Molecular Biology Commons^™

Combining Simulation And The Mspa Nanopore To Study P53 Dynamics And Interactions, Samantha A. Schultz

Masters Theses

p53 is a transcription factor and an important tumor suppressor protein that becomes activated due to DNA damage. Because of its role as a tumor suppressor, mutations in the gene that encodes it are found in over 50% of human cancers. The N-terminal transactivation domain (NTAD) of p53 is intrinsically disordered and modulates the function and interactions of p53 in the cell. Its disordered structure allows it to be controlled closely by post-translation modifications that regulate p53’s ability to bind DNA and interact with regulatory binding partners. p53 is an attractive target for developing cancer therapeutics, but its intrinsically disordered …

Exploring Topological Phonons In Different Length Scales: Microtubules And Acoustic Metamaterials, Ssu-Ying Chen

Dissertations

The topological concepts of electronic states have been extended to phononic systems, leading to the prediction of topological phonons in a variety of materials. These phonons play a crucial role in determining material properties such as thermal conductivity, thermoelectricity, superconductivity, and specific heat. The objective of this dissertation is to investigate the role of topological phonons at different length scales.

Firstly, the acoustic resonator properties of tubulin proteins, which form microtubules, will be explored The microtubule has been proposed as an analog of a topological phononic insulator due to its unique properties. One key characteristic of topological materials is the …

Small Gtpase Regulated Intracellular Protein Trafficking In Endothelium, Caitlin Francis

Electronic Theses and Dissertations

Intracellular protein trafficking is the movement of membrane-bound organelles to and from requisite locations within the cell. Small GTPases are a critical component to the spatiotemporal accuracy of intracellular trafficking pathways as they determine the specificity and direction of organelle transport. There exists over 150 small GTPases categorized into 5 sub-families and are employed across all cell types. Despite their universal expression and relevance to cellular function, small GTPases remain incompletely understood across tissue types. In various instances, the trafficking pathway of a particular Rab in one cell type may belong to a completely disparate pathway in another cell type. …

Probing Amyloid-Beta Protein Structure And Dynamics With A Selective Antibody, Shikha Grover

Dissertations

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The AD brain is characterized by significant neuronal loss and accumulation of insoluble fibrillar amyloid-β protein (Aβ) plaques and tau protein neurofibrillary tangles in the brain. However, over the last decade, many studies have shown that the neurodegenerative effect of Aβ may in fact be caused by various soluble oligomeric forms as opposed to the insoluble fibrils. Furthermore, the data suggest that a pre-fibrillar aggregated form, termed protofibrils, mediates direct neurotoxicity, and triggers a robust neuroinflammatory response.

Antibodies targeting the various conformation of Aβ are important therapeutic agents to prevent the progression …

A Novel Transmembrane Ligand Inhibits T Cell Receptor Activation, Yujie Ye

Doctoral Dissertations

T lymphocytes (T cells) play essential roles in the adaptive immune system. Each mature T cell expresses one type of functional T cell receptor (TCR). The TCR recognizes antigens bound to the major histocompatibility complex (MHC) in antigen presenting cells. The resulting stimulation signal crosses the transmembrane domain of TCR and initiates downstream signaling cascades. The human immune system relies on TCRs to recognize a variety of pathogens. Normally, TCR can distinguish the self-antigens from pathogenic antigens. However, dysfunction or aberrant expression of TCRs causes different inflammatory and autoimmune diseases, which afflict millions of people annually (Chapter I). Current treatments …

Comparative Analysis Of Conformational Transition Pathways In Homologous Proteins, Dylan Sebastien Ogden

Graduate Theses and Dissertations

Molecular dynamics (MD) simulations are routinely used to study the dynamics of proteins. However, conventional MD limited to the sampling of local conformational changes as the functionally important conformational transitions of proteins often extend beyond the timescales of the simulations employed, for example, membrane transport proteins. We have determined the combination of multiple MD based techniques that allows for a rigorous characterization of energetics and kinetics of large-scale conformational changes in membrane proteins. The methodology is based on biased, nonequilibrium collective variable based simulations including nonequilibrium pulling, string method with swarms of trajectories, bias-exchange umbrella sampling, and rate estimation techniques. …

Cell Division Dynamics Of Escherichia Coli In Extreme Environments, Steven P. Murray

