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From Bacteria To Human: Biophysical Studies Of Inward Rectifying Potassium Channels, Wayland Cheng
From Bacteria To Human: Biophysical Studies Of Inward Rectifying Potassium Channels, Wayland Cheng
All Theses and Dissertations (ETDs)
Inward rectifying potassium: Kir) channels are important in regulating cellular excitability in organs such as the heart, brain and pancreas. Prokaryotic KirBac channels are structurally similar to eukaryotic Kir channels, but distantly related and of unknown function. The goal of this thesis has been to investigate the functional properties of KirBac1.1 and to relate these findings to eukaryotic Kir channels. The approach was to use recombinantly-expressed, purified K+ channels--KirBac1.1, KcsA, Kir2.1 and Kir2.2--in order to integrate findings from functional studies, using liposomal flux assays and patch-clamping, with high-resolution crystal structures. By reconstituting KirBac1.1 into giant liposomes for patch-clamping, I show …
The Interaction Of Cofilin With The Actin Filament, Diana Wong
The Interaction Of Cofilin With The Actin Filament, Diana Wong
All Theses and Dissertations (ETDs)
The regulation of filamentous actin: F-actin) production from the polymerization of globular actin: G-actin) within the cell is critical for many cell functions. Since actin is found in all cells, understanding how actin-binding-proteins: ABPs) bind and how their regulating mechanisms work is not only important to the basics of cytoskeletal pathways, but also to understanding associated diseases and creating possible therapeutics to combat them. Cofilin is an ABP that plays an important part in the regulation process and in recent times, has come to be known as a player in maintaining a cell's homeostasis. It's activity has been shown to …
The Roles Of Vasoactive Intestinal Polypeptide In Circadian Entrainment Of Suprachiasmatic Nucleus, Sungwon An
The Roles Of Vasoactive Intestinal Polypeptide In Circadian Entrainment Of Suprachiasmatic Nucleus, Sungwon An
All Theses and Dissertations (ETDs)
In mammalian hypothalamus, the suprachiasmatic nucleus: SCN) generates daily behavioral and physiological rhythms as a circadian pacemaker. The 20,000 SCN neurons synchronize to each other and to the ambient cues to generate coherent daily rhythms. Vasoactive intestinal polypeptide: VIP), a neuropeptide produced by SCN neurons, plays a major role in synchronizing SCN neurons to each other. Whether VIP mediates synchrony to environmental cues and how synchrony within the SCN is achieved has not been examined extensively. We recorded PERIOD::LUCIFERASE: PER2::LUC) expression from SCN explant cultures over multiple days following VIP application at different circadian time points to generate a phase …
Delineating The Steps Of Bax Pore Activation, Eric Christenson
Delineating The Steps Of Bax Pore Activation, Eric Christenson
All Theses and Dissertations (ETDs)
The BCL2 protein family is the primary gatekeeper of mitochondrial apoptosis and governs integrity of the organelles' outer membranes. Permeabilization of mitochondrial outer membranes permits egress of cytochrome c and other apoptogenic factors, resulting in apoptosome formation, caspase activation, and subsequent proteolytic demolition of cells. Proapoptotic BAX & BAK effect the release of cytochrome c while their antiapoptotic counterparts like BCL-2, BCL-XL, & MCL-1 oppose this permeabilization. A third class of the BCL2 family, the prodeath BH3-only proteins, act as sentinels of cell stress and exert their influences by occupying antiapoptotic BCL2 members and/or activating BAX/BAK. Cell-free reconstitution …
The Structural Basis Of Flaviviridae Interaction With Antibodies And Receptors, Vincent Luca
The Structural Basis Of Flaviviridae Interaction With Antibodies And Receptors, Vincent Luca
All Theses and Dissertations (ETDs)
Flaviviridae are a family of enveloped, positive-stranded RNA viruses responsible for a variety of diseases including encephalitis, hemorrhagic fever and hepatocellular carcinoma. The envelope: E) proteins that coat the outer surface of these viruses provide the molecular machinery that drives receptor interaction and membrane fusion. The assignment of biological functions to specific structural elements of these E proteins has proven crucial to the understanding of viral entry into host cells. Clearance is dependent upon the presence of neutralizing antibodies that are able to disrupt several stages of this process. Given their fundamental role in the viral life cycle, we sought …
