Biochemistry Commons^™

The Disordered Regulation Of Calcineurin: How Calmodulin-Induced Regulatory Domain Structural Changes Lead To The Activation Of Calcineurin, Victoria B. Dunlap

Theses and Dissertations--Molecular and Cellular Biochemistry

Calcineurin (CaN) is a highly regulated Ser/Thr protein phosphatase that plays critical roles in learning and memory, cardiac development and function, and immune system activation. Alterations in CaN regulation contribute to multiple disease states such as Down syndrome, cardiac hypertrophy, Alzheimer’s disease, and autoimmune disease. In addition, CaN is the target of the immunosuppressant drugs FK506 and cyclosporin A. Despite its importance, CaN regulation is not well understood on a molecular level. Full CaN activation requires binding of calcium-loaded calmodulin (CaM), however little is known about how CaM binding releases CaN’s autoinhibitory domain from the active site. Previous work has …

Structural Basis For Ternary Complex Formation Between Tau, Hsp90, And Fkbp51, Alexander Steven Barrett

USF Tampa Graduate Theses and Dissertations

The accumulation of the microtubule associated protein tau has been implicated in several neurological disorders; however, its interaction with chaperones along its normal degradation pathway remains largely uncharacterized at single residue resolution. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to probe the interaction between tau, the molecular chaperone Hsp90, and the immunophilin FKBP51. Resonance intensity changes were observed for specific residues in the heteronuclear single quantum coherence (HSQC) spectra of 15N-labeled tau in the presence of Hsp90 and/or FKBP51. Analysis of the HSQC spectra identified the two hydrophobic hexapeptide motifs located at residues V275 - K280 and …

Investigations Of Protein-Lipid Interactions In Model Membranes: Influence Of Aromatic Anchoring Residues And Buried Polar Residues, Nick Gleason

Graduate Theses and Dissertations

To investigate in detail the interactions between transmembrane proteins and the lipid bilayers in which they are constituted, designed model peptides with selective isotopic labels were synthesized and analyzed by means of solid-state deuterium NMR spectroscopy. Starting from the well-characterized model peptide GWALP23, acetyl-GGALW(LA)6LWLAGA-amide, several Trp to Tyr mutations were compared to evaluate their respective interfacial anchoring abilities. It was found that Tyr, substituted on either or both termini, can effectively anchor the transmembrane alpha-helix, which then adopts a similar transmembrane topology in a range of bilayer thicknesses. Nevertheless, a consistent ~10° shift in tilt direction (helix rotation) is observed …

"Fine-Tuning" Of Ribosomal Structure And Functions By Pseudouridylation And Rna-Protein Interactions, Jun Jiang

Wayne State University Dissertations

ABSTRACT

"Fine-tuning" of ribosomal structure and functions by pseudouridylation and RNA-protein interactions

by

JUN JIANG

AUGUST 2012

Advisor: Prof. John SantaLucia Jr.

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Ribosomal structure and functions appear to be "fine-tuned" by pseudouridylation and RNA-protein interactions. Pseudouridylation may promote base stacking interactions by mediating the base stacking between residues on both sides. In the RNA duplex region, this enhanced stacking interaction contributes to stabilization of duplex folding. In the loop region, enhanced stacking in one structural motif may destabilize the conformation of adjacent structural residues. This hypothesis is supported by both UV-melting experiments, where …

The Spliceosomal Protein Prp8 Stabilizes A Compact Conformation Of The U2-U6 Complex, Subasinghe Appuhamilage Lemintha Imali Subasinghe

Wayne State University Theses

The spliceosome is a large, RNA-protein complex that catalyzes pre-mRNA splicing during mRNA maturation. The RNA components (small nuclear RNA; snRNAs) of the spliceosome have been well studied and are believed to be involved in the splicing catalysis. Although proteins are essential for splicing, they may not be directly involved in catalysis. Among hundreds of proteins, Prp8 is the only protein that interacts with all of the catalytically important snRNAs. Therefore, it is hypothesized that Prp8 may catalyze splicing either by directly participating in catalysis or by stabilizing the conformation of the catalytically active spliceosome. In order to test whether …

Structural And Functional Characterization Of Dna Polymerase Ss Mutator Mutants, Sneha Rangarajan

Legacy Theses & Dissertations (2009 - 2024)

