Biochemistry, Biophysics, and Structural Biology Commons^™

The Phosphoramidase Competency Of Prototypical Phosphatase Catalytic Motifs, Mark P. Haney

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Phosphorylation of proteins is ubiquitous. Phosphorylation can activate proteins, deactivate proteins, assist in signaling, or serve other roles depending upon the biochemical pathway. Attaching phosphate to proteins is accomplished by enzymes called kinases; removing phosphate from proteins is accomplished by enzymes called phosphatases. Cells must regulate their biochemical pathways, and the antipodal roles of kinases and phosphatases represent the yin-yang of phosphorylation.

Phosphorylation of proteins is known to occur on serine, threonine, and tyrosine. This creates a phosphoester bond. Phosphoester bonds have a phosphorus-oxygen (P-O) bond. The ability of phosphatases to cleave such phosphoester bonds is well studied. Phosphorylation of …

Evaluation Of The Tgf-Ss Inhibitor Repsox On The Expression Of Pluripotency Pathways In Murine And Bovine Cells, Davin M. Larsen

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Embryonic stem cells are cells which are isolated from early stage embryos and have the theoretical ability to become any adult cell type in the body. In the past few years much research has focused on the use of embryonic stem cells for clinical applications in the treatment of degenerative diseases. Consequently they are a promising tool in the treatment of diseases such as Diabetes and Parkinson’s disease, and could potentially be used in the repair of permanently damaged tissue. However, since these cells are isolated from early stage embryos their successful isolation often results in the destruction of the …

Increased Production And Extraction Efficiency Of Triacylglycerides From Microorganisms And An Enhanced Understanding Of The Pathways Involved In The Production Of Triacylglycerides And Fatty Alcohols, Robert M. Willis

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The continued increase in the demand for fossil fuels combined with their ever dwindling supply has prompted the search for a suitable alternative fuel. The research contained within this dissertation seeks to increase the lipid (fat) content of cellular feedstocks, improve extraction efficiencies of lipids, and to understand the pathways involved in the production of fatty alcohols and triacylglycerides, compounds commonly used in many industrial processes, from microbial feedstocks. This work has been done in an attempt to increase the overall economic viability of microbial biofuels production.

The production of biofuels from microalgae used as a feedstock allows for the …

Characterization And Potential Utility Of Porcine Trophoblast-Derived Stem-Like Cells, Edison A. Suasnavas

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

In mammalian pregnancy, the placenta is a very important organ which helps to establish a healthy pregnancy. Its functions could be described in four points: 1) It allows the fetus to receive gases and nutrients from the mother in a safe way; 2) it lets the fetus get rid of waste through the mother's kidneys; 3) it releases essential pregnancy-related hormones and growth factors that let the uterus hold the fetus; 4) it secretes immune response regulators to give the fetus immune protection against the mother. Abnormalities in the placenta can be the cause of death to the fetus. Mammalian …

Characterization Of The Product Specificity And Kinetic Mechanism Of Protein Arginine Methyltransferase 1, Shanying Gui

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Protein enzymes perform a vast array of functions within living organisms, catalyzing various metabolic reactions including DNA replication, DNA repair, protein synthesis, etc. In order to maintain proper cellular functions, enzymes need to be accurately regulated under different circumstances. Specifically, enzymes can be modified after their creation to give them additional functions. These modifications can do a variety of things including activating (turning on) or inactivating (turning off) an enzyme, changing what proteins or molecules can interact with the enzyme, changing the enzyme’s location in the cell, and/or targeting the enzyme for destruction. This dissertation focuses on a single class …

Investigation Of The Oxidation/Reduction Of Prmt1, Substrate Interaction With Prmt1, And The Role Of Arginine Methylation In Rna Surveillance, Damon V. Nitzel

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Enzymes are the machines of our cells. Just like machines, it takes a lot of energy to create them, and they then serve only the function they were created for. If we want to change the function of a machine, we need to modify it. Similarly, enzymes can be modified after their creation to give them additional function. These modifications can do a variety of things including activating (on) or inactivating (off) an enzyme, changing the enzyme’s location in the cell, and targeting the enzyme for destruction. This thesis focuses on a single class of enzymes, protein arginine methyltransferases (PRMTs), …

Biochemical Characterization Of Sac9, A Putative Phosphoinositide Phosphatase In Arabidopsis Thaliana, And Its Role In Cellular Abnormalities, Almut H. Vollmer

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Since the early colonization of land by fungi and plants some 700 million years ago, plants have been continuously faced with changes in their environment. Unlike animals, plants are not free to move about, and can therefore not evade many stress factors. How plants sense and respond to their environment has been of interest not only to scientific research but also in more practical applications such as agriculture.

