Biochemistry Commons^™

Determining The Full-Length Structure Of Collagenase H Using Small-Angle X-Ray Scattering, Josie Carson

Chemistry & Biochemistry Undergraduate Honors Theses

Known to cause gas gangrene, Hathewaya histolytica secretes two sister collagenases, collagenase G (Col G) and collagenase H (Col H), to degrade the triple helical structure of collagen to further infection in a host. Individual domains of Col H have been crystalized in previous studies, but methods in x-ray crystallization of full-length Col H have been unsuccessful. Using Small Angle X-Ray Scattering (SAXS) data, atomistic modeling was used to generate multiple conformations of Col H while accounting for flexibility between domains. Full-length Col H was found to adopt a two-state conformational model exhibiting a majority compact and a minority elongated …

Retro-Structural Analysis Of The Four Helix Bundle Motif In Binuclear Proteins, Walker Pedigo, Maggie Smith

Honors Theses

Protein structure is directly related to protein function. There are four levels of protein structure: primary, secondary, tertiary, and quaternary. The interactions amongst the structural components of a protein give rise to its unique characteristics. The four helix bundle motif is a common structural trait in a variety of binuclear proteins. In this study, PyMOL, a molecular visualization system, was used to analyze binuclear proteins that possess a four helix bundle. Images of proteins containing dicopper, diiron, and dimanganese sites were captured. The images were compiled into figures for each individual protein. After creating the figures, each protein was further …

Kinetic And Structural Influences Of Acetylation On Ubiquitin Processing, Rachel E. Lacoursiere

Electronic Thesis and Dissertation Repository

Ubiquitin (Ub) is a small modifying protein abundant in cells where it serves numerous regulatory roles including immune signaling, transcriptional regulation, and proteostasis. To exert its function, Ub covalently interacts with a series of E1, E2, and E3 enzymes before final substrate modification. Dysregulation of Ub signaling has implications in human maladies such as cancer, autoimmune disorders, and neurodegenerative diseases. In these diseases and associated in cellulo models, modifications to Ub serve an additional role in Ub regulation. Post-translational modifications like acetylation or phosphorylation modulate protein-protein interactions and Ub signaling. To understand how acetylation of Ub alters the central E2 …

Dual Control Of One Component Signaling: Mechanistic And Structural Insights Into El222 Active States, Uthama Phani R. Edupuganti

Dissertations, Theses, and Capstone Projects

Photoreceptors play a crucial role in signal transduction as specialized proteins which sense light as environmental stimuli and transduce the signal to control of downstream functions. Here we focus our attention on one class of these proteins, the Light-Oxygen-Voltage (LOV) domain, which is sensitive to blue light via an internally-bound flavin chromophore. Since their initial discovery in plant phototropins, many details of their photochemistry, chromophore interactions, and use with a diverse set of functional effectors have been described. However, several key details, especially a comprehensive understanding of signaling mechanism and its regulation, still remain elusive due in part to the …

Characterization Of Avidin And Case9 Single Protein Molecules By A Solid-State Nanopore Device, Haopeng Li

Graduate Theses and Dissertations

The shape and charge of a protein play significant roles in protein dynamics in the biological system of humans and animals. Characterizing and quantifying the shape and charge of a protein at the single-molecule level remains a challenge. Solid-state nanopores made of silicon nitride (SiNx) have emerged as novel platforms for biosensing such as diagnostics for single-molecule detection and DNA sequencing. SSN detection is based on measuring the variations in ionic conductance as charged biomolecules translocate through nanometer-sized channels driven by an external voltage applied across the membrane. In this paper, we observe the translocation of asymmetric cylindrical structure CRISPR-Cas9 …

Single Molecule Fluorescence Studies Of Protein Structure And Dynamics Underlying The Chloroplast Signal Recognition Particle Targeting Pathway, Dustin R. Baucom

Graduate Theses and Dissertations

The work presented in this dissertation explores the structural dynamics in the chloroplast signal recognition particle pathway. Findings include cpSRP shows scanning functionality similar to that in the cytosolic SRP with the ribosome. The intrinsically disordered C-terminal tail of the Albino3 protein has some transient secondary structure. Upon binding to cpSRP43 in solution, separate secondary structure formation was identified in the C-terminal tail of Albino3. Finally, to increase efficiency of analyzing fluorescence time traces for this work, a modular software was produced.

