Biochemistry Commons^™

The C. Neoformans Cell Wall: A Scaffold For Virulence, Christine Chrissian

Dissertations, Theses, and Capstone Projects

Cryptococcus neoformans is a globally distributed opportunistic fungal pathogen and the causative agent of life threatening cryptococcal meningoencephalitis in immunocompromised individuals, resulting in ~180,000 deaths each year worldwide. A primary virulence-associated trait of this organism is the production of melanin. Melanins are a class of diverse pigments produced via the oxidation and polymerization of aromatic ring compounds that have a characteristically complex, heterogenous, and amorphous structure. They are synthesized by representatives of all biological kingdoms and share a multitude of remarkable properties such as the ability to absorb ultraviolet (UV) light and protect against ionizing radiation. Melanin production in fungi …

Design Of Fibril Forming Collagen Mimetic Peptides: Heterotrimers And Nucleation Domains, Sally Tan

Theses and Dissertations

This paper attempts to design collagen mimetic peptides where the triple-helical region mimics that of human Type I Collagen. With consideration for chain selection and chain register, we utilize the NC2 domain of heterotrimeric Type IX Collagen as a nucleation domain for triple-helix folding.

Synthesis And Biological Evaluation Of Grp94 Selective Inhibitors, Zach Priebe, Kyler Pugh, Brian S. J. Blagg

Summer Research

Grp94 is one of four chaperones which belong to the Heat Shock Protein 90 (Hsp90) family. Hsp90 isoforms exist as homodimers and assist in the folding and maturation of over 400 client proteins. These proteins are highly sought-after therapeutic targets as they are known to chaperone misfolded proteins involved in various signaling pathways. During the protein folding cycle, ATP binds to the N-Terminal domain of Hsp90, which allows nascent polypeptides to undergo maturation. To prevent the folding and maturation of oncogenic proteins, researchers developed N-terminal inhibitors to prevent ATP binding. Unfortunately, these inhibitors (termed pan-inhibitors) were not isoform selective, which …

Photoreceptor Phosphodiesterase (Pde6): Activation And Inactivation Mechanisms During Visual Transduction In Rods And Cones, Rick H. Cote

Faculty Publications

Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary

intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes …

Photoreceptor Phosphodiesterase (Pde6): Structure, Regulatory Mechanisms, And Implications For Treatment Of Retinal Diseases, Rick H. Cote, Richa Gupta, Michael J. Irwin, Xin Wang

Faculty Publications

The photoreceptor phosphodiesterase (PDE6) is a member of large family of Class I phosphodiesterases responsible for hydrolyzing the second messengers cAMP and cGMP. PDE6 consists of two catalytic subunits and two inhibitory subunits that form a tetrameric protein. PDE6 is a peripheral membrane protein that is localized to the signaling-transducing compartment of rod and cone photoreceptors. As the central effector enzyme of the G-protein coupled visual transduction pathway, activation of PDE6 catalysis causes in a rapid decrease in cGMP levels that results in closure of cGMP-gated ion channels in the photoreceptor plasma membrane. Because of its importance in the phototransduction …

Computational Analysis And Prediction Of Intrinsic Disorder And Intrinsic Disorder Functions In Proteins, Akila I. Katuwawala

Theses and Dissertations

COMPUTATIONAL ANALYSIS AND PREDICTION OF INTRINSIC DISORDER AND INTRINSIC DISORDER FUNCTIONS IN PROTEINS

By Akila Imesha Katuwawala

A dissertation submitted in partial fulfillment of the requirements for the degree of Engineering, Doctor of Philosophy with a concentration in Computer Science at Virginia Commonwealth University.

Virginia Commonwealth University, 2021

Director: Lukasz Kurgan, Professor, Department of Computer Science

Proteins, as a fundamental class of biomolecules, have been studied from various perspectives over the past two centuries. The traditional notion is that proteins require fixed and stable three-dimensional structures to carry out biological functions. However, there is mounting evidence regarding a “special” class …

Interactions Of Post-Pks Enzymes Of The Mithramycin Biosynthetic Pathway, Ryan Wheeler

Theses and Dissertations--Pharmacy

Combinatorial biosynthesis is a powerful tool for generating new, more active drug analogues to combat disease. But in order for combinatorial biosynthesis to be employed to its full potential, a deep understanding of the enzymes that produce the parent molecule must be had. The goals of the work presented in this thesis are to characterize the reaction catalyzed by MtmW, the final enzyme in the mithramycin (MTM) biosynthetic pathway, and to discover the interaction between MtmW and MtmOIV.