Graduate Theses and Dissertations

Life is remarkable in how resilient it can be. Many organism, classified as ex- tremophiles, can not only survive in extreme environments, but they can thrive in them. In the search for extraterrestrial life, the best candidates to harbor life exist with some kind of extreme condition. Europa, for example, is a favorite for the possibility of accommodating life as we know it within our solar system. Thought there is believed to be a liquid ocean under its icy surface, this habitat would be under immense pressures and high salinity. To best know where to look for extraterrestrial life, it …

Controlling Myosin’S Function Via Interactions Between The Substrate And The Active Site, Mike K. Woodward

Doctoral Dissertations

Molecular motors, such as myosin, have evolved to transduce chemical energy from ATP into mechanical work to drive essential cellular processes, from muscle contraction to vesicular transport. Dysfunction in these motors is a root cause of many pathologies necessitating the application of intrinsic control over molecular motor function. We hypothesized that altering the myosin’s energy substrate via minor positional changes to the triphosphate portion of the molecule will allow us to control the protein and affect its in vitro function. We utilized positional isomers of a synthetic non-nucleoside triphosphate, azobenzene triphosphate, and assessed whether myosin’s force- and motion-generating capacity could …

Improving The Ribozyme Toolbox: From Structure-Function Insights To Synthetic Biology Applications, Jessica Michelle Roberts

Boise State University Theses and Dissertations

Self-cleaving ribozymes are a naturally occurring class of catalytically active RNA molecules which cleave their own phosphate backbone. In nature, self-cleaving ribozymes are best known for their role in processing concatamers of viral genomes into monomers during viral replication in some RNA viruses, but to a lesser degree have also been implicated in mRNA regulation and processing in bacteria and eukaryotes. In addition to their biological relevance, these RNA enzymes have been harnessed as important biomolecular tools with a variety of applications in fields such as bioengineering. Self-cleaving ribozymes are relatively small and easy to generate in the lab using …

Modeling Electrostatics In Molecular Biology And Its Relevance With Molecular Mechanisms Of Diseases, Mahesh Koirala

All Dissertations

Electrostatics plays an essential role in molecular biology. Modeling electrostatics in molecular biology is complicated due to the water phase, mobile ions, and irregularly shaped inhomogeneous biological macromolecules. This dissertation presents the popular DelPhi package that solves PBE and delivers the electrostatic potential distribution of biomolecules. We used the newly developed DelPhiForce steered Molecular Dynamics (DFMD) approach to model the binding of barstar to barnase and demonstrated that the first-principles method could also model the binding. This dissertation also reflects the use of existing computational approaches to model the effects of Single Amino Acid Variations (SAVs) to reveal molecular mechanisms …

Investigating Structures And Functions Of Apoptotic Caspases, Ishankumar V. Soni

Doctoral Dissertations

Caspases are cysteine aspartate proteases involved in various cellular pathways including apoptosis, inflammation, and neurodegeneration. Caspase-9 is classified as an initiator apoptotic caspase that is activated upon intrinsic stress. Caspase-9 is composed of two domains: an N- terminal CARD domain and a catalytic core domain. We have employed hydrogen deuterium exchange mass spectrometry (H/DX-MS) to determine the 1) dynamics of the full-length caspase- 9, 2) dynamic impacts on caspase-9 upon substrate-induced dimerization, and 3) regions involved in the CARD: catalytic core domains interactions. Upon intrinsic stress, caspase-9 activates executioners, procaspase-3 and -7 but not procaspase-6. We have employed site-directed mutagenesis …

Supertertiary Structural Dynamics Modulate Function In Postsynaptic Density Protein 95, George L. Hamilton Iii

All Dissertations

Proteins, RNA, and DNA serve as the primary sub-cellular machinery that give rise to the necessary functions of life. The long-standing paradigm has been that the structures of biomolecules, or the arrangement of the subunits that make up a biomolecule, determine biological function. However, biomolecules are not static objects. Instead, they often undergo structural rearrangements that are crucial to enabling and regulating their functions. In my thesis I present several studies of the interplay between the structures, dynamics, and functions of biomolecules that combine experimental fluorescence spectroscopy and computational methods to probe these systems at the single-molecule level. In particular, …

Eukaryotic Initiation Factor 4e (Eif4e) In Complex With Eif4e Binding Protein 1 (4e-Bp1) Binds With Higher Affinity To M7gpppn Cap Of A Subset Of Human Mrnas, Izza F. Nawaz

Theses and Dissertations

Fluorescence anisotropy binding assays were used to analyze the binding of eIF4E in complex with 4E-BP1 onto the 5’ m7G cap of a subset of mRNA that are known to carry cap-independent translation. These studies suggest that 4E-BP1 increases eIF4E binding affinity to 5’cap of both FGF-9 and HIF-1𝝰.

