Life Sciences Commons^™

Real-Time Sensing Of Single-Ligand Delivery With Nanoaperture-Integrated Microfluidic Devices, W. Elliott Martin, Ning Ge, Bernadeta R. Srijanto, Emily Furnish, C. Patrick Collier, Christine A. Trinkle, Christopher I. Richards

Chemistry Faculty Publications

The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor. We observed single ligands binding to EGFRs, …

Fret-Based Investigations Of The Structure-Function Relationships In The Nmda Receptor, Drew M. Dolino

Dissertations & Theses (Open Access)

The N-methyl-D-aspartate (NMDA) receptor is one member of a class of proteins known as the ionotropic glutamate receptors. Ionotropic glutamate receptors mediate the majority of excitatory neurotransmission in the central nervous system, with the NMDA receptor standing out among these receptors for its requirement of a co-agonist, its magnesium-block-based coincidence detection, its slow kinetics, its calcium permeability, its allosteric modulation, and its especially important functional roles in synaptic plasticity, excitotoxicity, and more. In recent years, a wealth of structural information has come about describing endpoint structures to high resolution, but such structures are unable to fully resolve the movements …

Using Fluorescence Lifetimes To Characterize Lipid Behavior In Nanodiscs, Cynthia Janku

Undergraduate Theses, Professional Papers, and Capstone Artifacts

Cellular uptake of molecules, including drugs, can be affected by the fluidity of the membrane. Nanoparticles have been hypothesized to alter membrane fluidity resulting in inflammation and its related clinical effects. Variations in phospholipids can alter membrane structure and its interaction with drugs or nanoparticles. To study membrane lipid differences and dynamics, we are using nanodiscs and liposomes as model systems. Nanodiscs are a lipid bilayer surrounded by a membrane scaffold protein, which is a derivative of Apolipoprotein A1, a protein involved in the removal of cholesterol from the body. There are important unresolved questions about how the belt protein …

Extraction, Purification And Partial Characterization Of A Carotenoid Binding Protein (Cbp) From The Epidermis Of The Monarch Butterfly Larvae (Danaus Plexippus), Nan Fang

FIU Electronic Theses and Dissertations

This dissertation describes the purification and partial characterization of CBP from the epidermis of the monarch butterfly larvae (Danaus plexippus). A yellow protein-carotenoid complex was extracted from the yellow pigmented epidermal tissue from monarch butterfly larvae by homogenization. Additional steps in the purification process included differential precipitation with ammonium sulfate, cation and anion chromatography, and lastly size exclusion chromatography. Polyacrylamide gel electrophoresis demonstrates that a single protein was isolated (M-LBP) having a ~60 kDa molecular weight, the value has subsequently been confirmed by HR-tandem MS. Lutein is the sole carotenoid bound by M-LBP with a stoichiometry of the …

Targeting Cancer: The Ph-Responsive Binding And Insertion Of Roxy7, Kristen Rae Booth

Chancellor’s Honors Program Projects

No abstract provided.

A Fret Investigation Into Molecular Mechanisms Of Cardiac Troponin Activation In Reconstituted Thin Filaments, Maria Eleni Moutsoglou

Electronic Theses and Dissertations

Cardiomyopathies (CM) are the leading cause of death in America, and can develop from mutations in sarcomeric proteins, leading to altered protein structure and function. Current therapies target upstream signaling pathways to treat the symptoms of heart failure, but are associated with increased mortality by affecting downstream signaling pathways and other muscle types. Rational drug design can develop therapies to treat CM at the protein level. However, a detailed knowledge of how sarcomeric proteins regulate muscle contraction is required. Muscle contraction occurs through a cyclic interaction between actin thin and myosin thick filaments, regulated by intracellular Ca2+ concentration. Troponin (Tn), …

Biophysical Characterization Of The Folding, Membrane Topology And Ion Transport Activity Of Ucp2 Using Selective Trp Mutants, Tyler C. Auld

