Biochemistry, Biophysics, and Structural Biology Commons^™

Intrinsically Disordered Proteins And Their Role In Biomolecular Condensates, Danielle Latham

All Dissertations

Proteins are biomacromolecules responsible for the functions of life. While classically proteins are thought to be well structured in order to perform a specific function, 50% of proteins within Eukaryotic cells contain intrinsically disordered regions (IDRs), regions with no well-defined structure. IDRs are often used for cell signaling, responding to external factors such as temperature changes or the presence of small molecules. To understand how IDRs can function without structure, it is important to understand the dynamics of such systems. Understanding IDR intramolecular and intermolecular interactions will shed light on IDR dynamics. Intramolecular interactions are first explored using fluorescence spectroscopy …

Synthesis And Biophysical Analysis Of Modified Cell-Penetrating Peptides, Joel Mitchell

Theses and Dissertations (Comprehensive)

Cell-penetrating peptides (CPPs) are a family of peptides that have the ability to penetrate biological membranes. They were discovered in the late 1980s and have been the topic of many studies. Much of the interest in CPPs has been due to their ability to translocate biological membranes, and the possibility that they could offer a novel drug delivery method by conjugation to biologically active molecules. Linear CPPs can be modified to form cyclic structures. This change in structure has been observed to enhance the stability and penetrative ability of the CPPs which have been studied. The current thesis focuses on …

Binding Interactions Of Biologically Relevant Molecules Studied Using Surface-Modified And Nanostructured Surfaces, Palak Sondhi

Dissertations

This research focuses on the field of surface nanobioscience, wherein different nanosurfaces that will be used as working electrodes in the electrochemical cell are manufactured and surface modified to understand the critical binding interactions between biologically significant molecules like proteins, carbohydrates, small drug molecules, and glycoproteins. This research is essential if we are to determine whether a synthetic molecule can serve as a therapeutic candidate or diagnose a disease in its early stages. In order to fully understand the binding interactions, the study begins with defining some of the fundamental concepts, principles, and analytical tools for biosensing.

Afterwards, we addressed …

Structural Integrity And Stability Of Dna In Ionic Liquid And Near-Infrared Indolizine Squaraine Dye, Ember Yeji Suh

Honors Theses

Luminol, the most common presumptive test for blood at a crime scene, has multiple issues, such as false positive results with chemical agents, no luminescence due to “active oxygen” cleaning agents on bloodstains, and inability to penetrate textile materials. A combination of indolizine squaraine dye and ionic liquid (IL), or Dye Enhanced Textile Emission for Crime Tracking (DETECT), have shown potential to address these issues. The purpose of this study was to assess the binding mechanism of CG (1:1) and SO₃SQ dye to HSA and how the mechanism can explain the W214 fluorescence quenching effect and to determine …

Novel Mechanistic Insight Into Ciliary Regulation: Old Pathways Yield New Mechanisms, Larissa L. Dougherty

Dartmouth College Ph.D Dissertations

Cilia are structures present on most eukaryotic cells which provide important signaling and motile components to cells from early development to fully differentiated and matured cells. Regulation of these structures is critical to proper functioning of the cell and is known to be tied to the cell cycle. Preparation for ciliary assembly following cell cycle exit and ciliary disassembly following cell cycle reentry requires components throughout the cell body and within the cilium to facilitate this process. Here I identify how the cell adapts to ensure modifications to cilia occur for assembly or disassembly using the model organism Chlamydomonas reinhardtii. …

Mechanism Of Sila- And Germafluorenes For Biological Applications, Shelby Jarrett

Dissertations

2,7-disubstituted silafluorenes and germafluorenes, originally designed for OLED applications, are a class of fluorescent dyes that have gained recent interest as probes for bioimaging and as biosensors to monitor cellular dynamics and interactions. Desirable biological probes absorb in the visible region, have high extinction coefficients, high quantum yield and excellent photostability. Here, their spectral properties are investigated under aqueous conditions for relevant biological applications. These molecules display intense blue fluorescence in the solid state and in solution, have high extinction coefficients, and exhibit appreciable solubility in aqueous solution. To better understand potential applications, the mechanism of fluorescence was investigated. It …

