Biochemistry, Biophysics, and Structural Biology Commons^™

Utilizing In Silico And/Or Native Esi Approaches To Provide New Insights On Haptoglobin/Globin And Haptoglobin/Receptor Interactions, Ololade Fatunmbi

Doctoral Dissertations

Haptoglobin (Hp), an acute phase protein, binds free hemoglobin (Hb) dimers in one of the strongest non-covalent interactions known in biology. This interaction protects Hb from causing potentially severe oxidative damage and limiting nitric oxide bioavailability. Once Hb/Hp complexes are formed, they proceed to bind CD163, a cell surface receptor on macrophages leading to complex internalization and catabolism. Myoglobin, (Mb) a monomeric protein, that is normally found in the muscle but can be released into the blood in high concentrations during myocardial injury, is homologous to Hb and shares many conserved Hb/Hp interface residues. Both monomeric Hb and Mb species …

Biophysical Characterization Of Katanin’S Regulation Of Microtubules, Megan E. Bailey

Doctoral Dissertations

Microtubules, as an essential part of the cytoskeleton, require proper function as well as correct spatial and temporal localization. In order to achieve correct organization, microtubule-associated proteins (MAPs) regulate microtubule dynamics. Katanin, a known microtubule-severing enzyme from the AAA family of proteins, plays a role in regulating microtubules, but the mechanisms of microtubule control and the mechanism of severing activity remain to be elucidated. In the following studies I examine mechanisms of katanin-based regulation of microtubule dynamics using a single molecule biophysics approach. I use this simplified in vitro approach to change specific parameters to investigate how katanin targets microtubules …

New Insights Into The Role Of The Udp-Glucose: Glycoprotein Glucosyltransferase 1 In The Endoplasmic Reticulum Quality Control, Abla Tannous

Doctoral Dissertations

The UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a central quality control factor in the Endoplasmic Reticulum (ER). It surveys the folding status of proteins in the ER and redirects them, via its reglucosylation activity, to bind to the ER carbohydrate binding (lectin) chaperones calreticulin (CRT) and calnexin (CNX). However, the cellular mechanism of UGT1 is not completely understood. Using a cell based reglucosylation assay, we found that UGT1 reglucosylated proteins that eventually fold. This modification was transient and resulted in delay of protein trafficking in the secretory pathway and prolonged binding to lectin chaperones in the ER. In addition, terminally misfolded …

Responsive Supramolecular Assemblies Based On Amphiphilic Polymers And Hybrid Materials, Longyu Li

Doctoral Dissertations

The design and synthesis of responsive supramolecular assemblies are of great interest due to their applications in a variety of areas such as drug delivery and sensing. We have developed a facile method to prepare self-crosslinking disulfide-based nanogels derived from an amphiphilic random copolymer containing a hydrophilic oligo-(ethylene glycol)-based side-chain functionality and a hydrophobic pyridyl disulfide functional group. This thesis first provides a concept of studying the influence of Hofmeister ions on the size and guest encapsulation stability of a polymeric nanogel. The size and core density of nanogel can be fine-tuned through the addition of both chaotropes and kosmotropes …

Chemical Biology-Based Probes For The Labeling Of Targets On Live Cells, Amanda M. Hussey

Doctoral Dissertations

Proper detection is the key to studying any processes on the cellular scale. Nowhere is this more evident than in the tight space which confines the synaptic cleft. Being able to ascertain the location of receptors on live neurons is fundamental to our understanding of not only how these receptors interact and move inside the cell but also how neurons function. Most detection methods rely on significantly altering the receptor; both tagging with a fluorescent protein or targeting the receptor by a fluorescent reporter in the form of a small molecule causes significant difficulties. These localization techniques often result in …

Molecular Mechanisms Underlying The Contralateral Repeated Bout Effect (Crbe) In Human Skeletal Muscle, Ling Xin

Doctoral Dissertations

Eccentric (muscle lengthening) exercise induces temporary muscle damage that can lead to long-term muscle adaptation, a process known as the repeated bout effect where subsequent exercise results in less damage. The existence of a contralateral repeated bout effect (CRBE) has been controversial. The primary goals of this study were to: 1) validate the existence of the CRBE; and 2) define the underlying molecular mechanisms. Thirty-six young men performed 100 maximal eccentric actions of the knee extensors using one leg (bout 1) and repeated the exercise with the contralateral leg five weeks later (bout 2). Vastus lateralis muscle biopsies were …