Signals (such as light or salinity) from the outside of plant cells trigger a flow of information to the inside of the cell. The final target for most of the information is …

1. Improving The Yield Of Biodiesel From Microalgae And Other Lipids. 2. Studies Of The Wax Ester Biosynthetic Pathway And Potential Biotechnological Application, Bradley D. Wahlen

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The production of biofuels and oleochemicals from renewable sources offers an opportunity to reduce our dependence on fossil fuels. The work contained in this dissertation has focused on developing and improving methods for the production of biodiesel from non-traditional feedstocks and understanding biosynthetic pathways that result in the production of oleochemicals and fuels.

Pure vegetable oil can account for 70-80% of the total cost of biodiesel production. Many low-cost oils contain high amounts of free fatty acids, which are unsuitable for base-catalyzed transesterification. Herein an approach is described that efficiently accomplishes the simultaneous esterification and transesterification of both free fatty …

Structural And Mechanistic Investigations Of Phosphothreonine Lyase Class Of Enzymes, Alok Gopalkrishna Shenoy

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Phosphorylation and dephosphorylation are a highly pervasive mechanism in biology that is used by the cell to modulate enzymes and proteins. The presence of a phosphate group can activate or deactivate an enzyme. The phosphate group is linked to a protein by a phosphoester bond that is known to be highly stable in cytoplasmic pH range. Thus the breaking and formation of these bonds need to be effected by enzymes.

Recent discovery of the activity carried out by certain virulence related proteins (OspF released by Shigella and SpvC released by Salmonella) have resulted in a necessity to create a new …

Studies Of The Distinguishing Features Of Nadph:2-Ketopropyl-Coenzyme M Oxidoreductase/Carboxylase, An Atypical Member Of The Disulfide/ Oxidoreductase Family Of Enzymes, Melissa A. Beighley-Kofoed

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The metabolism of propylene in Xanthobacter autotrophicus occurs via epoxypropane formation and subsequent metabolism by a three-step, four-enzyme pathway, utilizing the atypical cofactor Coenzyme M (CoM) to form acetoacetate. The last step in the epoxide carboxylase pathway is catalyzed by a distinctive member of the disulfide oxidoreductase (DSOR) family of enzymes, NADPH:2-ketopropyl CoM oxidoreductase/carboxylase (2-KPCC). 2-KPCC catalyzes the unorthodox cleavage of a thioether bond and successive carboxylation of the substrate. The focus of the research presented in this dissertation aims to elucidate the details of 2-KPCC that allow it to perform chemistry unconventional for typical DSOR members. Sitedirected mutagenesis was …

Kinetic, Mechanistic, And Structural Investigation Of Features Controlling Stereoselectivity Of (R)- And (S)-Hydroxypropyl Com Dehydrogenases From Xanthobacter Autrophicus Strain Py2, Dariusz Adam Sliwa

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Enantiopure alcohols are valuable intermediates in fine organic synthesis, in particular for preparation of biologically active compounds. The necessity of preparing single enantiomer drugs in an optically pure form has triggered much research, especially in the pharmaceutical industry. The biocatalytical production of chiral alcohols by alcohol dehydrogenase enzymes is characterized by the asymmetric reduction of the corresponding ketones, usually with high degree of stereoselectivity. The commercial value of the enzymes as stereoselective biocatalysts has been a significant driving force in understanding features that control their mechanism of catalysis and stereoselectivity. This work focuses on two enantiocomplementary dehydrogenase enzymes ((R …

Host Signaling Response To Adhesion Of Bifidobacterium Infantis, Reed N. Gann

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Investigations of the molecular binding partners of the probiotic bacterium Bifidobacterium longum subspecies infantis (B. infantis) and the pathogen Salmonella enterica subspecies enterica serovar Typhimurium LT2 (Salmonella ser. Typhimurium) found that these two very different bacteria bind gangliosides. However, these organisms lead to completely different host health outcomes when present in the gut. B. infantis is the founding microbial population in the intestinal tract of breast-fed infants. S. typhimurium is the most important food-borne pathogen that results in humans. This study used an in vitro gut epithelial cell model to examine the host cellular response to adhesion …

Physiological And Molecular Function Of Hap3b In Flowering Time Regulation And Cold Stress Response, Mingxiang Liang

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Heme-activated proteins (HAPs) are transcription factors that have multiple roles in plant growth and development, such as embryogenesis, flowering time control, and drought tolerance.

