Biochemistry Commons^™

Understanding The Molecular Mechanism Underlying The Great Thermal Stability Of Thermophilic Enzymes Using Aminoglycoside Nucleotidyltransferase 4' As A Model, Xiaomin Jing

Doctoral Dissertations

The aminoglycoside nucleotidyltransferase 4' (ANT) is a homodimeric enzyme that detoxifies antibiotics by nucleotidylating at the C4'-OH site. Two thermostable variants T130K and D80Y generated by direct evolution in laboratory differ by only a single residue replacement compared to the wild type mesophilic enzyme. Both variants display enhanced melting temperatures and execute catalysis at temperatures the wild type would be inactive. However, T130K variant still keeps molecular properties of mesophilic enzyme. T130àK130 does not trigger significant change in enzyme’s local flexibility or thermodynamics of ligand binding while D80Y variant has distinct properties in ligand recognition and dynamics. We hypothesize that …

Engineering Photosystem I Complexes For Use In Bio-Hybrid Dye-Sensitized Solar Cells, Richard Franklin Simmerman

Doctoral Dissertations

Increasing global population, growing per capita energy needs, diminishing fossil fuels, and climate change collectively will require new, innovative, and sustainable alternatives to meet the world’s growing energy needs. One of the most promising yet simple approaches are dye-sensitized solar cells (DSSCs). However, conventional DSSCs use semi-conductor anodes sensitized with complex synthetic organometallic dyes. Most dyes utilize ruthenium complexes to absorb photons, which upon excitation, inject electrons into the anode, while holes migrate to the cathode via liquid electrolyte. However, these dyes are expensive, difficult to make, and resource-limited. This dissertation focuses on replacing synthetic dyes with the naturally occurring, …

Quantitative And Functional Analysis Pipeline For Label-Free Metaproteomics Data And Its Applications, Lang Ho Lee

Doctoral Dissertations

Since the large-scale metaproteome was first reported in 2005, metaproteomics has advanced at a tremendous rate both in its quantitative and qualitative metrics. Furthermore metaproteomics is now being applied as a general tool in microbial ecology in a large variety of environmental studies. Though metaproteomics is becoming a useful and even a standard tool for the microbial ecologist, standardized bioinformatics pipelines are not readily available. Therefore, we developed quantitative and functional analysis pipeline for metaproteomics (QFAM) to help analyze large and complicated metaproteomics data in a robust and timely fashion with outputs designed to be simple and clearly understood by …

Calmodulin-Like Protein 38: A Component Of Ribonucleoprotein Particles During Hypoxic Stress Responses In Arabidopsis, Ansul Lokdarshi

Doctoral Dissertations

Waterlogging stress leads to a crisis in energy metabolism and the accumulation of toxic metabolites due to the hypoxic and/or anoxic environment associated with this condition. To respond and adapt to this situation, higher plants employ an integrated genetic program that leads to the induction of anaerobic response polypeptide genes that encode metabolic and signaling proteins involved in altering metabolic flow and other adaptive responses. The study presented here shows that the Arabidopsis thaliana calmodulin-like protein CML38 is calcium sensor protein that serves as a member of the core anaerobic response gene family and is involved in modulating the survival …

Characterization Of The Role Of Alpha-Arylphorin In The Heliothis Virescens Midgut Response To Cry1ac Toxin From Bacillus Thuringiensis, Jerreme Jamael Jackson

Doctoral Dissertations

Homeostasis of the intestinal epithelium in Heliothis virescens is mediated by the proliferation and differentiation of multipotent intestinal stem cells (ISCs) that lie adjacent to the basal lamina. In response to extrinsic and intrinsic signals, ISC proliferation and differentiation promotes epithelial growth and regeneration following the loss of integrity. We tested the in vivo effects of the ISC mitogen, a [alpha]-arylphorin, on ISC proliferation and the morphological changes of the midgut during larval development. Additionally, we examined how these changes affected the intestinal epithelium response to Cry1Ac toxin from Bacillus thuringiensis. Histological and in vitro evidence supported two distinct …

Effects Of N-Acetylcysteine Amide In Preventing/Treating Cataracts, Sri Krishna Yasaswi Maddirala

Doctoral Dissertations

"Cataract, the opacification of an eye lens, is a common pathological abnormality of the lens accounting for approximately 50% of all blindness. The only effective treatment currently available for a cataract is the surgical removal of the affected lens and replacement with an artificial lens for the restoration of vision. Although, cataract surgery is considered to be a very successful procedure in terms of visual outcome, the cost of surgery, need for trained personnel and surgeons, and postsurgical complications, limit the worldwide availability and accessibility of this procedure. Hence, alternative preventive and treatment procedures are worthy of investigation. The lens …

