Biochemistry, Biophysics, and Structural Biology Commons^™

Dissecting The Molecular Mechanism Of Familial Cardiomyopathies, Sarah Ruth Clippinger Schulte

Arts & Sciences Electronic Theses and Dissertations

Familial cardiomyopathies, including hypertrophic (HCM), restrictive (RCM) and dilated cardiomyopathy (DCM), are the leading cause of sudden cardiac death in young people. These diseases, which are characterized by altered cardiac contractility and remodeling of the heart, can lead to heart failure. These diseases are primarily caused by point mutations in sarcomeric proteins that generate or regulate heart contraction, such as troponin T. In the heart, the troponin complex together with tropomyosin lie along the actin filament and regulate myosin’s ability to bind actin and produce force. Here I show how mutations in troponin T affect contractility at the molecular level …

The Effects Of Molecular Chaperone Modulation On Protein Folding, Prion Formation, And Prion Propagation In Saccharomyces Cerevisiae, Leeran Blythe Dublin Ryan

Arts & Sciences Electronic Theses and Dissertations

Proper and efficient protein folding is vital for cell survival. Many factors affect protein folding fidelity and prion formation, including molecular chaperone availability and activity. Research has shown that modulating chaperone availability and function can affect protein misfolding and aggregation, as well as de novo prion formation and propagation. However, the factors involved and underlying mechanisms influencing prion formation and protein folding are largely unknown. The following work aims to elucidate these areas. The Nascent Polypeptide-Associated Complex (NAC) is the first point of chaperone contact for nascent polypeptides. Previous work has shown that disruption of the NAC leads to improved …

Computational Methods For Analysis Of Data For Conformational And Phase Equilibria Of Disordered Proteins, Jared Michael M Lalmansingh

Arts & Sciences Electronic Theses and Dissertations

Intrinsically disordered proteins and regions (IDPs / IDRs) are a class of proteins with diverse conformational heterogeneity that do not fold into a tertiary structure due to the lack of a native structural state. Consequently, disordered proteins are remarkably flexible and exhibit multivalent properties that enable them to adopt myriad functional roles within the cell such as: signaling transduction, transcription, enzymatic catalysis, translation, and many more. Due to their multivalency, some IDPs undergo monomeric and heterotypic interactions which can drive phase separation. Such IDPs can form membraneless organelles with specific regulatory roles within the cell which include, but are not …

Slo2.1 Channels: A New Molecular Mechanism To Regulate Uterine Excitability, Juan Jose Ferreira

Arts & Sciences Electronic Theses and Dissertations

At the end of pregnancy, the uterus transitions from a non-contractile state to a highly contractile state. Two processes primarily drive this transition. First, from the 28th week of pregnancy until labor, the resting membrane potential of uterine (myometrial) smooth muscle cells (MSMCs) gradually becomes more positive (depolarizes) (Parkington et al. 1999). Second, at the end of pregnancy, MSMCs express more oxytocin receptors and become more sensitive to oxytocin (Kimura et al. 1996). However, the detailed mechanisms by which these processes occur have not been determined. My central hypothesis was that the Na+-activated K+ channel SLO2.1 plays a key role …

On The Challenges And Rewards Of Analyzing Molecular Dynamics At The Terabyte And Millisecond Scale, Justin Roy Porter

Arts & Sciences Electronic Theses and Dissertations

Molecular Dynamics (MD) and Markov state models (MSMs) are powerful tools for estimating and concisely representing the conformational ensemble accessible to biological macromolecules, particularly proteins. Conformational ensembles are of special importance biological function, both in health and disease, because biology derives from molecules’ entire conformational distribution rather than any single structure. Consequently, MD is poised to become a powerful tool for personalized medicine and for the study of molecular sequence-function relationships generally. However, because of their hyperdimensionality and size, just generating MD datasets and Markov state models (MSMs) that represent biologically relevant molecules is a substantive technical challenge. Then, even …

Regulatory Effects Of The E. Coli Recbcd Nuclease Domain On Dna Unwinding Kinetics, Nicole Fazio

Arts & Sciences Electronic Theses and Dissertations

I have examined the effects of deleting the nuclease domain of the E. coli helicase RecBCD on the rates of ATP-independent DNA melting, single stranded (ss) DNA translocation, and double stranded (ds) DNA unwinding by RecBCD. The canonical role of the nuclease domain is DNA degradation, but the removal of this domain showed unexpected effects on other RecBCD activities including DNA binding, melting, and unwinding. This thesis presents a mechanistic study of DNA unwinding by RecBCD and a RecBCD variant with the nuclease domain deleted (RecBΔnucCD). I examined the rates of ssDNA translocation and dsDNA unwinding by RecBCD and RecBΔnucCD …