In the present study I found that HAP3b was also involved in controlling response to cold stress. Transcript profiling and gene expression analyses indicated that HAP3b repressed the CBF3 regulon under normal growth conditions. As a result, plants with HAP3b-overexpressed showed decreased survival rates while plants homozygous for the null allele hap3b showed an improved freezing tolerance compared to wild-type plants.

To understand the mechanism of HAP3b in Arabidopsis, i.e. whether it also acts …

Transesterification And Recovery Of Intracellular Lipids Using A Single Step Reactive Extraction, Daniel R. Nelson

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

A single-step, extractive reaction for extraction of lipids such as biodiesel components, omega-3 fatty acids, or other triglycerides from microbial cells was examined. Conventional methods for lipid extraction use toxic solvents, and require multiple steps and long processing times. When the goal is to produce fatty acid methyl esters or FAMEs, the extracted lipids are subjected to a separate transesterification reaction with simple alcohols in the presence of an acid or base catalyst. A simplified, single-step reactive extraction method can be applied that combines the sequential extraction followed by transesterification using acidified alcohols - a process known as in situ …

A Synthetic Biological Engineering Approach To Secretion- Based Recovery Of Polyhydroxyalkanoates And Other Cellular Products, Elisabeth Linton

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The costs associated with cellular product recovery commonly account for as much as 80% of the total production expense. As a specific example, significant recovery costs limit commercial use of polyhydroxyalkanoates (PHA), which comprise a class of microbially-accumulated polyesters. PHAs are biodegradable compounds that are of interest as a sustainable alternative to petrochemically-derived plastics. Secretion-based recovery of PHAs was studied to decrease PHA production costs. Type I and II secretory pathways are commonly used for the translocation of recombinant proteins out of the cytoplasm of E. coli. Proteins were targeted for translocation using four signal peptides (HlyA, TorA, GeneIII, …

Role Of Protein Kinase C Isotypes In 1,25-Dihydroxyvitamin D3 Mediated Signal Transduction Through The 1,25d3 Membrane Associated, Rapid Response Steroid Binding Receptors In Chick Intestinal Cells, Sakara Tunsophon

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

It is now accepted that 1,25(OH)2D3 mediates its rapid actions on the control of phosphate and calcium homeostasis through its membrane receptor termed the 1,25D3-MARRS (membrane associated rapid response steroid binding) protein. I determined the various PKC isotypes involved in the rapid regulation of phosphate uptake and calcium extrusion in chick intestinal cells. 1,25(OH)2D3-mediated phosphate uptake was stimulated within 1 min after addition of the hormone. Western blot analyses on isolated intestinal cells treated with steroid hormone resulted in dose-dependent increases in PKC alpha and PKC beta in postnuclear centrifugation fractions, but not in the low speed centrifugation fractions. The …

Development Of Novel Methods And Their Utilization In The Analysis Of The Effect Of The N-Terminus Of Human Protein Arginine Methyltransferase 1 Variant 1 On Enzymatic Activity, Protein-Protein Interactions, And Substrate Specificity, Brenda Bienka Suh-Lailam

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first …

Characterization Of The Substrate Specificity And Mechanism Of Protein Arginine Methyltransferase 1, Whitney Lyn Wooderchak

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Protein arginine methyltransferases (PRMTs) posttranslationally modify protein arginine residues. Type I PRMTs catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA) via methyl group transfer from S-adenosyl methionine onto protein arginine residues. Type II PRMTs generate MMA and symmetric dimethylarginine. PRMT-methylation affects many biological processes. Although PRMTs are vital to normal development and function, PRMT-methylation is also linked to cardiovascular disease, stroke, multiple sclerosis, and cancer.

Thus far, nine human PRMT isoforms have been identified with orthologues present in yeast, plants, and fish. PRMT1 predominates, performing an estimated 85% of all protein arginine methylation in vivo. Yet, the substrate …

A Comparative Study Of The Structural Features And Kinetic Properties Of The Mofe And Vfe Proteins From Azotobacter Vinelandii, Miguel Alejandro Pabon Sanclemente

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Biological nitrogen fixation is accomplished in the bacterium Azotobacter vinelandii by means of three metalloenzymes: The molybdenum, vanadium, and iron-only nitrogenase. The knowledge regarding biological nitrogen fixation has come from studies on the Mo-dependent reaction. However, the V- and Fe-only-dependent reduction of nitrogen remains largely unknown.