Theses and Dissertations (Comprehensive)

Human Uncoupling Protein 2 (hUCP2) is one of five known human UCPs which are found in the inner mitochondrial membrane and have been shown to facilitate the translocation of protons from the intermembrane space to the mitochondrial matrix. The detailed physiological role of UCP2 proton transport, the mechanism by which it mediates this proton transport, as well as its structure has also yet to be elucidated. In order to help determine the topology of UCP2 embedded in the membrane as well as its mechanism of proton transport, the intrinsic fluorescence properties of the two tryptophan residues (Trp) present in its …

Purification And Characterization Of Bcsc; An Integral Component Of Bacterial Cellulose Export, Emily D. Wilson Ms

Theses and Dissertations (Comprehensive)

Biofilms are a growing concern in the medical field due to their increased resistance to antibiotics. When found in a biofilm, bacteria can have antibiotic resistance 10-1000 times that of their planktonic counterparts. Therefore, it is important to study the formation of biofilms. Cellulose biofilms are formed by Enterobacteriaceae, such as many Escherichia coli and Salmonella spp. strains. Biofilms provide these species with benefits including antimicrobial protection, development of bacterial communities, promotion of DNA exchange, uptake of nutrients, and, in the case of cellulose biofilms, immune system evasion. Cellulose biofilms are controlled by the Bacterial cellulose synthesis (Bcs) complex located …

Amylin Structure, Aggregation, And Pancreatic Β Cell Toxicity, Sharadrao Patil

MCB Articles

In most type 2 diabetes patients, amyloid plaques have been found juxtaposed with membranes of pancreatic β-cells. These plaques are composed of amyloid fibrils of the 37 residue endocrine hormone amylin and cause distinct changes in cell membrane morphology associated with the destruction of β-cells. Research is still ongoing to identify the toxic species involved and the mechanisms by which mature fibrils or oligomers cause cytotoxicity. The projects undertaken were designed to study the molecular structural features of amylin, mechanism of amyloid aggregation and, to develop cytotoxicity inhibitors. We determined the structure of human amylin bound to SDS micelles using …

The Effect Of Docosahexaenoic Acid (Dha)-Containing Phosphatidylcholine (Pc) On Liquid-Ordered And Liquid-Disordered Coexistence, Yongwen Gu

Dissertations and Theses

Plasma membranes are essential to both the structure and function of mammalian cells. The first unifying paradigm of membrane structure, the Fluid Mosaic Model, is no longer considered adequate to describe the many non-homogeneous lipid structures that have been observed in both natural and model membranes over the past approximately thirty years. The field of membrane biophysics now appreciates that the complex mixture of different lipid species found in natural membranes produces a range of dynamic, laterally segregated, non-homogeneous structures which exist on time scales ranging from microseconds to minutes.

When sphingomyelin (SM), POPC and cholesterol are all present in …

Megadalton-Node Assembly By Binding Of Skb1 To The Membrane Anchor Slf1, Lin L. Deng, Ruth Kabeche, Ning Wang, Jian-Qiu Wu, James B. Moseley

Dartmouth Scholarship

The plasma membrane contains both dynamic and static microdomains. Given the growing appreciation of cortical microdomains in cell biology, it is important to determine the organizational principles that underlie assembly of compartmentalized structures at the plasma membrane. The fission yeast plasma membrane is highly compartmentalized by distinct sets of cortical nodes, which control signaling for cell cycle progression and cytokinesis. The mitotic inhibitor Skb1 localizes to a set of cortical nodes that provide spatial control over signaling for entry into mitosis. However, it has been unclear whether these nodes contain other proteins and how they might be organized and tethered …

Interactions Of Eukaryotic Translation Initiation Factors And 3' Untranslated Region Of Barley Yellow Dwarf Virus Mrna During Protein Synthesis: A Study Of Equilibrium Binding, Kinetics And Thermodynamics, Bidisha Banerjee