MtmW is an aldol-ketoreductase responsible for reducing the most distal carbonyl on the MTM pentyl side chain. It forms an octamer that …

Substrate Trafficking Within The Type Vii Secretion Systems Of Pathogenic Mycobacteria, Zachary A. Williamson

Theses and Dissertations--Molecular and Cellular Biochemistry

Tuberculosis (TB), primarily caused by infection of Mycobacterium tuberculosis (Mtb) in the lungs, is the deadliest infectious bacterial disease killing 1.5 million people annually. A major determinant of virulence is active secretion through three specialized type VII secretion (ESX) systems; ESX-1, ESX-3, and ESX-5. A large group of substrates exported by the ESX systems is the PE (Proline-Glutamine) and PPE (Proline-Proline-Glutamate) families of proteins, which are highly expanded in the pathogenic species of Mycobacteria and encompass over 7% of Mtb’s genome coding capacity. PE and PPE proteins interact together to form PE-PPE heterodimers, and are secreted through …

On The Structure And Function Of Mitochondrial Uncoupling Proteins: The Case Of Ucp2, Afshan Ardalan

Theses and Dissertations (Comprehensive)

Uncoupling proteins (UCPs) are regulated proton transporters of the mitochondrial inner membrane. UCP-mediated proton leak negatively impacts the rate of ATP synthesis. Despite the importance of their physiological role(s) in certain tissues, molecular aspects of UCPs’ structure-function relationships are not fully understood. The current study explores the tertiary and quaternary structure of UCP2, as well as its proton transport mechanism in lipid membranes. The proteins were expressed in the E. coli inner membrane, purified and reconstituted into liposomes. Proteins were characterized by semi-native SDS-PAGE. Circular dichroism spectroscopy (CD) and fluorescence quenching assays were utilized to study the conformation of proteins …

Investigation Of Multidrug Efflux Transporter Acrb In Escherichia Coli: Assembly, Degradation And Dynamics, Prasangi Irosha Rajapaksha

Theses and Dissertations--Chemistry

The Resistant Nodulation Division (RND) super family member, tripartite AcrA-AcrB-TolC efflux pump, is a major contributor in conferring multidrug-resistance in Escherichia coli. The structure of the pump complex, and drug translocation by functional rotation mechanism have been widely studied. Despite of all these data, the dynamics of the assembly process of the pump and AcrB during functional rotation in the process of drug efflux remains poorly understood. My thesis focuses on understanding the pump assembly process, dynamics of AcrB in functional rotation mechanism, and also investigate the mechanism of degradation of AcrB facilitated by a C-terminal ssrA tag.

In the …

Cryogenic Electron-Microscopy (Cryo-Em) Analysis Of Mscs, Ynai, Connexin 26 And Bioinformatic Analysis Of Cysteine And Serine In Experimental Protein Structures, Claudio Catalano

Theses and Dissertations

Functional insights into macromolecules lie in the arrangement of their molecular structures. Investigating the atomic framework of a biomolecule could unwind its biochemical mechanism, providing aid to many relevant biological questions, thus facilitating drug discovery processes. Two approaches have been used to gain knowledge at the atomic level of a protein function: experimental and computational methodologies.

From an experimental perspective, membrane proteins (MPs) were investigated. Such proteins represent ~20% of the human proteome, and ~60% of them are drug targets since they are exposed to the surface of cells and are responsible for a wide range of fundamental functions like …

Characterization Of Cytochrome P450 3a4 Structure And Ligand Binding Mechanism In Anionic Nanodiscs, David T. Sweeney

Theses and Dissertations

The Cytochromes P450 superfamily are ubiquitous throughout life and are present in some viruses. Humans encode for 57 of these enzymes that are involved in the metabolism of ~90% of all chemicals. These membrane-bound monooxygenase enzymes utilize a heme protoporphyrin at their active site to activate molecular oxygen and oxidize their substrates through an intermediate deemed nature's most powerful oxidizer. They are a powerful system at our disposal yet understanding of their structure and ligand binding mechanisms in the presence of a lipid bilayer remains poorly understood. The focus of this work is on Cytochrome P450 3A4 (CYP3A4). We set …

