Biochemistry, Biophysics, and Structural Biology Commons^™

Caulobacter Clpxp Adaptor Popa’S Domain Interactions In The Adaptor Hierarchy Of Ctra Degradation, Thomas P. Scudder

Masters Theses

The degradation and recycling of protein is a process essential for the maintenance and regulation of cellular function. More specifically, in Caulobacter crescentus, the ClpXP protease is responsible for driving progression through the cell cycle and protein quality control. This protease utilizes three known adaptors to selectively degrade proteins that initiate different stages of development. This thesis will elaborate on the specific binding interface on one of these adaptors, PopA, with another, RcdA, and focus in on specific residues on PopA and investigate their roles in adaptor binding and delivery of CtrA, the master regulator of Caulobacter. Finally, I …

Investigating The Helicase Activity Of Methylated Vs Unmethylated Ded1, Hannah Lukow

Honors Theses

Ded1 is an RNA helicase protein of the DEAD-box subfamily in eukaryotic organisms (Sharma & Jankowsky, 2014) which can act as an activator or initiation factor, during translation (Hilliker et al., 2011). Ded1 has several functions in yeast including assembly of translational initiation factors, scanning the mRNA for the start codon, and unwinding any double stranded segments of mRNA with its helicase ability. Ded1 was discovered to be methylated at four arginine sites in vivo (Low et al., 2013), with a fifth methylation site being discovered recently (Low et al., 2020), however the purpose of such post-translational modifications is still …

Characterizing The Roles Of The Variable Linker And Hub Domains In Camkii Activation, Noelle Dziedzic

Doctoral Dissertations

Learning and memory formation at the cellular level involves decoding complex electrochemical signals between nerve cells, or neurons. Understanding these processes at the molecular level requires a comprehensive study of calcium-sensitive proteins that serve as signal mediators within cells. More specifically, the protein calcium/calmodulin-dependent protein kinase II (CaMKII) is a key regulator of downstream cellular signaling events in the brain, playing an important role in long term memory formation. CaMKII is encoded in humans on four different genes: alpha, beta, gamma and delta. For added complexity, each of these gene products can be alternatively spliced and translated into multiple protein …

Mistranslating Trnas Alter The Heat Shock Activation By Hsf1, Rebecca Dib

Undergraduate Student Research Internships Conference

Translation, or the production of protein from an mRNA blueprint, is among the most fundamental processes to life as we know it. tRNAs are essential to accurate translation, as they decode the codons of mRNA and recruit corresponding amino acids. Variant tRNAs with anticodon mutations can decrease translational fidelity by recruiting the incorrect amino acid, an aberrant process known as mistranslation. When proteins are produced with incorrect amino acid sequences, they may misfold. The heat shock response functions to alleviate cellular stress caused by misfolded proteins, either by refolding or targeting misfolded proteins for degradation. Hsf1 acts as a transcriptional …

Expression And Purification Of E. Coli Yoaa, A Putative Helicase, Mark Gregory, Vincent Sutera Mr., Susan Lovett Dr.

Medical Student Research Symposium

All cells must maintain their genomic integrity to survive, which they achieve through several repair mechanisms that necessitate unwinding the damaged DNA by helicases. In Escherichia coli (E. coli), YoaA has been genetically shown to be involved in DNA repair and shares conserved sequences with helicase DinG. The goal of our study was to purify YoaA for further biochemical characterization. For expression, YoaA was fused to a His tag and overexpressed in MG1655 E.coli under the lacZ or T7 promoters for 2 hours, 4 hours, or overnight at 24oC, 30oC or 37oC. For purification, crude lysate was applied to a …

Computational Investigation Of Calmodulin Photocontrol With The Help Of An Azobenzene Derivative, Jeremy Wells