Preparation Of A Flavonol Specific Glucosyltransferase Found In Grapefruit And Site-Directed Mutants For Protein Crystallization, Aaron Birchfield

Electronic Theses and Dissertations

This research was designed to determine the conditions necessary to remove c-myc and 6x-His tags from a flavonol specific glucosyltransferase found in grapefruit (CP3GT) using thrombin in preparation for crystallization. X-ray crystallography of CP3GT crystals may elucidate structural features that account for flavonol specificity in some glucosyltransferase enzymes. A thrombin cleavage site was inserted into WT CP3GT and one mutant. Recombinant CP3GT was expressed in yeast and purified. Optimal conditions for thrombin digestion were explored. Digestion with 100U of thrombin for 2 hours at 4^o C was optimal for removing tags from CP3GT. Storage at 4^o C for …

Elucidation Of The Amino-Terminal Structure And Characteristics Of Perilpin 5, Derek T. Wei

Undergraduate Honors Thesis Projects

The United States of America, among many other countries around the world, is suffering from lipid-related disorders. Developing treatment options for these disorders that impact the lives of billions of individuals is an imperative task for scientists. In order to develop treatments and therapeutics, it is first necessary to understand the etiologies of these disorders and their molecular mechanisms. The perilipins are a heavily studied family of proteins found on the surface of lipid droplets that have an integral relevance in regulating lipolysis. Understanding the proteins involved in lipolysis can not only lead to a more complete understanding of the …

Structural Studies Of Acid Alpha Glucosidase And Pompe Disease, Derrick Deming

Doctoral Dissertations

Acid α-glucosidase (GAA) is required for the degradation of lysosomal glycogen. Pompe disease is an autosomal recessive disorder caused by reduced GAA activity, resulting in the accumulation of glycogen within lysosomes. The most severe form of the disease is characterized by a progressive deterioration of cardiac and skeletal muscle leading to death before two years of age. An intense interest from both the academic and pharmaceutical communities led us to determine the crystal structure of GAA. The structure provides insight into Pompe disease by examining how over 200 disease-associated point mutations perturb GAA function. To aid in the development of …

Elucidating Structure, Function, And Small Molecular Interactions Of Human Immunodeficiency Virus And Chikungunya Virus, Kristin Nicole Slater

Wayne State University Theses

Abstract HIV-1:

Human immunodeficiency virus-1 (HIV-1) is a widespread, incurable retrovirus known to cause

immunodeficiency and a shortened life span. Despite successful treatment methods, HIV-1

frequently mutates, resulting in antiviral resistance. Many therapies target the HIV-1 protease

(PR), which is responsible for cleaving the viral polyprotein essential for its life cycle. HIV-1 PR

often evades treatment by way of mutations and less commonly through residue insertions. We

have identified a clinical isolate with a five residue insertion between residues 28 and 29.

Through molecular dynamics simulations we analyzed the protease protein structure and

determined that the residue insertion created a …

Structure And Dynamics Of A Small Multidrug Resistance Transporter, Emre, Chao Wu

Arts & Sciences Electronic Theses and Dissertations

EmrE is a small multidrug resistance transporter in E. coli. It effluxes a wide range of antibiotics, thus contributing to the evolving epidemic of drug resistance. Despite its small size, EmrE is a fully functional transporter making it an ideal model system for a comprehensive study of the multidrug transport mechanism. In the transport cycle, EmrE must alternate between outward- and inward-facing conformations upon substrate binding to translocate substrates across the membrane. High-resolution structures of EmrE in complex with substrates facing different sides of the membrane will shed light on the coupling mechanism between substrate binding and transport. However, the …

Structure And Biological Activity Of A D3g Mutation In The Human Mitochondrial Chaperonin Hsp60, Jihui Li