Quantitative Imaging Of Tensile Forces At Cell-Cell Junction With Dna-Based Probes, Puspam Keshri

Doctoral Dissertations

Mechanical forces are an integral part in biology, they regulate several cellular properties, such as morphology, proliferation, migration. These forces are also involved in receptor signaling and the differentiation of different cell types. Different proteins and biomolecules such as cadherin, integrin, notch proteins are essential elements of these processes. Measuring these intercellular forces are challenging considering the minimal intensity (piconewton-level) of these molecular forces. In our lab, we have developed a membrane DNA tension probe (MDTP) that uses a DNA hairpin module to sense tensile forces and has a lipid anchor to modify onto live-cell membranes. The programmability of DNA …

Paddling Along The Voltage Gated Sodium Channel Galaxy With Sea Anemone Toxins: Structural Studies Of The Interaction Between The Paddle Motif From Nav1.5div And Sea Anemone Toxin, Adel K. Hussein

Dissertations, Theses, and Capstone Projects

Voltage gated sodium channels (VGSC) are membrane proteins that serve an important function in the central nervous system (CNS), peripheral nervous system (PNS), and cardiac muscles amongst others. The main function of VGSC is in the propagation of electrical signals by depolarizing excitable cells. Nine mammalian VGSC subtypes have been characterized, Na_V1.1 – Na_V1.9, that are expressed in a tissue specific manner, each with unique gating properties. Numerous diseases have been linked to defects in VGSC including epilepsy, mental retardation, long QT syndrome, and Brugada disease. Furthermore, these channels are one of the primary targets of …

The Role Of Charge On Dna Packaging And Integrity Within Reconstituted Peptide-Dna Assemblies, Ehigbai Oikeh

Theses and Dissertations--Chemistry

In nature, DNA exists primarily in a highly compacted form. The compaction of DNA in vivo is mediated by cationic proteins; histone in somatic nuclei and arginine-rich peptides called protamines in sperm chromatin. The packaging in the sperm nucleus is significantly higher than somatic nuclei resulting in a final volume roughly 1/20^th that of a somatic nucleus. This tight packaging results in a near crystalline packaging of the DNA helices. While the dense packaging of DNA in sperm nuclei is considered essential for both efficient genetic delivery as well as DNA protection against damage by mutagens and oxidative species, …

Elucidating The Proteasomal Regulatory Mechanism Of Proteasome Activator Pa28Γ /Regγ, Taylor Ann Thomas

Graduate Theses, Dissertations, and Problem Reports

Virtually all cellular processes are precisely regulated by the proteasome which is the primary enzyme responsible for the degradation of misfolded, damaged, or no longer necessary soluble proteins. To prevent any untimely degradation of these target protein substrates and protect the cell, the proteasome is tightly regulated via adaptor proteins, known as proteasomal regulators. There are many classes of proteasomal regulators each with their own unique structures, functions, and effects on protein degradation through the proteasome. One such class is the 11S family of proteasomal regulators which are also referred to as PA26/28, or REG. The 11S family are ATP-independent …

Intrinsically Disordered Protein Regions In Human Evolution And As Therapeutic Targets, Karen Paco Mendivil

KGI Theses and Dissertations

Intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) fail to form stable structures but have important biological functions via interacting with various molecular partners (proteins, DNA, RNA, glycosaminoglycans). We hypothesized that IDPRs are potential targets for therapeutics development because they are reservoirs of evolutionary innovation, and they play crucial roles in adaptation to pathogens.