Undergraduate Honors Theses

The ability to control the activity and binding capability of enzymes in a reversible manner offers tremendous control over biological processes. Photocontrol, in particular, is promising in that electromagnetic radiation can be fine-tuned in terms of its strength, location, and duration. Photosensitive compounds, such as the azobenzene family, experience an isomerization at certain wavelengths, and attaching these compounds to enzymes has the potential to alter their structure and activity. Calmodulin (CaM) is a Ca2+ -sensitive signaling protein that has the ability to affect several downstream processes in eukaryotes and is an excellent target for photocontrol due to its small size …

Retro-Structural Analysis Of The Four Helix Bundle Motif In Binuclear Proteins, Walker Pedigo, Maggie Smith

Honors Theses

Protein structure is directly related to protein function. There are four levels of protein structure: primary, secondary, tertiary, and quaternary. The interactions amongst the structural components of a protein give rise to its unique characteristics. The four helix bundle motif is a common structural trait in a variety of binuclear proteins. In this study, PyMOL, a molecular visualization system, was used to analyze binuclear proteins that possess a four helix bundle. Images of proteins containing dicopper, diiron, and dimanganese sites were captured. The images were compiled into figures for each individual protein. After creating the figures, each protein was further …

Small Heat Shock Protein 27 And Its Role In Human Disease, Bianka Andrea Holguin

Open Access Theses & Dissertations

Small heat shock protein 27 (Hsp27) is a ubiquitously expressed molecular chaperone with roles in many physiological processes. As an ATP-independent molecular chaperone, Hsp27 protects substrates from irreversible aggregation and holds them in a folding competent state for later recycling into the proteome. Hsp27 proteins form dimers that are assembled into large oligomeric complexes. Phosphorylation of Hsp27 dissembles the oligomers into chaperone active dimers. Several missense mutations of Hsp27 are causative for the neurodegenerative disorders Charcot-Marie-Tooth disease 2F and distal Hereditary Motor Neuropathy IIB. Here I show that the oligomerization and chaperoning ability of Hsp27 are altered by the Hsp27 …

Computational Algorithms For Predicting Membrane Protein Assembly From Angstrom To Micron Scale, Nandhini Rajagopal

Dissertations - ALL

Biological barriers in the human body are one of the most crucial interfaces perfected through evolution for diverse and unique functions. Of the wide range of barriers, the paracellular protein interfaces of epithelial and endothelial cells called tight junctions with high molecular specificities are vital for homeostasis and to maintain proper health. While the breakdown of these barriers is associated with serious pathological consequences, their intact presence also poses a challenge to effective delivery of therapeutic drugs. Complimenting a rigorous combination of in vitro and in vivo approaches to establishing the fundamental biological construct, in addition to elucidating pathological implications …

Computational Algorithms For Predicting Membrane Protein Assembly From Angstrom To Micron Scale, Nandhini Rajagopal

Dissertations - ALL

Biological barriers in the human body are one of the most crucial interfaces perfected through evolution for diverse and unique functions. Of the wide range of barriers, the paracellular protein interfaces of epithelial and endothelial cells called tight junctions with high molecular specificities are vital for homeostasis and to maintain proper health. While the breakdown of these barriers is associated with serious pathological consequences, their intact presence also poses a challenge to effective delivery of therapeutic drugs. Complimenting a rigorous combination of in vitro and in vivo approaches to establishing the fundamental biological construct, in addition to elucidating pathological implications …

The Role Of Nutrition And Hormone Signaling In Extended Larval Development And Obesity In Starvation-Selected Drosophila Melanogaster, Jennifer M. Clark

UNLV Theses, Dissertations, Professional Papers, and Capstones

Brief periods of starvation are a common stressor that most animals encounter in the wild and must be able to survive in order to maximize their fitness. Starvation resistance of the adult fruit fly, Drosophila melanogaster, is thought to be primarily conferred by adult fat stores, body size, metabolic rate, behavior, and activity levels. Additionally, flies selected for starvation resistance also often show delayed pupariation, which is usually indicative of altered hormone signaling. How starvation selection extends development and if it contributes to adult starvation resistance remains incompletely studied. Identifying the targets of starvation selection that cause extended development and …

Investigating Structural Proteins By Light Scattering, Uma Nudurupati

Masters Theses

This thesis evaluates the organization of the structural proteins, Human Gamma D crystallin and Collagen type II, into higher-order structures using light scattering. Specifically, it evaluates the natures of incipient aggregation in Human Gamma D crystallin and the nature of its interactions with CAPEGn, an electrostatic blocker. Additionally, this thesis evaluates whether Collagen type II growth kinetics follows Classical nucleation theory.