By using homology modeling techniques, the protein folds that contain the metal cluster active sites for the V- and Fe-only nitrogenases were constructed. The models uncovered similarities and differences existing among the nitrogenases regarding the identity of the amino acid residues lining pivotal structural features for the correct functioning of the proteins. These differences, …

Expression Analysis Of Plant Defense Responses During The Establishment Of Biotrophy And Role Of Abiotic Stress In The Infection Of Dyer’S Woad (Isatis Tinctoria) By Puccinia Thlaspeos, Elizabeth Thomas

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The kinetics and amplitude of the salicylic acid-responsive pathogenesis-related (PR) genes and the cytochrome P450 gene ItCYP79B2 in the compatible interaction between Puccinia thlaspeos and dyer’s woad (Isatis tinctoria) during the first 72 hours of inoculation were examined. Immediately following penetration of the host by the rust pathogen, there was a modest up-regulation of PR genes but a significant down-regulation of ItCYP79B2 expression. During haustoria formation, a significant pathogen-mediated suppression of PR genes was observed with a corresponding up-regulation of ItCYP79B2. This potentially facilitates haustoria formation by P. thlaspeos. After haustoria formation, a more …

Role Of Iron In The Mechanism Of Asbestos-Induced Apoptosis In Human Lung And Pleural Target Cells, Aleksander Baldys

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Occupational exposure to asbestos has been associated with increased incidence of pulmonary interstitial fibrosis, mesothelioma of the pleura, and bronchogenic carcinoma. Although the mechanism by which asbestos causes cancer remains unknown, iron associated with asbestos is thought to play a role in the pathogenic effects of fibers.

The aim of this research was to examine and compare the asbestos-induced signaling phenomena in relevant human lung and pleural target cells, and to determine the role of iron from asbestos fibers in these events. Exposure of human airway epithelial (A549) cells, human pleural mesothelial (MET5A) cells, and normal human small airway epithelial …

Studies On The Roles Of Atp In Nitrogenase Catalysis, Wei Wu

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Nitrogenase is the enzyme that catalyzes the reduction of nitrogen to ammonia in a reaction requiring MgATP hydrolysis. Two component proteins of nitrogenase are the iron protein (Fe protein) and the molybdenum-iron protein (MoFe protein).

Nitrogenase contains two nucleotide binding sites. During catalysis, the Fe protein binds two MgATP first. The conformational changes induced upon MgATP binding allow the Fe protein to associate with the MoFe protein. After the formation of the Fe protein-MoFe protein complex, a single electron is transferred from the Fe protein to the MoFe protein, an event that is coupled to MgATP hydrolysis in the Fe …

Phylogenetic Studies Of The United States Bluetongue Viruses And Characterization Of The Viral Vp4 Protein, I-Jen Huang

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Bluetongue virus (BTV) is transmitted by arthropod vectors and causes bluetongue disease with serious economic loss in many regions of the world. The replication mechanism of bluetongue virus is still not clear. To have a better understanding regarding the viral replication, the function of each individual protein has to be identified. This study used molecular biology techniques to investigate the function of the inner core protein VP4.

The M1 genes of United States bluetongue virus serotypes-2, -10, -11, -13, and -17 were cloned and sequenced. The length of each of the five M1 genes is 1981 nucleotides. The coding region …

Characterization Of An Axial Ligand Substitution In Sperm Whale Myoglobin, Michael J. Chen

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Of central importance to the study of heme proteins are the effects imposed by axial ligand(s) on the heme structure and, therefore, on the overall activity of the protein. In this study, we confirm and extend the spectroscopic characterization of a mutated sperm whale myoglobin in which the proximal Histidine is replaced with a Tyrosine residue (MbH93Y). The MbH93Y, as well as wild-type sperm whale myoglobin and horse erythrocyte catalase (HEC), was purified and characterized by optical absorption and x-ray absorption (XAS) spectroscopies. Optical absorption spectra of HEC and the metmyoglobin, cyanometmyoglobin, reduced, oxy, and carbon-monoxy forms of both sperm …

Extended X-Ray Absorption Fine Structure And Redox Potential Studies Of Heme-Substituted Horseradish Peroxidase And Myoglobin, Bing He