Single-Molecule Imaging Studies Of Protein Dynamics, Shannon Kian Zareh
Single-Molecule Imaging Studies Of Protein Dynamics, Shannon Kian Zareh
All Theses and Dissertations (ETDs)
Single-molecule fluorescence imaging is a powerful method for studying biological events. The work of this thesis primarily focuses on single molecule studies of the dynamics of Green Fluorescent Protein: GFP) and other fluorescent-labeled proteins by utilizing Total Internal Reflection Fluorescence: TIRF) microscopy and imaging. The single molecule experiments of this thesis covered three broad topics. First, the adsorption mechanisms of proteins onto hydrophobic and hydrophilic fused silica surfaces were imaged and reversible and irreversible adsorption mechanisms were observed. The second topic covered a new technique for measuring the diffusion coefficient of Brownian diffusing proteins, in particular GFP, in solution via …
Cellular And Cytoskeletal Responses Of Myofibroblasts In Three Dimensional Culture To Mechanical Stretch, Sheng-Lin Lee
Cellular And Cytoskeletal Responses Of Myofibroblasts In Three Dimensional Culture To Mechanical Stretch, Sheng-Lin Lee
All Theses and Dissertations (ETDs)
Myofibroblasts play important roles in wound healing and pathological organ remodeling, such as hypertensive cardiac fibrosis and promotion of metastasis. Differentiated myofibroblasts are characterized by increased production of extracellular matrix: ECM) proteins and by the development of α-smooth muscle actin: α-SMA) positive stress fibers that are connected to the ECM through focal adhesion assemblies. Moreover, mounting evidence suggests that development of myofibroblasts is profoundly influenced by the mechanical microenvironment, especially, by the structure, organization or stiffness of the ECM: Hinz and Gabbiani, 2003a). Myofibroblasts are likely signaled by mechanical changes in their environment transduced through their cytoskeletons, and the fundamental …
Digital Protein Analysis: Technologies For Protein Diagnostics And Proteomics Through Single-Molecule Detection, Lee Tessler
Digital Protein Analysis: Technologies For Protein Diagnostics And Proteomics Through Single-Molecule Detection, Lee Tessler
All Theses and Dissertations (ETDs)
Protein quantification tools are valuable in biological and biomedical research and in clinical applications because they enable measurements that elucidate physiological states, including disease states. Protein biomarker detection is likely to play a major role in patient health and aid in the personalization of medicine. However, protein detection has lagged behind other bio-analytical methods due to intrinsic properties of proteins as well as the complexity of biological fluids, such as blood. This thesis describes the design, development, and testing of several technologies for the advancement of protein detection in clinical and research settings. A common thread through these technologies is …
Mass Spectrometry For Determination Of Conformation And Dynamics Of Proteins And Structure And Biosynthesis Of Bacterial Peptidoglycan, Jiawei Chen
All Theses and Dissertations (ETDs)
Mass spectrometry: MS) has emerged as an important tool for analyzing and characterizing large biomolecules. In this dissertation, two aspects of the development and application of MS-based approaches are presented; they include: 1) protein conformation and folding dynamics: in Chapters 2 to 5) and bacterial peptidoglycan: PG) structure and biosynthesis: Chapters 6 to 8). Chapter 1 serves as the introduction for both aspects. Part I of the dissertation focuses on the development of analytical methods combining fast photochemical oxidation of proteins: FPOP) and mass spectrometry analysis. In chapter 2 to 4, we discuss protein folding with sub-millisecond time resolution by …
Molecular Mechanism Of The Allosteric Coupling For Ca(2+) Activation Of The Voltage- And Ca(2+)-Activated K(+) (Bk) Channels, Junqiu Yang
All Theses and Dissertations (ETDs)