DNA Polymerase ß (polß) plays a crucial role in repairing damaged DNA in a process called Base Excision Repair (BER). BER is a major pathway of DNA repair, making this system absolutely vital for maintaining genomic integrity. Recent studies estimate 30% of human tumors to contain polß variants that led us to believe that there is a high degree of association between mutations in polß and cancer. In this pathway, after recognition and excision of the damaged base, the DNA is cleaved at an apurinic (AP) site by AP endonuclease leaving behind a 3' hydroxyl and 5' deoxyribose phosphate (dRP). …

Microfluidic Impedance Spectroscopy As A Tool For Quantitative Biology And Biotechnology, Ahmet C. Sabuncu, Jie Zhuang, Juergen F. Kolb, Ali Beskok

Mechanical & Aerospace Engineering Faculty Publications

A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to …

Microfluidic Separation Of Live And Dead Yeast Cells Using Reservoir-Based Dielectrophoresis, Saurin Patel, Daniel Showers, Pallavi Vedantam, Tzuen-Rong Tzeng, Shizhi Qian, Xiangchun Xuan

Mechanical & Aerospace Engineering Faculty Publications

Separating live and dead cells is critical to the diagnosis of early stage diseases and to the efficacy test of drug screening, etc. This work demonstrates a novel microfluidic approach to dielectrophoretic separation of yeast cells by viability. It exploits the cell dielectrophoresis that is induced by the inherent electric field gradient at the reservoir-microchannel junction to selectively trap dead yeast cells and continuously separate them from live ones right inside the reservoir. This approach is therefore termed reservoir-based dielectrophoresis (rDEP). It has unique advantages as compared to existing dielectrophoretic approaches such as the occupation of zero channel space and …

Protein Folding By 'Levels Of Separation': A Hypothesis, Lesley H. Greene, Terri M. Grant

Chemistry & Biochemistry Faculty Publications

The protein folding process has been studied both computationally and experimentally for over 30 years. To date there is no detailed mechanism to explain the formation of long-range interactions between the transition and native states. Long-range interactions are the principle determinants of the tertiary structure. We present a theoretical model which proposes a mechanism for the acquisition of these interactions as they form in a modified version of ‘degrees of separation’, that we term ‘levels of separation’. It is based on the integration of network science and biochemistry. (C) 2012 Federation of European Biochemical Societies.

Lipid Raft Formation And Peptide-Lipid Interactions In Myelin Model Membranes, Ashtina R. Appadu

Theses and Dissertations (Comprehensive)

Multiple sclerosis (MS), a demyelinating disease affecting 75,000 Canadians and almost 400,000 Americans, is one of the most prevalent diseases in young adults. Unfortunately, there exist no known cures to date and the pathways involved in the progression of the disease remain relatively obscure. The demyelination process triggered by the onset of MS, affects the lipid composition of the myelin membrane and causes a loss in viable myelin which can in turn greatly impact the proper functioning of the central nervous system (CNS). The cholesterol content of myelin fluctuates during MS and consequently this could affect the fluidity as well …

Conformational Changes In The Extracellular Domain Of Glutamate Receptors, Anu Rambhadran

Dissertations and Theses (Open Access)

The family of membrane protein called glutamate receptors play an important role in the central nervous system in mediating signaling between neurons. Glutamate receptors are involved in the elaborate game that nerve cells play with each other in order to control movement, memory, and learning.

Neurons achieve this communication by rapidly converting electrical signals into chemical signals and then converting them back into electrical signals. To propagate an electrical impulse, neurons in the brain launch bursts of neurotransmitter molecules like glutamate at the junction between neurons, called the synapse. Glutamate receptors are found lodged in the membranes of the post-synaptic …

Determining A Method For Rendering Low Cost Cdse(Zns) Core(Shell) Quantum Dots Aqueous Soluble Via Amphiphilic Polymer Wrapping, Patrick Mcbride

Materials Engineering

Herein is described the procedure of two amphiphilic polymer wrapping techniques that may be employed for obtaining aqueous soluble quantum dots (QDs) for use in biological fluorescent imaging applications. The advent of QDs has led to new nanoscale fluorescent materials that exhibit unparalleled quantum yields (QYs), high resistance to photobleaching, tunable emissions, and
absorption over a large optical range. However, the QD synthesis employed here at Cal Poly to obtain bright, photostable CdSe(ZnS) core(shell) QDs involves the use of organic solvents and surfactants, leading to hydrophobic QDs. Since all of biology relies on aqueous solubility, this hydrophobicity creates a major …

Protein-Lipid Interactions: Influence Of Anchoring Groups And Buried Arginine On The Properties Of Membrane-Spanning Peptides, Vitaly V. Vostrikov

