Biochemistry, Biophysics, and Structural Biology Commons^™

Structural And Functional Investigation Of Φ-El-Chaperonin Mediated Protein Folding, Sudheer Kumar Molugu

Open Access Theses & Dissertations

Chaperonins are ubiquitous, sequence related protein complexes that aid in the folding of nascent and misfolded polypeptides in an ATP driven pathway. Recently a GroEL-like, 860 kilo Dalton chaperonin protein complex was identified and isolated from the bacteriophage EL, a virus that infects the Gram-negative bacterium Pseudomonas aeruginosa. The bacteriophage EL contains 201 predicted open reading frames and is the only known phage that encodes for its own chaperonin known as Φ-EL-chaperonin.

To understand the importance of Φ-EL-chaperonin in phage EL life cycle, the recombinant Φ-EL-chaperonin protein was expressed in Escherichia coli (E. coli) purified to homogeneity and the structure …

Identification And Characterization Of Fkbp52-Specific Inhibitors For The Treatment Of Prostate Cancer, Johanny Tonos De Leon

Open Access Theses & Dissertations

Steroid hormone receptors require the ordered assembly of various chaperone and cochaperone proteins in order to reach a functional state. The final stage in the receptor maturation process requires the formation of a mutimeric complex consisting of Hsp90 dimer, p23, and one of several large immunophilins. Studies conducted previously demonstrated that the large immunophilin FKBP52 acts to potentiate glucocorticoid, androgen, and progesterone receptor signaling pathways. The aim of these studies was to identify and characterize FKBP52-specific inhibitors that would not only serve as tools for the pharmacological analysis of FKBP52-receptor interactions, but may also lead to novel drugs with significant …

Targeted Identification Of Metastasis-Associated Cell-Surface Sialoglycoproteins In Prostate Cancer, Lifang Yang, Julius O. Nyalwidhe, Sigi Guo, Richard R. Drake, O. John Semmes

Bioelectrics Publications

Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC₄ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins …

An Apoptosis Targeted Stimulus With Nanosecond Pulsed Electric Fields (Nspefs) In E4 Squamous Cell Carcinoma, Wei Ren, Stephen J. Beebe

Bioelectrics Publications

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was [95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. …

Epigenetic Effects Of Benzo(A)Pyrene In Fundulus Heteroclitus And Danio Rerio, Xiefan Fang

Electronic Theses and Dissertations

DNA methylation is one of the epigenetic mechanisms that controls gene expression and is vulnerable to early life environmental toxicant exposures. Our goal was to use two fish models, Fundulus heteroclitus and Danio rerio (zebrafish), to study the benzo(a)pyrene (BaP) effects on DNA methylation status and whether the alterations could contribute to BaP-mediated reproductive and developmental toxicities. Initially, we used Fundulus to study BaP effects on glycine N-methyltransferase (GNMT) expression throughout development. Fundulus embryos were exposed to waterborne BaP at nominally 10 or 100 μg/L and both GNMT mRNA expression and enzyme activity were measured. Quantitative PCR and whole mount …

Differential Precipitation And Solubilization Of Proteins., Barry Ryan

Books/Book Chapters/ Proceedings

Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physico-chemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g. His-Tag, Size Exclusion etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during over-expression in the host cell. Although this phenomenon permits easier initial separation from native proteins, these inclusion bodies must carefully be differentially solubilised so as to reform functional, correctly folded …

Synthesis, Self-Assembly And Biological Properties Of Self-Folding Cu(Ii) Coordination Polymers, Alejandro Arzola-Rubio

Open Access Theses & Dissertations

The synthesis of materials via molecular self-assembly is a powerful bottom-up approach for fabricating matter in the nano- and micro-meter scales. This methodology involves the spontaneous and reversible organization of small molecules that interact with one another to create larger structures driven by non-covalent interactions such as hydrogen bonding, hydrophobic forces and metal-ligand coordination interactions. In this thesis, the design, synthesis and characterization of a new set of molecules that spontaneously self-organize in water to form three-dimensional nanoscale toroidal structures was investigated. These supramolecular structures were designed with an intrinsic affinity to bind and condense DNA strands with the intent …