Design And Synthesis Of Polymeric Nanoparticles For Drug And Protein Delivery, Judy A. Ventura

Doctoral Dissertations

Nanoparticles are emerging as carriers in biological applications due to advances in their preparation, size control, surface modification and encapsulation capabilities. In addition, nanomaterials improve bioavailability by enhancing aqueous solubility of the guest molecule and increasing resistance time in the body. However, the delivery of guest molecules is still challenging due to the intrinsic characteristics of the guest molecule including large size and propensity to denature or degradation in the case of biomolecules and the encapsulation stability of the small guest molecules. Our group recently reported the preparation of self-cross-linked polymeric nanogels possessing surface functionalization capabilities. In this dissertation we …

Inhibition And Cofactor Targeting Of Hypoxia-Sensing Proteins, Cornelius Y. Taabazuing

Doctoral Dissertations

Hypoxia Inducible Factor (HIF) is a transcription activator considered to be the main regulator of O₂ homeostasis in humans. The transcriptional ability of HIF is regulated by the Fe²⁺/αKG-dependent enzyme, Factor Inhibiting HIF (FIH). FIH uses molecular oxygen to catalyze hydroxylation of an asparagine residue (Asn⁸⁰³) in the C-terminal transactivation domain (CTAD) of the HIFα subunit, abrogating HIF target gene expression. The mechanism of FIH and other αKG-dependent oxygenases involves the ordered sequential binding of αKG, substrate, and O₂, which becomes activated to form a reactive ferryl intermediate that hydroxylates the substrate. The …

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, …

Elucidation Of Conformational Switching Mechanisms Of Sensing Proteins By Molecular Dynamics Perturbation Studies, Quentin Ramon Johnson

Doctoral Dissertations

Sensing proteins are a subclass of switchable proteins, which are biologically designed with multiple, purposeful, low-free-energy states and can interconvert between these states in the wake of some environmental perturbation. Note, that this phenomenon is no small feat. This is a preprogrammed response for regulatory purposes that requires no cognitive action and is reversible as the environment returns to normal. Sensing proteins often switch between active and non-active states, closed and open conformations or other particular dichotomous states. Therefore, understanding the mechanism by which these proteins sense a specific perturbation and how they switch between conformations is paramount. Addressing these …

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 …

Exploring Structure-Dynamics-Function Relationship In Proteins, Protein: Ligand And Protein: Protein Systems Through Computational Methods, Karan Pal Kapoor

Doctoral Dissertations

The study focuses on understanding the dynamic nature of interactions between molecules and macromolecules. Molecular modeling and simulation technologies are employed to understand how the chemical constitution of the protein, specific interactions and dynamics of its structure provide the basis of its mechanism of function. The structure-dynamics-function relationship is investigated from quantum to macromolecular-assembly level, with applications in the field of rationale drug discovery and in improving efficiency of renewable sources of energy. Results presented include investigating the role of dynamics in the following:

1) In interactions between molecules: analyzing dynamic nature of a specific non-covalent interaction known as “anion-π …

Development Of High Performance Molecular Dynamics With Application To Multimillion-Atom Biomass Simulations, Roland Schulz

Doctoral Dissertations

An understanding of the recalcitrance of plant biomass is important for efficient economic production of biofuel. Lignins are hydrophobic, branched polymers and form a residual barrier to effective hydrolysis of lignocellulosic biomass. Understanding lignin's structure, dynamics and its interaction and binding to cellulose will help with finding more efficient ways to reduce its contribution to the recalcitrance. Molecular dynamics (MD) using the GROMACS software is employed to study these properties in atomic detail. Studying complex, realistic models of pretreated plant cell walls, requires simulations significantly larger than was possible before. The most challenging part of such large simulations is the …

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 …

Evolutionary Dynamics Of Speciation And Extinction, Dawn Michelle King

Doctoral Dissertations

Presented here is an interdisciplinary study that draws connections between the fields of physics, mathematics, and evolutionary biology. Importantly, as we move through the Anthropocene Epoch, where human-driven climate change threatens biodiversity, understanding how an evolving population responds to extinction stress could be key to saving endangered ecosystems. With a neutral, agent-based model that incorporates the main principles of Darwinian evolution, such as heritability, variability, and competition, the dynamics of speciation and extinction is investigated. The simulated organisms evolve according to the reaction-diffusion rules of the 2D directed percolation universality class. Offspring are generated according to one of three reproduction …