Dissertations, Theses, and Capstone Projects

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley Yellow Dwarf Virus (BYDV) employs a cap-independent mechanism of translation initiation which is mediated by a structural element BTE (BYDV translation element) located in the 3’ UTR of its mRNA. eIF4F bound the BTE and a translational inactive mutant with high affinity; thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5-164% compared to WT were studied. Using fluorescence anisotropy …

Septin Assemblies Form By Diffusion-Driven Annealing On Membranes, Andrew A. Bridges, Huaiying Zhang, Shalin B. Mehta, Patricia Occhipinti, Tomomi Tani, Amy S. Gladfelter

Dartmouth Scholarship

Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make …

Detection Of Boronic Acids Through Excited-State Intramolecular Proton-Transfer Fluorescence, Matthew R. Aronoff, Brett Vanveller, Ronald T. Raines

Brett VanVeller

Boronic acids are versatile reagents for the chemical synthesis of organic molecules. They and other boron-containing compounds can be detected readily by the interruption of the excited-state intramolecular proton transfer (ESIPT) of 10-hydroxybenzo[h]quinolone. This method is highly sensitive and selective, and useful for monitoring synthetic reactions and detecting boron-containing compounds on a solid support.

From Loop To Strand: Characterization Of The Conformation And Dynamics Of The Human Plasminogen Activator Inhibitor-1 Reactive Center, Tihami Qureshi

Doctoral Dissertations

Plasminogen activator inhibitor-1 (PAI-1), with its cofactor vitronectin (VN), controls the rate of plasmin-mediated fibrin breakdown in blood clots by inhibiting tissue-plasminogen activator (tPA) and urokinase-plasminogen activator (uPA). The activity of PAI-1 is attributed to its reactive center loop (RCL), which is solvent-exposed in an active conformation, but inserts as an additional strand into its central β [beta]-sheet during transition to a latent state and during inhibition. VN slows the latency transition, and the rate at which PAI-1 inhibits the plasminogen activators (PAs) also differs. However, the steps during the latency transition, mechanism of VN stabilization, and basis for inhibitory …

Zinc Chemical Biology: The Pursuit Of The Intracellular Targets Of Zinquin, Andrew Nowakowski

Theses and Dissertations

Fluorescent sensors have been a main microscopic tools used to understand Zn2+ physiology on a cellular level. The use of the fluorescent Zn2+ sensor Zinquin (ZQ) and its analogues have revealed that transient Zn2+ is a chief component in a variety of biochemical pathways. Yet, little work has been performed to validate the exact targets of Zinquin in a cellular environment. The goals of this investigation are to determine the types of Zinquin reactions that take place in the cell as well as the identities of its cellular targets.

It has been hypothesized that Zinquin reacts with free Zn2+ within …

Investigating The Metal Binding Properties Of Plasminogen Activator Inhibitor Type 1 (Pai-1) With Intrinsic Tryptophan Fluorescence, Omar M. Alsharif

Chancellor’s Honors Program Projects

No abstract provided.

Biophysical Characterization Of Tryptophan Locales, Mg²+ Binding And Protein Folding In Gα Subunits, Matthew Najor

Dissertations

The objective of this study is to understand the structure of guanine nucleotide - binding (G) proteins using a variety of spectroscopic tools. G proteins are membrane-bound proteins consisting of α, β, and γ subunits required for the transduction of extracellular signals to various intracellular effectors. Activation of G protein coupled receptors by neurotransmitters or hormones result in a conformational change of a G protein that is triggered by the exchange of guanosine 5'- diphosphate (GDP) bound to the  subunit for guanosine 5'- triphosphate (GTP) and concomitant dissociation of the  dimer.

