Biochemistry, Biophysics, and Structural Biology Commons^™

Identification Of Covalent Catechin Adducts Of Enoyl-Acp Reductase (Fabi) From E. Coli Using Mass Spectrometry, Francisco G. Atwi, P. M. Joyner, Rustin Ghidari

Seaver College Research And Scholarly Achievement Symposium

Enoyl-ACP reductase (FabI), an enzyme which is inhibited by catechin, is essential for lipid biosynthesis in bacteria (E. coli). Another FabI inhibitor, triclosan, has strong antibacterial effects,1 but overuse can be detrimental to the environment. Flavonoids, a class of dietary compounds, are known to have potent inhibitory effects on FabI and other kinases.2,3,4 Adducts form as a result of the biochemical reaction between flavonoids and cysteine, a modification which can be verified by m/z shifts on the mass spectra.5 Samples of E. coli protein with overexpressed FabI are utilized: the wild type with ordinary primary structure, and mutant type lacking …

Development Of Cyan And Blue Thermostable Fluorescent Proteins: Characterization And Structural Analysis, Acacia J. Jurkowski

Graduate Theses/Dissertations

Fluorescent proteins are commonly used as cell markers in living organisms. Modifications by mutation can be used to improve the qualities of these proteins including fluorescence, thermostability, pH stability, and chemical stability. The goal of this project is to use mutagenesis to improve the fluorescence of thermostable cyan and blue proteins derived from the thermal green protein (TGP). The first cyan protein developed by the DeVore lab (CTP 0.0) shifted the fluorescence to cyan but decreased the quantum yield (Φ_F) to 0.056. Further mutations were incorporated to increase the quantum yield through incorporating hydrogen bonding interactions to the …

The Importance Of Solution Studies For The Structural Characterization Of The Enterovirus 5' Cloverleaf, Morgan G. Daniels, Meagan E. Werner, Xiaobing Zuo, Steven M. Pascal

Chemistry & Biochemistry Faculty Publications

Enteroviruses initiate genomic replication via a highly conserved mechanism that is controlled by an RNA platform, also known as the 5' cloverleaf (5'CL). Here, we present a biophysical analysis of the 5'CL conformation of three enterovirus serotypes under various ionic conditions, utilizing CD spectroscopy, size-exclusion chromatography, and small-angle X-ray scattering. In general, a tendency toward a smaller monomeric hydrodynamic radius in the presence of salts was observed, but the exact structural signature of each 5'CL varied depending upon the serotype. Rhinovirus B14 (RVB14) exhibited at least two monomeric conformations and a low propensity for dimerization, while poliovirus 1 (PV1) showed …

Design Of An Artificial Transcription Factor For Inducing Potent Interferon-Γ Signals, Ashley King

Theses and Dissertations

Cytokines are small immune signaling proteins responsible for cellular communication and their levels are tightly regulated throughout immune recognition and defense. Interferon gamma is a key cytokine responsible for upregulation of antigen presentation and supports the differentiation of pro-inflammatory effector cells. Due to its role in regulation, excess interferon gamma is cleared from the body limiting the success of interferon gamma immunotherapies. This study reports the design and validation of an artificial transcription factor (ATF) that induces endogenous interferon gamma expression. The transcription factor contains zinc finger proteins to recognize the interferon gamma gene and an activation domain to promote …

Computational Modelling And Design Of Antibodies: Benefits From Analysis Of Their Unique Structural Motifs, Katherine M. Mccoy

Dartmouth College Ph.D Dissertations

The complexes that antibodies make with their binding partners, or antigens, are especially valuable to be able to predict and modify due to their unique role in the immune system. Yet, they present significant challenges to computational methods of both modelling and design. Antibodies are unlike most other proteins in that they are composed of a scaffold region, which is a highly conserved structure that is largely the same between antibodies of the same class, and Complementary Determining Regions (CDRs), which are comprised of hypervariable loops that largely determine their binding motifs. Additionally, antibodies and their antigens do not co-evolve …

