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Title: PHOSPHOLIPASE A2MECHANISM OF INTERFACIAL ACTIVATION,AN INTERDISCLIPLINARY APPROACH.
Creator: Nemec, Kathleen, Tatulian, Suren, University of Central Florida
Abstract / Description: Phospholipase A2 (PLA2) is an enzyme that hydrolyzes the sn-2-ester bond of membrane phospholipids and liberates arachidonic acid, which is converted to eicosanoids that act as potent mediators of inflammation and allergy. As such this enzyme plays a crucial role in many homeostatic physiological and immunologic processes and disease progression. PLA2s undergo substantial increase in activity upon binding to cellular membranes. This effect of interfacial activation is well recognized, yet its...
Show morePhospholipase A2 (PLA2) is an enzyme that hydrolyzes the sn-2-ester bond of membrane phospholipids and liberates arachidonic acid, which is converted to eicosanoids that act as potent mediators of inflammation and allergy. As such this enzyme plays a crucial role in many homeostatic physiological and immunologic processes and disease progression. PLA2s undergo substantial increase in activity upon binding to cellular membranes. This effect of interfacial activation is well recognized, yet its structural and physical aspects are poorly understood. In this work, we have employed the interdisciplinary methods of molecular biology, biochemistry, biophysics, bioinformatics and computational biology, in order to elucidate the structure-function relationships mediating the interfacial activation of human group IIA and group IB PLA2 isoforms. We have evaluated the structural and functional consequences of two conservative, single residue substitutions, located at key membrane-binding and substrate-binding positions of hIIA PLA2. We have also evaluated a human group IB fragment (hIBdeltaN10), missing the first 10 N-terminal residues which make up the N-terminal alpha helix, as well as a chimeric enzyme substituting the N-terminal alpha helix of hIB PLA2 with that from hIIA PLA2 (hIIA/IB PLA2). We have compared the engineered proteins against both the hIIA and hIB PLA2 native enzymes and their N-terminal peptides, N10-hIB and N10-hIIA, respectively. We have developed and used a novel multidisciplinary approach in order to position the segmentally labeled hIB PLA2 and hIIA/IB chimeric PLA2s at the membrane surface. The results of this work provide significant insight into the understanding of the physical aspects of interfacial activation by determining the precise membrane binding modes of PLA2 isoforms and identifying certain amino acid residues and whole protein segments that play key roles in membrane binding, activation, and involved allosteric conformational effects in PLA2s.
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Date Issued: 2006
Identifier: CFE0001324, ucf:47015
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0001324

Title: STRUCTURAL TRANSITION DURING FIBRILLOGENESIS OF AMYLOID ? PEPTIDE.
Creator: Sidrak, George, Tatulian, Suren, University of Central Florida
Abstract / Description: Alzheimer's Disease (AD) is a neurodegenerative disease marked by progressive neuronal cell death, leading to dementia. AD is the most common disease that results in dementia and largely affects the elderly, with five million people in the United States diagnosed with the disease as of 2015 and approximately 35 million people worldwide. Diseases resulting in dementia cost the US healthcare system an estimated $172 billion in 2010 and that cost is expected to increase as the population ages...
Show moreAlzheimer's Disease (AD) is a neurodegenerative disease marked by progressive neuronal cell death, leading to dementia. AD is the most common disease that results in dementia and largely affects the elderly, with five million people in the United States diagnosed with the disease as of 2015 and approximately 35 million people worldwide. Diseases resulting in dementia cost the US healthcare system an estimated $172 billion in 2010 and that cost is expected to increase as the population ages and as diagnostic techniques improve so that more people are treated (Holtzman, 2011). The disease was first reported by psychiatrist Alois Alzheimer at the onset of the 20th century, when one of his patients "suffered memory loss, disorientation, hallucinations and delusions and died at the age of 55," then was found to have severe brain atrophy post-mortem (Cipriani, Dolciotti, Picchi, & Bonuccelli, 2011). There are palliative treatments available that marginally slow disease progression but there is currently no cure for the disease (Awasthi, Singh, Pandey, & Dwivedi, 2016). More research is needed to develop effective therapeutic strategies to combat the disease. Currently, AD cytotoxicity is believed to be caused by increased amyloid ? (A?) peptide plaque deposition in the brain, as described by the amyloid cascade hypothesis (Barage & Sonawane, 2015). The current understanding is that oligomers of A? peptide lead to neuronal death through multiple mechanisms, most notably hyper-phosphorylation of the tau protein. Having a better understanding of the structural changes in the fibrillization process of A? will provide a broader insight into mechanisms of cell death and open new possibilities for pharmacological treatments, which is what this research intends to provide.
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Date Issued: 2017
Identifier: CFH2000178, ucf:45994
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000178

Title: ROLE OF MEMBRANE LIPIDS IN MODULATING PROTEIN STRUCTURE & FUNCTION.
Creator: Ray, Supriyo, Tatulian, Suren, University of Central Florida
Abstract / Description: A-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated...