C. Elegans Response To Cadmium Toxicity, Brian James Earley

Arts & Sciences Electronic Theses and Dissertations

Cadmium is an environmental pollutant and significant health hazard that is similar to the physiological metal zinc. Residing in the same group of the periodic table, cadmium and zinc share chemical characteristics that are important for their industrial uses in electroplating, batteries, pigments, and metal alloys. The similarities of ionic cadmium and zinc have significant repercussions on biological systems. While it has long been clear that cadmium is toxic to biological systems, the mechanisms of cadmium toxicity remain poorly understood. In contrast, mechanisms of zinc homeostasis have been elucidated in growing detail. In C. elegans high zinc homeostasis is regulated …

Method Development For Enhancing Sensitivity Of Dynamic Nuclear Polarization Nuclear Magnetic Resonance Spectroscopy For Structural Studies Of Pkc-Drug Interactions, Patrick Terrence Judge

Arts & Sciences Electronic Theses and Dissertations

To perform the most relevant structural studies on biological systems, experiments need to be carried out when the target proteins are in their endogenous cellular environment. Nuclear magnetic resonance (NMR) is well-suited to probe the structure and dynamics of a wide variety of systems, including biologically relevant proteins. However, NMR suffers from an inherent lack of sensitivity. Dynamic nuclear polarization (DNP) NMR is a powerful technique that is used to enhance NMR sensitivity by transferring the greater polarization of exogenously doped electron spins to nuclear spins of interest though the use of a high-power microwave source. Solid effect radicals offer …

Chemical Damage To Mrna And Its Impact On Ribosome Quality-Control And Stress-Response Pathways In Eukaryotic Cells, Liewei Yan

Arts & Sciences Electronic Theses and Dissertations

Ribosome often faces defective adducts that disrupt its movement along the mRNA template. These adducts are primarily caused by chemical damage to mRNA and are highly detrimental to the decoding process on the ribosome. Hence, unless dealt with, chemical damage to RNA has been hypothesized to lead to the production of toxic protein products. Even more detrimental is the ability of damaged mRNA to drastically affect ribosome homeostasis through stalling. This in turn would lead to greatly diminished translation capacity of cells. Therefore, the inability of cells to recognize and resolve translational-stalling events is detrimental to proteostasis and could even …

The Role Of Excited States In Determining Β-Lactamase Function And Bacterial Fitness, Catherine Rae Knoverek

Arts & Sciences Electronic Theses and Dissertations

Proteins are macromolecular machines that play a role in nearly every biological process. They are dynamic molecules which adopt many different conformations as they fold into their 3D structures, interact with their binding partners, and perform their functions. The most probable (lowest energy) protein conformation is referred to as the ground state, and this is often assumed to be the state determined by experimental methods such as x-ray crystallography. However, proteins also adopt higher energy excited states which can have significant probabilities. As these excited states are notoriously difficult to find and study, it is unclear if excited states contribute …

Rna Polymerase Binding Protein A (Rbpa) Regulation Of Mycobacteria Transcription And Sensitivity To Fidaxomicin, Jerome Prusa

Arts & Sciences Electronic Theses and Dissertations

Mycobacterium tuberculosis is the causative agent of the disease tuberculosis (TB) and remains one of the deadliest microorganisms on the planet. The effort to eradicate M. tuberculosis would benefit from the development of novel therapeutics, which requires a detailed understanding of M. tuberculosis physiology. Like all living organisms, M. tuberculosis gene expression requires transcription. Transcription in the phylum Actinobacteria, which includes mycobacteria, is unique because it includes RNA Polymerase Binding Protein A (RbpA) that is essential in both M. tuberculosis and the nonpathogenic model organism Mycobacterium smegmatis. RbpA increases the housekeeping A and housekeeping like B interactions with the RNA …

Copper-Mediated Regulation Of A Traditional Iron Uptake System In Uropathogenic Escherichia Coli., George Lwanga Katumba

Arts & Sciences Electronic Theses and Dissertations

Transition metals constitute an important part of the host-pathogen interface. Iron is an essential nutrient that functions as a cofactor for numerous bacterial and host proteins, as either a ligand for oxygen in carrier proteins or an enzyme catalytic site due to its natural redox properties. As part of the innate immune response, infected hosts sequester iron from pathogens to limit their growth, a phenomenon known as nutritional immunity. On the other hand, copper ions are deployed at infection sites as a potent antimicrobial agent to kill bacteria. The ability to survive within multiple, often harsh, microenvironments is fundamental to …