We first studied the evolution of IDPRs in the human proteome and compared it with the proteome of non-human primates. We have found that evolutionary young protein-coding genes have included low conserved regions in the N-terminal part of proteins, and such regions are linked to high …

A Review Of Calcineurin Biophysics With Implications For Cardiac Physiology, Ryan B. Williams

Theses and Dissertations

Calmodulin is a prevalent calcium sensing protein found in all cells. Three genes exist for calmodulin and all three of these genes encode for the exact same protein sequence. Recently mutations in the amino acid sequence of calmodulin have been identified in living human patients. Thus far, patients harboring these mutations in the calmodulin sequence have only displayed an altered cardiac related phenotype. Calcineurin is involved in many key physiological processes and its activity is regulated by calcium and calmodulin. In order to assess whether or not calcineurin contributes to calmodulinopathy (a pathological state arising from dysfunctional calmodulin), a comprehensive …

Granulins In Norm And Neurodegenerative Pathologies, Anukool Bhopatkar

Dissertations

Granulins (GRNs) are small, cysteine-rich modules produced from the proteolytic cleavage of the precursor protein called progranulin (PGRN). GRNs are present in the form of seven tandem repeats within the precursor and are known to be produced in the extracellular and in lysosomal environments. In physiology, PGRN and GRNs plays pleiotropic roles such as neuronal growth and differentiation, immunomodulation, wound healing. Recent studies have implicated pathological role for PGRN in Alzheimer disease (AD) and frontotemporal dementia (FTD) but specific mechanism(s) remains unclear. However, potential interactions between GRNs and Ab42 and TDP-43 seem like a plausible underlying mechanism. Studies presented here …

Liposomes: Production And Applications For Controlled Drug Delivery, Gamid Abatchev

Boise State University Theses and Dissertations

This dissertation recognizes the enormous potential presented by the ever-evolving development of liposomes as drug carriers and seeks to offer further investigation into their useful production and utilization. The first chapter presents the basic principles governing their formation by self-assembly in water solutions, briefly describes the most common production methods, and points out essential past advances that led to their use as drug carriers. Chapter two exemplifies production of liposomes by the traditional methods of extrusion and sonication, detailing passive and active loading, as well as physical characterization by Dynamic Light Scattering, microscopy imaging, and fluorescence spectroscopy. In the next …

Engineering Fluorescently Labeled Human Fibroblast Growth Factor One Mutants And Characterizing Their Photophysics Properties Towards Designing Fret Assays, Mamello Mohale

Graduate Theses and Dissertations

Human fibroblast growth factor one (hFGF1) belongs to a family of 22 FGF members produced by fibroblast cells. Cell signaling during physiological processes of angiogenesis and wound healing occurs when hFGF1 binds to its receptor (FGFR). However, when heterogenous homeostasis is not maintained, fibroblast cells exhibit excessive proliferation which can lead to a myriad of cancers. smFRET is an ultrasensitive distant dependent (1-10 nm) technique capable of resolving such heterogeneity in structural dynamics and binding affinities (Kd). Therefore, we successfully designed and characterized fluorescently labeled hFGF1 tracers which span the visible light region of the electromagnetic spectrum for use in …

Understanding How Camkii Holoenzyme Dynamics Facilities Activation-Triggered Subunit Exchange, Ana P. Torres-Ocampo

Doctoral Dissertations

Long-term memory and learning are still poorly understood from a molecular and cellular standpoint. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric kinase that is involved in this remarkable process. However, the molecular details of its specific roles in these processes remains elusive. CaMKII activation-triggered subunit exchange presents a novel possible mechanism involved in long-term memory and learning by exchanging active subunits with other CaMKIIs. CaMKII subunit exchange also shows that exchanged CaMKIIs spread their phosphorylation state to newly synthesized CaMKIIs. This provides a long-lasting signal that might possibly be involved in long-term memory by escaping a cell’s …

Dual Control Of One Component Signaling: Mechanistic And Structural Insights Into El222 Active States, Uthama Phani R. Edupuganti

Dissertations, Theses, and Capstone Projects

Photoreceptors play a crucial role in signal transduction as specialized proteins which sense light as environmental stimuli and transduce the signal to control of downstream functions. Here we focus our attention on one class of these proteins, the Light-Oxygen-Voltage (LOV) domain, which is sensitive to blue light via an internally-bound flavin chromophore. Since their initial discovery in plant phototropins, many details of their photochemistry, chromophore interactions, and use with a diverse set of functional effectors have been described. However, several key details, especially a comprehensive understanding of signaling mechanism and its regulation, still remain elusive due in part to the …