Show moreA-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated degradation (ERAD). Most ERAD substrates are purged out of the ER into the cytosol for proteasomal degradation. However, the low content of lysine amino acid residues allows the toxin to evade polyubiquitination and subsequent proteasomal degradation. The toxin A subunit refolds into an active conformation in the cytosol, setting off downstream toxic events. In the first part of my thesis, the hypothesis was tested that inhibiting the unfolding of the toxin A subunit inside the ER will prevent ERAD activation, toxin export to the cytosol and intoxication. The chemical chaperones glycerol and sodium 4-phenyl butyrate (PBA) were used to inhibit the toxin A chain unfolding. In vitro biophysical experiments indicated that both chemical chaperones indeed stabilize the cholera toxin A subunit and prevent cytotoxicity. In case of ricin, both chaperones stabilized the toxin A chain but only glycerol prevented cytotoxicity. Additional experiments showed that PBA-treated ricin A chain is destabilized when exposed to anionic lipid membranes mimicking the properties of the ER membrane. In contrast, anionic lipid did not prevent ricin A chain stabilization by glycerol. This explains why glycerol but not PBA blocked ricin intoxication, as only glycerol stabilizes ricin A chain in the presence of ER membranes. Cholera toxin in contrast, remained either unaffected or slightly stabilized in presence of anionic lipids both in presence and absence of PBA. This shows that destabilization by anionic lipids is a toxin-specific rather than a general effect. In the second part of my thesis, the effect of inner leaflet of plasma membrane on the structure of cholera toxin A chain (CTA1) was studied. Since CTA1 refolds into an active conformation in the cytosol in association with unidentified host factors, I hypothesized that inner leaflet of the plasma membrane might play a role to stabilization and/or refolding of CTA1. CTA1 was shown to be a membrane interacting protein, and membranes mimicking lipid rafts had a significant stabilizing effect on its structure. Lipid rafts helped in the regaining of the tertiary and secondary structure of CTA1, while non-raft lipids had a smaller stabilizing effect on CTA1 structure. In the next part of my thesis, I studied the effect of membrane binding on the structure and function of human pancreatic phospholipase A2 (PLA2). Lipid thermal phase transition was found to have a dramatic effect on PLA2 activity. It was also established that although membrane binding and insertion was essential for of PLA2 activity, lipid structural heterogeneity was more important than the depth of membrane insertion for enzyme activation. Most importantly, significant changes in PLA2 secondary and tertiary structures were identified that evidently contribute to the interfacial activation of PLA2. Overall, we conclude that the function of membrane binding enzymes can be significantly modulated via conformational changes induced by interactions with membranes. Thus, we have elucidated various roles of membrane lipids from unfolding and refolding to activation and modulation of membrane binding enzymes. Physical properties of lipids help in regulating various aspects of protein structure and function and their analysis helped us in appreciating the influence wielded by the membrane lipids in the enzyme's surrounding environment.
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Date Issued: 2011
Identifier: CFE0004035, ucf:49184
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004035

Title: BIOPHYSICAL CHARACTERIZATION OF THE MEMBRANE BINDING DOMAIN OF THE PRO-APOPTOTIC PROTEIN BAX.
Creator: Garg, Pranav, Tatulian, Suren, University of Central Florida
Abstract / Description: The BCL-2 family of proteins tightly regulates the delicate balance between life and death. The pore forming Bax is a pro-apoptotic member belonging to this protein family. At the onset of apoptosis, monomeric cytoplasmic Bax translocates to the outer mitochondrial membrane, forms oligomeric pores thereby letting mitochondrial cytochrome c enter the cytosol and initiate the apoptotic cascade. The C-terminal "helix 9" is thought to mediate the membrane binding of BAX. A 20-amino acid peptide...
Show moreThe BCL-2 family of proteins tightly regulates the delicate balance between life and death. The pore forming Bax is a pro-apoptotic member belonging to this protein family. At the onset of apoptosis, monomeric cytoplasmic Bax translocates to the outer mitochondrial membrane, forms oligomeric pores thereby letting mitochondrial cytochrome c enter the cytosol and initiate the apoptotic cascade. The C-terminal "helix 9" is thought to mediate the membrane binding of BAX. A 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with Glu (charge reversal mutant, EE) or Leu (charge neutralization mutant, LL) have been studied to elucidate the pore formation capabilities of Bax C-terminus and the underlying molecular mechanism. Interactions of the wild-type and the two mutant peptides with zwitterionic and anionic phospholipid membranes caused efficient membrane permeabilization, as documented by release of vesicle-entrapped fluorescent indicator calcein. Light scattering experiments showed that vesicles maintained their integrity upon peptide binding, indicating that the content leakage was due to pore formation and not vesicle degradation. Kinetics of calcein release at various peptide concentrations were used to determine the peptide-peptide association constants and the oligomeric state of the pore. The structure of membrane-bound peptides was analyzed by circular dichroism (CD) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. CD data indicated all three peptides reconstituted in lipid vesicles contained [alpha]-helical and [beta]-strand structures. ATR-FTIR experiments indicated that the minimally hydrated samples of peptides in stacked lipid bilayers (absence of bulk water) were mostly [alpha]-helical but adopted mostly [beta]-sheet conformation in the presence of excess water. Finally, the depth of membrane insertion of the peptides was analyzed using tryptophan fluorescence quenching by dibromo-phosphatidylcholines brominated at various positions of their acyl chains. In case of zwitterionc phospholipid membranes, the single Trp16 was located at ~9 Å from membrane center. In case of membranes containing 30% of an anionic phospholipid, the depth of membrane insertion of the EE mutant was not affected but the wild-type and the LL mutant peptides were embedded much deeper into the membrane, with Trp16 located at 3-4 Å from membrane center. These results will help achieve a better understanding of the molecular mechanism of membrane pore formation of Bax protein. In addition, they provide insight into the molecular details of membrane pore formation by peptides and could facilitate the design and production of cytotoxic peptides with improved capabilities to lyse cells such as bacteria or cancer cells.