Changes In Environmental Conditions Affect The Two Isoforms Of Snap-25 Differently, Joseph Jackson, Dixon J. Woodbury, Tanner M. Blocker, Samuel W. Shumway, Jarom S. Sumsion, Thomas Weed, Jason Carlson, Nathan Mumford, Ryan Poland

Library/Life Sciences Undergraduate Poster Competition 2024

In the brain, neurons communicate via releasing and detecting neurotransmitters. Release occurs through exocytosis, following fusion of synaptic vesicles to neuronal cell membranes. This process is driven by formation of a dynamic quaternary protein structure known as the SNARE complex. SNAP-25 contributes two alpha helical domains to this complex. Neurons express SNAP-25 in two distinct isoforms, SNAP-25A (25A) and SNAP-25B (25B). These two isoforms vary by only 9 amino acids and are expressed differently depending on brain region and the developmental stage of the neuron [1]. The primary structures of 25A and 25B and their effect on SNARE complex formation …

Mechanical Properties Of Natural Osmia Lignaria Silk And Recombinant Expression Of The Silk Proteins, Jackson Morley

Undergraduate Honors Capstone Projects

The use of petroleum-based plastic and the subsequent waste has led researchers to explore alternatives in the form of biopolymers. Spider silk and hagfish intermediate filaments show the potential to be advantageously utilized in textile and biomedical industries. Bee silk is a sparsely studied biopolymer that has shown potential to be added to these other impressive biopolymers. Osmia lignaria, is a solitary bee native to the western United States. Silk from these bees can be isolated while the larvae are spinning their silk. The silk from these bees has been mechanically tested for strength, stress, strain, and elasticity. Cocoon …

Fundamental Study Of Ionic Liquid Physicochemical Effects On Thermal Stability Of Model Biological Macromolecules, Austin Keith Clark

Theses and Dissertations

Ionic Liquids (ILs) are substances with a unique physical attribute compared to that of solid ionic salts. At room temperature, ILs are molten salts that have a variety of physical effects that can play a role in their impact on other molecules, as solvents or solutes. They can play the role of the solvent in a variety of applications, from biofuels to organic catalysis or as excipients in pharmaceutical formulations. These ILs have a desirable use as solvents due to their ability to be tunable substances. Changing the cation or anion of the IL causes a change in its physical …

The Roles Of Estrogen Receptor Alpha-Interacting Rnas And Exosomes In Breast Cancer, And E-Cadherin In Carcinomas, Brihget Catalina Sicairos-Meza

Graduate Theses and Dissertations

Carcinomas are malignancies of epithelial cells and account for >80% of cancer-related deaths. Approximately 75% of breast cancers express estrogen receptor alpha (ERα). ERα promotes breast cancer progression by enhancing cancer cell proliferation. Tamoxifen is commonly used to treat ERα-positive breast cancers but can result in endocrine therapy resistance. The mechanisms underlying endocrine therapy resistance remain elusive. The Du lab recently found that ERα is an RNA-binding protein. However, the significance of the ERα-RNA interaction is unknown. We fractionated ERα-positive MCF7 cells into cytoplasmic and nuclear fractions, followed by immunoprecipitation of ERα with associated RNAs and RNA-sequencing. We identified many …

Mutating Tetur02g09850 Originated From Spider Mites To Enhance Crystallization, Hayley Cash

Senior Theses

This study aimed to explain and adequately utilize several common biochemical laboratory techniques to mutate a portion of the nucleic acid sequence within a specific protein. This protein, referred to as Tetur02g09850 is originated from Tetranychus Urticae, or the Two-Spotted Spider Mite. This mite causes excessive damage to crops and is known to be extremely resistant to most common pesticides. For this reason, understanding the protein structure could be used to explain this acaricide resistance and aid in the development of more effective pesticides. Some of the testing utilized was site-directed mutagenesis, IMAC and gel filtration for protein purification, gel …

