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Studies Of Intermediates In Snare-Induced Membrane Fusion , Fan Zhang Jan 2006

Studies Of Intermediates In Snare-Induced Membrane Fusion , Fan Zhang

Retrospective Theses and Dissertations

The purpose of this course of study is to understand the intermediates during SNAREs assembly and the membrane fusion mediated by SNAREs. SNAREs have been proposed to be the minimal machinery for the membrane fusion. According to the location distribution, SNAREs can be classified as either v-(vesicles) or t-(target) SNAREs. The assembly of three SNARE components from the opposing membranes into the ternary complex is believed to provide the ultimate driving force for merging the separate bilayers into a continuous entity. Before this step the t-SNAREs associate as the binary complex on the target membrane to serve as ...


Cloning, Expression, And Characterization Of A Recombinant Bifunctional Protein -- Porcine Air Carboxylase And Saicar Synthetase , Xin Gao Jan 2006

Cloning, Expression, And Characterization Of A Recombinant Bifunctional Protein -- Porcine Air Carboxylase And Saicar Synthetase , Xin Gao

Retrospective Theses and Dissertations

The conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR) by phosphoribosylaminoimidazole carboxylase (AIR carboxylase) and the conversion of conversion of ATP, L-aspartate and CAIR to 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR), ADP and phosphate by phosphoribosylaminoimidazolesuccinocarboxamide synthetase (SAICAR synthetase) represent the 7th and 8th steps respectively of de novo purine nucleotide biosynthesis. For the vertebrate enzyme system, AIR carboxylase and SAICAR synthetase are combined in a bifunctional protein encoded by a single gene. Here, a porcine gene encoding AIR carboxylase/SAICAR synthetase was cloned and expressed in Escherichia coli. The native molecular weight of the purified recombinant protein infers an octameric ...


Structural And Functional Studies On Snares-Mediated Membrane Fusion , Yong Chen Jan 2006

Structural And Functional Studies On Snares-Mediated Membrane Fusion , Yong Chen

Retrospective Theses and Dissertations

Membrane fusion is an essential biological process for vesicle trafficking in eukaryotic cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of proteins that have been recognized as key components of protein complexes that drive membrane fusion. The SNARE proteins localized in opposite membranes form a SNARE complex, which bridges the two membranes into close proximity and promotes fusion. Despite considerable sequence divergence among SNARE proteins, this fusion machinery is conserved structurally, mechanistically and evolutionally from yeast to human;Site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) are well established techniques to study the structures of membrane ...


Release Of Human Brain Hexokinase From The Mitochondrial Membrane , David Andrew Skaff Jan 2006

Release Of Human Brain Hexokinase From The Mitochondrial Membrane , David Andrew Skaff

Retrospective Theses and Dissertations

Type I hexokinase (HKI) is the pacemaker of glycolysis in the brain and red blood cell. Recent work has tied hexokinase association with the mitochondrial membrane to the prevention of apoptosis. With increased hexokinase activity and expression in tumor cells, the importance in understanding the release of HKI from the membrane has become extremely important. Glucose 6-phosphate and nucleotides have been shown to effectively release HKI from the membrane; however, the mechanisms by which these molecules can induce release have never been studied;Expressed recombinant HKI protein was lacking the N-terminus that is required for binding to the mitochondrial membrane ...


Molecular And Genetic Characterization Of A Biotin Biosynthetic Gene In Arabidopsis Encoding Both 7,8-Diaminopelargonic Acid Aminotransferase And Dethiobiotin Synthetase , Elve Chen Jan 2006

Molecular And Genetic Characterization Of A Biotin Biosynthetic Gene In Arabidopsis Encoding Both 7,8-Diaminopelargonic Acid Aminotransferase And Dethiobiotin Synthetase , Elve Chen

Retrospective Theses and Dissertations

Biotin is an essential enzyme cofactor required for different metabolic processes. Plants are a major source of biotin, however, the genes and the protein products of biotin biosynthetic pathway are not fully characterized. We have molecularly characterized the genetically defined Arabidopsis bio1 locus as encoding 7,8-diaminopelargonic acid (DAPA) aminotransferase. Molecular and genetic analysis revealed that the full length BIO1 cDNA is capable of producing a fusion protein that catalyzes not only DAPA aminotransferase, but also dethiobiotin synthetase (BIO3) which is another enzymatic step in the biotin biosynthetic pathway. We refer to this fusion-gene as BIO3/BIO1. This bifunctional enzyme ...


Signaling Capabilities Of A Novel H-Ras Mutant From The Golgi Apparatus , Daniel R. Zamzow Jan 2006

Signaling Capabilities Of A Novel H-Ras Mutant From The Golgi Apparatus , Daniel R. Zamzow

Retrospective Theses and Dissertations

The paradigm that H-Ras needs to be attached to the plasma membrane to signal has been put into question in the last decade. New insights into H-Ras signaling point to a biological requirement for signaling from endomembranes.;This thesis will demonstrate the abilities of a unique H-Ras mutant that accumulates on the Golgi.


An Examination Of Hexacoordinate Hemoglobins Using The Techniques Of Biochemistry, Biophysics And Molecular Biology , James Thomas Trent Iii Jan 2006

An Examination Of Hexacoordinate Hemoglobins Using The Techniques Of Biochemistry, Biophysics And Molecular Biology , James Thomas Trent Iii

Retrospective Theses and Dissertations

Hexacoordinate hemoglobins are found in a truly diverse array of organisms, ranging from cyanobacteria to humans. The prevalence of these proteins in nature coupled with very high sequence identity between species homologues suggests they have a role vital to life. The high sequence identity also implies that hxHb structural features, including the hexacoordination phenomenon, are critical aspects of the physiological function. A detailed understanding of the ligand binding behavior of these proteins will not only facilitate efforts towards discovering their physiological role(s), but will also help explain how hxHbs perform their function. This dissertation consists of four published papers ...


Development Of Aptamer Based Targeted Reversibly Attenuated Probes , Xiangyu Cong Jan 2006

Development Of Aptamer Based Targeted Reversibly Attenuated Probes , Xiangyu Cong

Retrospective Theses and Dissertations

The targeted reversely attenuated probe (TRAP) is an aptamer-based biosensor in which the aptamer activity can be regulated by a specific nucleic acid sequence such as an mRNA. The TRAP has the potential of being developed for imaging gene expression in vivo. The central portion of the TRAP, between the aptamer and the attenuator, is complementary to a target nucleic acid, such as an mRNA, which is referred to as a regulatory nucleic acid (regDNA);I developed 8att-cmRas20 and 9att-cmRas15 ATP DNA TRAPs with regDNA-dependent aptamer activity. The results suggested that, as well as inhibiting the aptamer, the attenuator also ...


Structural Characterization Of Ligand Binding In Hexacoordinate Hemoglobins , Julie Anne Hoy Jan 2006

Structural Characterization Of Ligand Binding In Hexacoordinate Hemoglobins , Julie Anne Hoy

Retrospective Theses and Dissertations

Hemoglobins are ancient proteins that predate the divergence of prokaryotes and eukaryotes. Although the most thoroughly studied hemoglobins are mammalian oxygen transporters, it has become clear that this function is a recent evolutionary advancement coinciding with the "oxygen revolution" and the development of multicellular organisms. Hemoglobins capable of oxygen transport are highly specialized structurally to maintain pentacoordination of the heme and appropriate ligand rate and equilibrium constants, while preventing autooxidation due to bound oxygen. Their predecessors however, adopt a more energetically favorable hexacoordinate conformation that precludes oxygen transport;Despite the fact that hexacoordinate hemoglobins are ubiquitous in biological organisms, little ...


From Endomembrane To The Plasma Membrane: The Traffic, Signal Transduction And Micro-Localization Of H-Ras , Hui Zheng Jan 2006

From Endomembrane To The Plasma Membrane: The Traffic, Signal Transduction And Micro-Localization Of H-Ras , Hui Zheng

Retrospective Theses and Dissertations

Ras proteins are small G proteins that play key roles in many aspect of cell signal transduction. H-Ras is one of three isoforms of mammalian Ras protein that is best studied. The work presented in this dissertation describes how the carboxyl terminal lipid modifications affect the transportation, signal transduction and micro-localizations of H-Ras protein. The already known traffic pathway for H-Ras to the plasma membrane is Golgi mediated classical vesicular Transportation; However, we found the H-Ras transportation has many different features other than the classical pathway, and thereby discovered a new pathway for H-Ras targeting to the plasma membrane. This ...


Genetic And Molecular Analysis Of Starch Synthases Functions In Maize And Arabidopsis , Xiaoli Zhang Jan 2006

Genetic And Molecular Analysis Of Starch Synthases Functions In Maize And Arabidopsis , Xiaoli Zhang

Retrospective Theses and Dissertations

Understanding the specific functions played by individual starch synthase isoforms in maize and Arabidopsis will provide important evidence for how highly organized starch structure is made. Starch synthases (SS) catalyze the transfer of the glucosyl moiety from ADP-Glc to the terminus of a growing alpha-(1, 4)-linked glucan linear chain. At least five classes of SSs are identified in higher species, referred to as GBSS, SSI, SSII, SSIII, and SSIVN. They have high similarity in the catalytic and starch-binding domains of the C-termini but differ at their N-termini. All of these enzymes are highly conserved in plant kingdom, which ...