Graduate Theses and Dissertations

Designed transmembrane peptides were employed for investigations of protein-lipid interactions by means of oriented solid-state deuterium NMR spectroscopy using isotope-enriched alanine residues. Using the model GWALP23 sequence (GGALW(LA)6LWLAGA) as a host peptide having single interfacial tryptophan anchor residues, the effects of different guest mutations were explored. Replacements of glycine residues 2 and 22 to positively charged lysine or arginine on both termini had little influence on the peptide average orientation. Conversely, glycine to tryptophan substitutions had profound effects, manifested in the increased dynamics and altered tilt direction of the peptide. While the charged residues at the peptide termini did not …

Functional Studies Of Human Cellular Detoxification Enzymes, Melanie Neely Willis

Department of Biochemistry: Dissertations, Theses, and Student Research

Cellular detoxification allows for the maintenance of cellular homeostasis and prevention of abnormal cell growth by clearing harmful xenobiotics and endobiotics. After oxygenation by phase I enzymes, phase II enzymes such as glucuronosyltransferases and glutathione-s-transferases conjugate a small molecule to the compound, marking it for subsequent export. Many up-stream enzymes are also essential to cellular detoxification by supplying the small compounds for conjugation. These up-stream enzymes include UDP-glucose dehydrogenase, which synthesizes UDP-glucuronate, and glutamate cysteine ligase, which catalyzes the first and rate-limiting step in the synthesis of glutathione.
UDP-glucose dehydrogenase (UGDH) is an important enzyme in human development and in …

Bioinformatics, Thermodynamics And Kinetics Analysis Of An All Alpha Helical Protein With A Gree-Key Topology, Hai Li

Chemistry & Biochemistry Theses & Dissertations

Computational and experimental studies focusing on the role of conserved residues for folding and stability is an active and promising area of research. To further expand our understanding we present the results of a bioinformatics analysis of the death domain superfamily. The death domain superfamily fold consists of six α-helices arranged in a Greek-key topology, which is shared by the all β-sheet immunoglobulin and mixed α/β-plait superfamilies. Our sequence and structural studies have identified a group of conserved hydrophobic residues and corresponding long-range interactions, which we propose are important in the formation and stabilization of the hydrophobic core and native …

Characterization Of Splicing Mechanisms By Single-Molecule Fluorescence, Krishanthi Sanjeewani Karunatilaka

Wayne State University Dissertations

Group II introns rank amongst the largest self-splicing ribozymes found in bacteria and organellar genomes of various eukaryotes. Despite the diversity in primary sequences, group II introns posses highly conserved secondary structures consisting of six domains (D1-D6). To perform its function, the large multidomain group II intron RNA must adopt the correctly folded structure. As a result, in vitro splicing of these introns requires high ionic strength and elevated temperatures. In vivo, this process is mainly assisted by protein cofactors. However, the exact mechanism of protein-mediated splicing of group II intron RNA is still not known.

In order to …

A Cell Electrofusion Microfluidic Device Integrated With 3d Thin-Film Microelectrode Arrays, Ning Hu, Jun Yang, Shizhi Qian, Sang W. Joo, Xiaolin Zheng

Mechanical & Aerospace Engineering Faculty Publications

A microfluidic device integrated with 3D thin film microelectrode arrays wrapped around serpentine-shaped microchannel walls has been designed, fabricated and tested for cell electrofusion. Each microelectrode array has 1015 discrete microelectrodes patterned on each side wall, and the adjacent microelectrodes are separated by coplanar dielectric channel wall. The device was tested to electrofuse K562 cells under a relatively low voltage. Under an AC electric field applied between the pair of the microelectrode arrays, cells are paired at the edge of each discrete microelectrode due to the induced positive dielectrophoresis. Subsequently, electric pulse signals are sequentially applied between the microelectrode arrays …

Homologous And Heterologous Crystallin Protein Interactions As The Molecular Basis Of Inherited Cataract, Priya Ranjan Banerjee

Legacy Theses & Dissertations (2009 - 2024)

Age-related cataract is the most common cause of blindness worldwide. Nearly fifty percent of Americans above the age of 75 are diagnosed with this disease (http://www.nei.nih.gov/eyedata/pbd_tables.asp), and surgical intervention is the sole method of treatment at present (http://www.nei.nih.gov/healthyvision/objective/cataracts.asp). In the developing world, even this treatment is not readily available. These are compelling reasons to search for better treatments to delay, prevent or arrest cataract formation. Recent evidence suggests that age-related cataracts also have a genetic component. Therefore, determining the mechanisms underlying genetic cataracts with a known association to a protein-mutation is one important strategy towards understanding the molecular basis for …