The large conductance voltage- and Ca2+-activated K+: BK) channel is important for many physiological functions. The BK channel is activated by both membrane depolarization and intracellular Ca2+ so that it integrates these two important cellular signals. Ca2+ sensing of the BK channel is mediated by two binding sites, both located on the cytosolic domain. The structure of the cytosolic domain indicates that both binding sites are away from the pore-gate domain, suggesting that a coupling mechanism between the binding sites and the gate is required for Ca2+-dependent activation. The work in this dissertation focuses on the coupling mechanism, which can …
Mass Spectrometry-Based Structural Proteomics: Bottom-Up Protein Footprinting And Top-Down Native Electrospray Of Protein Assemblies, Hao Zhang
All Theses and Dissertations (ETDs)
Converting gene-sequence information into functional information about a protein is a major challenge of post-genomic biology. Proteins have a variety of functions from serving as catalysts to acting as structural components; all these functions are closely related to protein structure. The first step to understand protein function is often a structural study of that protein. Two major approaches, NMR spectroscopy and X-ray crystallography, can provide an atomic-level, 3D structural model of a protein. The applications of these high resolution approaches, however, are limited by protein size, conformational flexibility, and aggregation propensity. To obtain complementary structural information about proteins, a variety …
Structural And Functional Studies On Thrombin Allostery, Prafull Gandhi
Structural And Functional Studies On Thrombin Allostery, Prafull Gandhi
All Theses and Dissertations (ETDs)
Thrombin is an allosteric serine protease endowed with important physiological functions in the blood. Once activated, α-thrombin plays procoagulant, prothrombotic, and anticoagulant functions in blood. Its important role in blood coagulation can be emphasized by the fact that thrombin interacts with numerous substrates, cofactors, ligands, and inhibitors. Two well-documented allosteric pathways, involving the Na+ binding site and the exosite I, regulate thrombin catalytic efficiency and substrate specificity. Identifying structural epitopes of substrate binding, the exact pathway and the molecular basis of allosteric regulation remains challenging. The case of protease-activated receptor: PAR1) is particularly relevant in view of the plethora of …
Ultrasonic Characterization Of Human Coronary Arteries And Atherosclerotic Plaques, Joseph Hoffman
Ultrasonic Characterization Of Human Coronary Arteries And Atherosclerotic Plaques, Joseph Hoffman
All Theses and Dissertations (ETDs)
The development, execution, and interpretation of studies investigating the physics of the interaction of ultrasound with normal and pathological coronary artery tissues are described in this dissertation. Ultrasound is a modality capable of visualizing and characterizing the lesions that define atherosclerosis. A better understanding of the physics underlying the mechanisms by which ultrasound interacts with arterial tissue and plaques may provide benefit to patients with coronary artery disease. The two-fold goal of the studies presented in this thesis was to better understand the fundamental physics of the relationship between ultrasound and coronary artery tissue and to use this knowledge to …
Determination Of The Structural And Functional Mechanisms Of Perfluorocarbon-Based Nanoemulsion Particle Interactions With Model Biomembrane Systems, Sun Joo Lee
All Theses and Dissertations (ETDs)
Perfluorocarbon based nanoemulsion particles: PFC-NEPs) are very small sized: ~ 250 nm in diameter) greasy droplets that are enclosed by emulsifying phospholipid monolayer. PFC-NEPs have been extensively developed for target-specific delivery of therapeutic agents such as imaging agents and drug molecules. Because of the extremely small sizes of PFC-NEPs and fluid nature of their surface, the structure of these particles at atomic resolutions has yet to be determined by experimental approaches. The aim of this thesis is to determine the atomistic structure of PFC-NEP interfaces with a particular focus on their interaction with model target bilayers. The goal of this …
Fcs Analysis Of Pore Formation By The Human Protein Bax In Lipid Membranes, Olena Ivashyna
Fcs Analysis Of Pore Formation By The Human Protein Bax In Lipid Membranes, Olena Ivashyna
All Theses and Dissertations (ETDs)