Magel2 Truncation Alters Select Behavioral And Physiological Outcomes In A Rat Model Of Schaaf-Yang Syndrome, Derek L Reznik, Mingxiao V Yang, Pedro Albelda De La Haza, Antrix Jain, Melanie Spanjaard, Susanne Theiss, Christian P Schaaf, Anna Malovannaya, Theresa V Strong, Surabi Veeraragavan, Rodney C Samaco

Faculty, Staff and Students Publications

Previous studies in mice have utilized Magel2 gene deletion models to examine the consequences of its absence. We report the generation, molecular validation and phenotypic characterization of a novel rat model with a truncating Magel2 mutation modeling variants associated with Schaaf-Yang syndrome-causing mutations. Within the hypothalamus, a brain region in which human MAGEL2 is paternally expressed, we demonstrated, at the level of transcript and peptide detection, that rat Magel2 exhibits a paternal, parent-of-origin effect. In evaluations of behavioral features across several domains, juvenile Magel2 mutant rats displayed alterations in anxiety-like behavior and sociability measures. Moreover, the analysis of peripheral organ …

Automating The High-Throughput Screening Of Protein-Based Optical Indicators And Actuators, Jihwan Lee, Beatriz Campillo, Shaminta Hamidian, Zhuohe Liu, Matthew Shorey, François St-Pierre

Faculty, Staff and Students Publications

Over the last 25 years, protein engineers have developed an impressive collection of optical tools to interface with biological systems: indicators to eavesdrop on cellular activity and actuators to poke and prod native processes. To reach the performance level required for their downstream applications, protein-based tools are usually sculpted by iterative rounds of mutagenesis. In each round, libraries of variants are made and evaluated, and the most promising hits are then retrieved, sequenced, and further characterized. Early efforts to engineer protein-based optical tools were largely manual, suffering from low throughput, human error, and tedium. Here, we describe approaches to automating …

Quantitative Measurement Of Protac Intracellular Accumulation, Xin Yu, Jin Wang

Faculty, Staff and Students Publications

In recent years, Proteolysis Targeting Chimera (PROTAC) technology has emerged as one of the most promising approaches to remove disease-associated proteins by utilizing cells' own destruction machinery. To achieve successful degradation of a protein of interest (POI), the heterobifunctional PROTAC molecules must penetrate into the cells first, followed by target engagement and formation of the POI-PROTAC-E3 ligase complex. Based on this understanding, the assessment of cell permeability and in cell target engagement are of great importance to evaluate the efficacy of PROTAC candidates. PROTAC molecules can be classified as non-covalent and covalent, and covalent PROTACs can be further divided into …

Enhanced Structural Control Of Soft-Templated Mesoporous Inorganic Thin Films By Inert Processing Conditions, Maximilliano Jesus Jara Fornerod, Alberto Alvarez-Fernandez, Eric R. Williams, Miximillian W.A. Skoda, Beatriz Prieto-Simon, Nicolas H. Voelcker, Morgan Stefik, Marc-Oliver Coppens, Stefan Guldin

Faculty Publications

Mesoporous thin films are widely used for applications in need of high surface area and efficient mass and charge transport properties. A well-established fabrication process involves the supramolecular assembly of organic molecules (e.g., block copolymers and surfactants) with inorganic materials obtained by sol-gel chemistry. Typically, subsequent calcination in air removes the organic template and reveals the porous inorganic network. A significant challenge for such coatings is the anisotropic shrinkage due to the volume contraction related to solvent evaporation, inorganic condensation, and template removal, affecting the final porosity as well as pore shape, size, arrangement, and accessibility. Here, we show that …

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, …

Determining The Nucleic Acid Binding Affinities Of Crispr-Associated Ding (Casding), Matt Armbrust