Constitutive Activity In Orphan G Protein Coupled Receptors, Adam Lee Martin

Doctoral Dissertations

"The goal of this research was to use a distal signaling pathway analysis to evaluate the extent of agonist independent constitutive signaling among orphan class-A G protein coupled receptors (GPCRs). These receptors translate extracellular signals via conformational change into intracellular activation of different G proteins and subsequent second messenger synthesis. These small molecules regulate cellular biochemistry, eventually leading to nuclear signaling that results in changes in gene expression. Some GPCRs are capable of signaling in the absence of an activating ligand, a phenomenon called constitutive activity that is inhibited via an "inverse-agonist". The use of cAMP dependent Luciferase expression is …

Pore Selectivity And Gating Of Arabidopsis Nodulin 26 Intrinsic Proteins And Roles In Boric Acid Transport In Reproductive Growth, Tian Li

Doctoral Dissertations

Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional channels of uncharged metabolites and water. They share the same canonical hourglass fold as the aquaporin family. The aromatic arginine (ar/R) selectivity filter controls transport selectivity based on size, hydrophobicity, and hydrogen bonding with substrates. In Arabidopsis thaliana, NIP II subclass proteins contain a conserved ar/R “pore signature” that is composed of Alanine at the helix 2 position (H2), Valine/Isoleucine at the helix 5 position (H5), and an Alanine (LE1) and an invariant Arginine (LE2) at the two loop E positions. In this study, …

Comparative Genomics Of Microbial Chemoreceptor Sequence, Structure, And Function, Aaron Daniel Fleetwood

Doctoral Dissertations

Microbial chemotaxis receptors (chemoreceptors) are complex proteins that sense the external environment and signal for flagella-mediated motility, serving as the GPS of the cell. In order to sense a myriad of physicochemical signals and adapt to diverse environmental niches, sensory regions of chemoreceptors are frenetically duplicated, mutated, or lost. Conversely, the chemoreceptor signaling region is a highly conserved protein domain. Extreme conservation of this domain is necessary because it determines very specific helical secondary, tertiary, and quaternary structures of the protein while simultaneously choreographing a network of interactions with the adaptor protein CheW and the histidine kinase CheA. This dichotomous …

Nanoparticle Building Blocks For Functional Structures, Youngdo Jeong

Doctoral Dissertations

A major goal in material science is achieving a desired function using structures fabricated with designed building blocks. Advanced synthetic and self-assembly techniques allow various nanomaterials to become promising building blocks, providing the control of the interaction between building blocks. The unique properties of nanomaterials can be transferred to structured systems. Among nanomaterials, inorganic nanoparticles such as gold nanoparticles (AuNPs), magnetic particles, and quantum dots (QDs) provide useful physical properties stemming from their inorganic core, large surface areas, and oriented surface functionalities. My research has focused on fabricating functional systems using gold nanoparticles (AuNPs), manipulating the interaction between AuNPs, bio-entities, …

Protein Behavior Directed By Heparin Charge And Chain Length, Burcu Baykal Minsky

Doctoral Dissertations

Glycosaminoglycans (GAGs), highly charged biological polyelectrolytes, are of growing importance as biomaterials and pharmaceutical drugs due to their immense range of physiological functions. They bind to many proteins; however, the degree of structural selectivity in GAG-protein interactions is largely unknown .Our studies have focused on the importance of heparin (a model GAG) charge and chain length in protein binding in order to explore its potential applications in biofunctional tissue scaffold materials, as polysaccharide drugs in anticoagulation, and as inhibitory agents in protein aggregation. We used electrospray ionization mass spectrometry, capillary electrophoresis, size exclusion chromatography, dynamic/static light scattering and electrostatic protein …

Structural Biology And Pharmacology Of Human Cathepsin A And Neuraminidase 1, Nilima Kolli

Doctoral Dissertations

Human cathepsin A (also known as Protective Protein/Cathepsin A, PPCA; E.C. 3.4.16.5) is a lysosomal serine carboxypeptidase. Cathepsin A is also involved in a complex with two other lysosomal enzymes: lysosomal neuraminidase (NEU1, E.C. 3.2.1.18) and β-galactosidase (GLB1, E.C. 3.2.1.23). Deficiency in cathepsin A and NEU1 result in the lysosomal storage diseases, galactosialidosis and sialidosis respectively. Deficiency in GLB1 results in G_M1 gangliosidosis and Morquio B diseases. Cathepsin A protease activity is spatially regulated by activation of the inactive precursor form to the mature form in the lysosome. Structural studies on the mature form of cathepsin A were performed …

