Biochemistry, Biophysics, and Structural Biology Commons^™

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 …

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 …

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 …

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 …

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 …

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 …

Simulated Breathing: Application Of Molecular Dynamics Simulations To Pulmonary Lung Surfactant, Maksymilian Dziura, Basel Mansour, Mitchell Dipasquale, P. Charukeshi Chandrasekera, James W. Gauld, Drew Marquardt

Chemistry and Biochemistry Publications

In this review, we delve into the topic of the pulmonary surfactant (PS) system, which is present in the respiratory system. The total composition of the PS has been presented and explored, from the types of cells involved in its synthesis and secretion, down to the specific building blocks used, such as the various lipid and protein components. The lipid and protein composition varies across species and between individuals, but ultimately produces a PS monolayer with the same role. As such, the composition has been investigated for the ways in which it imposes function and confers peculiar biophysical characteristics to …

Protein Composition: Translating Amino Acid Sequences Into Music, Melody Campbell

WWU Honors College Senior Projects

Proteins are composed of individual building blocks (amino acids) assembled in a chain, resembling beads on a string. This string – or sequence of amino acids – folds into a unique three-dimensional shape to form a fully functional protein. In nature, there are 20 different naturally occurring amino acids. I assigned specific musical chords to each different amino acid and arranged the chords sequentially in an order that mirrored the sequence of amino acids. The resulting composition contains a pattern of chords representative of the protein’s amino acid sequence. For example, if Glycine (one of the 20 natural amino acids) …

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, …

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. …

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 …

Characterizing The Behavior Of Mutated Proteins With Emcap: The Energy Minimization Curve Analysis Pipeline, Matthew Lee, Bodi Van Roy, Filip Jagodzinski

WWU Honors College Senior Projects

Studies of protein mutants in wet laboratory experiments are expensive and time consuming. Computational experiments that simulate the motions of protein with amino acid substitutions can complement wet lab experiments for studying the effects of mutations. In this work we present a computational pipeline that performs exhaustive single-point amino acid substitutions in silico. We perform energy minimization as part of molecular dynamics (MD) of our generated mutant proteins, and the wild type, and log the energy potentials for each step of the simulations. We motivate several metrics that rely on the energy minimization curves of the wild type and mutant, …

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 …

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 …

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 …

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 …

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- …

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 …

Role Of Protein Charge Density On Hepatitis B Virus Capsid Formation, Xinyu Sun, Dong Li, Zhaoshuai Wang, Panchao Yin, Rundong Hu, Rundong Hu, Hui Li, Qiao Liu, Yunyi Gao, Baiping Ren, Jie Zheng, Yinan Wei, Tianbo Liu

Chemistry Faculty Publications

The role of electrostatic interactions in the viral capsid assembly process was studied by comparing the assembly process of a truncated hepatitis B virus capsid protein Cp149 with its mutant protein D2N/D4N, which has the same conformational structure but four fewer charges per dimer. The capsid protein self-assembly was investigated under a wide range of protein surface charge densities by changing the protein concentration, buffer pH, and solution ionic strength. Lowering the protein charge density favored the capsid formation. However, lowering charge beyond a certain point resulted in capsid aggregation and precipitation. Interestingly, both the wild-type and D2N/D4N mutant displayed …

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 …