Exploration Of The Active Site Specificity Of Mala, A Glucosidase From The Predatory Bacterium Bdellovibrio Bacteriovorus, Christine Isabella

Summer Research

Sequencing of the HD100 genome of Bdellovibrio Bacteriovorus in 2005 revealed a gene for the putative maltase, MalA. However, given the bacterium’s observed disuse of prey carbohydrates as an energy source, this enzyme is seemingly out of place. In this study, the specificity and activity of MalA were explored through various enzymatic assays. Using p-nitrophenol-α-D-glucopyranoside (p-NPG) as a colorimetric substrate to allow for rapid and accurate detection of enzymatic activity through spectrophotometry, enzyme stability inhibition of p-NPG cleavage were explored. While numerous alpha-linked disaccharides were shown to inhibit MalA, only maltose was shown to be cleaved into glucose.

Developing A Protocol For Purifying The Mala Enzyme In Bdellovibrio Bacteriovorus, John Jared Trecker

Summer Research

The sequenced genome of the gram-negative predatory bacterium Bdellovibrio bacteriovorus contains a gene that encodes for malA, a putative maltase. Given the bacterium's observed disuse of prey carbohydrates, the gene's presence is mysterious. That characterization of the enzyme and studies of its activity and specificity can be better carried out, it is necessary to obtain pure enzyme. Protein was collected from lysed cultures of Top10/pmalA E. coli. Attempted purification by ion-exchange chromatography with DEAE columns produced significantly purer protein; SP ion exchange columns were unsuccessful, as were heparin and hydroxyapatite affinity columns. Gel filtration chromatography should prove a useful method …

Reactive Sulfur: Redox Reactions Of Cysteines And Methionines In The Cytoskeletal Protein Tubulin, Tara Dawn Hagedorn

Dissertations, Theses, and Masters Projects

No abstract provided.

An Investigation Of The Polymorphic Arss Gene Of Helicobacter Pylori, Daniel Ross Hallinger

Dissertations, Theses, and Masters Projects

No abstract provided.

Physical Models Of Amyloid Fibril Assembly, Shannon Elizabeth Hill

USF Tampa Graduate Theses and Dissertations

Formation of large fibers and plaques by amyloid proteins is recognized as the molecular hallmark of an increasing number of human disorders, including Parkinson's disease, Alzheimer's disease and even type II diabetes. The broader objective of my research is to unravel the basic mechanisms that initiate and regulate fibril formation by amyloidogenic proteins. This objective is significant because even basic aspects of how fibril formation proceeds from a soluble, monomeric protein to an insoluble amyloid fibril remain much debated. Furthermore, there is increasingly strong evidence suggesting that intermediates of the aggregation process, with properties distinct from those of mature fibrils, …

Probing Molecules In Confined Space, Carissa Marie Vetromile

USF Tampa Graduate Theses and Dissertations

Despite the plethora of information regarding cellular crowding and its importance on modulating protein function the effects of confinement on biological molecules are often overlooked when investigating their physiological function. Recently however, the encapsulation of biomolecules in solid state matrices (NafionTM, sol-gels, zirconium phosphate,etc.) has increased in importance as a method for examining protein conformation and dynamics in confined space as well as novel applications in biotechnology. Biotechnological applications include, but are not limited to, bioremediation, biosensors, biocatalysts, etc. In order to better utilize solid state materials as substrates for biological molecules an understanding of the effects of encapsulation on …

Analysis Of Ahr Expression And Stability In A Recombinant Yeast Model System, Sarah Elizabeth Cuccinello

USF Tampa Graduate Theses and Dissertations

The aryl hydrocarbon receptor (Ahr) and the aryl hydrocarbon receptor nuclear translocator (Arnt) are well characterized bHLH-PAS transcription factors shown to regulate expression of xenobiotic metabolism genes. Extensive study has shown that upon treatment with certain aromatic hydrocarbons, mammalian cells rapidly activate the Ahr signaling pathway in order to stimulate gene expression and attempt to metabolize the xenobiotic compounds. It has been shown that after DNA-binding, the Ahr but not the Arnt protein, is quickly eliminated from the nuclear compartment thereby attenuating the dose of gene regulation administered by the Ahr*Arnt transcription factor complex. Previous studies have implicated involvement of …