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Date Issued: 2011
Identifier: CFE0003983, ucf:48674
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0003983

Title: Structure of reflectin protein probed by solid-state nuclear magnetic resonance.
Creator: Boykin, Tommy, Chen, Bo, Schulte, Alfons, Tatulian, Suren, Teter, Kenneth, University of Central Florida
Abstract / Description: An unusual protein found in squids, termed reflectin, possesses the unique ability for optical reflectivity and proton conductivity. Reflectin has the potential to become optically and electronically tunable, however, the molecular structure of reflectin has yet to be determined. Previous literature has considered using reflectin proteins as diffraction gratings, protonic transistors, and substrates for neural stem cell growth. Applying recombinant protein techniques, we purified the...
Show moreAn unusual protein found in squids, termed reflectin, possesses the unique ability for optical reflectivity and proton conductivity. Reflectin has the potential to become optically and electronically tunable, however, the molecular structure of reflectin has yet to be determined. Previous literature has considered using reflectin proteins as diffraction gratings, protonic transistors, and substrates for neural stem cell growth. Applying recombinant protein techniques, we purified the reflectin 4Ax4 protein. We determined the leucine, tryptophan, and threonine amino acids are in an ordered state by solid-state nuclear magnetic resonance (NMR). Knowing these ordered amino acids is possibly the key to understanding reflectin's natural optical and electrical properties. Understanding the link between reflectin's structure and electrical properties is essential to make the next generation of bioelectronic materials based on inexpensive, natural resources.
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Date Issued: 2019
Identifier: CFE0007433, ucf:52712
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007433

Title: Nanoscale Functional Imaging by Tailoring Light-matter Interaction to Explore Organic and Biological Systems.
Creator: Otrooshi, Negar, Tetard, Laurene, Tatulian, Suren, Peale, Robert, Santra, Swadeshmukul, University of Central Florida
Abstract / Description: ABSTRACTProbing molecular systems with light has been critical to deepen our understanding of life sciences. However, conventional analytical methods fail to resolve small quantities of molecules or the heterogeneity in molecules assembled into complex systems. This bottleneck is mostly attributed to light diffraction limit. In recent years, the successful implementation of new approaches to achieve sub-wavelength chemical speciation with an Atomic Force Microscope (AFM) has paved the way to...
Show moreABSTRACTProbing molecular systems with light has been critical to deepen our understanding of life sciences. However, conventional analytical methods fail to resolve small quantities of molecules or the heterogeneity in molecules assembled into complex systems. This bottleneck is mostly attributed to light diffraction limit. In recent years, the successful implementation of new approaches to achieve sub-wavelength chemical speciation with an Atomic Force Microscope (AFM) has paved the way to a deeper understanding of the effect of local composition and structure on the functional properties of a larger scale system. The combination of infrared light, to excite the vibrational modes of a sample, and AFM detection to monitor the resulting local photothermal expansion has emerged as a powerful approach. In this work, we explore new applications of AFM-infrared (IR) to further the understanding of proteins and bacterial cells. We first consider the vibrational modes and secondary structure of proteins. We show that beyond the localized IR fingerprint of the system, light polarization could affect the response of the protein. To investigate this further, we combine the AFM-IR measurements with plasmonic substrates to tune the electromagnetic field. Using plasmonic structures, we map the electromagnetic field confinement using nanomechanical infrared spectroscopy. We detect and quantify, in the near field, the energy transferred to the lattice in the form of thermal expansion resulting from the heat generated. We compare the photothermal expansion patterns in the structures under linearly and circular polarized illumination. The results suggest the formation of hot spots, of great interest for biomolecules detection. Using a model system, poly-L-lysine, we show that the IR spectrum and the vibrational circular dichroism fingerprint of a chiral biological system can be probed at the nanoscale, far beyond the conventional limits of detection. The second part of the study focuses on utilizing the capabilities of AFM-IR to investigate bacterial cells and their responses to nanoparticle-based treatments. We highlight the potential of these new capabilities to further dive into the fundamental molecular mechanism of antibacterial activity and of development of drug resistance. We conclude this work by providing a perspective on the impact nanoscale functional imaging and spectroscopy can have on life sciences and beyond.
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Date Issued: 2019
Identifier: CFE0007897, ucf:52750
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007897

Title: Unraveling PDI and its Interaction with AB Toxins.
