Biochemistry, Biophysics, and Structural Biology Commons^™

On The Origins Of Life — Modelling The Initial Stages Of Complex Coacervate Droplet Formation, Yixuan Wu

Western Libraries Undergraduate Research Awards (WLURAs)

Coacervate droplets are considered a plausible model for protocells due to their spontaneous formation and ability to compartmentalize macromolecules such as nucleic acid and peptides. Although experimental studies have observed and synthesized coacervates under different laboratory conditions, little is known about their structure. Here we present atomistic molecular dynamic simulations of the interactions between water and oppositely charged proteins that cluster together in a salt-dependent process. Observing such liquid-liquid phase separation on an atomic level would serve as a model for the initial stages of complex coacervate formation. Molecular Dynamics was used to compute diagnostics of the structure at different …

Short-Term Versus Long-Term Effects Of Nitrogen Addition And Warming On Ecosystem N Dynamics In A Grass-Dominated Temperate Old Field, Benjamin F. A. Souriol

Electronic Thesis and Dissertation Repository

Increased atmospheric nitrogen (N) deposition and climate warming are both anticipated to influence the ecosystem N dynamics of northern temperate ecosystems substantially over the next century. Nevertheless, in field experiments with N addition and warming treatments, temporal scale can play an important role in determining the extent of treatment effects on N dynamics, and it is unclear to what extent the results of short-term studies can be extrapolated to responses over longer time scales. I compared the short-term versus long-term effects of N addition and warming on net N mineralization, N leaching, and N retention in a grass-dominated old field. …

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 …

Towards Clinical Microscopic Fractional Anisotropy Imaging, Nico Jj Arezza

Electronic Thesis and Dissertation Repository

Microscopic fractional anisotropy (µFA) is a diffusion-weighted magnetic resonance imaging (dMRI) metric that is sensitive to neuron microstructural features without being confounded by the orientation dispersion of axons and dendrites. µFA may potentially act as a surrogate biomarker for neurodegeneration, demyelination, and other pathological changes to neuron microstructure with greater specificity than other dMRI techniques that are sensitive to orientation dispersion, such as diffusion tensor imaging. As with many advanced imaging techniques, µFA is primarily used in research studies and has not seen use in clinical settings.

The primary goal of this Thesis was to assess the clinical viability of …

A Highly Charged Topic: Intrinsically Disordered Proteins And Protein Pka Values, Carter J. Wilson

Electronic Thesis and Dissertation Repository

Intrinsically disordered proteins (IDPs) are known not only for their roles in disease but also for their conformational flexibility, which makes them elusive for experimentation. We consider the role played by theory and simulation in resolving important questions pertaining to IDP structure and dynamics, as well as the nature of the charged residues (e.g., glutamate, lysine, etc.) that enrich them. Specifically, we investigated how the deep learning trained AlphaFold2 (AF2) predictor estimates disorder content, revealing both strong performance in relation to conventional approaches and an important relationship between the AF2 confidence metric and IDP dynamics. We also assessed how modern …

The Synthesis And Characterization Studies Of Modified Nucleobase In Pna And Dna, Gyeongsu Park

Electronic Thesis and Dissertation Repository

Nucleic acids have been extensively studied not only for their importance in biological systems as the medium of genetic information but also for their potential uses in therapeutic, diagnostic and other biological applications. As such, modified oligonucleotides and oligonucleotide analogues have drawn the attention of researchers from various disciplines. Modification of oligonucleotides can enhance their desired characteristics and engender unique properties, such as fluorescence, giving rise to a variety of applications. Peptide nucleic acid (PNA) is an oligonucleotide mimic with a pseudo-peptide backbone based on N-(2-aminoethyl)glycine that is renowned for high target binding affinity and resistance to enzyme degradation. …

Conformational Dynamics And Aggregation Of Thermally Stressed Proteins Studied By Hydrogen/Deuterium Exchange Mass Spectrometry, Nastaran Nosrat Tajoddin

Electronic Thesis and Dissertation Repository

Proteins perform various biological functions, e.g., as enzymes or transporters. In addition to naturally occurring proteins, the use of protein therapeutic drugs for treating cancer and other diseases is a rapidly growing area. A thorough biophysical characterization of proteins and protein therapeutics opens the door to a more comprehensive understanding of their role in health and disease. This dissertation aims to expand the capabilities of an existing technique (Hydrogen Deuterium Exchange Mass Spectrometry, HDX-MS), which is widely used for probing protein structure and dynamics. Conventionally, HDX-MS experiments are performed as a function of labelling time. Here we aim to establish …

Synthesis Of Nucleotide Prodrugs As Potential Antiviral Agents, Mohammed Attaelmanan

Undergraduate Student Research Internships Conference

No abstract provided.