Fall Student Research Symposium 2021

CRISPR-Cas systems are adaptive prokaryotic immune systems that enable host cells to defend against attack from foreign nucleic acids such as phage infections or plasmids. CRISPR-Cas systems are diverse and encompass 2 classes, 6 types, and 33 subtypes. The Type IV-A CRISPR-Cas system from Pseudomonas aeruginosa strain 83 is composed of five different genes (csf1, csf2, csf3, cas6, and dinG). Type IV-A systems are poorly understood, and currently there is little research detailing their biological and biochemical mechanism of immunity. CasDinG, an ancillary protein within the Type IV-A system, is required for an immune response in vivo. However, the role …

Molecular Simulation Of Rna Conformational Dynamics : An Example Of Micro-Rna Targeting Messenger Rna : Mir-34a-Msirt1, Parisa Ebrahimi

Legacy Theses & Dissertations (2009 - 2024)

MicroRNA (miRNA), as a distinct class of biological regulators and a ”guide” member of non-coding RNA-protein complexes (RNPs), regulates more than 60% of protein-coding genes expression through base-pairing with targeted messenger RNA (mRNA) in the RNA-Induced Silencing Complex (RISC). Most of miRNAs identified in human, are conserved in other animals, which have preferentially conserved interaction sites particularly in 3’ untranslated regions (3’UTRs) of many human messenger mRNAs.The capability of a single miRNA to target more than hundreds of mRNAs, suggests that miRNAs influence essentially all developmental process and diseases, which also makes them interesting candidates as therapeutics agents. The primary …

Molecular Simulation Of Rna Conformational Dynamics : An Example Of Micro-Rna Targeting Messenger Rna : Mir-34a-Msirt1, Parisa Ebrahimi

Legacy Theses & Dissertations (2009 - 2024)

MicroRNA (miRNA), as a distinct class of biological regulators and a ”guide” member of non-coding RNA-protein complexes (RNPs), regulates more than 60% of protein-coding genes expression through base-pairing with targeted messenger RNA (mRNA) in the RNA-Induced Silencing Complex (RISC). Most of miRNAs identified in human, are conserved in other animals, which have preferentially conserved interaction sites particularly in 3’ untranslated regions (3’UTRs) of many human messenger mRNAs.The capability of a single miRNA to target more than hundreds of mRNAs, suggests that miRNAs influence essentially all developmental process and diseases, which also makes them interesting candidates as therapeutics agents. The primary …

Anatomy And Physiology Preparatory Course Textbook (2nd Edition), Carlos Liachovitzky

Open Educational Resources

The goal of this preparatory textbook is to give students a chance to become familiar with some terms and some basic concepts they will find later on in the Anatomy and Physiology course, especially during the first few weeks of the course.

Organization and functioning of the human organism are generally presented starting from the simplest building blocks, and then moving into levels of increasing complexity. This textbook follows the same presentation. It begins introducing the concept of homeostasis, then covers the chemical level, and later on a basic introduction to cellular level, organ level, and organ system level. This …

Investigations Of The Structure And Protein-Protein Interactions Of Chlamydia Trachomatis Scc4, Thilini Oshadhi Senarath Ukwaththage

LSU Doctoral Dissertations

Chlamydia trachomatis (CT) is the most common, sexually transmitted bacterial disease (STD) in the world. In the developmental cycle of CT, specific chlamydia chaperone 4 (Scc4) is a unique protein with essential and multiple roles. Hence, Scc4 is significant as a virulence target for therapeutic approaches to treat chlamydial infections. A novel approach was discovered to purify tag free Scc4 by utilizing a 6X-histidine-tag on Scc1 in the co-expressed Scc4:Scc1 complex by capturing the complex on nickel-charged immobilized metal affinity chromatography resin, followed by dissociation of Scc4 with sarkosyl. Using triple resonance NMR experiments, backbone and sidechain resonances …

Development Of Chemical Methods For Oligonucleotide Purification, Paramagnetic Labeling And Synthesis Of Dna-Based Advanced Materials, Muhan He