Creator: Guyette, Jessica, Teter, Kenneth, Self, William, Jewett, Travis, Tatulian, Suren, University of Central Florida
Abstract / Description: Protein disulfide isomerase (PDI) is an essential endoplasmic reticulum (ER) protein that acts as both an oxidoreductase and chaperone. It exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin (Ctx), which is a unique property of PDI. This unfolding allows PDI to disassemble the Ctx holotoxin, which is required for Ctx activity. Here, we investigated the unfolding and refolding property of PDI and how this affects its...
Show moreProtein disulfide isomerase (PDI) is an essential endoplasmic reticulum (ER) protein that acts as both an oxidoreductase and chaperone. It exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin (Ctx), which is a unique property of PDI. This unfolding allows PDI to disassemble the Ctx holotoxin, which is required for Ctx activity. Here, we investigated the unfolding and refolding property of PDI and how this affects its interaction with bacterial toxins. PDI showed remarkable redox-linked conformational resilience that allows it to refold after being thermally stressed. Deletion constructs of PDI showed that both active domains play opposing roles in stability, and can both refold from an unfolded state, indicating that either domain could unfold during its interaction with Ctx. Its ability to refold suggests that the cycle of unfolding and refolding with Ctx is a normal mechanism that prevents protein aggregation. Disruption of this cycle with the polyphenol, quercetin-3-rutinoside, prevented the disassembly of Ctx, which blocked Ctx intoxication of cultured cells. Loss of PDI function was also found to inhibit intoxication with Escherichia coli heat-labile toxin but not with ricin and Shiga toxins. Toxin structure also contributes to efficiency of PDI binding and disassembly, which may explain the differential potencies between toxins. While Ctx and Ltx share similar structures, Ctx is more potent and efficiently disassembled compared to Ltx. We believe that PDI-mediated disassembly is the rate-limiting step in intoxication, thus dictating toxin potency. Overall, PDI can be targeted for a potential therapeutic for many bacterial toxins because of its unique unfolding properties and its key role in cell intoxication.
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Date Issued: 2019
Identifier: CFE0007646, ucf:52511
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007646

Title: The Dynamic Functions of Bax are Dependent on Key Structural and Regulatory Features.
Creator: Boohaker, Rebecca, Khaled, Annette, Cole, Alexander, Zervos, Antonis, Tatulian, Suren, University of Central Florida
Abstract / Description: Bax is an essential mediator of cell fate. Since its discovery in 1985 as a protein that interacts with the anti-apoptotic protein, Bcl-2, key elements related to its function, structure and regulation remains to be determined. To this end, mitochondrial metabolism was examined in non-apoptotic Bax-deficient HCT-116 cells as well as primary hepatocytes from Bax-deficient mice. Although mitochondrial density and mitochondrial DNA content was the same in Bax-containing and Bax -deficient cells,...
Show moreBax is an essential mediator of cell fate. Since its discovery in 1985 as a protein that interacts with the anti-apoptotic protein, Bcl-2, key elements related to its function, structure and regulation remains to be determined. To this end, mitochondrial metabolism was examined in non-apoptotic Bax-deficient HCT-116 cells as well as primary hepatocytes from Bax-deficient mice. Although mitochondrial density and mitochondrial DNA content was the same in Bax-containing and Bax -deficient cells, MitoTracker staining patterns differed, suggesting the existence of Bax -dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in Bax -deficient cells, while glycolysis was increased. These results suggest that cells lacking Bax have a deficiency in the ability to generate ATP through cellular respiration, supported by detection of reduced citrate synthase activity in Bax -deficient cells. Expression of either full length or C-terminal truncated Bax in Bax -deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of Bax was not dependent upon its C-terminal helix. Expression of BCL-2 in Bax-containing cells resulted in a subsequent loss of ATP measured, implying that, even under non-apoptotic conditions, an antagonistic interaction exists between the two proteins. Bax is composed of nine alpha-helices. While three of these helices have features of a trans-membrane region, the contribution of each domain to the apoptotic or non-apoptotic functions of Bax remains unknown. To examine this, we focused on the C-terminal alpha-9 helix, an amphipathic domain with putative membrane binding properties and discovered that it has an inherent membrane-binding and cytotoxic capacity. A peptide based on the last twenty amino acids (CT20p) of the alpha-9 helix was synthesized and proved a potent inducer of cell death independent of any apoptotic stimuli. The solubility of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs), and these CT20p-NPs caused the death of colon and breast cancer cells in vitro and induced tumor regression in vivo, using a murine breast cancer tumor model. CT20p caused increased mitochondrial membrane potential followed by cell death via membrane rupture, without the characteristic membrane asymmetry associated with apoptosis. Hence, while CT20p is based on Bax, its innate cytotoxic activity is unlike the parent protein and could be a powerful anti-cancer agent that bypasses drug resistance, can be encapsulated in tumor-targeted nanoparticles and has potential application in combination therapies to activate multiple death pathways in cancer cells. While previous work revealed novel aspects of the biology of Bax that were unrecognized, new structural information is needed to fully elucidate the complexity of Bax's function. One approach is to use computational modeling to assess the solved structure of Bax and provide insight into the structural components involved in the activity of the protein. Use of molecular dynamics simulators such as GROMACS, as well as other computational tools provides a powerful means by which to test the feasibility of certain modifications in defined parameters. Such work revealed that the removal of the C-terminal alpha-9 helix of Bax, which normally resides within a hydrophobic pocket, significantly destabilized the protein, perhaps explaining how the protein transitions from soluble to membrane-bound form and maintain energy production via aerobic respiration or, conversely, how the C-terminus helix conveys cytotoxicity. Collectively, this work reveals that Bax is more than an inducer of cell death but has complex activities yet to be determined.