The Regulation Of Plasmodium Falciparum Metabolism By Haloacid Dehalogenase Proteins, Philip Frasse

Arts & Sciences Electronic Theses and Dissertations

Malaria is an enormous financial and public health burden for much of the world, infecting over 200 million and killing over 400,000 people every year. While much progress has been made combating malaria in the past few decades, those advances have slowed in recent years, partially due to the emergence of resistance to all known antimalarials used to date. To achieve the goal of eliminating malaria as a major global health problem, new therapeutics need to be developed, targeting novel categories of parasite biology. One poorly understood area of parasite biology is the regulation of various metabolic pathways. We have …

Potentiation Of Tmem16a Currents By Clca1 In Cystic Fibrosis Airway, Kayla Berry

Arts & Sciences Electronic Theses and Dissertations

In the airway, proper activity of the anion channel cystic fibrosis transmembrane conductance regulator (CFTR) contributes to innate immune defense by maintaining a hydratedand alkaline mucus layer through the conductance of chloride and bicarbonate ions. This allows potentially pathogenic microorganisms to be trapped, quickly killed, and cleared via mucociliary clearance, thus preventing microbial colonization of the lungs. In cystic fibrosis (CF), this activity is impaired, resulting in repeated pulmonary infections that damage the lung and, if severe and prolonged, may lead to premature death without lung transplantation. Available therapies remain focused on targeted rescue of the CFTR mutation. However, given …

Etv2/Myct1 Axis In The Regulation Of Tumor Angiogenesis And Anti-Tumor Immunity, Ashraf Ul Kabir

Arts & Sciences Electronic Theses and Dissertations

Angiogenesis is a critical determinant of neoplastic growth and metastatic spread. As such, anti-angiogenic approaches have long been tried to throttle down tumor progression. However, current anti-angiogenic treatments so far have produced modest clinical benefits. Further in-depth research has provided rationales behind these disappointing and apparent perplexing clinical outcomes. It is now established that VEGF (vascular endothelial growth factor) and other prominent current angiogenic targets are neither specific to the vascular system nor the pathological conditions explaining the sub-optimal angiogenic control following the existing treatments. This suggests that anti-angiogenesis could still be a viable strategy for cancer patients should there …

Deep Learning Deleterious Small Molecule Biochemistry From The Lab To The Clinic, Matthew Matlock

Arts & Sciences Electronic Theses and Dissertations

Small molecules are key tools in biology and medicine. In biology, small molecules are used to probe biological systems and gain insight into their structure and function. In medicine, this role is further refined to reverse the biological conditions that contribute to human disease. Developing new small molecules into biological probes or drugs can be a daunting scientific task. These projects often begin with many thousands of potential candidates, which are progressively screened and eliminated from consideration by high-throughput experimental assays. Those molecules that emerge from this process as candidate drugs are also subject to extensive in vitro, in vivo, …

Single-Cell Resolution Mechanistic Analyses Of Direct Lineage Reprogramming, Chuner Guo

Arts & Sciences Electronic Theses and Dissertations

End-stage organ failures remain a clinical challenge with an unmet need for medical therapies, with transplantation often being the only curative option. Despite advances in transplantation outcomes, organ shortage continues to limit the availability of cures to patients in need. The direct lineage reprogramming of one cell type to another is a promising avenue for therapy with the following advantages: (1) patient-specific cell sources, (2) direct conversion without reverting to pluripotency and the associated risk of teratoma formation, and (3) utilization of the cell type responsible for fibrotic scar formation for the engineering towards the desired cell fate. Nonetheless, many …

Rhoa Mediated Juxtacrine Regulation Of Glucagon Secretion, Yong Hee Chung

Arts & Sciences Electronic Theses and Dissertations

Glucagon is secreted from pancreatic islet alpha-cells in response to hypoglycemia. The regulation of this secretion likely involves multiple interacting molecular pathways. There are three general types of proposed models for glucose-dependent regulation of glucagon secretion: direct regulation by glucose mediated modulation of cell electrophysiology, paracrine regulation by other endocrine cell types within the islets of Langerhans, and juxtacrine regulation by surface protein interactions from neighboring beta-cells. This work is focused on one pathway of juxtacrine regulation that occurs through signaling from EphA4 receptors on the surface of α-cells upon interaction with ephrin-A5 ligands on the surface of beta-cells. In …

The Role Of Cd53 In Hematopoietic Development, Stress, And Malignancy, Zev Joshua Greenberg