Engineering Probes To Detect Cholesterol Accessibility On Membranes Using Perfringolysin O, Benjamin B. Johnson

Doctoral Dissertations

Cholesterol is an essential component of mammalian cell membranes and it is important to regulate the structure and function of lipid bilayers. Changes in cholesterol levels are involved in many physiological and pathological events such as the formation of arterial plaques, viral entry into cells, sperm capacitation, and receptor organization. Determination of cholesterol trafficking and distribution is essential for understanding how cells regulate cholesterol. A key factor in the regulation of cholesterol is cholesterol accessibility. Through it interactions in the membrane, cholesterol is sequestered below the surface of the membrane. Based on the composition of the membrane, a certain amount …

Ligand-Receptor Interactions For Supramolecular Disassembly With Applications In Screening And Drug Delivery, Diego Amado Torres

Doctoral Dissertations

Proteins have the capacity to bind specific sets of compounds known as ligands, these are small molecules with a recurrent theme in their molecular design that is a characteristic exploited here to (i) identify particular affinities of small molecules for proteins with the aim of using them as ligands, inhibitors, or targeting moieties in more complex systems by means of a methodology that screens small molecules based on protein affinity; (ii) decorate a self-assembling supramolecular system at different positions, making it responsive to a complementary protein with the aim of exploring differences in disassembly and sensitivity of the release of …

Applications And Improvements In The Molecular Modeling Of Protein And Ligand Interactions, Jason Bret Harris

Doctoral Dissertations

Understanding protein and ligand interactions is fundamental to treat disease and avoid toxicity in biological organisms. Molecular modeling is a helpful but imperfect tool used in computer-aided toxicology and drug discovery. In this work, molecular docking and structural informatics have been integrated with other modeling methods and physical experiments to better understand and improve predictions for protein and ligand interactions. Results presented as part of this research include:

1.) an application of single-protein docking for an intermediate state structure, specifically, modeling an intermediate state structure of alpha-1-antitrypsin and using the resulting model to virtually screen for chemical inhibitors that can …

Structure, Function And Regulation Of Two Isoforms Of Glutamine Synthetase From Soybean Root Nodules, Pintu Daulatrao Masalkar

Doctoral Dissertations

Glutamine synthetase (GS) is a major ammonia assimilatory enzyme in soybean nodules. The four isoforms of cytosolic glutamine synthetase (GS₁[glutamine synthetase 1]β[beta]1, GS₁β2, GS₁γ[gamma]1 and GS₁γ2) present in soybean nodules are 80% identical with respect to amino acid sequence, and share similar kinetic properties. It is shown all major GS₁ isoforms interact with nodulin 26, a member of the aquaporin family of membrane channels. Nodulin 26 is the major protein component of the symbiosome membrane (SM), where it serves a function as an ammonia and water channel. The site of interaction …

Toward Direct Biosynthesis Of Drop-In Ready Biofuels In Plants: Rapid Screening And Functional Genomic Characterization Of Plant-Derived Advanced Biofuels And Implications For Coproduction In Lignocellulosic Feedstocks, Blake Lee Joyce

Doctoral Dissertations

Advanced biofuels that are “drop-in” ready, completely fungible with petroleum fuels, and require minimal infrastructure to process a finished fuel could provide transportation fuels in rural or developing areas. Five oils extracted from Pittosporum resiniferum, Copaifera reticulata, and surrogate oils for Cymbopogon flexuosus, C. martinii, and Dictamnus albus in B20 blends were sent for ASTM International biodiesel testing and run in homogenous charge combustion ignition engines to determine combustion properties and emissions. All oils tested lowered cloud point. Oils derived from Copaifera reticulata also lowered indicated specific fuel consumption and had emissions similar to the ultra-low sulfur diesel control. Characterization …

Chromatin Insulators: Master Regulators Of The Eukaryotic Genome, Todd Andrew Schoborg

Doctoral Dissertations

Proper organization of the chromatin fiber within the three dimensional space of the eukaryotic nucleus relies on a number of DNA elements and their interacting proteins whose structural and functional consequences exert significant influence on genome behavior. Chromatin insulators are one such example, where it is thought that these elements assist in the formation of higher order chromatin loop structures by mediating long-range contacts between distant sites scattered throughout the genome. Such looping serves a dual role, helping to satisfy both the physical constraints needed to package the linear DNA polymer within the small volume of the nucleus while simultaneously …