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 …

Functional Diversification Of Thylakoidal Processing Peptidases In Arabidopsis Thaliana, Shih-Chi Hsu, Joshua K. Endow, Nicholas J. Ruppel, Rebecca Roston, Amy J. Baldwin, Kentaro Inoue

Department of Biochemistry: Faculty Publications

Thylakoidal processing peptidase (TPP) is responsible for removing amino-terminal thylakoid-transfer signals from several proteins in the thylakoid lumen. Three TPP isoforms are encoded by the nuclear genome of Arabidopsis thaliana. Previous studies showed that one of them termed plastidic type I signal peptidase 1 (Plsp1) was necessary for processing three thylakoidal proteins and one protein in the chloroplast envelope in vivo. The lack of Plsp1 resulted in seedling lethality, apparently due to disruption of proper thylakoid development. The physiological roles of the other two TPP homologs remain unknown. Here we show that the three A. thaliana TPP isoforms …

Enzymatic Defects Underlying Hereditary Glutamate Cysteine Ligase Deficiency Are Mitigated By Association Of The Catalytic And Regulatory Subunits, Melanie Neely Willis, Yilin Liu, Ekaterina I. Biterova, Melanie A. Simpson, Heejeong Kim, Jaekwon Lee, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises

production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly

reduced levels of erythrocyte glutathione, leading to hemolytic anemia and in some cases,

impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a

catalytic (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate limiting step

in glutathione production. Four clinical missense mutations have been identified within GCLC:

Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of

these mutations on enzymatic function in vivo and in vitro …

Mne1 Is A Novel Component Of The Mitochondrial Splicing Apparatus Responsible For Processing Of A Cox1 Group I Intron In Yeast, Talina Watts, Oleh Khalimonchuk, Rachel Z. Wolf, Edward M. Turk, Georg Mohr, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Saccharomyces cerevisiae cells lacking Mne1 are deficient in

intron splicing in the gene encoding the Cox1 subunit of cytochrome

oxidase but contain wild-type levels of the bc1 complex.

Thus, Mne1 has no role in splicing of COB introns or expression

of the COB gene. Northern experiments suggest that splicing of

the COX1 aI5β intron is dependent on Mne1 in addition to the

previously known Mrs1, Mss116, Pet54, and Suv3 factors. Processing

of the aI5_ intron is similarly impaired in mne1∆ and

mrs1∆ cells and overexpression of Mrs1 partially restores the

respiratory function of mne1∆ cells. Mrs1 …

Rpir Homologues May Link Staphylococcus Aureus Rnaiii Synthesis And Pentose Phosphate Pathway Regulation, Yefei Zhu, Nandakumar Madayiputhiya, Marat R. Sadykov, Nandakumar Madayiputhiya, Thanh T. Luong, Rosmarie Gaupp, Chia Y. Lee, Greg Somerville

Department of Biochemistry: Faculty Publications

Staphylococcus aureus is a medically important pathogen that synthesizes a wide range of virulence determinants. The synthesis of many staphylococcal virulence determinants is regulated in part by stress-induced changes in the activity of the tricarboxylic acid (TCA) cycle. One metabolic change associated with TCA cycle stress is an increased concentration of ribose, leading us to hypothesize that a pentose phosphate pathway (PPP)-responsive regulator mediates some of the TCA cycle-dependent regulatory effects. Using bioinformatics, we identified three potential ribose-responsive regulators that belong to the RpiR family of transcriptional regulators. To determine whether these RpiR homologues affect PPP activity and virulence determinant …

Quantifying Agonist Activity At G Protein-Coupled Receptors, Frederick J. Ehlert, Hinako Suga, Michael T. Griffin

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.

Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence …