Browsing by Author "Tatyana Smirnova, Committee Member"

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Binding and Local Dynamics of Iso-1-cytochrome c to Substrate-Supported Lipid Nanotube Arrays
(2004-01-15) Smith, Shani Jomella; Edmond Bowden, Committee Member; Alex Smirnov, Committee Chair; Tatyana Smirnova, Committee Member
A study of the binding and local dynamics of iso-1-cytochrome c to lipid nanotube arrays composed of 1,2-Diheptadecanoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DOPG), an anionic phospholipid on an nanoporous anodic aluminum oxide (AAO) substrate was undertaken to gain an understanding of the effects of nanoscale confinement on protein binding. Iso-1-cytochrome c was spin-labeled with 1-oxyl-2,2,3,3-tetramethylpyrolline-3-methylmethanesulfonate (MTSL) at cysteine 102. The binding and local dynamics of the spin-labeled iso-1-cytochrome c bound to multilamellar liposomes was compared with the binding to lipid nanotube arrays. Location of spin-labeled cysteine 102 of iso-1-cytochrome c with respect to the bilayer was probed using an EPR oxygen accessibility experiment. CD monitored changes in the heme coordination in iso-1-cytochrome c upon binding to DOPG.
Characterization and analytical applications of dye-encapsulated zwitterionic liposomes
(2010-02-16) Yitbarek, Emnet; Morteza Khaledi, Committee Chair; Edmond Bowden, Committee Member; Tatyana Smirnova, Committee Member; Alexander Nevzorov, Committee Member
The aim of this project was to use marker encapsulated liposomes as biomembrane mimicking entities in order to study membrane properties like permeability and to better understand the interaction of biological lipid bilayers with membrane-active molecules, like beta blocker drugs and antimicrobial peptides (AMP). The physical characteristics of liposomes, such as size, surface charge and encapsulation capacity were also studied using electrophoretic, fluorescence and light scattering techniques. In addition, marker-encapsulated and self-lysing liposomes were used to study antigen-antibody binding. The immunoassay application of these self-lysing liposomes was also investigated. The first area of research is focused on investigating the effect of the liposome lipid composition on the size and the electrical properties of zwitterionic liposomes. The cholesterol composition of phosphatidylcholine (PC) and sphingomyelin (Sph) liposomes is varied and the effect on their size, zeta potential and electrophoretic mobility is monitored using dynamic light scattering (DLS), laser doppler velocimetry (LDV), and capillary zone electrophoresis (CZE) techniques, respectively. In addition, the permeability and the encapsulation capacity of large unilamellar vesicles (LUV), or liposomes that are made by extrusion, were compared as their lipid and cholesterol composition varied. The size and electrophoretic mobility of zwitterionic liposomes was found to increase with the cholesterol composition. The interaction of indolicidin, a 13-mer cationic AMP, with (dye-encapsulated) liposomes that were made of different lipid and cholesterol composition was investigated by DLS, fluorescence and capillary electrophoresis (CE) methods. DLS results show a change in liposome size, and size distribution index (PI), after indolicidin interaction. Fluorescence leakage experiments show the extent of membrane perturbation caused by the AMP and the AMPÃ¢â‚¬â„¢s innate tryptophan fluorescence provided qualitative information regarding the type (polar/non-polar) and nature of the liposome-AMP interaction, as lipid composition of the liposomes varied. In addition, CZE and liposome electrokinetic chromatography (LEKC) techniques were also used to further probe the (polar/non-polar/electrostatic) nature of this interaction. The immunoassay application of the marker encapsulated liposomes was investigated using a combination of fluorescence, DLS, and CE-LIF (capillary electrophoresis with laser induced fluorescence detector) techniques. The liposomes were made from a non-lamellar lipid DOPE (dioleoylphosphatidylethanolamine) that was stabilized with a 20% bilayer lipid DPPC (dipalmitoylphosphatidylcholine) and a 1% hapten-attached DPPE lipid. Small hapten molecules, like biotin and DNP (dinitrophenyl), were attached to the liposome surface via the DPPE lipid, and used to detect their conjugate molecules (avidin and anti-DNP antibody) in a homogeneous solution. The biotin-attached DOPE liposomes aggregate and leaked their marker content in standard avidin solution. The extent of liposome aggregation and the fluorescence intensity of the leaked dye are dependent on the concentration of avidin present in solution. The different parameters that affect the quality of the assay were also investigated.