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Date Issued: 2012
Identifier: CFE0004521, ucf:49285
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004521

Title: Cholera toxin activates the unfolded protein response through an adenylate cyclase-independent mechanism.
Creator: Vanbennekom, Neyda, Teter, Kenneth, Tatulian, Suren, Jewett, Mollie, Zervos, Antonis, University of Central Florida
Abstract / Description: Cholera toxin (CT) is a bacterial protein toxin responsible for the gastrointestinal disease known as cholera. CT stimulates its own entry into intestinal cells after binding to cell surface receptors. Once internalized, CT is delivered via vesicle-mediated transport to the endoplasmic reticulum (ER), where the CTA1 subunit dissociates from the rest of the toxin and is exported (or translocated) into the cytosol. CTA1 translocates from the ER lumen into the host cytosol by exploiting a host...
Show moreCholera toxin (CT) is a bacterial protein toxin responsible for the gastrointestinal disease known as cholera. CT stimulates its own entry into intestinal cells after binding to cell surface receptors. Once internalized, CT is delivered via vesicle-mediated transport to the endoplasmic reticulum (ER), where the CTA1 subunit dissociates from the rest of the toxin and is exported (or translocated) into the cytosol. CTA1 translocates from the ER lumen into the host cytosol by exploiting a host quality control mechanism called ER-associated degradation (ERAD) that facilitates the translocation of misfolded proteins into the cytosol for degradation. Cytosolic CTA1, however, escapes this fate and is then free to activate its target, heterotrimeric G-protein subunit alpha (Gs?), leading to adenlyate cyclase (AC) hyperactivation and increased cAMP concentrations. This causes the secretion of chloride ions and water into the intestinal lumen. The result is severe diarrhea and dehydration which are the major symptoms of cholera. CTA1's ability to exploit vesicle-mediated transport and ERAD for cytosolic entry demonstrates a potential link between cholera intoxication and a separate quality control mechanism called the unfolded protein response (UPR), which up-regulates vesicle-mediated transport and ERAD during ER stress. Other toxins in the same family such as ricin and Shiga toxin were shown to regulate the UPR, resulting in enhanced intoxication.Here, we show UPR activation by CT, which coincides with a marked increase in cytosolic CTA1 after 4 hours of toxin exposure. Drug induced-UPR activation also increases CTA1 delivery to the cytosol and increases cAMP concentrations during intoxication. We investigated whether CT stimulated UPR activation through Gs? or AC. Chemical activation of Gs? induced the UPR and increased CTA1 delivery to the cytosol. However, AC activation did not increase cytosolic CTA1 nor did it activate the UPR. These data provide further insight into the molecular mechanisms that cause cholera intoxication and suggest a novel role for Gs? during intoxication, which is UPR activation via an AC-independent mechanism.
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Date Issued: 2013
Identifier: CFE0004951, ucf:49560
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004951

Title: Structural and functional characterization of enzymes of a novel group of tryptophylquinone cofactor containing oxidases.
Creator: Sehanobish, Esha, Davidson, Victor, Khaled, Annette, Self, William, Tatulian, Suren, University of Central Florida
Abstract / Description: Protein-derived cofactors are redox and catalytic centers that are generally formed by the posttranslational modifications of one or more amino acids. An important class of these cofactors are the quinones derived from tyrosine and tryptophan. Amongst redox proteins, it has been known till now that oxidases either contain a flavin or a tyrosylquinone cofactor, whereas tryptophylquinone ones are present within the dehydrogenases. In recent times, oxidases from a marine bacterium, Marinomonas...