Investigation Of The Binding Domain Interfaces Of The C-Terminus Of The Albino3 Insertase And The 43kda Chloroplast Signal Recognition Particle Subunit Via Single Molecule Förster Resonance Energy Transfer, Amanda Tomanek

Chemistry & Biochemistry Undergraduate Honors Theses

Fluorescent labeling is a technique used for visualizing functional groups contained in biomolecules by fluorescence imaging. This technique was used in this project to analyze post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCP), which are the core complexes that harvest sunlight to drive photosynthetic electron transfer. This protein is synthesized in the cytosol and post-translationally targeted to the stroma of chloroplasts. CpSRP43 is a signal recognition particle (SRP) subunit unique to chloroplasts, which has been shown to interact with the stroma-soluble C-terminus of the thylakoid-bound Albino3 insertase (Alb3-Cterm). In the chloroplast stroma, targeting to thylakoids is performed via the cpSRP pathway …

Investigating The Main Protease (Mpro) Of Sars-Cov-2 As A Potential Drug Target, Valerie Giovina Pascetta

Honors Theses and Capstones

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 19 (COVID-19) pandemic has claimed the lives of roughly 6.2 million people worldwide as of May 2022. The virus’s main protease (M^pro ) has been identified as an attractive drug target due to the critical role it plays in the viral life cycle. The roughly 34 kDa M^pro cleaves functional viral polypeptides out of two long polyproteins at conserved cut sites, allowing them to fulfill their role in processes like transcription and replication. Here, we have studied the enzymatic activity …

Investigation Of G Protein-Coupled Receptor Quaternary Structure Through Fluorescence Micro-Spectroscopy And Theoretical Modeling: Interdependence Between Receptor-Receptor And Receptor-Ligand Interactions, Joel David Paprocki

Theses and Dissertations

Proteins are of high interest in biophysics research due to the important roles they play within cells, such as sensing of chemical (ions and small molecules) and physical (e.g., light) stimuli, providing structure, transporting ions/molecules, signaling, and intercellular communication. The studies described in this dissertation focus on a particular type of membrane proteins known as G protein-coupled receptors (GPCR), which play a key role in cellular response to external stimuli. We used the sterile 2 α-factor mating pheromone receptor (Ste2), a prototypical class D GPCR present within Saccharomyces cerevisiae (baker’s yeast). Ste2 is responsible for initiating the second messenger signal …

Part I: Development Of Small-Molecule-Based Probes For The Vitamin D Receptor; Part Ii: Development Of A Scalable Manufacturing Process For Orcein Dye, Tania Roseann Mutchie

Theses and Dissertations

PART I:The vitamin D receptor (VDR) is a ligand-dependent transcription factor and member of the nuclear hormone receptor superfamily. VDR is expressed in the epithelia of endocrine organs, digestive system, bronchi, kidneys, and thymus, as well as being present in leukocytes and bone cells. Cell proliferation, cell differentiation, and immunomodulation, along with calcium and phosphate homeostasis, are all processes regulated by the receptor. Within the cell, VDR can be membrane-bound or located in the nucleus. Nuclear localization of VDR transpires following the binding of vitamin D metabolites, the most active of which is 1α,25-dihydroxyvitamin D3 (calcitriol). Within the nucleus, interactions …

Indolizine Donor-Based Dyes For Applications In Fluorescence Biological Imaging, William Meador

Honors Theses

NIR emissive fluorophores are intensely researched due to their potential to replace modern imaging procedures. Many molecular strategies have been employed in the literature to optimize fluorophores for deeper NIR absorption and emission, biocompatibility, and higher fluorescence quantum yields. Amongst the fluorophores studied to date, proaromatic indolizine donors are attractive alternatives to traditional alkyl amine and indoline based donors due to their 1) lower energy absorption and emission facilitated by proaromaticity, 2) large Stokes shifts due to increased dihedral angles about the π-system, 3) ease of functionalization and capacity for bioconjugation at the phenyl ring, and 4) potential for further …

On The Structure And Function Of Mitochondrial Uncoupling Proteins: The Case Of Ucp2, Afshan Ardalan

Theses and Dissertations (Comprehensive)