Functional Analysis Of Corazonin And Its Receptor In Drosophila Melanogaster, Kai Sha

Doctoral Dissertations

Corazonin (Crz) is an amidated undecapeptide originally isolated from the American cockroach. It has been shown to affect diverse physiological functions in a species-specific manner. However, the functionality of Crz in Drosophila melanogaster has not yet been determined. To gain insight into the role of Crz signaling in vivo, Crz and CrzR null alleles were obtained by transposable element mobilization. Flies carrying a deficiency uncovering Crz and pr-set7 loci were generated via P-element excision, and the latter was rescued by wild-type pr-set7 transgene. A mutation of Crz receptor (CrzR) was generated by Minos-element mobilization from …

The N-Terminus Of The Saccharomyces Cerevisiae G Protein-Coupled Receptor Ste2p: Formation Of Dimer Interfaces And Negative Regulation, Mohammad Seraj Uddin

Doctoral Dissertations

G protein-coupled receptors (GPCRs), the largest family of membrane proteins on the cell surface, play essential roles in signal transduction in all eukaryotic organisms. These proteins are responsible for sensing and detecting a wide range of extracellular stimuli and translating them to intracellular responses. This signaling requires a tight control for receptor activation without which abnormal signal leads to diseases. In fact, malfunctions of these receptors are associated with numerous pathological conditions and currently an estimated 40-50% of therapeutic drugs are designed to target these receptors suggesting that further increases in understanding of GPCRs and the signaling pathways they initiate …

Understanding The Origins Of Product Specificity Of Protein Methyltransferases From Qm/Mm Md And Free Energy Simulations, Yuzhuo Chu

Doctoral Dissertations

Protein lysine methyltransferases (PKMTs) catalyze the methylation of certain lysine residues on histone tails using S-adenosyl-L-methionine (AdoMet) as the methyl donor. Regulation of chromatin structure and gene expression through histone lysine methylation depends on the degree of methylation. Therefore, it is of importance to understand the features of PKMTs that control how many methyl groups would be added to the target lysine (product specificity). In my dissertation, I have applied quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations to understand the origin of the product specificities of SET8, GLP and their mutants. The relative free energy barriers …

Role Of Retinoids In The Regulation Of Hepatic Glucose And Lipid Metabolism, Rui Li

Doctoral Dissertations

The liver plays an important role in controlling glucose and lipid homeostasis. Metabolic abnormalities such as obesity and type 2 diabetes are often associated with profound changes in the expression of genes involved in hepatic glucose and lipid metabolism. Dietary nutrients provide us with macronutrients for energy and micronutrients for maintenance of general health. However, the effects of individual micronutrients on the development of metabolic diseases are unknown. Sterol regulatory element binding protein-1c (SREBP-1c) is the master regulator of fatty acid synthesis, and glucokinase (GK) is the key enzyme in glucose metabolism. Based on the preliminary results from our laboratory …

Quorum Sensing And Metabolism In Marine Environments, Amanda May

Doctoral Dissertations

Quorum sensing (QS) is a phenomenon that allows bacteria to communicate with each other. Small molecules known as autoinducers are synthesized and released by bacteria, and once enough members of the community are around to ensure survival, i.e. quorum, a phenotype, e.g. bioluminescence, is expressed. There are two types of QS molecules, intra- and inter-species.

S-4,5-Dihydroxy-2,3-pentanedione (DPD) is a byproduct of the activated methyl cycle which recycles methionine. This has led to the discussion as to whether DPD is a metabolic byproduct or is the interspecies signal as proposed previously. The detection and quantitation of DPD however, has not …

Experimental And Computational Analysis Of Chloroplast Transit Peptide Domain Architecture And Function, Prakitchai Chotewutmontri

Doctoral Dissertations

The Majority of chloroplast proteins are nuclear-encoded and utilize an N-terminal transit peptide (TP) to target into chloroplasts via the general import pathway. Bioinformatic and proteomic analyses provide thousands of predicted TPs, which show low sequence similarity. How the common chloroplast translocon components recognize these diverse TPs is not well understood. Previous results support either sequence- or physicochemical-specific recognitions. To further address this question, a reverse sequence approach was utilized such that the reverse TP contains the same amino acid composition as wild-type TP but lack similar sequence motifs. Using both native and reverse TPs of the two well-studied precursors, …

Soybean Nodulin 26: A Channel For Water And Ammonia At The Symbiotic Interface Of Legumes And Nitrogen-Fixing Rhizobia Bacteria, Jin Ha Hwang