Open Access Theses & Dissertations

Proteins are essential components in all cellular activities, including signal transduction, gene expression regulation, immune response, structural support and many others. Following their synThesis on the ribosome, proteins need to fold into their precise three-dimensional structure to obtain functionality. If they fold incorrectly they are prone to aggregation and cause a wide range of neurodegenerative diseases. Some proteins are capable of folding without assistance but others need help from protein complexes known as chaperonins. Chaperonins are a family of proteins that assemble into barrel-shaped cages the create a protected chamber for misfolded proteins to refold into their biologically active state. …

The Characterization Of A Recombinant Virophage Integrase, Martin Christopher Chacon

Open Access Theses & Dissertations

Virophages are satellite-like dsDNA viruses that parasitize giant viruses of the family Mimiviridae. Mavirus is the second virophage discovered that associates with its host virus Cafeteria roenbergensis Virus (CroV). When co-infecting their common host cell Cafeteria roenbergensis, a marine zooplankton that is widely spread throughout the oceans, mavirus will inhibit CroV's replication. In addition, mavirus was shown to share high similarities to the Maverick/Polinton eukaryotic DNA transposons. A coding sequence in mavirus genome (MV02) reveals high homology to retroviral integrases such as those found in HIVs. The putative integrase MV02 is predicted to integrate mavirus DNA into the host genome. …

Structural Elucidation Of Aggr-Activated Regulator, Aar, In Enteroaggregative Escherichia Coli, Andrew Heindel

Senior Honors Projects, 2010-2019

Travelers’ Diarrhea is the number one cause of childhood death in the world. Enteroaggregative Escherichia coli (EAEC) is one of the main causes of this disease. EAEC adhere to the surface of the intestine and stack in a brick-like pattern. Via an unstudied quorum-sensing mechanism, these bacteria express a variety of virulence factors that lead to diarrhea. The long-term goal of this research is to elucidate the mechanism by which EAEC changes from benign to virulent. A previously-unstudied open-reading frame in EAEC, AggR activated repressor (Aar), has recently been hypothesized to act as one of the major transcription factors influencing …

Development, Validation, And Application Of Analytical Methods For Characterizing Adsorbed Protein Orientation, Conformation, And Bioactivity, Aby Thyparambil

All Dissertations

The structure and bioactivity of adsorbed proteins are tightly interrelated and play a key role in their interaction with the surrounding environment. These factors are of critical importance in many biotechnological applications. However, because the bioactive state of an adsorbed protein is a function of the orientation, conformation, and accessibility of its bioactive site(s), the isolated determination of just one or two of these factors will typically not be sufficient to understand the structure-function relationships of the adsorbed layer. Rather a combination of methods is needed to address each of these factors in a synergistic manner to provide a complementary …

Structure And Molecular Mechanism Of A Plp/Gaba Dependent Transcription Regulator Gabr, Rui Wu

Dissertations

GabR is a member of the MocR/GabR subfamily of the GntR family of bacterial transcription regulators. It regulates the metabolism of γ-aminobutyric acid (GABA), an important nitrogen and carbon source in many bacteria. The crystal structures reported here show that this protein has evolved from the fusion of a type I aminotransferase and a winged helix-turn-helix (wHTH) DNA binding protein to form a chimeric protein that adopts a dimeric head-to-tail configuration. The pyridoxal 5′-phosphate (PLP)-binding regulatory domain of GabR is therefore an example of a coenzyme playing a role in transcription regulation rather than in enzymatic catalysis. Our structural and …

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 …

Conformational Changes In The Extracellular Domain Of Glutamate Receptors, Anu Rambhadran

Dissertations & Theses (Open Access)

The family of membrane protein called glutamate receptors play an important role in the central nervous system in mediating signaling between neurons. Glutamate receptors are involved in the elaborate game that nerve cells play with each other in order to control movement, memory, and learning.

Neurons achieve this communication by rapidly converting electrical signals into chemical signals and then converting them back into electrical signals. To propagate an electrical impulse, neurons in the brain launch bursts of neurotransmitter molecules like glutamate at the junction between neurons, called the synapse. Glutamate receptors are found lodged in the membranes of the post-synaptic …