Biochemistry, Biophysics, and Structural Biology Commons^™

Protein Stability In Solution And In The Gas Phase., Yousef Haidar

Electronic Thesis and Dissertation Repository

Electrospray Ionization mass spectrometry (ESI-MS) is widely used for probing proteins, yet many aspects of this technique remain elusive. Using MS, ion mobility spectrometry (IMS), and circular dichroism (CD) spectroscopy, this thesis sheds light on the stability differences of proteins in the gas phase and solution. After a general introduction (Chapter 1), Chapter 2 scrutinizes some aspects of native ESI. Our data highlight the significance of cone voltage in maintaining a native-like fold and show the advantage of using NH₄Ac in protein experiments. Chapter 3 focuses on hydrogen/deuterium exchange (HDX)-MS. Several studies have reported that D₂O …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

Probing Large Intrinsically Disordered Regions Through Novel Sortase-Mediated Ligation, Leah Kjormoe

Scholars Week

In the realm of proteins, it is widely accepted that structure informs function. However, there are many proteins that contain intrinsically disordered regions (IDRs). These regions are areas in which the protein lacks defined structure, and IDPs are also often unstable, which complicates structural studies. NMR spectroscopy is an established method for probing protein structure and has been applied to that end in small IDRs. However, larger IDRs often have spectral overlap that makes data difficult to interpret. Furthermore, low-concentration samples limit spectral clarity. One method to address these difficulties is to use sortase ligation and segmental labeling, which increases …

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 …

An Exploration Of Protein And Dna Components In Fingerprint Residue, Ashley Borrego

Student Theses

The main focus of this project was to investigate the protein and DNA components in both sebaceous and eccrine fingerprints. This study investigated the relative content of DNA and proteins in eccrine fingerprints to sebaceous fingerprints. All volunteers were instructed to wash and dry their hands prior to depositing parallel thumbprints. Twenty volunteers were instructed to touch their face to produce sebaceous prints, and 5 volunteers were instructed to wear gloves over a heat source to produce sweaty or eccrine prints. Microscopy was used to score the cellular debris of the right fingerprint on a scale of 1-4 based on …

Developing Tale Proteins As A Biosensor For Detecting Pathogen Specific Double-Stranded Dna, Kathrine Gaiko

Mahurin Honors College Capstone Experience/Thesis Projects

Transcription activator-like effector (TALE) proteins are important for DNA binding. They bind to specific nucleotide sequences by the use of two residues in each repeat allowing them to target specific DNA sequences. Their modular structure makes TALEs advantageous over other DNA binding proteins such as zinc finger proteins. Zinc finger proteins (ZFP) use a finger like projection to bind 3-4 subsequence base pairs while TALE proteins use two amino acid residues in each repeat to bind one nucleotide. ZFP can use SEER-Lac system for colorimetric detection, while TALE proteins can use fluorescence labeling with Alexa for detection of binding to …

Sers For Protein Detection At A Single Molecule Level For Developing A New Medical Diagnostics Platform, Lamyaa Almehmadi

Legacy Theses & Dissertations (2009 - 2024)

A two-step process of protein detection at a single molecule level using Surface Enhanced Raman Spectroscopy (SERS) was developed as a new platform for medical diagnostics in this proof-of-concept study. First, a protein molecule was bound to a linker in the bulk solution and then this adduct was chemically reacted with the SERS substrate. Traut’s Reagent (TR) was used to thiolate Bovine serum albumin (BSA) in solution followed by chemical cross linking to a gold surface through a sulfhydryl group. A Glycine-TR adduct was used as a control sample to identify the protein contribution to the SER spectra. Gold SERS …

Characterization Of The Microbial Phosphonate-Activating Pntc Enzymes, Kyle Rice

Theses and Dissertations (Comprehensive)

New strategies are urgently needed to combat infectious diseases in an era of rising antibiotic resistance. Furthermore, an emerging appreciation for the human microbiome’s role in maintaining health motivates discovery of species-specific antibiotics that minimally disrupt our native bacterial communities. Small molecule modifications to bacterial cell surfaces represent a potentially rich source of new targets for next generation antibiotics, as these molecules mediate virulence and evasion of the host immune response. Phosphocholine (PCho) is a rare cell surface modification that contributes to virulence, and modifications with phosphonates like 2-aminoethylphosphonate (AEP) are even more unusual and therefore provide opportunities for species- …

The Dissemination, Regulatory Role, And Evolution Of Mycobacterial Inteins, Danielle Skye Kelley

Legacy Theses & Dissertations (2009 - 2024)