Bax is a pro-apoptotic member of the BCL-2 protein family and its paramount function during apoptosis is to form pores in the mitochondrial outer membrane: MOM). In a simplified apoptosis model, upon apoptotic stimulation, Bax is activated via an unresolved mechanism, translocates to the MOM, where it changes conformation, inserts into the membrane as a monomer, and finally undergoes in-membrane oligomerization to form a pore. Once the Bax pore is formed, cytochrome c and other mitochondria-resident proteins escape into the cytoplasm where they activate the cascade of caspases, which dismantle the cell. To date, the structure of the inactive cytoplasmic …
Examination Of Molecular Recognition In Protein-Ligand Interactions, Yat Tang
Examination Of Molecular Recognition In Protein-Ligand Interactions, Yat Tang
All Theses and Dissertations (ETDs)
This dissertation is a compilation of two main projects that were investigated during my thesis research. The first project was a prospective study which identified and characterized drug-like inhibitors of a prototype of bacterial two-component signal transduction response regulator using computational and experimental methods. The second project was the development and validation of a scoring function, PHOENIX, derived using high-resolution structures and calorimetry measurements to predict binding affinities of protein-ligand interactions. Collectively, my thesis research aimed to better understand the underlying driving forces and principles which govern molecular recognition and molecular design. A prospective study coupled computational predictions with experimental …
Transient-State Kinetic Studies Of The Mechanisms Of Dna Unwinding And Translocation Of The E. Coli Recbc And Recbcd Helicases, Colin Wu
All Theses and Dissertations (ETDs)
This thesis presents mechanistic studies of the E. coli RecBC and RecBCD helicases using transient-state kinetic approaches to understand the relationship between DNA unwinding and ssDNA translocation. RecBC initiates unwinding from duplex DNA ends with pre-existing 5'-(dT)6 and 3'-(dT)6 ssDNA tails using a series of repeated rate-limiting steps with a rate of 345 ┬▒ 8 bp/sec and an average kinetic step-size of ~4 bp: 20 mM Mops-KOH, pH 7.0 at 25°C, 30 mM NaCl, 10 mM MgCl2, 5% glycerol, 1 mM 2-mercaptoethanol) while RecBCD unwinds these same DNA substrates with a rate of 745 ┬▒ 18 bp/sec using a more …
Structural And Functional Studies On Bk(Ca) Channels, Akansha Saxena
Structural And Functional Studies On Bk(Ca) Channels, Akansha Saxena
All Theses and Dissertations (ETDs)
The long term goal of this research is to study the structure and function of the BKCa channels, by focusing on the effect of a single residue mutation, the epilepsy mutation. BKCa channels are potassium channels, activated by voltage, Ca2+ and Mg2+ ions. These factors control the opening and closing of the channel pore and thus regulate the large K+ current passing through them. Recently, a mutation D434G in humans, was found to make the channel hyperactive and more sensitive to the Ca2+ ions. The single residue mutation, resulting from a substitution of an Asp to Gly, was …
An Examination Of Oxysterol Effects On Membrane Bilayers Using Molecular Dynamics Simulations, Brett Olsen
An Examination Of Oxysterol Effects On Membrane Bilayers Using Molecular Dynamics Simulations, Brett Olsen
All Theses and Dissertations (ETDs)
My thesis work focuses on understanding the role of biological membranes in cellular processes. I am particularly interested in how small molecules perturb membrane structure and regulate membrane function. Membranes are primarily responsible for segregating different compartments of biological cells from each other. However, they have other roles as well, including influencing signal transduction in the cell, both through associated membrane proteins and directly through alterations in membrane organization. In order to understand how small molecules change membranes, I use molecular dynamics simulations of small simplified membranes to examine, at an atomic-level detail, how the addition of small molecules to …
Structure And Function Of The Fmo Protein From The Photosynthetic Green Sulfur Bacteria, Jianzhong Wen
Structure And Function Of The Fmo Protein From The Photosynthetic Green Sulfur Bacteria, Jianzhong Wen
All Theses and Dissertations (ETDs)