Arts & Sciences Electronic Theses and Dissertations

For a cell to function properly, it must be able to interact with and respond to environmental cues; however, expression of surface molecules, proteins, and receptors is not always sufficient to execute a cellular response. Proper organization of the plasma membrane is necessary to facilitate these highly regulated protein interactions, such that a cell can respond to stressors, growth factors, and other signaling molecules. Tetraspanins are a family of transmembrane proteins which help correctly orient surface molecules on the cell membrane, often through tetraspanin enriched microdomains, a membrane structure similar to lipid rafts. As a family, tetraspanins are known to …

The Role Of Subclonal Gene Mutations During Progression From Myelodysplastic Syndrome To Secondary Acute Myeloid Leukemia, Andrew John Menssen

Arts & Sciences Electronic Theses and Dissertations

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal bone marrow disorders characterized by ineffective hematopoiesis. Approximately 30% of MDS patients progress to secondary acute myeloid leukemia (AML). MDS is caused by somatic mutations in hematopoietic stem/progenitor cells and progression to secondary AML is associated with the acquisition and/or expansion of at least one subclone. We hypothesized that specific gene mutations would be enriched in subclones compared to founding clones, and that the order of mutation acquisition would be critical for clonal evolution and progression from MDS to secondary AML. Sequencing of paired MDS and secondary AML samples from 44 …

Mechanisms Of Natural Killer Cell Anti-Tumor Function And Homeostasis, Julia Alexandra Wagner

Arts & Sciences Electronic Theses and Dissertations

Natural killer (NK) cells are innate lymphoid cells (ILCs) that mediate anti-tumor and anti-viral immune responses. They do so via two primary effector functions: cytokine production and direct cytotoxicity. Unlike adaptive T and B lymphocytes, NK cells do not rearrange their DNA to express a predominant antigen-specific receptor, and instead express a variety of stochastically-expressed, germline DNA-encoded activating and inhibitory receptors whose signals integrate to govern their functional responses. What results is a diverse repertoire of NK cells capable of recognizing a variety of malignantly-transformed or virally-infected cells. Studies from several groups have established the anti-tumor potential of NK cells, …

The Role Of Enampt-Mediated Systemic Nad+ Biosynthesis In Aging And Lifespan, Mitsukuni Yoshida

Arts & Sciences Electronic Theses and Dissertations

Aging is a systemic decline in tissue functions that occurs during the course of our life leading to the development of age-associated health deteriorations and ultimately to the expiration of life. Over the past few decades, population aging has become increasingly evident in many parts of the world, leading to increased prevalence of age-related disabilities and diseases. Because age is a major risk factor for many chronic diseases, extensive research effort has been spent to identify common age-associated molecular changes contributing to the pathogenesis and progression of these health issues. One such molecule identified to play a central role in …

Protein Synthesis Adaptation To The Au-Rich Transcriptome Of Plasmodium Falciparum, Jessey Lee Erath

Arts & Sciences Electronic Theses and Dissertations

The process of protein synthesis whereby a messenger RNA is decoded into an amino acid chainis conserved among the domains. Fastidious protein synthesis is necessary for organism survival. However, exceptions negatively affecting the mRNA translation cycle – inadvertently or by design – may occur. Polyadenosine tracts are one such motif causing ribosomal stalling and frameshifting in almost all organisms tested thus far; save Plasmodium spp. Thus, with ~60% of their protein-coding genome harboring polyadenosine tracts, the elucidation of such paradigm-breaking adaptations enabling Plasmodium spp. to translate this typically problematic motif without issue is salient from both basic science and clinical …

Prodrug Activation In Staphylococci And The Implications For Antimicrobial Development, Justin J. Miller

Arts & Sciences Electronic Theses and Dissertations

Antibiotic resistance is an increasing concern for global health care, with some estimates suggesting that 10 million people will die from antibiotic resistant infections in the year 2050. Fueling this prospect, few antimicrobials are being actively developed and recently commercial entities have fled from the development of new anti-infectives. New antimicrobials and drug development strategies are urgently needed to revitalize this critical pipeline. While many putative antibiotics demonstrate promising in vitro potency, they routinely fail in vivo due to poor drug-like properties (e.g. oral bioavailability, serum-half life, toxicity) resulting in overly expensive drug development pipelines. Fortunately, drug-like properties can be …

