Molecular Biology Commons^™

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 …

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 …

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 …

Biophysical Characterization Of The Par-4 Tumor Suppressor: Evidence Of Structure Outside The Coiled Coil Domain And Interactions With Platinum Chemotherapeutics, Andrea Megan Clark

Chemistry & Biochemistry Theses & Dissertations

Prostate apoptosis response-4 (Par-4) is an apoptosis-inducing tumor suppressor protein. Full-length Par-4 has previously been shown to be a predominantly intrinsically disordered protein (IDP) under neutral conditions, with significant regular secondary structure evident only within the C-terminal coiled coil domain. However, IDPs can gain ordered structure through the process of induced folding, which often occurs under non-neutral conditions. Previous work has shown that the Par-4 leucine zipper, which is a subset of the C-terminal coiled coil domain, is disordered under neutral conditions, but forms a dimeric coiled coil at acidic pH. Increase in ionic strength was also shown to increase …

Direct Recruitment Of Eif4gi And/Dap5 To The 5' Utr Of A Subset Of Human Mrna Drives Their Cap-Independent Translation, Solomon A. Haizel

Dissertations, Theses, and Capstone Projects

During unfavorable cellular conditions (e.g., tumor hypoxia, viral infection, nutrient deprivation, etc.), the canonical, cap-dependent translation initiation pathway in human cells is suppressed by sequestration of the cap-binding protein, eukaryotic initiation factor(eIF) 4E, by 4E-binding proteins. Circumvention of cap-dependent translation shutdown has been linked to tumor development and cancer progression. The stress-induced repression of cap-dependent translation has also been correlated with increased eIF4GI and its homolog, Death Associated Protein 5 (DAP5) expression levels, suggesting these factors have a role in cap-independent translation. Despite several evidence pointing towards a link upregulation of eIF4GI and /DAP5 levels during stress conditions, and the …

Using The Marcus Inverted Region And Artificial Cofactors To Create A Charge Separated State In De Novo Designed Proteins, Eskil Me Andersen

Dissertations, Theses, and Capstone Projects

To create an efficient de novo photosynthetic protein it is important to create long lived charge separated states. Achieving stable charge separation leads to an increase in the efficiency of the photosynthetic reaction which in turn leads to higher yields of end products, such as biofuels, electrical charge, or synthetic chemicals. In an attempt to create charge separated states in de novo proteins we hypothesized that we could engineer the free energy gaps in the proteins from excited primary donor (PD) to acceptor (A), and A back to ground state PD such that the forward electron transfer (ET) would be …

Measuring And Manipulating Tension-Dependent Behavior Of Mechanosensitive Ion Channels, Angela M. Schlegel

Arts & Sciences Electronic Theses and Dissertations

Mechanical forces play critical roles throughout the lives of all organisms and, as such, diverse arrays of mechanotransduction systems have evolved to detect and initiate responses to force. Many mechanotransduction systems consist of mechanosensitive (MS) ion channels, membrane pores that open in response to sufficient mechanical force. My dissertation focuses on both the study and application of force-dependent conformational changes of MS channels. I tested whether charged pore-lining residues R326 and D327 of the Arabidopsis thaliana mitochondrial MS channel MSL1 function in inward rectification or gating kinetics. Mutating these residues showed no effect on MSL1 rectification; however, these residues are …

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 …

The Effects Of Increasing Positively Charged Metal Ions Within Synovial Fluid, Kandisi Anyabwile

Williams Honors College, Honors Research Projects

Osteoarthritis is a degenerative joint disease that affects 10% of men and 13% of women over age of 60. It is the degradation of the cartilage between two bones; obesity, age, overuse, or injury are major contributors to the development of this disease. The joint is incapsulated by the synovial sac filled with a viscous solution that aids in lubrication referred to as synovial fluid. If the synovial sac is ruptured due to injury, positive ions (K⁺, Na⁺, Ca²⁺, and Fe³⁺) may affect viscoelastic properties within the sac. The purpose of this …

Understanding The Relationship Between Local Environmental Changes And The Function Of The Ph Low Insertion Peptide, Violetta Burns Casamayor

Graduate Theses, Dissertations, and Problem Reports

Cancer is the second leading cause of death in the US with over 1.7 million new cases each year. Current cancer treatments tend to also target healthy tissues due to similarities with cancerous ones, resulting in acute side effects. Early detection is the best approach towards defeating cancer, however, modern imaging techniques require sizeable samples, often implying a late stage in the disease. One common attribute of tumors is their acidic microenvironment, which can be taken advantage of.

The pH Low Insertion Peptide (pHLIP) is a membrane-active peptide that can take advantage of the acidic microenvironment surrounding cancer cells. pHLIP …

Simulation Of A Rat Muscle-Tendon Unit With Hill-Type Model Dynamics And The Study Of Viscoelasticity In A Collagen Molecule Via Molecular Dynamics, Veronica Siko

West Chester University Master’s Theses

The field of biological science has established that tendons transfer muscular forces to adjacent bones, but there is a dearth of information about the underlying physical principles of these interactions and how the property of viscoelasticity (displayed in tendons as a difference in mechanical response to stress with differing stretching rates) is encompassed in the collagen of tendons. This thesis details the results of concentric and eccentric contractions of the rat muscle-tendon unit (MTU) with and without viscoelasticity (concentric contraction requires active shortening of the muscle, while eccentric contraction requires active lengthening of the muscle) (Lovering & Brooks, 2014). Once …

Flavin Modification And Redox Tuning In The Bifurcating Electron Transfer Flavoprotein From Rhodopseudomonas Palustris: Two Arginines With Different Roles, Nishya Mohamed-Raseek

Theses and Dissertations--Chemistry

Electron bifurcation is considered as a third fundamental mode of energy conservation mechanism in addition to two well-known mechanisms, substrate level phosphorylation and Oxidative phosphorylation, in electron bifurcation endergonic and exergonic redox reactions are coupled. The newly discovered flavin based electron bifurcation in electron transfer flavoproteins (ETFs) helps to reduce low potential ferredoxin, which provides electrons to drive biologically demanding reactions such as atmospheric dinitrogen fixation in diazotroph and methane production in methanogens.

Current research demonstrates the capacity for electron bifurcation in the Rhodopseudomonas palustris ETF (RpalETF) system. RpalETF contains two chemically identical but functionally different FADs: …

Fxr Agonists Induce Distinct H-12 Structural States, Vikash Kumar

Graduate Student Theses, Dissertations, & Professional Papers

The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity. The FXR LBD shows multiple low-energy conformational states of the activation function-2 (AF-2) coregulator binding surface upon ligand binding, indicating the complexity of FXR activation. However, it is unknown how ligand binding leads to different conformational states within the AF-2 region centered on helix 12 (H-12) of the LBD. Here we observe the conformation of the coregulator binding surface (H-12 specifically) of FXR upon ligand binding in solution using fluorine-19 (19F) nuclear magnetic resonance (NMR) and simulations of this surface using molecular dynamics. Fluorescence anisotropy of fluorescein-labeled coregulator peptides …