleic-Acid Dynamics, Eric Muthuri Patrick

Wayne State University Dissertations

Nucleic acids undergo both global and local conformational changes that are important for their function. Structural studies have over the decades been invaluable in elucidation of various biomolecular mechanisms, hence contributing significantly to the understanding of biological events. However, a clear understanding of how molecules function in the cellular context requires investigation of their interconversion between multiple conformations, including mapping the folding landscape and any coupled changes in conformation. Work in this thesis focuses on fluorescence experiments, mainly at a single-molecule level to investigate such processes.

First, a novel single-molecule approach is described focusing on local dynamics within nucleic acids …

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 …

Elucidation Of The Functional Role Of Pcbp-The Cytosolic Iron Chaperone Protein Family, In Cellular Iron Homeostasis, Poorna Subramanian

Wayne State University Dissertations

Biological Role of Ferritin - Iron is essential for life and often utilized as a cofactor in many proteins. In humans, iron accumulation causes cirrhosis, arthritis, cardiomyopathy and diabetes mellitus, and it is associated with increased risk of cancer and heart disease. In contrast, decreased brain iron content results in permanent neurocognitive and motor impairment. Intracellular iron content must be maintained within a narrow range to avoid the adverse effects of iron depletion or excess, and this function is performed by the protein ferritin. Ferritins are iron storage proteins that are ubiquitously expressed in animals, plants and bacteria. They serve …

Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T, Sandamali Amarasingha Ekanayaka

Wayne State University Dissertations

DEVELOPMENT AND OPTIMIZATION OF AN IN VITRO FRET ASSAY TO CHARACTERIZE THE SACCHAROMYCES CEREVISIAE GPI TRANSAMIDASE

By

SANDAMALI AMARASINGHA EKANAYAKA

December 2013

Advisor: Dr. Tamara L. Hendrickson

Major: Biochemistry

Degree: Doctor of Philosophy

The enzyme glycosylphosphatidylinositol transamidase (GPI-T) mediates the attachment of a glycosylphosphatidylinositol (GPI) anchor to the C-terminus of specific proteins to produce GPI anchored proteins. This post-translational modification is essential for viability of eukaryotic organisms. However, very little is known about GPI-T and its catalytic activity. Thus, the research described in this abstract was conducted to develop an in vitro assay to monitor GPI-T. A high-throughput assay for …

Biochemical, Structural, And Drug Design Studies Of Multi-Drug Resistant Hiv-1 Therapeutic Targets, Tamaria Grace Dewdney

Wayne State University Dissertations

Protein point mutations acquired as a mechanism of survival against therapeutics cause structural changes that effect protein function and inhibitor binding. This work investigates the structural mechanisms that lead to multi-drug resistance to HIV-1 protease and integrase inhibitors.

Proper proteolytic processing of the HIV-1 Gag/Pol polyprotein is required for HIV infection and viral replication. This feature has made HIV-1 protease an attractive target for antiretroviral drug design for the treatment of HIV-1 infected patients, thus the development of drug resistance has arisen as a major therapeutic and drug design challenge. To understand the molecular mechanisms leading to drug resistance we …

Synthesis And Application Of Atp Analogs For Phosphorylation-Dependent Kinase-Substrate Crosslinking, Satish Kumar Garre Venkata Raghavendra

Wayne State University Dissertations

Phosphorylation is an important post-translational modification that plays a key role in a variety of signaling cascades and cellular functions. Kinases phosphorylate protein substrates in a highly regulated manner and are promiscuous. Understanding kinase-substrate specificity has been challenging and there is a need for new chemical tools. To this end we developed -phosphate modified ATP photocrosslinking analogs ATP-ArN3 and ATP-BP, that crosslink substrate and kinase in a phosphorylation dependent manner. We have successfully demonstrated that ATP-ArN3 and ATP-BP can be used with natural kinase and substrates using cell lysates in vitro. We used our approach to identify novel kinases of …

Computational Approaches To Anti-Toxin Therapies And Biomarker Identification, Rebecca Jane Swett

Wayne State University Dissertations

This work describes the fundamental study of two bacterial toxins with computational methods, the rational design of a potent inhibitor using molecular dynamics, as well as the development of two bioinformatic methods for mining genomic data.

Clostridium difficile is an opportunistic bacillus which produces two large glucosylating toxins. These toxins, TcdA and TcdB cause severe intestinal damage. As Clostridium difficile harbors considerable antibiotic resistance, one treatment strategy is to prevent the tissue damage that the toxins cause. The catalytic glucosyltransferase domain of TcdA and TcdB was studied using molecular dynamics in the presence of both a protein-protein binding partner and …

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 …

Histone Deacetylase 1: Mutagenesis And Small Molecule Studies, Magdalene Wambua

Wayne State University Dissertations

Histone deacetylase 1 (HDAC1) has been linked to cell growth and cell cycle regulation, which makes it a widely recognized target for anticancer drugs. The 14 Å channel of Class 1 HDAC isotypes has long being hypothesized to be the exit cavity for acetate following deacetylation. The amino acids lining this cavity are very similar among the HDAC isoforms, suggesting the role of the cavity is relevant to all HDACs proteins Importanly, HDAC1 selective inhibitors designed to fit the 14Å channel have been designed. To understand the importance of the 14Å channel to HDAC1 activity, we used an alanine scan …

The Role Of Cardiolipin In Iron Homeostasis And Glutathione Metabolism, Vinay A. Patil

Wayne State University Dissertations

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. Inside the mitochondria, CL is a critical target of mitochondrial generated reactive oxygen species (ROS) and regulates signaling events related to apoptosis and aging. CL deficiency causes perturbation of signaling pathways outside the mitochondria, including the PKC-Slt2 cell integrity pathway and the high osmolarity glycerol (HOG) pathway, and is a key player in the cross-talk between the mitochondria and the vacuole. The importance of CL in human health is underscored by the observation that perturbation of CL …