Doctoral Dissertations

During the infection and nodulation of legume roots by soil bacteria of the Rhizobiaceae family, the invading endosymbiont becomes enclosed within a specialized nitrogen-fixing organelle known as the "symbiosome". In mature nodules the host infected cells are occupied by thousands of symbiosomes, which constitute the major organelle within this specialized cell type. The symbiosome membrane is the outer boundary of this organelle which controls the transport of metabolites between the symbiont and the plant host. These transport activities include the efflux of the primary metabolic product of nitrogen fixation and the uptake of dicarboxylates as an energy source to support …

Development And Application Of Mass Spectrometry-Based Proteomics To Generate And Navigate The Proteomes Of The Genus Populus, Paul Edward Abraham

Doctoral Dissertations

Historically, there has been tremendous synergy between biology and analytical technology, such that one drives the development of the other. Over the past two decades, their interrelatedness has catalyzed entirely new experimental approaches and unlocked new types of biological questions, as exemplified by the advancements of the field of mass spectrometry (MS)-based proteomics. MS-based proteomics, which provides a more complete measurement of all the proteins in a cell, has revolutionized a variety of scientific fields, ranging from characterizing proteins expressed by a microorganism to tracking cancer-related biomarkers. Though MS technology has advanced significantly, the analysis of complicated proteomes, such as …

Multiscale Modeling Of Enzyme-Catalyzed Methanol Production By Particulate Methane Monooxygenase, Katherine K. Bearden

Doctoral Dissertations

In this work, the conversion of methane to methanol by the particulate Methane Monooxygenase (pMMO) enzyme is investigated using a multi-scale modeling approach. This enzyme participates in carbon cycling and aids in the removal of harmful atmospheric methane, converting it to methanol. The interaction between pMMO and a neighboring enzyme that is present in the same organism is studied, and the unknown pMMO active site is elucidated and tested for methane oxidation towards the production of methanol.

Fundamental knowledge of pMMO's mechanism is not fully understood. Understanding how this enzyme works in nature will provide information towards designing efficient synthetic …

The Contribution Of Oxidative Stress In The Protein Damage And Dna Lesion In Alzheimer's Disease Neuropathology, Cheng Zhang

Doctoral Dissertations

Glutathione (GSH) plays an essential role in the intracellular antioxidant defense against the oxidant radicals, especially the ·OH radical. To understand the early and progressive cellular changes in Alzheimer's disease (AD) development, we investigated reduced glutathione/oxidized glutathione (GSH/GSSG) status in a double mutated AD transgenic mouse model (B6.Cg-Tg), which carries Swedish amyloid precursor protein mutation (APPswe) and exon 9 deletion of the PSEN1 gene. Likewise, S-glutathionylation (Pr-SSG) is a specific post-translational modification (PTM) of cysteine residues by the addition of glutathione. S-glutathionylated proteins induced by oxidative stress play an essential role in understanding the pathogenesis of the aging …

Synthesis, Characterization And Self-Assembly Of Amphiphilic Block Copolymers, Xiaojun Wang

Doctoral Dissertations

This dissertation presents a review on state-of-the-art research of well-defined charged block copolymers, including synthesis, characterization, bulk morphology and self-assembly in aqueous solution of amphiphilic block polyelectrolytes. In Chapter 1, as a general introduction, experimental observations and theoretical calculations devoted towards understanding morphological behavior in charged block copolymer systems are reviewed along with some of the new emerging research directions. Further investigation of charged systems is urged in order to fully understand their morphological behavior and to directly target structures for the tremendous potential in technological applications. Following this background, in Chapters 2, 3, 4 and 5 are presented the …

Development And Application Of Liquid Chromatography-Tandem Mass Spectrometry Methods To The Understanding Of Metabolism And Cell-Cell Signaling In Several Biological Systems, Jessica Renee Gooding

Doctoral Dissertations

Liquid chromatography tandem mass spectrometry has become a powerful tool for investigating biological systems. Herein we describe the development of both isotope dilution mass spectrometry methods and targeted metabolomics methods for the study of metabolic and cell-cell signaling applications.

A putative yeast enzyme was characterized by discovery metabolite profiling, kinetic flux profiling, transcriptomics and structural biology. These experiments demonstrated that the enzyme shb17 was a sedoheptulose bisphosphatase that provides a thermodynamically dedicated step towards riboneogenesis, leading to the redefinition of the canonical pentose phosphate pathway.

An extension of metabolic profiling and kinetic flux profiling methods was developed for a set …