The Structural And Functional Role Of Photosensing In Rgs-Lov Proteins, Zaynab Jaber

Dissertations, Theses, and Capstone Projects

Light provides organisms with energy and spatiotemporal information. To survive and adapt, organisms have developed the ability to sense light to drive biochemical effects that underlie vision, entrainment of circadian rhythm, stress response, virulence, and many other important molecularly driven responses. Blue-light sensing Light-Oxygen-Voltage (LOV) domains are ubiquitous across multiple kingdoms of life and modulate various physiological events via diverse effector domains. Using a small molecule flavin chromophore, the LOV domain undergoes light-dependent structural changes leading to activation or repression of these catalytic and non-catalytic effectors. In silico analyses of high-throughput genomic sequencing data has led to the marked expansion …

Modulation Of Protein Dynamics By Ligand Binding And Solvent Composition, Richard J. Lindsay

Doctoral Dissertations

Many proteins undergo conformational switching in order to perform their cellular functions. A multitude of factors may shift the energy landscape and alter protein dynamics with varying effects on the conformations they explore. We apply atomistic molecular dynamics simulations to a variety of biomolecular systems in order to investigate how factors such as pressure, the chemical environment, and ligand binding at distant binding pockets affect the structure and dynamics of these protein systems. Further, we examine how such changes should be characterized. We first investigate how pressure and solvent modulate ligand access to the active site of a bacterial lipase …

Characterizing Multivalent Interactions Between Folded Protein Domains And Intrinsically Disordered Regions Or Peptide Substrates, Tongyin Zheng

Dissertations - ALL

Protein-protein interactions (PPIs) play central roles in most biological processes. Studying PPIs is a fundamental step in understanding the molecular basis of cellular processes such as cell-cell contact, enzyme activity, and transient assembly of signaling complexes or cellular structures. In this work, we employed a combination of biophysical and biochemical methods to characterize PPIs, with a focus on interactions between structured domains and intrinsically disordered regions or peptide substrates.The subject of Chapter two is prolyl isomerase Ess1, which is an essential enzyme found in Saccharomyces cerevisiae. Ess1 regulates the transcription and co-transcriptional RNA processing by catalyzing the isomerization of serine-proline …

Novel Approaches Towards Improved Purity In High Yield Transcription Reactions, Elvan Cavac

Doctoral Dissertations

High yields of RNA (e.g., mRNA, gRNA, lncRNA) are routinely prepared following a two-step approach: high yield in vitro transcription using T7 RNA polymerase, followed by extensive purification using gel or chromatic methods. In high yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer than desired, (partially) double stranded impurities. Current purification methods often fail to fully eliminate these impurities which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. This study establishes …

Abhd5 Induced Morphological Changes On Model Membrane Systems, Nasser S. Junedi

Honors College Theses

Proper regulation of neutral lipid storage (lipogenesis) and release (lipolysis) are critical molecular processes localized to an organelle called the Lipid Droplet (LD). The LD consists of a core with neutral lipids such as triacylglycerols (TAGs) and sterol esters surrounded by a phospholipid monolayer. Dysregulation of the processes localized to the LD are involved in the pathology of various diseases such as Neutral Lipid Storage Disease, diabetes, stroke and cancer. The non-enzymatic protein ABHD5 (α-β Hydrolase Domain-Containing Protein 5), is thought to play a key role in the process of lipolysis by forming homo-oligomers on the surface of the LD …

Lions, Tigers, And Hemes - Oh My! A Dynamic Look At The Electronic Effects Of Porphyrin Substitution On Cytochrome P450 Olet, Alexis J. Holwerda

Senior Theses

OleT, a member of the CYP152 family of cytochrome P450s (CYPs), decarboxylates fatty acids using hydrogen peroxide as an oxidant. The resultant products are a terminal alkene and carbon dioxide. This C–C cleavage reaction is highly atypical for CYPs, which prototypically oxygenate substrates, and provides a potential means to enzymatically produce drop-in fuels. OleT contains a heme-iron cofactor that facilitates decarboxylation through the activation of hydrogen peroxide. The catalytic cycle, as determined by transient kinetics, includes two ferryl intermediates known as Compound I (Ole-I) and Compound II (Ole-II). Ole-I performs substrate hydrogen abstraction and subsequent single electron transfer to Ole-II …