Experimental And Computational Observations Of Immunogenic Cobalt Porphyrin Lipid Bilayers: Nanodomain-Enhanced Antigen Association., Jasmin Federizon, Conrard Giresse Tetsassi Feugmo, Wei-Chiao Huang, Xuedan He, Kazutoyo Miura, Aida Razi, Joaquin Ortega, Mikko Karttunen, Jonathan F Lovell

Chemistry Publications

Cobalt porphyrin phospholipid (CoPoP) can incorporate within bilayers to enable non-covalent surface-display of antigens on liposomes by mixing with proteins bearing a polyhistidine tag (his-tag); however, the mechanisms for how this occurs are poorly understood. These were investigated using the his-tagged model antigen Pfs25, a protein antigen candidate for malaria transmission-blocking vaccines. Pfs25 was found to associate with the small molecule aquocobalamin, a form of vitamin B12 and a cobalt-containing corrin macrocycle, but without particle formation, enabling comparative assessment. Relative to CoPoP liposomes, binding and serum stability studies indicated a weaker association of Pfs25 to aquocobalamin or cobalt nitrilotriacetic acid …

Effects Of Oxidative Modifications On The Structure And Non-Canonical Functions Of Cytochrome C Studied By Mass Spectrometry, Victor Yin

Electronic Thesis and Dissertation Repository

The peroxidase activity of the mitochondrial protein cytochrome c (cyt c) plays a critical role in triggering programmed cell death, or apoptosis. However, the native structure of cyt c should render this activity impossible due to the lack of open iron coordination sites at its heme cofactor. Despite its key biological importance, the molecular mechanisms underlying this structure-function mismatch remain enigmatic. The work detailed in this dissertation fills this knowledge gap by using mass spectrometry (MS) to decipher the central role that protein oxidative modifications and their associated structural changes play in activating the peroxidase function of cyt c …

Synthesis Of Crosslinkable Poly(Ester Amide)S For Cell Encapsulation And Delivery, Yu Ting (Natalie) Liang

Electronic Thesis and Dissertation Repository

Tissue engineering using adipose-derived stromal cells (ASCs) shows promise for soft tissue regeneration. Biodegradable polymers are potential biomaterials as they support the growth and delivery of cells. Specifically, poly(ester amide)s (PEAs) are a class of biodegradable polymers with tunable structures that have been shown to exhibit low cytotoxicity and support the growth of various cell types. This thesis involved the development of new water soluble amino acid-based PEAs with crosslinkable moieties to enable formation of hydrogel scaffolds for ASC encapsulation. These hydrophilic phenylalanine-based and alanine-based PEAs were synthesized by solution polycondensation and photo-crosslinked into a series of hydrogels with and …

Investigating The Rotary Mechanism Of Atp Synthase Using Molecular Dynamics Simulations, Angela Marcela Murcia Rios

Electronic Thesis and Dissertation Repository

F₁-ATPase is a motor protein that can use ATP hydrolysis to drive rotation of the central subunit. The γ C-terminal helix constitutes of the rotor tip that is seated in an apical bearing formed by the α₃β₃ head. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we develop a molecular dynamics (MD) strategy to model the off-axis forces acting on γ in F₁-ATPase. MD runs showed stalling of the …

Investigation Of Flow Disturbances And Multi-Directional Wall Shear Stress In The Stenosed Carotid Artery Bifurcation Using Particle Image Velocimetry, Amanda L. Dicarlo

Electronic Thesis and Dissertation Repository

Hemodynamics and shear forces are associated with pathological changes in the vascular wall and its function, resulting in the focal development of atherosclerosis. Flow complexities that develop in the presence of established plaques create environments favourable to thrombosis formation and potentially plaque rupture leading to stroke. The carotid artery bifurcation is a common site of atherosclerosis development. Recently, the multi-directional nature of shear stress acting on the endothelial layer has been highlighted as a risk factor for atherogenesis, emphasizing the need for accurate measurements of shear stress magnitude as well direction. In the absence of comprehensive patient specific datasets numerical …

Detection Of Amyloid Beta (Aβ) Oligomeric Composition Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (Maldi Ms)., Jasmine S-H Wang, Shawn N Whitehead, Ken K-C Yeung

Chemistry Publications

The use of MALDI MS as a fast and direct method to detect the Aβ oligomers of different masses is examined in this paper. Experimental results suggest that Aβ oligomers are ionized and detected as singly charged ions, and thus, the resulting mass spectrum directly reports the oligomer size distribution. Validation experiments were performed to verify the MS data against artifacts. Mass spectra collected from modified Aβ peptides with different propensities for aggregation were compared. Generally, the relative intensities of multimers were higher from samples where oligomerization was expected to be more favorable, and vice versa. MALDI MS was also …