Show moreProtein-derived cofactors are redox and catalytic centers that are generally formed by the posttranslational modifications of one or more amino acids. An important class of these cofactors are the quinones derived from tyrosine and tryptophan. Amongst redox proteins, it has been known till now that oxidases either contain a flavin or a tyrosylquinone cofactor, whereas tryptophylquinone ones are present within the dehydrogenases. In recent times, oxidases from a marine bacterium, Marinomonas mediterranea, have been shown to possess the latter. This study involves the characterization of two such proteins, lysine-?-oxidase (LodA) and glycine oxidase (GoxA). They have been reported to contain the same cysteine tryptophylquinone (CTQ) cofactor. Both require the co-expression of a second protein, LodB and GoxB respectively to generate matured CTQ containing active protein. Kinetic analysis of the reaction catalyzed by LodA showed that it followed the usual Michaelis-menten mode of interaction with its substrates. GoxA on the other hand exhibited allosteric cooperativity for its substrate glycine. This was attributed to the dimeric conformation of the wildtype GoxA based on size exclusion chromatographic studies. Mutagenesis study of amino acid residues based on the crystal structure of LodA and a homology model of GoxA, have given a detailed idea about their structure-function relationship. Kinetic studies on mutants of Tyr211 of LodA along with Lys530 present at the substrate channel, showed effects on both Km for the substrates and kcat for the reaction. As a result these residues have shown their involvement in forming a gate-like structure to control the to and fro movement of the substrate and products. Corresponding to this residue, the Phe237 of GoxA has proved to be important in maintaining the allostericity, by mediating the stable dimer formation. From the kinetic parameters, Cys448 of LodA was found to be responsible for substrate specificity and affinity. Whereas, mutants of His466 of GoxA that correspond to the Cys448 residue, were unable to yield CTQ containing active GoxA. On the other hand, Asp512 of LodA and Asp547 of GoxA that correspond to each other, have been implicated for their involvement in CTQ biogenesis. This study therefore highlights how even though this new pool of enzymes have great degrees of similarity in terms of the cofactors and conserved active site residues, there are major differences in the mechanism of the reaction that they catalyze which on a broader sense could influence the overall physiological importance of the enzyme in the biological system.
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Date Issued: 2016
Identifier: CFE0006390, ucf:51494
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006390

Title: Spherical self-assembly of rous sarcoma virus CA, probed by solid-state NMR and the structure of prostate acidic phosphatase and reflectin protein.
Creator: Qiao, Xin, Chen, Bo, Tatulian, Suren, Schulte, Alfons, Cole, Alexander, University of Central Florida
Abstract / Description: In this dissertation, we investigate the morphology of three different protein assemblies, which are formed by prostate acidic phosphatase, residues 248-286 (PAP39), reflectin and rous sarcoma virus capsid (RSV CA). First of all, the main aim of this research is to study the structure of PAP39 which is derived from protease cleavage of Prostate Acidic Phosphatase. The PAP39 can form fibrils of different morphologies in phosphate-buffered saline (PBS) and NaBiCarb (25 mM sodium bicarbonate and...
Show moreIn this dissertation, we investigate the morphology of three different protein assemblies, which are formed by prostate acidic phosphatase, residues 248-286 (PAP39), reflectin and rous sarcoma virus capsid (RSV CA). First of all, the main aim of this research is to study the structure of PAP39 which is derived from protease cleavage of Prostate Acidic Phosphatase. The PAP39 can form fibrils of different morphologies in phosphate-buffered saline (PBS) and NaBiCarb (25 mM sodium bicarbonate and 40 mM sodium phosphate, pH=8.83) buffer conditions, each exhibiting different potentials to enhance the infection of HIV in vitro due to different assemble pathways. In this project, we use solid state nuclear magnetic resonance (ssNMR) and Transmission electron microscopy (TEM) to probe the molecular structure and the fibril morphology in those two buffers. In the second part, we study the optical property of reflectin and also apply ssNMR to evaluate its structure. Reflectin is a protein derived from in flat, structural platelets in reflective tissues of the Hawaiian bobtail squid which obtain unique self-assembling and optical properties. Its self- assembly manifest tunable iridescence and modulate incident light. It can be easily processed into thin films, photonic grating structures and fibrils. SsNMR and TEM are used to determine the structure of reflectin assembly and elucidate the mechanism for the iridescence.In the last part, we study the spherical assembly of RSV CA, which consists of 12 CA pentamers. Therefore, the high resolution structural information of this RSV CA spherical assembly can provide essential information to understand how the same CA protein switches into conformation suitable for pentameric assembly and causes sharp curvature in authentic capsid. We use transmission electron microscopy (TEM) to screen appropriate assembly and obtain spherical assembly sample contains predominantly of T=1 capsid. A series of 2D and 3D spectra were acquired. The ssNMR spectra of the assembly exhibit similar resolution and resonance patterns to those of the RSV CA tubular assembly in our previous work. By referring to the assignments of the tubular assembly, we assign 200 residues of the 237-residue RSV CA in its spherical assembly. The assignments show some regions of RSV CA adopt different chemical shifts, in spite of overall similar resonances, which implies the structural rearrangements upon the spherical assembly and conformation variation of CA to switch between hexameric and pentameric state.
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Date Issued: 2017
Identifier: CFE0006781, ucf:51814
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006781

Title: Conformations and Dynamics of Semi-Flexible Polymers.
Creator: Huang, Aiqun, Bhattacharya, Aniket, Kokoouline, Viatcheslav, Tatulian, Suren, Campiglia, Andres, University of Central Florida
Abstract / Description: In this dissertation, we investigate the conformations, transverse fluctuations and dynamics of two-dimensional (2D) semi-flexible polymers both in the bulk and under channel confinement. We present unified scaling relations in regard to various quantities of interest for a broad range of combinations of chain length and chain stiffness using Langevin dynamics simulation. We also present a three-dimensional (3D) heterogeneous semi-flexible chain model for a double stranded DNA (dsDNA). Our...