Applied Molecular Dynamics: From Targeting Viral Helicases, To Understanding The Interactions Of Cucurbituril Complexes In Ionic Solutions, Bryan Raubenolt

LSU New Orleans Theses and Dissertations

Molecular Dynamics simulations are a highly useful tool in helping understand the fundamental interactions present in a variety of chemical systems. The work discussed here illustrates it’s use in determining the conformational dynamics of the Zika and SARS-Cov-2 helicase in a physiological environment, largely in an effort to discover inhibitors capable of rendering the protein inert. Additionally, we show how it can be used to understand paradoxical trends in the anion-induced precipitation of Cucurbituril cavitands.

Viral helicases are motor proteins tasked with unwinding the viral dsRNA, a crucial step in preparing the strand to be translatable by host cells. By …

Rna Structure And Function: Biological Relevance In Neurodegenerative And Infectious Disease Pathogenesis, Joshua Imperatore

Electronic Theses and Dissertations

The studies outlined in this dissertation encompass a broad focus, relating to the pathogenesis of neurological disorders such as Alzheimer’s disease, fragile X syndrome, and amyotrophic lateral sclerosis, as well as a novel infectious disease, COVID-19. However, all four studies detailed within this dissertation contain similar elements, namely, the coordinated role of RNA structure and function in essential molecular mechanisms. The results of these investigations allow us to speculate on various disease- related mechanisms, including dysregulated microRNA processing pathways, nucleocytoplasmic shuttling via distinct RNA-protein interactions, and fine-tuned molecular switches controlling genomic dimerization and viral hijacking of host cellular microRNAs. Taken …

Deciphering The Catalytic Mechanism Of Human Manganese Superoxide Dismutase, Jahaun Azadmanesh

Theses & Dissertations

The livelihood of human cells is heavily dependent on the ability to modulate the presence of highly reactive oxygen-based molecules termed reactive oxygen species (ROS). In excess, ROS facilitate oxidative damage to the macromolecules of cellular life. SODs are the major family of antioxidant proteins that prevent the buildup of overwhelming amounts of ROS within cells. Sometimes dubbed the “first line of defense” against oxidative damage, SODs defend against the harmful accumulation of ROS by eliminating superoxide. Superoxide is a ROS itself that is also a precursor to much more harmful ROS molecules. MnSOD is the manganese containing form of …

Single-Fluorophore Sensors For Mechanical Force In Living Cells, Sarah Kricheff

Honors Scholar Theses

Mechanotransduction is the process by which a mechanical stimulus is converted to a cellular signal. This process is heavily influential of cell morphology, differentiation, and behavior. However, altered levels of mechanical stimuli are also found in many pathological contexts. For example, cancerous cells have stiffer surrounding tissue than healthy cells, and research suggests that this alters cell behavior and promotes metastasis. Despite these findings, the cellular processes behind these signaling alterations remain widely unknown. Understanding these cascades is critical, as involved proteins can give us a deeper understanding of the role of mechanotransduction, and certain proteins can potentially be targeted …

Degree Of Conservation Of Methionines Found To Be Oxidized In The Human Urinary Proteome, Alexis Hall

Graduate Theses and Dissertations

In previous work from this laboratory, methionine containing peptides from the human urinary proteome were examined by mass spectrometry for the degree of methionine oxidation to the sulfoxide form. While this demonstrated that many of the methionines detected were capable of being oxidized, the question of whether these methionines are important in the structure and/or function of the parent proteins came about. In some proteins, methionine oxidation has been linked to conformational changes and alteration of function and thus can serve as a mechanism for reversible regulation of activity. It is hypothesized that methionines which might serve a regulatory purpose …

Engineering And Application Of A Biosensor With Focused Ligand Specificity, Dennis Della Corte, Hugo L. Van Beek, Falk Syberg, Marcus Schallmey, Felix Tobola, Kai U. Cormann, Christine Schlicker, Philipp T. Baumann, Karin Krumbach, Sascha Sokolowsky, Connor J. Morris, Alexander Grünberger

Faculty Publications

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering …

Investigating Photosynthetic Stability: Relation Between Thylakoid Lipid Content And The Stability Of The Cytochrome B6f Complex, Marina Mehling