A Chimeric Nucleobase - Phenylazo Derivative As An Intrinsic Nucleobase Quencher, Gyeongsu Park, Timothy Martin-Chan, Amer El Samm, Robert H.E. Hudson

Western Research Forum

Molecular beacons are important bioanalytical probes which are most often

constructed from a single-stranded oligonucleotide which has been labeled at

opposite termini with a fluorophore and a quencher. When the fluorophore and

quencher are in close proximity, no fluorescence is observed due to FRET

(Fluorescence Resonance Energy Transfer). DABCYL (4-dimethylaminoazobenzene-

4'-carboxylic acid) has been used as a quencher in the molecular beacon to absorbs

excitation energy from a fluorophore and to dissipate the energy as heat. However,

DABCYL is unable to form a base-pair and is conventionally placed as an overhanging

residue. This produces a derivative wherein the chromophore has …

Nbpmf: Novel Network-Based Inference Methods For Peptide Mass Fingerprinting, Zhewei Liang

Electronic Thesis and Dissertation Repository

Proteins are large, complex molecules that perform a vast array of functions in every living cell. A proteome is a set of proteins produced in an organism, and proteomics is the large-scale study of proteomes. Several high-throughput technologies have been developed in proteomics, where the most commonly applied are mass spectrometry (MS) based approaches. MS is an analytical technique for determining the composition of a sample. Recently it has become a primary tool for protein identification, quantification, and post translational modification (PTM) characterization in proteomics research. There are usually two different ways to identify proteins: top-down and bottom-up. Top-down approaches …

Metalation And Structural Properties Of Apo-Metallothioneins, Gordon W. Irvine

Electronic Thesis and Dissertation Repository

Metals are required by a quarter of all proteins to achieve their biological function, whether in an active site involved in catalytic chemistry or in a structural capacity. Metals are tightly regulated at the cellular level due to their propensity to cause unwanted side reactions and to be scavenged for use by pathogens. One of the proteins involved in this regulation of metal homeostasis is metallothionein (MT) which is a small, cysteine rich protein primarily involved in the regulation of zinc and copper homeostasis and heavy metal detoxification. MT is unique in its high cysteine content (~30% of the residues), …

Hyperpolarized 129xe Magnetic Resonance Imaging Of Radiation-Induced Lung Injury, Ozkan Doganay

Electronic Thesis and Dissertation Repository

Lung cancer is the largest contributor to cancer-related mortality worldwide. Only 20% of stage III non-small cell lung cancer patients survive after 5-years post radiation therapy (RT). Although RT is an important treatment modality for lung cancer, it is limited by Radiation-Induced Lung Injury (RILI). RILI develops in two phases: (i) the early phase (days-weeks) referred to radiation pneumonitis (RP), and (ii) the late phase (months). There is a strong interest in early detection of RP using imaging to improve outcomes of RT for lung cancer. This thesis describes a promising approach based on ¹²⁹Xe gas as a contrast …

Reactions Between Zinc Metallothionein And Carbonic Anhydrase, Tyler B. J. Pinter

Electronic Thesis and Dissertation Repository

More than 25% of proteins require metal ion cofactors for structure or function. The interactions between metalloproteins have largely been overlooked, though these interactions ultimately govern metal localization and control metal ion homeostasis. Mammalian metallothionein (MT) is a small, cysteine-rich metalloprotein that binds numerous metal ions per protein strand. Up to seven divalent metals, such as zinc or cadmium, are wrapped into a clustered two-domain structure. This unusually high metal content places MT as an attractive candidate for studying interactions with other metal-binding proteins. This present study investigates the metal transfer reactions between MTs and other metalloproteins, using carbonic anhydrase …

Complementary Mass Spectrometry Methods For Characterizing Protein Folding, Structure, And Dynamics, Siavash Vahidi

Electronic Thesis and Dissertation Repository

Proteins are involved in virtually every biochemical process. A comprehensive characterization of factors that govern protein function is essential for understanding the biomedical aspects of human health. This dissertation aims to develop complementary mass spectrometry-based methods and apply them to solve problems pertaining to the area of protein structure, folding and dynamics.

‎Chapter 1 uses fast photochemical oxidation of proteins (FPOP) to characterize partially disordered conformers populated under semi-denaturing conditions. In FPOP, ·OH generated by laser photolysis of H₂O₂ introduces oxidative modifications at solvent accessible side chains. By contrast, buried sites are protected from radical attack. Using …

Fabricating Cost-Effective Nanostructures For Biomedical Applications, Erden Ertorer

Electronic Thesis and Dissertation Repository

In this thesis we described inexpensive alternatives to fabricate nanostructures on planar substrates and provided example applications to discuss the efficiency of fabricated nanostructures.