Control of Biological Processes with Modified Nucleosides, Amino Acids, and Small-Molecule Probes
(2010-07-29) Lusic, Hrvoje; Paul Agris, Committee Member; Tatyana Smirnova, Committee Member; Jonathan Lindsey, Committee Member; Bruce Novak, Committee Member; Alex Deiters, Committee Chair
Several novel caging groups with a wide range of applications were designed. The caging groups were applied to the synthesis of molecular tools for control of gene expression. A number of RNA, DNA, and antisense-oligomer subunits were prepared. The caged nucleosides were site-specifically incorporated into corresponding oligonucleotide strands, using standard automated-synthesis protocols. The caged nucleosides were found to readily photolyse under biologically non-damaging UV-light (>365 nm). Spatio-temporal control over oligomersâ€™ biological and/or catalytic activity was demonstrated with excellent on(or off) photo-triggered switches. Additionally, non-standard nucleosides 5-formylcytidine and N-[(9-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine were synthesized for incorporation into tRNA to elucidate the importance of the non-standard nucleoside modifications on codon recognition in RNA translation. Furthermore, tools for control over protein functions were designed. A set of novel unnatural amino acids were successfully synthesized and genetically (in vivo) incorporated into proteins using evolved orthogonal tRNA/synthetase pairs. Lastly, several caged protein kinase inhibitors were prepared, and used to study importance of kinase enzymatic activity in embryonic development of Xenopus laevis.
Donor-Acceptor Contributions to Ferromagnetic Exchange Coupling within Heterospin Biradicals
(2006-12-08) Brannen, Candice Leigh; Paul Maggard, Committee Member; David A. Shultz, Committee Chair; T. Brent Gunnoe, Committee Member; Tatyana Smirnova, Committee Member
The evaluation of new Donor-Acceptor heterospin biradicals is carried out within this thesis: SQ-Gal, SQ-Ph-Gal, SQ-E-NN, and SQ-TNN. A molecular orbital and spin density analysis is performed in order to predict the exchange coupling within these biradicals. A Valence Bond Configuration Interaction model is used to describe exchange coupling in a Donor-Acceptor-type Semiquinone-NitronylNitroxide (SQNN) biradical ligand. Within this framework, a SQ → NN charge transfer state is responsible for the experimentally determined ferromagnetic coupling in the ground state, and the intraligand exchange parameter is correlated with the single-site Coulomb repulsion integral (U), the singlet-triplet gap (2K) of the CT excited state, and the electronic coupling matrix element, Hab. This charge transfer transition is observed spectroscopically, and probed using resonance Raman spectroscopy. Importantly, if the crystal structure of the Donor-Acceptor biradical is known, this Hab can be directly linked to the molecular conformation determined by X-ray crystallography. We suggest how our methods could be extended to any bridge fragment for SQ-Bride-NN complexes. This model is useful for designing new strongly exchange coupled systems, for using electronic structure theory to study exchange in organic systems, and for correlating exchange coupling parameters with elements of electron transfer theory.
Structural Studies of Inhibitor and Substrate Binding in the Hemoglobin Dehaloperoxidase
(2010-08-07) Davis, Michael Foster; Tatyana Smirnova, Committee Member; Steven Lommel, Committee Member; Edmond Bowden, Committee Member; Stefan Franzen, Committee Chair
Dehaloperoxidase (DHP) is a dual function heme protein found in the marine polychaete Amphitrite ornata. A. ornata is filter feeding worm that inhabits estuary inlets alongside other annelids such as Notomastus lobatus and Thelepus crispus, which secrete various haloaromatics theoretically as a means of territorial protection. N. lobatus, in particular, expels mono-, di-, and trihalogentated phenols. Even though DHP is one of two hemoglobins found in A. ornata, the protein possesses significant peroxidase activity and is capable of oxidatively dehalogenating certain halophenols found in its environment. The ability of DHP to bind monohalogenated phenols in an internal distal cavity separates the protein from any other known globin. A variety of spectroscopic and enzymatic techniques have been utilized to probe halophenol binding in DHP. In order to perform these techniques, codon optimization of the DHP gene was first performed. 1H NMR experiments on low-spin metcyano DHP revealed separate modes of binding between mono-, di-, and trihalogentated phenols. Specifically it was found that binding of mono- and dihalogenated phenols occurs in the internal binding pocket, while trihalogenated phenols bind at a second, external site. This led to enzymatic studies that revealed inhibition of DHP peroxidase activity upon monohalogenated phenol binding in the internal pocket. NMR experiments on the protein backbone of 13C and 15N labeled DHP show trihalogenated substrates induce chemical shift deviations in the distal histidine H55 NÎµH and amide protons near tryptophan 120. This indicates binding of trihalophenols may occur on the external side of H55 resulting in allosteric changes at the dimer interface, or that binding may occur directly at W120.
The Synthesis and Characterization of Ester-Bearing Polycarbodiimides.
(2010-06-18) Clark, Joe; Bruce Novak, Committee Chair; Marian McCord, Committee Member; Tatyana Smirnova, Committee Member; Lin He, Committee Member