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 …

Living With It: A Patient’S And A Biochemist’S Perspective On Kidney Disease; A Historical Review Of Alport Syndrome, Jacob Olson

WWU Honors College Senior Projects

A review paper on the origin of health studies around Alport Syndrome, including aspects of genetics, pharmacy, and biochemistry, from the past to today and beyond. This report deals with important aspects of health development with regards to kidney disease overall, but narrows its focus on Alport Syndrome specifically due to the personal nature of the topic for the author. While this paper includes no personal testimony, as it is strictly meant to be formal, the author shares a deep connection with the material.

Single-Fluorophore Sensors For Mechanical Force In Living Cells, Sarah Kricheff

Honors Scholar Theses

Mechanotransduction is the process by which a mechanical stimulus is converted to a cellular signal. This process is heavily influential of cell morphology, differentiation, and behavior. However, altered levels of mechanical stimuli are also found in many pathological contexts. For example, cancerous cells have stiffer surrounding tissue than healthy cells, and research suggests that this alters cell behavior and promotes metastasis. Despite these findings, the cellular processes behind these signaling alterations remain widely unknown. Understanding these cascades is critical, as involved proteins can give us a deeper understanding of the role of mechanotransduction, and certain proteins can potentially be targeted …

A Chemical Interpretation Of Protein Electron Density Maps In The Worldwide Protein Data Bank, Sen Yao, Hunter N. B. Moseley

Molecular and Cellular Biochemistry Faculty Publications

High-quality three-dimensional structural data is of great value for the functional interpretation of biomacromolecules, especially proteins; however, structural quality varies greatly across the entries in the worldwide Protein Data Bank (wwPDB). Since 2008, the wwPDB has required the inclusion of structure factors with the deposition of x-ray crystallographic structures to support the independent evaluation of structures with respect to the underlying experimental data used to derive those structures. However, interpreting the discrepancies between the structural model and its underlying electron density data is difficult, since derived sigma-scaled electron density maps use arbitrary electron density units which are inconsistent between maps …

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 …

Crystallization Efforts For An Engineered Nickel-Binding Protein, Gold-Bovine Serum Albumin Nanoclusters, And An Artificial De Novo Tetramer Hydrogenase Mimic, Skyler Crane

Honors Theses

Protein crystallization is fundamental to modern research efforts given its ability to determine a protein’s structure as well as the interactions that structure allows and relies upon. This process, though lacking direct application, provides necessary information for subsequent research efforts for which applicationsmay be explored. As such, efforts were taken to crystallize nickel-binding protein (NBP) reengineered from Copper Storage Protein 1 (Csp1) in its apo and metal bound form, Bovine Serum Albumin (BSA) in its apo and gold bound form (Au-BSA), and an artificial de novo tetramer hydrogenase mimic peptide to better inform future research actions for these respective molecules. …

Investigation Of Potentially Catalytic Residues Of Uba5 Through Mutagenesis, Purification, And Structural Characterization, Grant Bradley

Senior Honors Projects, 2020-current

Ubiquitin-fold modifier 1 (Ufm1) is a member of the Ubiquitin (Ub) family of proteins whose primary function is degradation of proteins through a sequential mechanism of chemical reactions. Though Ufm1’s specific roles are largely unknown, this family of proteins has shown to play a part in a wide variety of processes, including regulation of the cell cycle¹, secretory functions of cells^2,3, and blood clotting⁴. Ufm1’s mechanism of action proceeds with the aid of three enzymes: an E1, E2, and E3. Uba5 is the E1 activating enzyme that is specific to Ufm1, and its mechanism of …