Show moreIn this dissertation, we investigate the conformations, transverse fluctuations and dynamics of two-dimensional (2D) semi-flexible polymers both in the bulk and under channel confinement. We present unified scaling relations in regard to various quantities of interest for a broad range of combinations of chain length and chain stiffness using Langevin dynamics simulation. We also present a three-dimensional (3D) heterogeneous semi-flexible chain model for a double stranded DNA (dsDNA). Our model not only confirms the established findings for homogeneous dsDNA, but also predicts new physical phenomenon for heterogeneous dsDNA. The problems studied in this dissertation are relevant to analysis of the conformations and dynamics of biopolymers (such as DNA) in living organisms, and also offer insights for developing devices which operate on the single-molecule level.In particular, we present a unified description for the dynamics of building-blocks (monomers) of a semi-flexible chain. We consider the full range of flexibility from the case where the chain is fully flexible (no stiffness at all) to the case where the chain behaves like a rod (infinite stiffness). Our theory predicts qualitatively different sub-diffusive regimes for the monomer dynamics originating from the chain stiffness by studying the mean square displacement (MSD) of the monomers before the chain dynamics become purely diffusive.For the conformations in the bulk, we present results confirmed and agreed by two completely different models of semi-flexible polymers, with one of which is the bead-spring model (studied by Langevin dynamics) in the continuum space, the other (studied by Monte Carlo) is a self-avoiding walk chain on the square lattice, where only discrete bond angles are possible. We point out the universal features of chain conformations and fluctuations which are independent of the models.For the conformations under channel confinement, we discover qualitatively different conformations and dynamics of the chain as a function of the channel width and chain stiffness, and show how globule like shapes ((")de Gennes blobs(")) for more flexible chains continuously go over to shapes in the form of deflections from the wall ((")Odijk limit(")) for more stiff chains. We provide theoretical arguments how these regimes occur and interpolate among each other as one varies different parameters of the model. We also demonstrate the effect of physical dimensions (either 2D or 3D) on these regimes and argue that since in 2D the excluded volume (EV) effect is more severe compared to 3D, certain regimes do not exist in 2D.Finally, we study a model of a dsDNA , where both base-pairing and base-stacking interactions are accounted for albeit at a low computational cost compared to the other existing models. Our model correctly recovers the stiffness for dsDNA and ssDNA at different temperatures. Under most conditions of interest, a dsDNA can locally denature and form bubbles due to thermal fluctuations. At a critical temperature, a dsDNA undergoes a phase transition, in which the two strands of dsDNA completely melt to two single strands (two ssDNA). By considering EV interactions and calculating the bubble size distribution, recent studies have shown that this denaturation process is a first order transition. We show that for a homogeneous dsDNA made of only AT or GC pairs, our simulation results agree with the previous conclusion of first order transition, however, for sequences of periodic AT and GC regions, when the periodic size is relatively large compared to the sequence length, we show that the bubble size distribution exhibits peaks expressing the sequence pattern, and more importantly, the denaturation is no longer a first order transition.All these studies reported in the dissertation are relevant to the physics of living systems.
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Date Issued: 2016
Identifier: CFE0006464, ucf:51429
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006464

Title: The mauC gene encodes a versatile signal sequence and redox protein that can be utilized in native and non-native protein expression and electron trnasfer systems.
Creator: Dow, Brian, Davidson, Victor, Self, William, Rohde, Kyle, Tatulian, Suren, University of Central Florida
Abstract / Description: The redox-active type 1 copper site of amicyanin is composed of a single copper ion that is coordinated by two histidines, a methionine, and a cysteine residue. This redox site has a potential of +265 mV at pH7.5. Over ten angstroms away from the copper site resides a tryptophan residue whose fluorescence is quenched by the copper. The effects of the tryptophan on the electron transfer (ET) properties were investigated by site-directed mutagenesis. Lessons learned about the hydrogen bonding...