Mechanisms Of Decreased Cholesterol Absorption Mediated By Phytosterols In The Intestinal Lumen, Andrew W. Brown

Department of Nutrition and Health Sciences: Dissertations, Theses, and Student Research

Phytosterols and their fatty acyl esters have been known for decades to lower LDL cholesterol, making them powerful nutraceuticals in lowering cardiovascular disease risk. The mechanisms by which phytosterols lower cholesterol, though, have been incompletely characterized. Three studies were executed to examine three aspects of cholesterol and phytosterol interactions in the intestinal lumen. In the first study, the ability of pancreatic cholesterol esterase to hydrolyze phytosterol esters was examined. Pancreatic cholesterol esterase hydrolyzed phytosterol esters, but the rate of hydrolysis proved sensitive to the structures of both the sterol and ester components. In the second study, cholesterol micellarization was challenged …

Conformation And Ion Transport Of Neuronal Uncoupling Proteins: Ucp2, Ucp4, And Ucp5, Tuan Hoang

Theses and Dissertations (Comprehensive)

Located in the inner mitochondrial membrane of brown adipose tissue, uncoupling protein -1 (UCP1) dissipates the proton electrochemical gradient, causing reduction in the rate of ATP synthesis, and generates heat by non-shivering thermogenesis. Three other UCP homologs (UCP2, UCP4 and UCP5), expressed in neurons, are suggested to have potential roles in the function and protection of the central nervous system (CNS). Up to date, structural information for UCPs still remains limited. Extensive biochemical studies on UCP2 have provided adequate evidence for its participation in proton and anion transport. So far, no functional studies in proteoliposome systems have been performed on …

Tryptophan Anchored Peptides In Lipid Bilayer Membranes: Control Of Peptide Orientation And The Phase Behavior Of Cholesterol-Containing Ternary Lipid Mixtures, Johanna Maria Rankenberg

Graduate Theses and Dissertations

Model WALP peptides and "next generation" WALP-derived hydrophobic model peptides were employed to discover principles that govern protein-lipid interactions in biological membranes.

Ternary cholesterol-containing lipid mixtures were examined in the presence of WALP peptides of different lengths (acetyl-GWW(LA)_nLWWA-ethanolamide, with n between 3 and 8). Deuterium NMR spectra from labeled lipids reveal that WALP peptides may stabilize lipid ordered "raft" domains and therefore promote lipid phase separation, albeit to a minor extent. The results depend upon whether dioleolyl- or diphytanoyl-phosphatidylcholine is present as the fluid lipid component.

Several WALP peptides were modified to remove anchoring Trp residues from one …

Systematic Approach To Electrostatically Induced 2d Crystallization Of Nanoparticles At Liquid Interfaces, Sumit Kewalramani, Suntao Wang, Yuan Lin, Huong Giang Nguyen, Qian Wang, Masafumi Fukuto, Lin Yang

Faculty Publications

We report an experimental demonstration of a strategy for inducing two-dimensional (2D)crystallization of charged nanoparticles on oppositely charged fluid interfaces. This strategy aims to maximize the interfacial adsorption of nanoparticles, and hence their lateral packing density, by utilizing a combination of weakly charged particles and a high surface charge density on the planar interface. In order to test this approach, we investigated the assembly of cowpea mosaic virus (CPMV) on positively charged lipid monolayers at the aqueous solution surface, by means of in situX-ray scattering measurements at the liquid–vapor interface. Theassembly was studied as a function of the solution …

Negative Dielectrophoretic Capture Of Bacterial Spores In Food Matrices, Mehti Koklu, Seungkyung Park, Suresh D. Pillai, Ali Beskok

Mechanical & Aerospace Engineering Faculty Publications

A microfluidic device with planar square electrodes is developed for capturing particles from high conductivity media using negative dielectrophoresis (n-DEP). Specifically, Bacillus subtilis and Clostridium sporogenes spores, and polystyrene particles are tested in NaCl solution (0.05 and 0.225 S/m), apple juice (0.225 S/m), and milk (0.525 S/m). Depending on the conductivity of the medium, the Joule heating produces electrothermal flow (ETF), which continuously circulates and transports the particles to the DEP capture sites. Combination of the ETF and n-DEP results in different particle capture efficiencies as a function of the conductivity. Utilizing 20 μm height DEP chambers, “almost complete” and …