Cloning And Expression Of Hydra Innexin 2, A Gap Junction Protein Required For Coordinated Contraction Of The Body Column, Ashley O'Brien

Biology Theses

In invertebrates gap junctions are formed by the innexin family of proteins. Remarkably, the genome of Hydra magnipapillata contains 17 innexin genes. This study focused on Hydra innexin-2 (h-Inx2) which is expressed in nerve cells and plays a role in contraction of the body column. The gene sequence of H-Inx2 was obtained from the National Center for Biotechnology Information (NCBI), the gene was synthesized externally and transferred to a vector suitable for expression in Xenopus oocytes (pcDNA3.1 CT-GFP TOPO). The TOPO CT-GFP vector includes a priming site for RNA polymerase which allows in vitro preparation of RNA. Another advantage is …

Mechanisms Of Statin Effects On Muscle And Neuronal Proteostasis, Daniel Yu

University Scholar Projects

Statins are widely prescribed and used chronically, but we know little about the effects on long-term protein homeostasis during stress and aging. Our aim was to quantify the effect of statins on stress-induced protein damage. We administered atorvastatin in a dose-response curve in Caenorhabditis elegans under naïve control conditions and in conditions of hypertonic and heat stress known to induce muscle damage measurable as countable puncta in a polyglutamine aggregation model of damage. We observed that there is significant polyglutamine aggregation variability among worms at baseline and thus further study requires within experiment baseline controls, per worm. Our results are …

Characterization Of Avidin And Case9 Single Protein Molecules By A Solid-State Nanopore Device, Haopeng Li

Graduate Theses and Dissertations

The shape and charge of a protein play significant roles in protein dynamics in the biological system of humans and animals. Characterizing and quantifying the shape and charge of a protein at the single-molecule level remains a challenge. Solid-state nanopores made of silicon nitride (SiNx) have emerged as novel platforms for biosensing such as diagnostics for single-molecule detection and DNA sequencing. SSN detection is based on measuring the variations in ionic conductance as charged biomolecules translocate through nanometer-sized channels driven by an external voltage applied across the membrane. In this paper, we observe the translocation of asymmetric cylindrical structure CRISPR-Cas9 …

The Classification Of An Unknown Protein 3h04, Britney Dyszel

Biochemistry and Molecular Biology Presentations

An unclassified protein in the Protein Data Bank, 3H04, was selected as a protein of interest. Research was performed in silico to gain key information on the structure, sequence, and homology of protein 3H04. This research was guided using the BASIL Project’s protocols on in silico research. Several databases were utilized to study protein 3H04. Based on the data gathered, unclassified protein 3H04 is an aminopeptidase that cleaves prolyl-dipeptidyl peptide bonds through alpha beta hydrolase function.

A Proteomic Analysis Of Corydoras Sterbai Secretions And Tissues, Erik Powell Wictor

University of the Pacific Theses and Dissertations

Defensive mechanisms vary widely in the animal kingdom ranging from physical defenses like spines to chemical defenses such as toxins. Toxins in these secretions and tissues can fluctuate from enzymes to lipids to uncharacterized chemicals. Next generation -omics technology and mass spectrometry are extremely important in analyzing these samples because of their ability to distinguish minute amounts of toxic substance within a complicated sample. The goal of this experiment was to look at secretions and tissues from Corydoras sterbai. All samples in this study were proteolyzed using a mixture of Trypsin and Lys-C, fractionated, and run through nanoLC-MS/MS analysis using …

Single Molecule Fluorescence Studies Of Protein Structure And Dynamics Underlying The Chloroplast Signal Recognition Particle Targeting Pathway, Dustin R. Baucom

Graduate Theses and Dissertations

The work presented in this dissertation explores the structural dynamics in the chloroplast signal recognition particle pathway. Findings include cpSRP shows scanning functionality similar to that in the cytosolic SRP with the ribosome. The intrinsically disordered C-terminal tail of the Albino3 protein has some transient secondary structure. Upon binding to cpSRP43 in solution, separate secondary structure formation was identified in the C-terminal tail of Albino3. Finally, to increase efficiency of analyzing fluorescence time traces for this work, a modular software was produced.