Uncoupling proteins (UCPs) are regulated proton transporters of the mitochondrial inner membrane. UCP-mediated proton leak negatively impacts the rate of ATP synthesis. Despite the importance of their physiological role(s) in certain tissues, molecular aspects of UCPs’ structure-function relationships are not fully understood. The current study explores the tertiary and quaternary structure of UCP2, as well as its proton transport mechanism in lipid membranes. The proteins were expressed in the E. coli inner membrane, purified and reconstituted into liposomes. Proteins were characterized by semi-native SDS-PAGE. Circular dichroism spectroscopy (CD) and fluorescence quenching assays were utilized to study the conformation of proteins …

Origins Of Amyloid Oligomers And Novel Approaches For Their Detection, Jeremy Barton

USF Tampa Graduate Theses and Dissertations

Alzheimer’s disease, type II diabetes, and other amyloid diseases are known to be associated with the formation of amyloid aggregates. It has been thoroughly researched whether amyloid fibrils or oligomers are the main culprit for these diseases, and recent evidence has connected oligomers as the most disease relevant aggregate species. However, many difficulties have arose in confirming this hypothesis. Techniques for oligomer detection are often limited in their sensitivity, and in many cases are unable to distinguish oligomers from rigid fibrils. Additionally, the role oligomer splay in fibril assembly is still unclear, and has led to the belief that different …

Investigatin Actin-Myosin Mechanics To Model Heart Disease Using Fluorescence Microscopy And Optical Trapping, Justin Edward Reynolds

Honors Theses

Hypertrophic cardiomyopathy (HCM) is a hereditary disease in which the myocardium becomes hypertrophied, making it more difficult for the heart to pump blood. HCM is commonly caused by a mutation in the β-cardiac myosin II heavy chain. Myosin is a motor protein that facilitates muscle contraction by converting chemical energy from ATP hydrolysis into mechanical work and concomitantly moving along actin filaments. Optical tweezers have been used previously to analyze single myosin biophysical properties; however, myosin does not work as a single unit within the heart. Multiple myosin interacts to displace actin filaments and do not have the same properties …

Novel Substrate-Free Cholinesterase Based Sensing Of Organophosphorus Compounds, Hailey J. Marini

Legacy Theses & Dissertations (2009 - 2024)

The increasing concerns of chemical weapons used by terrorists is growing, creating a need for infield detection methods that can rapidly detect these compounds that are a major health concern for civilians and military personnel. Displacement is a novel concept for the detection of cholinesterase inhibitors, including organophosphorus compounds, that could replace the classical instrumentation methods currently being used for diagnostics.

Autofluorescence To Study The Effects Of Acid Concentration On Cellular Metabolism In Vitro, Robin L. Raley

Chemistry & Biochemistry Undergraduate Honors Theses

Ultraviolet (UV) radiation-induced sunburns and their accompanying afflictions are a growing public health concern in the United States. There is a need for techniques that can accurately and non-invasively characterize the physiology of sunburned skin tissue directly after UV-damage and applying a topical skin treatment to relieve pain and promote healing. Two-photon excited fluorescence (TPEF) microscopy and fluorescence lifetime imaging (FLIM) can be used to investigate metabolic processes in live cells through endogenous fluorescence of the cofactors, NADH and FAD. These methods employ the optical redox ratio of FAD/(NADH+FAD), mean NADH lifetime, and the separation of the free and bound …

Effects Of Electrical Stimulation On Glioma Cells In Vitro With Implications For Treating Chronic Pain: Development Of A Model System, David C. Platt

Theses and Dissertations

Glial cells comprise over 70% of the central nervous system cells and exhibit diverse functions including regulation of synaptic transmission, neuron protection/repair, maintenance of neuronal metabolism, and are implicated in the development of persistent neuropathic pain. In addition, a perturbation in the concentration of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) has likewise been associated with the development of a chronic pain state. This perturbation in ROS/RNS creates an environment of oxidative stress. However, the mechanism by which the pain signal transmission is modulated, and the roles ROS play in the perpetuation of the pain state are …

Significance Of Rna 2'-5' Linkage And Metal-Ion Mediated Base Pairs, Fusheng Shen

Legacy Theses & Dissertations (2009 - 2024)

It has been known that the template-directed RNA chemical replication reaction produces mixture of backbones containing both 3’-5’ and 2’-5’ linkages. This backbone heterogeneity has been a significant problem in studying the emergence of RNA World from the prebiotic chemistry. However, very recently, it is reported that FMN binding aptamer and a hammerhead ribozyme are still able to retain considerable functions in the presence of certain 2’-5’ linkages, indicating that RNA backbones may be quite flexible and this backbone heterogeneity problem may not be as severe as originally thought. This finding also brings two related important questions: First, how does …