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Heme-substituted horseradish peroxidases and myoglobins were reconstituted from the apoenzyme using mesoheme and diacetyldeuteroheme. X-ray absorption spectroscopy was used to determine the dimensions of the active sites of these heme-substituted proteins, and were compared with those of the proto-hemeproteins. The change in the active-site structure corresponded with the electron withdrawing and donating effects of the different side chains. The oxidation-reduction potentials of Fe⁴⁺/Fe³⁺ couples of the heme-substituted proteins were measured at pH 7 with K₂IrCl₆. The oxidation-reduction potential sequence for compound I/compound II was diacetyldeutero- > proto- > meso- in horseradish peroxidase. The oxidation-reduction potential …

Mobilization Of Iron Enhances The Iron-Dependent Biochemical Reactivity Of Asbestos In Vitro And Contributes To The Cytotoxicity Of Asbestos In Cultured Cells, Loren Glen Lund

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Asbestos related research began approximately 60 years ago, yet, the mechanism(s) by which asbestos exerts its biological effects is not well understood. The hypothesis upon which this dissertation is based is that mobilization of iron from asbestos enhances the iron-dependent biochemical reactivity of asbestos in vitro and contributes to asbestos-dependent cytotoxicity. The specific aims for this hypothesis were, 1) to determine whether iron was responsible for the biochemical reactivity of asbestos in vitro and asbestos-induced cytotoxicity in cultured cells, and 2) to determine whether mobilization of iron from asbestos enhanced the reactions catalyzed by asbestos in vitro and contributes to …

Heterologous Expression And Characterization Of Three Active Recombinant Fungal Peroxidases From Phanerochaete Chrysosporium Using Baculovirus, Todd M. Johnson

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

In a collaborative effort with Dr. Steven D. Aust's group at Utah State University, this research is the first to describe the heterologous expression of active peroxidases from the filamentous fungus Panerochaete chrysosporium. The cDNA clones λML-1, λML-6, λML-5 and λMP-1, which had been tentatively identified as fungal peroxidase clones, were subcloned into baculovirus transfer vectors and recombinant Autographa californica nuclear polyhedrosis viruses were constructed which contained copies of these potential peroxidase genes. The recombinant baculovirus clones were used to infect insect cells for the heterologous production of active recombinant enzymes. Three recombinant lignin peroxidases (H2, H6 and H8) …

Energetic Requirements For Bacterial Protein Export, John Andrew Corbett

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Bacterial protein export involves the translocation of precursor proteins across the inner cytoplasmic membrane. Over 100 proteins are exported from Escherichia coli. This study showed that energy in the form of ATP and membrane gradient energy is essential for the export of leucine specific binding protein and β-lactamase precursors. Ionophores or combinations of ionophores (SF6847, valinomycin/nigericin and valinomycin/monensin) which dissipate protonmotive force inhibit protein export. Valinomycin alone also inhibits export, but not as well as reagents which dissipate protonmotive force. Nigericin or monensin alone slightly stimulate protein export. These results suggest that the transmembrane electrical potential (∆Ψ) is the …

A Study Of Denitrosation Of N-Nitroso Compounds By Irradiation With Long-Wavelength Uv Light, Zhenyu J. Wang

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

N-Nitrosamines and N-nitrosamides have been reported to be mutagenic. N-Nitrosamides are direct mutagens that need no activation to be mutagenic, whereas N-nitrosamines need to be enzymatically activated to exert their mutagenicity. Oxidative demethylation of nitrosamines is a commonly accepted activation mechanism. Another pathway of nitroso compounds, denitrosation, has recently been proposed. The mechanism of denitrosation, however, is still unknown.

The purpose of this study was to use a photo -reaction model to explore the possible denitrosation mechanism of N-nitroso compounds. An N-nitrosamine, N-nitrosomorpholi ne (NMOR), and an N-nitrosamide, N-methyl-N'-nitrosoguanidine (MMNG), were irradiated with long-wavelength UV light in the presence of …

Peptide Mapping Analysis Of Vp5 And Vp7 From Five Bluetongue Viruses Isolated In The United States, Yi-Yuan Yang

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

BTV-2, 10, 11, 13 and 17 were purified by three discontinuous sucrose gradients. Viral proteins were separated from the genomic double-stranded RNA using a newly developed KCI-SDS (sodium dodecyl sulfate) precipitation method. Cyanogen bromide (CNBr) peptide mapping of VP5 and VP7 from bluetongue virus (BTV) serotypes 2, 10, 11, 13 and 17 was then done using silver-stained SDS-polyacrylamide gels. The antigenic epitopes of VP5 and VP7 digested with CNBr were also investigated with polyclonal antibodies produced against KCl-SCS precipitated BTV-13 viral proteins. The peptide maps and the antigenic epitopes of VP7 were similar among the five American serotypes with very …