The Journal of Purdue Undergraduate Research

The cytochrome b₆f complex is an enzyme found in plants, cyanobacteria, and green algae that catalyzes the transport of electrons in the rate-limiting step of oxygenic photosynthesis. This dimeric complex has an extensive lipid architecture that is primarily composed of five distinct lipid classes: monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), phosphatidyl glycerol (PG), monoglucosyl diacylglycerol (GlcDG), and sulfoquinovosyl diacylglycerol (SQDG). While these lipid classes have been identified, their precise role in the function of the cytochrome complex are only beginning to be understood. Mechanisms describing the relation between thylakoid lipid content on the stability of the b_{6 …}

Characterization Of Human Pyrroline-5-Carboxylate Reductase Enzymes Responsible For L-Proline Biosynthesis, Sagar Patel

Department of Biochemistry: Dissertations, Theses, and Student Research

Pyrroline-5-carboxylate reductases (EC 1.5.1.2) are important housekeeping enzymes of L-proline biosynthesis, which generate L-proline and influence redox cycling of NAD(P)H/NAD(P)⁺ to support cellular growth in all domains of life. Structural evidence from X-ray crystal structures of HsPYCR1 (PDB codes 5UAT, 5UAU, and 5UAV) shows both NADPH bound in the N-terminal Rao-Rossmann fold motif and an important hydrogen bond or proton donor role for Thr238 with L-P5C. The Thr238Ala mutation results in 10-fold loss in catalytic efficiency with varied L-P5C relative to the wild-type enzyme, thus indicating Thr238’s potential hydrogen bond and proton donation to L-P5C is critical for catalysis. …

Insights Into Desiccation Tolerance: Properties Of Late Embryogenesis Abundant Proteins From Embryos Of Artemia Franciscana, Blase Matthew Leblanc

LSU Doctoral Dissertations

LEA proteins are a family of intrinsically disordered proteins that are expressed in various life stages of anhydrobiotic organisms and have been strongly associated with survival during water stress. The brine shrimp Artemia franciscana is the only known anhydrobiotic animal that expresses LEA proteins from Groups 1, 3, and 6. Here, I report that AfrLEA6, a novel Group 6 LEA protein, is most highly expressed in embryos during diapause and decreases throughout pre-emergence development. Notably, there is an acute drop in expression upon termination of the diapause state and the titer of AfrLEA6 during diapause is 10-fold lower than values …

Analysis Of Biofilm Remediation Capacity For Octenyl Succinic Anhydride (Osa), A Bioactive Food Starch Modifier Compound, Matthew R. Borglin

Master's Theses

Matthew R. Borglin

This thesis demonstrates efficacy of Octenyl Succinic Anhydride (OSA), as a biofilm sanitizer. Biofilms allow bacteria to adhere to solid surfaces with the use of excreted polymeric compounds. For example, surfaces found in food production or processing facilities such as the interior of a raw milk holding tank, are some of the most susceptible to biofilm contamination. When present, biofilms can cause a variety of negative effects, which include; reduction of product shelf life, corrosion, and outbreaks of foodborne illnesses. The close association of biofilms with the majority of foodborne illness cases led the US Environmental Protection …

Dna Damage Recognition And Uvrb Loading By Uvra Within The Nucleotide Excision Repair Pathway, Silas Hartley

Dissertations, Theses, and Capstone Projects

Maintaining the cellular genome is paramount to survival by any organism. A mutated genome can have detrimental effects on different cellular processes, especially replication and transcription. Cells maintain their genome using different deoxyribonucleic acid (DNA) repair pathways. The nucleotide excision repair (NER) pathway has a unique capability of repairing the genome from several different mutations, deletions, and adducts. In bacteria, the NER pathway accomplishes repair through four important steps: damage recognition by UvrA, damage verification by UvrB, DNA incision by UvrC, and repair synthesis using various cellular machinery.