The Fenna-Matthews-Olson: FMO) bacteriochlorophyll a protein has served as a model antenna system for understanding pigment-protein interaction and the energy transfer mechanism. The FMO protein has been extensively studied by a wide range of spectroscopic and theoretical techniques due to its stability, spectral resolution of pigments, water-soluble nature and availability of high-resolution structural information. A new 1.3 ├à FMO structure: PDB: 3EOJ) revealed an 8th pigment at the monomer connection region with partial occupancy. To understand the nature and stoichiometry of this new pigment, the molecular weight of the whole FMO complex was measured by the recently developed mass spectrometry: …
Unique Features Of Ptb Rrms: Insight Into Protein Motions And Rna Binding, Caroline Maynard
Unique Features Of Ptb Rrms: Insight Into Protein Motions And Rna Binding, Caroline Maynard
All Theses and Dissertations (ETDs)
Polypyrimidine tract binding protein: PTB) is a highly conserved RNA binding protein comprised of four RRMs: RNA recognition motifs). RRMs are extremely prevalent in all kingdoms of life, and have been very well characterized in terms of structure and RNA binding properties. However, all four RRMs of PTB exhibit unique features that can be exploited to learn more about the RNA selection and binding strategy of PTB, as well more general features involving structure/function relationships and protein unfolding mechanisms. PTB participates in a variety of functions in eukaryotic cells, including alternative splicing, mRNA stabilization, and internal ribosomal entry site: IRES) …
Probing The Early Stages Of Polyglutamine Aggregation With Computational Methods, Andreas Vitalis
Probing The Early Stages Of Polyglutamine Aggregation With Computational Methods, Andreas Vitalis
All Theses and Dissertations (ETDs)
Exonic CAG repeat diseases are a class of neurodegenerative age-of-onset diseases caused by an unstable trinucleotide expansion in a coding region of a gene. The most prominent example is Huntington's disease: HD) whose symptoms are characterized by loss of motor control and cognitive deficits. For all nine of the known CAG repeat diseases, pathology is ascribed to the mutant proteins which carry expanded stretches of glutamine residues: polyglutamine). The length of the polyglutamine segment is inversely correlated with the disease age-of-onset. Protein aggregates are routinely found in postmortem tissue samples of brains of HD patients. These findings suggest a prominent …
Advances In Computational Solvation Thermodynamics, Matthew Wyczalkowski
Advances In Computational Solvation Thermodynamics, Matthew Wyczalkowski
All Theses and Dissertations (ETDs)
The aim of this thesis is to develop improved methods for calculating the free energy, entropy and enthalpy of solvation from molecular simulations. Solvation thermodynamics of model compounds provides quantitative measurements used to analyze the stability of protein conformations in aqueous milieus. Solvation free energies govern the favorability of the solvation process, while entropy and enthalpy decompositions give insight into the molecular mechanisms by which the process occurs. Computationally, a coupling parameter λ modulates solute-solvent interactions to simulate an insertion process, and multiple lengthy simulations at a fixed λ value are typically required for free energy calculations to converge; entropy …
Computational And Experimental Investigation Of Allosteric Communication In The Transcriptional Regulator Nikr, Michael Bradley
Computational And Experimental Investigation Of Allosteric Communication In The Transcriptional Regulator Nikr, Michael Bradley
All Theses and Dissertations (ETDs)
The Ni2+ and DNA binding protein NikR is involved in nickel regulation in Escherichia coli through transcriptional repression of the NikABCDE nickel permease. NikR is a homotetramer and each chain contains both a DNA binding ribbon-helix-helix: RHH) domain and a Ni2+ binding regulatory ACT: aspartokinase, chorismate mutase, TyrA) fold. Work herein combines computational modeling of NikR structure with experimental studies aimed at understanding allosteric communication between the ACT and RHH domains. Hydrogen/deuterium exchange mass spectrometry shows a Ni2+ specific NikR conformational change relative to bound Cu2+, Co2+, and Zn2+. Concurrent coordination geometry and in vivo repressor function studies show that …