Inteins are intervening protein elements, capable of coordinating escape from a host protein through a self-catalyzed mechanism, called protein splicing. This results in free intein and a mature host protein product. Inteins are also mobile elements and many contain homing endonucleases that enable the targeting to ectopic sites and invasion of novel niches. Inteins have been found across all three domains of life and are often present in replication, recombination, and repair proteins. However, it is unclear if the observed distribution is simply a factor of endonuclease preference or if inteins have been selectively maintained due to an adaptation that …

2-D Electrophoresis Modeling Of Multienzyme Cutting Of Polypeptides, Howard Mayes

Theses

2-Dimensional Electrophoresis is one of the tools in the identification of proteins by molecular weight and pH. The display of molecular weight allows the researcher to quickly identify whether a specific protein or peptide string is in the sample. The pH measurement allows even better resolution between different species in the sample. The MultiEnzyme ElectroPhoresis (MEEP) program tries to model that by providing a graph that displays separated protein strings by both molecular weight and pH. The ability to cleave the protein with 43 different enzyme variations allows the researcher to analyze appropriate enzymes to isolate a protein subsequence before …

Study Of Biologically Important Macromolecules By Nuclear Magnetic Resonance, Christopher Michael Demott

Legacy Theses & Dissertations (2009 - 2024)

Intrinsically disordered proteins or unstructured segments within proteins play an important role in cellular physiology and pathology. A combination of peptide aptamers selected by using the yeast-two-hybrid scheme, and in-cell NMR identified high affinity binders to a transiently structured intrinsically disordered proteins (IDP). This method was validated using the prokaryotic ubiquitin-like protein, Pup, of the Mycobacterium proteasome. We discover two peptide aptamers that bind to opposite sites of a transient helix in Pup, an intrinsically disordered protein, that have vastly different effects on the survival of Mycobacterium bovis BCG.

Purification, Optimization, And Growth Of New Delhi Metallo-Β-Lactamase-1 Protein Crystals Mixed With Nz218 Inhibitor, Brandon M. Wills

Celebration of Learning

New Delhi metallo-β-lactamase-1 is a problematic gene found in certain strains of bacteria that cause them to become antibiotic resistant to nearly all known antibiotics. While some antibiotics are available to treat patients with a bacterial infection, most are toxic or do not have 100% success rates. With that being said, it is imperative that we search for a molecule that is successfully able to inhibit the effects of this gene every time. Such a discovery would help tremendously with new antibiotic drug development and also prevent further damage by these dangerous bacteria. In this presentation, I will describe the …

Using Simple Self-Assembling Peptides To Attain Novel Protein-Like Functions, Tyler Smith

Honors Capstone Projects - All

Proteins carry out many extremely efficient functions, including catalysis and biomolecule recognition. Underlying this efficiency is their extraordinary complexity and ability to fold into unique three-dimensional structures. Attempts to replicate this efficiency through de novo design have only shown moderate success, and it is unclear how modern-day proteins may have evolved. However, short peptides that alternate hydrophobic and hydrophilic residues can self-assemble into amyloid fibrils to achieve well-defined secondary structure. These aggregates may have served as a template from which the first proteins were derived. We designed self-assembling seven-residue peptides that are able to act as Zn²⁺-dependent esterases. …

The Application Of Hydrogen/Deuterium Exchange And Covalent Labeling Coupled With Mass Spectrometry To Examine Protein Structure, Nicholas B. Borotto

Doctoral Dissertations

Thorough insight into a protein’s structure is necessary to understand how it functions and what goes wrong when it malfunctions. The structure of proteins, however, is not easily analyzed. The analysis must take place under a narrow range of conditions or risk perturbing the very structure being probed. Furthermore, the wide diversity in size and chemistry possible in proteins significantly complicates this analysis. Despite this numerous methods have been developed in order to analyze protein structure. In this work, we demonstrate that mass spectrometry (MS)-based techniques are capable of characterizing the structure of particularly challenging proteins. This is done through …

A Survey Of The Current Drug Screening Techniques To Obtain Rational Design And Study Drug-Target Interactions, Stephen Dansereau

Legacy Theses & Dissertations (2009 - 2024)

Different techniques have been developed over the years for the purpose of studying proteins and understanding their functions. Early techniques typically employed bioluminescence or fluorescence such such as the firefly protein luciferase and the jellyfish green fluorescent protein (GFP), respectively, to localize proteins within the cell. X-ray crystallography has also provided valuable structural details of many different proteins in vitro. Yet, nuclear magnetic resonance (NMR) spectroscopy offers the most realistic insight into proteins' physiologic structures and how proteins function in their native, cellular environments.