The first method we described is forming large area systematically changing multi-shape nanoscale structures on a chip by laser interference lithography. We analyzed the fabricated structures at different substrate positions with respect to exposure time, exposure angle and associated light intensity profile. We presented experimental details related to the fabrication of symmetric and biaxial periodic nanostructures on photoresist, silicon surfaces, and ion-milled glass substrates. Behavior of osteoblasts and osteoclasts on the nanostructures was investigated. These results …

The Role Of Metallothionein In Zinc Homeostasis, Kelly L. Summers

Electronic Thesis and Dissertation Repository

The structure of the unique metal-binding protein, metallothionein (MT), consists of two metal-thiolate-clustered binding domains; the β-domain binds up to three divalent metals and the α-domain binds four. The mechanisms through which the metals are bound and arranged into domains, as well as the function of MT in metal ion homeostasis, remains largely unknown. By utilizing electrospray ionization mass spectrometry (ESI MS) to identify each species, and by comparing the data with simulations, MT 1a was found to bind Zn²⁺ non-cooperatively. Through a competition experiment between MT and its individual domain peptides, MT was proposed to bind Zn^{2+ …}

Integrated Development And Parallelization Of Automated Dicentric Chromosome Identification Software To Expedite Biodosimetry Analysis, Yanxin Li

Electronic Thesis and Dissertation Repository

Manual cytogenetic biodosimetry lacks the ability to handle mass casualty events. We present an automated dicentric chromosome identification (ADCI) software utilizing parallel computing technology. A parallelization strategy combining data and task parallelism, as well as optimization of I/O operations, has been designed, implemented, and incorporated in ADCI. Experiments on an eight-core desktop show that our algorithm can expedite the process of ADCI by at least four folds. Experiments on Symmetric Computing, SHARCNET, Blue Gene/Q multi-processor computers demonstrate the capability of parallelized ADCI to process thousands of samples for cytogenetic biodosimetry in a few hours. This increase in speed underscores the …

Structural Motifs Of Novel Metallothionein Proteins, Duncan E K Sutherland

Electronic Thesis and Dissertation Repository

Metallothioneins (MT) are a family of small cysteine rich proteins, which have been implicated in toxic metal detoxification, protection against oxidative stress, and as a metallochaperone. The most well studied member of the family is the mammalian MT, which consists of two domains: a β-domain with 9 cysteine residues, which sequesters 3 Cd²⁺/Zn²⁺, and an α-domain with 11 cysteine residues, which sequesters 4 Cd²⁺/Zn²⁺. The exact functions of MT are unknown but must relate to its metalation status. Several areas that could lead to the assignment of function include 1) the determination …

Molecular Dynamics Simulations Of Peptide-Mineral Interactions, Susanna Hug

Electronic Thesis and Dissertation Repository

We present molecular dynamics (MD) simulations providing information about the mechanisms of biomineralization. We focus on osteopontin-related peptides, which inhibit the growth of calcium oxalate monohydrate (COM) the primary constituent of kidney stones.

First, we performed two ab initio MD simulations: aspartic acid (Asp) and the dimer of aspartic acid and phosphoserine (Asp-pSer) interacting with a fully hydrated COM crystal slab exposing the {100} face. For Asp we found that one of the carboxyl and the amine group both interact with the crystal surface but neither forms a stable contact during the simulation. Asp-pSer interacts preferably with its carboxyl groups …

Structure And Dynamics Of The Membrane Protein Bacteriorhodopsin Studied By Mass Spectrometry, Yan Pan

Electronic Thesis and Dissertation Repository

Membrane proteins continue to represent a major challenge for most analytical techniques. Using bacteriorhodopsin (BR) as model system, this work aims to develop mass spectrometry (MS)-based approaches for exploring the structure, dynamics and folding of membrane proteins.

As the first step, BR in its native lipid environment was exposed to hydroxyl radicals, which were produced by laser photolysis of hydrogen peroxide. It was found that the resulting methionine (Met) labeling pattern was consistent with the known BR structure. This finding demonstrates that laser-induced oxidative Met labeling can provide structural information on membrane proteins. In subsequent experiments, the effects of different …

Development Of An Unbiased Statistical Method For The Analysis Of Unigenic Evolution, Colleen D. Behrsin, Chris J. Brandl, David W. Litchfield, Brian H. Shilton, Lindi M. Wahl

Biochemistry Publications

Background: Unigenic evolution is a powerful genetic strategy involving random mutagenesis of a single gene product to delineate functionally important domains of a protein. This method involves selection of variants of the protein which retain function, followed by statistical analysis comparing expected and observed mutation frequencies of each residue. Resultant mutability indices for each residue are averaged across a specified window of codons to identify hypomutable regions of the protein. As originally described, the effect of changes to the length of this averaging window was not fully eludicated. In addition, it was unclear when sufficient functional variants had been examined …