UvrA forms a head-to-head dimer (UvrA₂) with two ATPase sites …

Intrinsic Buffer Hydroxyl Radical Dosimetry For Hydroxyl Radical Protein Footprinting, Addison Roush

Honors Theses

Hydroxyl radical protein footprinting (HRPF) coupled to mass spectrometry is a powerful technique for the analysis of protein topography as it generates covalent mass labels that can survive downstream sample handling, and it is sensitive to the solvent accessibility of amino acid sidechains. Of the multiple platforms for HRPF, fast photochemical oxidation of proteins (FPOP) utilizes a pulsed 248 nm KrF excimer laser to label proteins by photolyzing hydrogen peroxide. FPOP is the most widely used HRPF platform because it labels proteins faster than unfolding can occur. Variations in FPOP sample conditions make it difficult to compare results between experiments …

Chemoenzymatic Study Of Coa-Linked Rna In Bacteria, Krishna Sapkota

Dissertations

The ability of RNA to store genetic information and to catalyze biochemical transformations led to the speculation of the existence of RNA world before the evolution of contemporary ribonucleoprotein (RNP) world. Recent discovery of RNA molecules containing metabolic cofactors including coenzyme A and its various thioesters at their 5’ end further supported the RNA world hypothesis as these CoA-linked RNA molecules could be the molecular fossils with very ancient origin. As both RNA and Coenzyme A are believed to have co-existed since last universal common ancestor (LUCA) or even before, the CoA-RNA conjugates in current biology may reveal fundamental molecular …

Pedagogical Approaches To Enhance Q2s Conversion Teaching And Learning: Application Of The Acue Effective Practice Framework To Upper Division Biochemistry, Jason Burke

Q2S Enhancing Pedagogy

The Effective Practice Framework course by the Association of College and University Educators (ACUE), and offered through the CSUSB Teaching Resource Center (TRC), was applied to the upper division biochemistry course series (CHEM 436A and 437A) in preparation for the Q2S conversion. This essay includes five reflections on the application of enhanced pedagogical approaches curated from a total of twenty-five. The enhanced pedagogies covered in the course are: designing and effective course and class; establishing a productive learning environment; using active learning techniques; promoting higher-order thinking; and assessing to inform instruction and promote learning. The reflections that follow have been …

The Role Of Conformational Dynamics In Isocyanide Hydratase Catalysis, Medhanjali Dasgupta

Department of Biochemistry: Dissertations, Theses, and Student Research

Post-translational modification of cysteine residues can regulate protein function and is essential for catalysis by cysteine-dependent enzymes. Covalent modifications neutralize charge on the reactive cysteine thiolate anion and thus alter the active site electrostatic environment. Although a vast number of enzymes rely on cysteine modification for function, precisely how altered structural and electrostatic states of cysteine affect protein dynamics, which in turn, affects catalysis, remains poorly understood.

Here we use X-ray crystallography, computer simulations, site directed mutagenesis and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in the enzyme, isocyanide hydratase (ICH), affects the protein …

Optimization Of Dapoxyl Aptamer For Label-Free Bioanalysis, Jack E. Mordeson, Ryan P. Connelly, Pedro F. Madalozzo, Yulia Gerasimova

Digital Repository: Showcase of Undergraduate Research Excellence

No abstract provided.

Physicochemical Characterization, Structural Determination, And Molecular Dynamic Modeling Of Proso Millet Proteins For Enhanced Food Functionality, Felix Akharume

Theses and Dissertations--Biosystems and Agricultural Engineering

More than one-third of Americans today incorporate plant-based protein into their diet and about 40% believed that plant-based protein is healthier than animal protein, especially Millennials. The increasing global demand for plant-based proteins driven by the high cost of animal proteins, consumers’ desire for lean protein, vegetarianism, and the need for more sustainable green protein products have necessitated research into alternate emerging and underutilized sources of protein to complement or supplement the major plant protein in the market- soy, pea, and gluten. Therefore, this dissertation is focused on the valorization of the proteins in proso millet. Specifically, this work focused …

The Exploration Of Nanotoxicological Copper And Interspecific Saccharomyces Hybrids, Matthew Joseph Winans Phd

Graduate Theses, Dissertations, and Problem Reports (ETD)

Nanotechnology takes advantage of cellular biology’s natural nanoscale operations by interacting with biomolecules differently than soluble or bulk materials, often altering normal cellular processes such as metabolism or growth. To gain a better understanding of how copper nanoparticles hybridized on cellulose fibers called carboxymethyl cellulose (CMC) affected growth of Saccharomyces cerevisiae, the mechanisms of toxicity were explored. Multiple methodologies covering genetics, proteomics, metallomics, and metabolomics were used during this investigation. The work that lead to this dissertation discovered that these cellulosic copper nanoparticles had a unique toxicity compared to copper. Further investigation suggested a possible ionic or molecular mimicry …