Cloning The Vision Related G Protein Transducin For Live Cell Fluorescence Studies, Deanna M. Bowman

Williams Honors College, Honors Research Projects

G coupled protein receptors (GCPR) are one of the largest families of receptors and mediate a variety of biological responses. Rhodopsin is the largest family and aids in sight, the α-subunit of the GCPR complex in extremely important to the activation and downstream signaling effects of GCPR. The α-subunit contains a small trans-domain portion and in this project the sequence of that portion will be inserted into a vector containing a fluorescent tag. These vectors will then be used to collect fluorescent cross correlation spectroscopy or FCCS data. The unit was cloned using assembly methods that include PCR and purification …

Harnessing The Physical Properties Of Zno Nanoparticles For Biological Applications And Factors That Impact Zno Nanoparticle Toxicity, Joshua Eixenberger

Boise State University Theses and Dissertations

The production of materials at the nanoscale leads to novel properties and has made the field of nanotechnology a part of everyday life. Numerous applications of nanomaterials have led to their use in electronics, optics, and medicine. However, creating materials at such a small size brings them on the same scale as many biomolecules and cellular components, altering their interactions with biological systems. This can lead to unintended biological impacts as many nanomaterials are considerably more toxic than their bulk counterpart material. ZnO nanoparticles (nZnO) are particularly interesting in this context. The FDA classifies ZnO as a generally recognized as …

Fluorescently Labeled Sirnas And Their Theranostic Applications In Cancer Gene Therapy, Stephen David Kozuch

Seton Hall University Dissertations and Theses (ETDs)

Gene therapy has emerged as a promising precision nano-medicine strategy in the treatment of numerous diseases including cancer. At the forefront of its utility are the applications of short-interfering RNA (siRNA), that silence oncogenic mRNA expression leading to cancer cell death through the RNA interference (RNAi) pathway. Despite the therapeutic potential, siRNAs are limited by poor pharmacological properties, which has hindered their translation into the clinic. Recent studies, however, have highlighted the applications of modified siRNAs, including the use of fluorescent probes and siRNA nanostructures in cancer detection and treatment. The siRNAs reported in this thesis are designed to target …

Fluorescent Probes And Functionalized Nanoparticles For Bioimaging: Synthesis, Photophysical Properties And Applications, Xinglei Liu

Dissertations

The development of new organic molecular probes with excellent photophysical properties and high fluorescence quantum yields is of considerable interest to many research areas including one- and two-photon fluorescence microscopy, fluorescence-based sensing methodologies, and cancer therapy. Series of organic linear-/non-linear optical molecules including squaraine derivatives, and fluorene derivatives as well as other bioconjugates are designed and synthesized during the doctoral study for the aim of ion detection (Chapter 5), photo dynamic therapy, and deep-tissue imaging (Chapter 4). These optical probes are capable of absorbing light in the near infrared (NIR) window and thus have deeper penetration and cause less photodamage …

Fret-Based Investigations Of The Structure-Function Relationships In The Nmda Receptor, Drew M. Dolino

Dissertations & Theses (Open Access)

The N-methyl-D-aspartate (NMDA) receptor is one member of a class of proteins known as the ionotropic glutamate receptors. Ionotropic glutamate receptors mediate the majority of excitatory neurotransmission in the central nervous system, with the NMDA receptor standing out among these receptors for its requirement of a co-agonist, its magnesium-block-based coincidence detection, its slow kinetics, its calcium permeability, its allosteric modulation, and its especially important functional roles in synaptic plasticity, excitotoxicity, and more. In recent years, a wealth of structural information has come about describing endpoint structures to high resolution, but such structures are unable to fully resolve the movements …

Using Fluorescence Lifetimes To Characterize Lipid Behavior In Nanodiscs, Cynthia Janku

Undergraduate Theses, Professional Papers, and Capstone Artifacts

Cellular uptake of molecules, including drugs, can be affected by the fluidity of the membrane. Nanoparticles have been hypothesized to alter membrane fluidity resulting in inflammation and its related clinical effects. Variations in phospholipids can alter membrane structure and its interaction with drugs or nanoparticles. To study membrane lipid differences and dynamics, we are using nanodiscs and liposomes as model systems. Nanodiscs are a lipid bilayer surrounded by a membrane scaffold protein, which is a derivative of Apolipoprotein A1, a protein involved in the removal of cholesterol from the body. There are important unresolved questions about how the belt protein …

Targeting Cancer: The Ph-Responsive Binding And Insertion Of Roxy7, Kristen Rae Booth

Chancellor’s Honors Program Projects

No abstract provided.