Wild type (WT) Giα1 has three tryptophan …

A Human Phospholipid Phosphatase Activated By A Transmembrane Control Module, Christian R. Halaszovich, Michael G. Leitner, Angeliki Mavrantoni, Audrey Le, Ludivine Frezza, Anja Feuer, Daniela N. Schreiber, Carlos A. Villalba-Galea, Dominik Oliver

School of Pharmacy Faculty Articles

In voltage-sensitive phosphatases (VSPs), a transmembrane voltage sensor domain (VSD) controls an intracellular phosphoinositide phosphatase domain, thereby enabling immediate initiation of intracellular signals by membrane depolarization. The existence of such a mechanism in mammals has remained elusive, despite the presence of VSP-homologous proteins in mammalian cells, in particular in sperm precursor cells. Here we demonstrate activation of a human VSP (hVSP1/TPIP) by an intramolecular switch. By engineering a chimeric hVSP1 with enhanced plasma membrane targeting containing the VSD of a prototypic invertebrate VSP, we show that hVSP1 is a phosphoinositide-5-phosphatase whose predominant substrate is PI(4,5)P(2). In the chimera, enzymatic activity …

Structural Studies Of Membrane-Assembled Popd And Popb, The Pseudomonas Aeruginosa Type 3 Secretion Translocators, Fabian B Romano Chernac

Doctoral Dissertations 1896 - February 2014

Transport of proteins across membranes is essential during many stages of pathogen infection and colonization of human cells. Many Gram-negative pathogens use a Type 3 Secretion (T3S) system to inject proteins into the target cell during infection. Substantial genetic and biochemical evidence suggest that proteins are translocated across the host plasma membrane through a proteinaceous pore or translocon formed by two bacterial secreted proteins: the T3S translocators. Despite its key role in pathogenesis, virtually nothing is known about the assembly mechanism, structure, and composition of this critical transmembrane complex.

To this end, a cell-free system for the structural and functional …

Conformational Properties Of Cardiolipin-Bound Cytochrome C, Jonas Hanske, Jason R. Toffey, Anna M. Morenz, Amber J. Bonilla, Katherine H. Schiavoni;, Ekaterina V. Pletneva

Dartmouth Scholarship

Interactions of cytochrome c (cyt c) with cardiolipin (CL) are important for both electron transfer and apoptotic functions of this protein. A sluggish peroxidase in its native state, when bound to CL, cyt c catalyzes CL peroxidation, which contributes to the protein apoptotic release. The heterogeneous CL-bound cyt c ensemble is difficult to characterize with traditional structural methods and ensemble-averaged probes. We have employed time-resolved FRET measurements to evaluate structural properties of the CL-bound protein in four dansyl (Dns)-labeled variants of horse heart cyt c. The Dns decay curves and extracted Dns-to-heme distance distributions P(r) …

Characterization Of The Desorption Electrospray Ionization Mechanism Using Microscopic Imaging Of The Sample Surface, Michael Craig Wood

Theses and Dissertations

Desorption electrospray ionization (DESI) is an ambient ionization technique for mass spectrometry. This solvent based desorption ion source has wide applicability in surface analysis with minimal sample preparation. Interest in improving detection limits, broadening applications, and increasing the spatial resolution for chemical imaging has led to studies of the DESI mechanism. An inverted microscope has been used to image interactions between the DESI spray and test analytes on a glass surface. Microscopic images recorded with millisecond time resolution have provided important insights into the processes governing analyte transport and desorption. These insights are the basis of a rivulet-based model for …

Identification Of The Allosteric Regulatory Site Of Insulysin, Nicholas Noinaj, Sonia K. Bhasin, Eun Suk Song, Kirsten E. Scoggin, Maria A. Juliano, Luiz Juliano, Louis B. Hersh, David W. Rodgers

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the Aβ peptide associated with Alzheimer's disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP.