Conformational Change And Topological Stability Of Proteins, Jeffrey Andrew Tibbitt

Chemistry & Biochemistry Theses & Dissertations

The conformation and topology of a protein changes when stabilizing forces are absent, but the mechanisms by which these changes occur remains elusive. This dissertation aims to broaden the understandings. On the conformational level, the M20 loop conformers of E. coli dihydrofolate reductase are interrogated to identify factors responsible for their stability as well as to determine how one conformer might change into another. Molecular dynamics is used to simulate the open, closed and occluded conformers (observed in X-ray crystal structures) under a series of different single ligand conditions. Analysis shows that all open conformers move to a similar new …

Dielectrophoretic Choking Phenomenon In A Converging-Diverging Microchannel, Ye Ai, Shizhi Qian, Sheng Liu, Sang W. Joo

Mechanical & Aerospace Engineering Faculty Publications

Experiments show that particles smaller than the throat size of converging-diverging microchannels can sometimes be trapped near the throat. This critical phenomenon is associated with the negative dc dielectrophoresis arising from nonuniform electric fields in the microchannels. A finite-element model, accounting for the particle-fluid-electric field interactions, is employed to investigate the conditions for this dielectrophoretic (DEP) choking in a converging-diverging microchannel for the first time. It is shown quantitatively that the DEP choking occurs for high nonuniformity of electric fields, high ratio of particle size to throat size, and high ratio of particle's zeta potential to that of microchannel. © …

Human Cerebral Neuropathology Of Type 2 Diabetes Mellitus, Peter T. Nelson, Charles D. Smith, Erin L. Abner, Frederick A. Schmitt, Stephen W. Scheff, Gregory J. Davis, Jeffrey N. Keller, Gregory A. Jicha, Daron Davis, Wang-Xia Wang, Adria Hartman, Douglas G. Katz, William R. Markesbery

Pathology and Laboratory Medicine Faculty Publications

The cerebral neuropathology of Type 2 diabetes (CNDM2) has not been positively defined. This review includes a description of CNDM2 research from before the ‘Pubmed Era’. Recent neuroimaging studies have focused on cerebrovascular and white matter pathology. These and prior studies about cerebrovascular histopathology in diabetes are reviewed. Evidence is also described for and against the link between CNDM2 and Alzheimer's disease pathogenesis. To study this matter directly, we evaluated data from University of Kentucky Alzheimer's Disease Center (UK ADC) patients recruited while non-demented and followed longitudinally. Of patients who had come to autopsy (N = 234), 139 met …

Molecular Dynamics Study Of Single Stranded Peptide Nucleic Acids, Anna K. Manukyan

Theses and Dissertations in Biomedical Sciences

A PNA molecule is a DNA strand where the sugar-phosphate backbone has been replaced by a structurally homomorphous pseudopeptide chain consisting of N(2-aminoethyl)-glycine units. PNA binds strongly to both DNA and RNA. However, an analysis of the X-ray and NMR data show that the dihedral angles of PNA/DNA or PNA/DNA complexes are very different from those of DNA:DNA or RNA:RNA complexes. In addition, the PNA strand is very flexible. One way to improve the binding affinity of PNA for DNA/RNA is to design a more pre-organized PNA structure. An effective way to rigidify the PNA strand is to introduce …

Regulation Of Intracellular Calcium Concentration By Nanosecond Pulsed Electric Fields, Shaka S. Scarlett, Jody A. White, Peter F. Blackmore, Karl H. Schoenbach, Juergen Kolb

Bioelectrics Publications

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma …

Letter From The Dean, Lalit Verma

Discovery, The Student Journal of Dale Bumpers College of Agricultural, Food and Life Sciences

No abstract provided.

Pressure-Driven Transport Of Particles Through A Converging-Diverging Microchannel, Ye Ai, Sang W. Joo, Xiangchun Xuan, Shizhi Qian

Mechanical & Aerospace Engineering Faculty Publications

Pressure-driven transport of particles through a symmetric converging-diverging microchannel is studied by solving a coupled nonlinear system, which is composed of the Navier-Stokes and continuity equations using the arbitrary Lagrangian-Eulerian finite-element technique. The predicted particle translation is in good agreement with existing experimental observations. The effects of pressure gradient, particle size, channel geometry, and a particle's initial location on the particle transport are investigated. The pressure gradient has no effect on the ratio of the translational velocity of particles through a converging-diverging channel to that in the upstream straight channel. Particles are generally accelerated in the converging region and then …