A Fret Investigation Into Molecular Mechanisms Of Cardiac Troponin Activation In Reconstituted Thin Filaments, Maria Eleni Moutsoglou

Electronic Theses and Dissertations

Cardiomyopathies (CM) are the leading cause of death in America, and can develop from mutations in sarcomeric proteins, leading to altered protein structure and function. Current therapies target upstream signaling pathways to treat the symptoms of heart failure, but are associated with increased mortality by affecting downstream signaling pathways and other muscle types. Rational drug design can develop therapies to treat CM at the protein level. However, a detailed knowledge of how sarcomeric proteins regulate muscle contraction is required. Muscle contraction occurs through a cyclic interaction between actin thin and myosin thick filaments, regulated by intracellular Ca2+ concentration. Troponin (Tn), …

Biophysical Characterization Of The Folding, Membrane Topology And Ion Transport Activity Of Ucp2 Using Selective Trp Mutants, Tyler C. Auld

Theses and Dissertations (Comprehensive)

Human Uncoupling Protein 2 (hUCP2) is one of five known human UCPs which are found in the inner mitochondrial membrane and have been shown to facilitate the translocation of protons from the intermembrane space to the mitochondrial matrix. The detailed physiological role of UCP2 proton transport, the mechanism by which it mediates this proton transport, as well as its structure has also yet to be elucidated. In order to help determine the topology of UCP2 embedded in the membrane as well as its mechanism of proton transport, the intrinsic fluorescence properties of the two tryptophan residues (Trp) present in its …

Purification And Characterization Of Bcsc; An Integral Component Of Bacterial Cellulose Export, Emily D. Wilson Ms

Theses and Dissertations (Comprehensive)

Biofilms are a growing concern in the medical field due to their increased resistance to antibiotics. When found in a biofilm, bacteria can have antibiotic resistance 10-1000 times that of their planktonic counterparts. Therefore, it is important to study the formation of biofilms. Cellulose biofilms are formed by Enterobacteriaceae, such as many Escherichia coli and Salmonella spp. strains. Biofilms provide these species with benefits including antimicrobial protection, development of bacterial communities, promotion of DNA exchange, uptake of nutrients, and, in the case of cellulose biofilms, immune system evasion. Cellulose biofilms are controlled by the Bacterial cellulose synthesis (Bcs) complex located …

The Effect Of Docosahexaenoic Acid (Dha)-Containing Phosphatidylcholine (Pc) On Liquid-Ordered And Liquid-Disordered Coexistence, Yongwen Gu

Dissertations and Theses

Plasma membranes are essential to both the structure and function of mammalian cells. The first unifying paradigm of membrane structure, the Fluid Mosaic Model, is no longer considered adequate to describe the many non-homogeneous lipid structures that have been observed in both natural and model membranes over the past approximately thirty years. The field of membrane biophysics now appreciates that the complex mixture of different lipid species found in natural membranes produces a range of dynamic, laterally segregated, non-homogeneous structures which exist on time scales ranging from microseconds to minutes.

When sphingomyelin (SM), POPC and cholesterol are all present in …

Interactions Of Eukaryotic Translation Initiation Factors And 3' Untranslated Region Of Barley Yellow Dwarf Virus Mrna During Protein Synthesis: A Study Of Equilibrium Binding, Kinetics And Thermodynamics, Bidisha Banerjee

Dissertations, Theses, and Capstone Projects

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley Yellow Dwarf Virus (BYDV) employs a cap-independent mechanism of translation initiation which is mediated by a structural element BTE (BYDV translation element) located in the 3’ UTR of its mRNA. eIF4F bound the BTE and a translational inactive mutant with high affinity; thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5-164% compared to WT were studied. Using fluorescence anisotropy …