Binding Interactions Of (R)- And (S)-Hydroxypropyl-Com Dehydrogenases And The Zinc Knuckle Proteins Air1 And Air2, Jeremy W. Bakelar

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

A thorough understanding of protein function requires knowledge of how proteins interact with their substrates and with other proteins. The work entailed in this dissertation describes the binding interactions of proteins from two different model systems: (1) the dehydrogenase enzymes R- and S-HPCDH and (2) the zinc knuckle proteins Air1 and Air2.

R- and S-HPCDH are highly similar enzymes (42% identical) that function in a unique metabolic pathway found in the soil bacterium Xanthobacter autotrophicus. The bacterium produces R- and S-HPCDH simultaneously to facilitate the transformation of two different forms of the organic …

Distinguishing Macrophage Activation States By Mass Spectrometry, Matthias Manfred Knust

Graduate Theses and Dissertations

Macrophages are versatile and highly adaptive cells that are involved in a wide range of physiological processes including host defense, homeostasis or regeneration, as well as pathogenesis. They react to their microenvironment, assuming various roles based on chemical and/or physical cues, and can reversibly shift between these so-called activation states. Concurrently, the technique of immunohistochemistry is used to gain spatial information on activated macrophages on tissue sections. The aim of this work was to find mass spectral biomarkers that allow the differentiation of activation states, and establish conditions that can be used in imaging mass spectrometry (IMS) experiments to investigate …

Nonenzymatic Glycosylation Of Erythrocyte Membrane Proteins. Relevance To Diabetes, J A. Miller, Ellen M. Gravallese, H F. Bunn

Ellen M. Gravallese

Nonenzymatic glycosylation of proteins of the erythrocyte membrane was determined by incubating erythrocyte ghosts with [3H]borohydride. The incorporation of tritium into protein provides a reliable assay of ketoamine linkages. The membrane proteins from 18 patients with diabetes incorporated twice as much radioactivity as membrane proteins from normal erythrocytes. After acid hydrolysis, amino acid analysis showed that the majority of radioactivity was localized to glucosyllysine. Autoradiograms showed that all of the major proteins of the erythrocyte membrane, separated by electrophoresis on sodium dodecyl sulfate gels, contained ketoamine linkages. No protein bands in either normal or diabetic erythrocytes showed significant preferential labeling. …

Novel Nmr Based Technologies To Study Macromolecular Structures, Subhabrata Majumder

Legacy Theses & Dissertations (2009 - 2024)

Nuclear Magnetic Resonance Spectroscopy (NMR) is one of the principle tools in structural biology to probe macromolecular structures and interactions. The atomic resolution afforded by this technique has been widely used to probe protein-protein, and protein-ligand interactions in-vitro. However, the natural milieu of the proteins is the living cell and the cellular cytoplasm is extremely heterogeneous. The NMR studies of folded protein in-cell, till now, have been limited by non-specific interactions of the cytosol. This thesis outlays a general methodology to study protein structure/interactions inside the living cells using NMR. In a closely related objective, it also describes the use …

Automatic Animation Of Molecular Motion Using Python And Cinema 4d, Diana Zajac, Nathaniel Smith, Dan Gurnon

Annual Student Research Poster Session

No abstract provided.

Stoichiometries And Affinities Of Interacting Proteins From Concentration Series Of Solution Scattering Data: Decomposition By Least Squares And Quadratic Optimization, Himanshu Chandola, Tim E. Williamson, Bruce A. Craig, Alan M. Friedman, Chris Bailey-Kellogg

Dartmouth Scholarship

In studying interacting proteins, complementary insights are provided by analyzing both the association model (the stoichiometry and affinity constants of the intermediate and final complexes) and the quaternary structure of the resulting complexes. Many current methods for analyzing protein interactions either give a binary answer to the question of association and no information about quaternary structure or at best provide only part of the complete picture. Presented here is a method to extract both types of information from X-ray or neutron scattering data for a series of equilibrium mixtures containing the initial components at different concentrations. The method determines the …

Primary Microrna Processing Assay Reconstituted Using Recombinant Drosha And Dgcr8., Ian Barr, Feng Guo

Natural Sciences and Mathematics | Faculty Scholarship

In animals, the Microprocessor complex cleaves primary transcripts of microRNAs (pri-miRNAs) to produce precursor microRNAs in the nucleus. The core components of Microprocessor include the Drosha ribonuclease and its RNA-binding partner protein DiGeorge critical region 8 (DGCR8). DGCR8 has been shown to tightly bind an Fe(III) heme cofactor, which activates its pri-miRNA processing activity. Here we describe how to reconstitute pri-miRNA processing using recombinant human Drosha and DGCR8 proteins. In particular, we present the procedures for expressing and purifying DGCR8 as an Fe(III) heme-bound dimer, the most active form of this protein, and for estimating its heme content.