Legacy Theses & Dissertations (2009 - 2024)

This thesis describes a chemical method for alternative oligonucleotide purification that is non-chromatographic and gel-free and allows to routinely synthesize and purify long functional RNA strands. The purification of long RNAs is based on the bio-orthogonal inverse electron demand Diels-Alder (IEDDA) chemistry between trans-cyclooctene (TCO) and tetrazine (Tz). Target oligonucleotide strands are selectively tagged with Tz and can be captured and purified from the failure sequences with immobilized TCO. RNA strands are synthesized on solid support through a photolabile linker to avoid the loss of Tz tag. Purity of the isolated oligonucleotides was evaluated using gel electrophoresis, HPLC and mass …

Identification Of Ires Activity In Cellular Mrnas And Viral Rna Using A Circular Rna Construct, Priyanka Sehta

Legacy Theses & Dissertations (2009 - 2024)

Translation initiation is a critical step in the process of protein synthesis. The canonical way of translation initiation involves ribosomes being recruited to the 7-methyl guanosine cap present at the 5’end of the untranslated region (5’ UTR) of the RNAs. However, viral RNAs and some cellular mRNAs lack this 5’ cap structure and thus deploy an alternate non-canonical translation initiation mechanism. In non-canonical translation initiation, ribosome recruitment is facilitated by the RNA secondary structures called Internal Ribosome Entry Site (IRES) present most often in the 5’ UTR. To measure IRES-mediated translation, the dual luciferase assay has been the gold standard. …

Missense Mutations In The Gamma Crystallins And Mechanisms Of Lens Opacity, Wenjuan Hou

Legacy Theses & Dissertations (2009 - 2024)

Cataract, or clouding of the ocular lens, among the most common types of eye diseases, is the leading cause of blindness worldwide. With the opacity or clouding of the lens, light incident on the lens is scattered rather than being transmitted and is thus prevented from focusing on the retina. The lens becomes cataractous due to a large number of reasons, among which aging and genetic mutations are two of the most common factors. Clouding of the center of the lens or nuclear opacity, is the most frequently observed type of age-onset cataract, as well as inherited, congenital cataract [1, …

Degree Of Conservation Of Methionines Found To Be Oxidized In The Human Urinary Proteome, Alexis Hall

Graduate Theses and Dissertations

In previous work from this laboratory, methionine containing peptides from the human urinary proteome were examined by mass spectrometry for the degree of methionine oxidation to the sulfoxide form. While this demonstrated that many of the methionines detected were capable of being oxidized, the question of whether these methionines are important in the structure and/or function of the parent proteins came about. In some proteins, methionine oxidation has been linked to conformational changes and alteration of function and thus can serve as a mechanism for reversible regulation of activity. It is hypothesized that methionines which might serve a regulatory purpose …

Tactviz: A Vmd Plugin For Tactile Visualization Of Protein Structures, Olivia R. Shaw, Jodi A. Hadden-Perilla

Journal of Science Education for Students with Disabilities

Scientific disciplines spanning biology, biochemistry, and biophysics involve the study of proteins and their functions. Visualization of protein structures represents a barrier to education and research in these disciplines for students who are blind or visually impaired. Here, we present a software plugin for readily producing variable-height tactile graphics of proteins using the free biomolecular visualization software Visual Molecular Dynamics (VMD) and protein structure data that is publicly available through the Protein Data Bank. Our method also supports interactive tactile visualization of proteins with VMD on electronic refreshable tactile display devices. Employing our method in an academic laboratory has enabled …

A Highly Elastic And Fatigue-Resistant Natural Protein-Reinforced Hydrogel Electrolyte For Reversible-Compressible Quasi-Solid-State Supercapacitors, Jingya Nan, Gaitong Zhang, Tianyu Zhu, Zhongkai Wang, Lijun Wang, Hongsheng Wang, Fuxiang Chu, Chunpeng Wang, Chuanbing Tang