PRINCIPAL FINDINGS: The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric …

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 …

Determining The Composition Of The Dwelling Tubes Of Antarctic Pterobranchs, Lukasz J. Sewera

Honors Projects

Pterobranchs are a group of marine invertebrates within the Hemichordata, which share characteristics with both chordates and echinoderms. Pterobranchs live in colonies of secreted tubes, coenicia, which are composed of a gelatinous material of unknown composition. Visually, the tubes appear similar to the tunic of tunicates, a group of invertebrates within the Chordata. The nonproteinaceous tunic of tunicates is composed of cellulose, which is unusual. The goal of this study was to determine the composition of the pterobranch coenicium. Some aspects of pterobranch phylogeny are still unclear even after multiple molecular and morphological studies. Identification of any new shared characteristics …

Identification Of Regions Responsible For The Open Conformation Of S100a10 Using Chimaeric S100a11/S100a10 Proteins, Liliana Santamaria-Kisiel

Electronic Thesis and Dissertation Repository

S100A11 is a dimeric, EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2), and facilitate membrane vesiculation events. In contrast to other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an “open” conformation that is very similar to S100A11 in its calcium-bound state (Ca2+-S100A11). To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimeric proteins were constructed where regions …

Requirements For Transitional Endoplasmic Reticulum Site Structure And Function In Saccharomyces Cerevisiae, Polina Shindiapina, Charles Barlowe

Dartmouth Scholarship

Secretory proteins are exported from the endoplasmic reticulum (ER) at specialized regions known as the transitional ER (tER). Coat protein complex II (COPII) proteins are enriched at tER sites, although the mechanisms underlying tER site assembly and maintenance are not understood. Here, we investigated the dynamic properties of tER sites in Saccharomyces cerevisiae and probed protein and lipid requirements for tER site structure and function. Thermosensitive sec12 and sec16 mutations caused a collapse of tER sites in a manner that depended on nascent secretory cargo. Continual fatty acid synthesis was required for ER export and for normal tER site structure, …

Determining The Rate Of Transcription Of T7 Rna Polymerase Using Single Molecule Fluorescence Imaging, Dawn Renee Nichola

Theses, Dissertations and Capstones

It is important to understand the many factors impacting the rate at which an RNA polymerase incorporates nucleotides. The transcription rate of T7 RNA polymerase has been determined using single molecule fluorescence microscopy. A Cy3 labeled circular 45nt ssDNA molecule was used to monitor the transcription process. T7 RNA polymerase was used because it is a single subunit polymerase that does not need any cofactors and will transcribe single-stranded DNA circles that do not contain a promoter. The transcription was monitored by measuring the quasi-periodic change in intensity associated with the transit of the probe through the polymerase as the …

Development Of Ultra-Sensitive Fluorescence Photoamplification Assays For The Detection Of Molecular Recognition Events, Tiffany Priscilla Gustafson

Electronic Theses and Dissertations

During the course of this research a novel method which couples the molecular recognition-triggered photoamplification chain in diaryl ketone adducts of dithiane with a "turn-off" or "turn-on" fluorescence-based assay for the detection of biological targets and ligands, regardless of their nature, through a molecular recognition event has been developed. This research has included several key steps, the most significant being: (1) the design of fluorophore adducts or dyads which recover fluorescence upon photocleavage for a "turn-on" assay and the identification of fluorophores which are quenched upon the photochemical release of a quencher for a "turn off" assay; (2) Optimization of …

Dendrimer Supramolecular Assembly For Gene Delivery, Karthikeyan Pasupathy

All Theses

Dendrimers have found many applications in the fields of polymer science, biophysics, nanomedicine and the petroleum industry. Poly(amidoamine) (PAMAM) was studied as a model dendrimer and squalane as a model hydrocarbon. The interaction between PAMAM and squalane is pH dependent. Specifically, at low or neutral pH the squalane is found on the periphery of the PAMAM while at high pH the hydrocarbon is entrapped inside the PAMAM molecules.

Single-molecule fluorescence revealed that the interaction between PAMAM and squalane is reversible. At a pH value of 8, the time constants for the approaching, binding and dissociation of single PAMAM to squalane …