Multiple Cytosolic Dna Sensors Bind Plasmid Dna After Transfection, Nina Semenova, Masa Bosnjak, Katarina Znidar, Maja Cemazar, Loree Heller

Bioelectrics Publications

Mammalian cells express a variety of nucleic acid sensors as one of the first lines of defense against infection. Despite extensive progress in the study of sensor signaling pathways during the last decade, the detailed mechanisms remain unclear. In our previous studies, we reported increased type I interferon expression and the upregulation of several proposed cytosolic DNA sensors after transfection of several tumor cell types with plasmid DNA (pDNA). In the present study, we sought to reveal the early events in the cytosolic sensing of this nucleic acid in a myoblast cell line. We demonstrated that DNA-dependent activator of interferon …

Synthesis, Stabilization, And Modification Of Zinc Oxide Nanoparticles For Biological Applications, Allison Kimberly Freese

MSU Graduate Theses

Nanoparticles have become very useful as delivery systems in biomedicine. The nanoparticles can be layered with different compounds to produce a vessel for transport of biological materials. Specifically, gold nanoparticles layered with a reducing agent, lysozyme, and polyelectrolytes can be synthesized to transport lysozyme into a cell. However, zinc oxide nanoparticles are cheaper, biocompatible nanoparticles that can be used for the same process. Here in, zinc oxide nanoparticle conjugates were synthesized, modified, and analyzed to be used as a biological material delivery system. The zinc oxide nanoparticles were synthesized using zinc chloride and sodium hydroxide. The particles were then layered …

Protein-Protein Interactions In Perilpin 5, Erin K. Hughes

Undergraduate Honors Thesis Projects

Many metabolic diseases contribute to a major part of the health crisis in the US. Type II diabetes mellitus and non-alcoholic fatty liver disease are two examples of metabolic diseases that are contributing to the current health crisis. Key to understanding these diseases and their progression is an understanding of neutral lipid metabolism. Perilipins are a class of five conserved proteins that are key regulators of lipid metabolism and are potentially involved in lipid trafficking within cells. The most recently discovered member of the family is perilipin 5, which is expressed most strongly in tissues that are highly oxidative such …

Computational Analysis Of Large-Scale Trends And Dynamics In Eukaryotic Protein Family Evolution, Joseph Boehm Ahrens

FIU Electronic Theses and Dissertations

The myriad protein-coding genes found in present-day eukaryotes arose from a combination of speciation and gene duplication events, spanning more than one billion years of evolution. Notably, as these proteins evolved, the individual residues at each site in their amino acid sequences were replaced at markedly different rates. The relationship between protein structure, protein function, and site-specific rates of amino acid replacement is a topic of ongoing research. Additionally, there is much interest in the different evolutionary constraints imposed on sequences related by speciation (orthologs) versus sequences related by gene duplication (paralogs). A principal aim of this dissertation is to …

Automatic 13C Chemical Shift Reference Correction Of Protein Nmr Spectral Data Using Data Mining And Bayesian Statistical Modeling, Xi Chen

Theses and Dissertations--Molecular and Cellular Biochemistry

Nuclear magnetic resonance (NMR) is a highly versatile analytical technique for studying molecular configuration, conformation, and dynamics, especially of biomacromolecules such as proteins. However, due to the intrinsic properties of NMR experiments, results from the NMR instruments require a refencing step before the down-the-line analysis. Poor chemical shift referencing, especially for ¹³C in protein Nuclear Magnetic Resonance (NMR) experiments, fundamentally limits and even prevents effective study of biomacromolecules via NMR. There is no available method that can rereference carbon chemical shifts from protein NMR without secondary experimental information such as structure or resonance assignment.

To solve this problem, we …