Faculty Publications

Compressible solid-state supercapacitors are emerging as promising power sources for next-generation flexible electronics with enhanced safety and mechanical integrity. Highly elastic and compressible solid electrolytes are in great demand to achieve reversible compressibility and excellent capacitive stability of these supercapacitor devices. Here, a lithium ion-conducting hydrogel electrolyte by integrating natural protein nanoparticles into polyacrylamide network is reported. Due to the synergistic effect of natural protein nanoparticles and polyacrylamide chains, the obtained hydrogel shows remarkable elasticity, high compressibility, and fatigue resistance properties. More significantly, the supercapacitor device based on this hydrogel electrolyte exhibits reversible compressibility under multiple cyclic compressions, working well …

Cloning, Protein Expression, And Characterization Of Interleukin 1 Alpha, Musaab Habeeb Ali Al Ameer

Graduate Theses and Dissertations

Recombinant DNA technology and the ability to produce recombinant proteins have significantly changed the world of pharmaceutical market. Recombinant DNA technology using E. coli cells has successfully produced many therapeutic proteins. In this study, the designed tag Ark-RUBY-tag facilitates rapid, and reproducible purification of recombinant proteins expressed as inclusion bodies in E. coli cells. Purification of Ark-RUBY-fused recombinant protein(s) can be obtained by using imidazole fractions. Target protein can be easily removed from the Ark-RUBY-tag by enzymatic cleavage. Ark-RUBY-fused recombinant proteins can be quantitatively detected using polyclonal antibodies. Ark-RUBY-tag can be used to purify small peptides. By using Ark-RUBY-tag, purified …

Methylation Of Salmonella Typhimurium Flagella Promotes Bacterial Adhesion And Host Cell Invasion, Julia A Horstmann, Michele Lunelli, Hélène Cazzola, Johannes Heidemann, Caroline Kühne, Pascal Steffen, Sandra Szefs, Claire Rossi, Ravi K Lokareddy, Chu Wang, Laurine Lemaire, Kelly T Hughes, Charlotte Uetrecht, Hartmut Schlüter, Guntram A Grassl, Theresia E B Stradal, Yannick Rossez, Michael Kolbe, Marc Erhardt

Department of Biochemistry and Molecular Biology Faculty Papers

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of …

Protein Footprinting: Auxiliary Engine To Power The Structural Biology Revolution, Mark R. Chance, Erik R. Farquhar, Sichun Yang, David T. Lodowski, Janna G. Kiselar

Faculty Scholarship

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins are determined regularly. These advances have been driven by over fifteen years of technology advancements, first in macromolecular crystallography, and recently in Cryo-electron microscopy. These structures are allowing detailed questions about functional mechanisms of the structures, and the biology enabled by these structures, to be addressed for the first time. At the same time, mass spectrometry technologies for protein structure analysis, “footprinting” studies, have improved their sensitivity and resolution dramatically and can provide detailed sub-peptide and residue level information for validating structures …

A Broad-Spectrum Antiviral Molecule, Ql47, Selectively Inhibits Eukaryotic Translation, Mélissanne De Wispelaere, Margot Carocci, Dominique J Burri, William J Neidermyer, Calla M Olson, Imme Roggenbach, Yanke Liang, Jinhua Wang, Sean P J Whelan, Nathanael S Gray, Priscilla L Yang

Faculty, Staff and Students Publications

Small-molecule inhibitors of translation are critical tools to study the molecular mechanisms of protein synthesis. In this study, we sought to characterize how QL47, a host-targeted, small-molecule antiviral agent, inhibits steady-state viral protein expression. We demonstrate that this small molecule broadly inhibits both viral and host protein synthesis and targets a translation step specific to eukaryotic cells. We show that QL47 inhibits protein neosynthesis initiated by both canonical cap-driven and noncanonical initiation strategies, most likely by targeting an early step in translation elongation. Our findings thus establish QL47 as a new small-molecule inhibitor that can be utilized to probe the …