Molecular Strategies To Overcome Antibiotic Resistance, Luting Fang

Arts & Sciences Electronic Theses and Dissertations

Antibiotics have greatly reduced the number of deaths caused by infectious diseases for the last 70 years, but as a result of overuse, antimicrobial resistance emerged. Resistance is the major reason why most traditional antibiotics currently have reduced clinical efficacy. However, there is still a drive to overcome this resistance in a variety of ways. There are two main avenues of research to overcome resistance; to discover and develop new drug candidates acting on new targets and pathways, and to determine the mechanisms of antibiotic resistance in order to combat it through multi-drug therapies, bringing back the effectiveness of antibiotics …

Thermodynamics And Conformational Heterogeneity Of Recbcd Binding To Dna Ends, Linxuan Hao

Arts & Sciences Electronic Theses and Dissertations

E. coli RecBCD is crucial in initiating repair of double stranded (ds) DNA breaks. It is a heterotrimeric helicase and nuclease complex possessing two ATPase motors, RecB and RecD, and a regulatory subunit without ATPase activity, RecC. The RecB subunit also contains a 30kDa nuclease domain (RecBNuc) that, according to published structural data, is situated over 60Å away from the site of dsDNA binding. Surprisingly, we have shown in previous studies that deletion of RecBNuc to form RecBΔNucCD affects its dsDNA unwinding properties. The mechanism by which RecBNuc influences RecBCD dsDNA unwinding is unclear. In this thesis, equilibrium binding techniques, …

Understanding The Molecular Mechanisms Of Photoferrotrophy And Phototrophic Extracellular Electron Uptake, Dinesh Gupta

Arts & Sciences Electronic Theses and Dissertations

Several anoxygenic phototrophs grow by utilizing soluble iron or insoluble mixed-valence iron minerals (such as rust) as electron donors to fix carbon dioxide using light energy, a process called photoferrotrophy. Photoferrotrophs can also use electron donors such as poised electrodes that serve as proxies for rust via phototrophic extracellular electron uptake (EEU). Despite the recognition that these two related microbial processes contribute to various biogeochemical cycles such as iron and carbon, the electron uptake mechanisms underlying photoferrotrophy and phototrophic EEU are poorly understood. To address the key knowledge gaps in our understanding of these microbial metabolisms, here we characterized Rhodopseudomonas …

The Role Of Mature Secretory Cells In Gastrointestinal Regeneration, Megan Deanna Radyk

Arts & Sciences Electronic Theses and Dissertations

Differentiated cells exhibit the ability to adjust their cell fate and become more progenitor-like after wide-scale tissue injury. This inherent cell plasticity is shown across many tissues and organisms and is a conserved behavior that ensures organ function even in a chronic injury setting. At the tissue level, the change in cell fate from a differentiated cell to one with more progenitor properties can be identified as metaplasia. Importantly, metaplasias, like Spasmolytic Polypeptide-Expressing Metaplasia (SPEM) in the stomach and Acinar-to-Ductal Metaplasia (ADM) in the pancreas, are risk factors for the development of adenocarcinoma. Thus, understanding the cellular and molecular mechanisms …

Homeostatic T Cell Receptor Interactions With Self-Peptide Tune Cd4+ T Cell Function, Juliet Marie Bartleson

Arts & Sciences Electronic Theses and Dissertations

Homeostatic T Cell Receptor Interactions with Self-Peptide Tune CD4+ T Cell Function

by

Juliet Marie Bartleson

Doctor of Philosophy in Biology and Biomedical Sciences

Immunology

Washington University in St. Louis, 2021

Professor Paul M. Allen, Chair

Mature CD4+ T cells circulate throughout peripheral secondary lymphoid organs using their T cell receptor (TCR) to surveil peptide presented on major histocompatibility complex class II molecules (pMHC) in search of cognate, antigenic peptide. In the absence of an immune challenge, however, the TCR is continuously interacting with self-pMHC, which induces a relatively weak TCR signal known as tonic signaling. These homeostatic TCR:self-pMHC interactions …

Understanding And Exploiting Protein Allostery And Dynamics Using Molecular Simulations, Sukrit Singh

Arts & Sciences Electronic Theses and Dissertations

Protein conformational landscapes contain much of the functionally relevant information that is useful for understanding biological processes at the chemical scale. Understanding and mapping out these conformational landscapescan provide valuable insight into protein behaviors and biological phenomena, and has relevance to the process of therapeutic design.

While structural biology methods have been transformative in studying protein dynamics, they are limited by technicallimitations and have inherent resolution limits. Molecular dynamics (MD) simulations are a powerful tool for exploring conformational landscapes, and provide atomic-scale information that is useful in understanding protein behaviors. With recent advances in generating datasets of large timescale simulations …