Show moreThe redox-active type 1 copper site of amicyanin is composed of a single copper ion that is coordinated by two histidines, a methionine, and a cysteine residue. This redox site has a potential of +265 mV at pH7.5. Over ten angstroms away from the copper site resides a tryptophan residue whose fluorescence is quenched by the copper. The effects of the tryptophan on the electron transfer (ET) properties were investigated by site-directed mutagenesis. Lessons learned about the hydrogen bonding network of amicyanin from the aforementioned study were attempted to be used as a model to increase the stability of another beta barrel protein, the immunoglobulin light chain variable domain (VL). In addition, amicyanin was used as an alternative redox partner with MauG. MauG is a diheme protein from the mau gene cluster that catalyzes the biogenesis of the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase (MADH). The amicyanin-MauG complex was used to study the free energy dependence and impact of reorganization energy in biological electron transfer reactions.The sole tryptophan of amicyanin was converted to a tyrosine via site-directed mutagenesis. This mutation had no effect on the electron transfer parameters with its redox partners, methylamine dehydrogenase and cytochrome c-551i. However, the pKa of the pH-dependence of the redox potential of the copper site was shifted +0.5 pH units. This was a result of an additional hydrogen bond between Met51 and the copper coordinating residue His95 in the reduced form of amicyanin. This additional hydrogen bond stabilizes the reduced form. Also, the stability of the copper site and the protein overall was significantly decreased, as seen by the temperature dependence of the visible spectrum of the copper site and the circular dichroism spectrum of the protein. This destabilization is attributed to the loss of an interior, cross-barrel hydrogen bond.The VL is structurally similar to amicyanin, but it does not contain any cross-barrel hydrogen bonds. The importance of the cross-barrel hydrogen bond in stabilizing amicyanin is evident. A homologous bond in VL was attempted to be engineered by using site-directed mutagenesis to insert neutral residues with protonatable groups into the core of the protein. Wild-type (WT) VL was purified from the periplasm and found to be properly folded as determined by circular dichroism and size exclusion chromatography. Mutants were expressed in E. coli using the amicyanin signal sequence for periplasmic expression. Folded mutant protein could not be purified from the periplasm.When amicyanin is used in complex with MauG, it retains the pH-dependence of the redox potential of its copper site due to the looseness of the interprotein interface. The free energy of the reaction was manipulated by variation in pH from pH 5.8 to 8.0. The ET parameters are reorganization energy of 2.34 eV and an electronic coupling constant of 0.6 cm-1. P94A amicyanin has a potential that is 120 mV higher than WT amicyanin and was used to extend the range of the free energy dependence studied. The ET parameters of the reaction of WT and P94A amicyanin with MauG were within error of each other. This is significant because the ET reaction of P94A amicyanin with its natural electron acceptor was not able to be studied due to a kinetic coupling of the ET step with a non-ET step. This kinetic coupling obscured the parameters of the ET step because it is not kinetically distinguishable from the ET step.A Y294H MauG mutant was also studied. This mutation replaced the axial tyrosine ligand of the six-coordinate heme of MauG with a histidine. No reaction is observed with Y294H MauG in its native reaction. However, the high valent oxidation state of the five-coordinate heme of Y294H MauG reacts with reduced amicyanin. The ET rate was analyzed by ET theory to study the high valent heme in Y294H MauG. The reorganization energy of Y294H MauG was calculated to be nearly 20% lower as compared to the same reaction with WT MauG. These results provide insight into the obscured nature of reorganization energy of large redox cofactors in proteins, particularly heme cofactors, as well as to how the active sites of enzymes are optimized to perform long range ET vs catalysis with regard to balancing redox potential and reorganization energy.
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Date Issued: 2016
Identifier: CFE0006100, ucf:51192
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006100

Title: Pyroglutamylated amyloid beta peptides enhance non-fibrillogenic aggregation of the unmodified peptide.
Creator: Goldblatt, Gregory, Tatulian, Suren, Chen, Bo, Teter, Kenneth, King, Stephen, University of Central Florida
Abstract / Description: Alzheimer's disease (AD) is accompanied by abnormal extracellular deposition of amyloid b (Ab) peptide. This has led to the amyloid cascade hypothesis, causatively relating Ab with AD. While Ab deposits assume a fibrillar cross-b structure, prefibrillar oligomers of Ab have been identified as the main cytotoxic agents in AD. Pyroglutamylated amyloid beta (AbpE) peptides are N-terminally truncated and pyroglutamylated (at Glu3 or Glu11) Ab molecules that display enhanced cytotoxicity and...
Show moreAlzheimer's disease (AD) is accompanied by abnormal extracellular deposition of amyloid b (Ab) peptide. This has led to the amyloid cascade hypothesis, causatively relating Ab with AD. While Ab deposits assume a fibrillar cross-b structure, prefibrillar oligomers of Ab have been identified as the main cytotoxic agents in AD. Pyroglutamylated amyloid beta (AbpE) peptides are N-terminally truncated and pyroglutamylated (at Glu3 or Glu11) Ab molecules that display enhanced cytotoxicity and represent up to 50% of total Ab in AD brains. AbpE significantly enhances the toxicity of unmodified Ab by an unknown mechanism. Although in situ Ab populations are heterogeneous, the majority of studies have been conducted on single Ab species. Here, we examined the structural and morphological changes that occur in mixed Ab/AbpE samples. Circular dichroism and transmission electron microscopy data indicate that AbpE3-42 forms b-sheet structure and undergoes delayed fibrillogenesis compared to unmodified Ab1-42. Further, AbpE3-42 decelerates b-sheet formation in mixed Ab1-42/AbpE3-42 samples. FTIR measurements, using 13C-labeled Ab1-42 and unlabeled AbpE3-42, indicate that AbpE3-42 inhibits cross-b-sheet formation by Ab1-42, which explains the retardation of fibrillogenesis. FTIR on peptides 13C-labeled at specific segments provided site specific structural information. Based on these data, the monomeric Ab structure has been modeled as a b-hairpin stabilized by intramolecular H-bonding with an N-terminal a-helix. These hairpins likely form higher order aggregates through ionic and hydrophobic interactions between the C-terminus of one hairpin and the N-terminus of another. Utilizing a novel technique, hydration from gas phase, we examined the a-helix to b-sheet transitions of these peptides. When combined, AbpE3-42 and Ab1-42 mutually inhibit intermolecular b-sheet formation, instead promoting formation of AbpE3-42/Ab1-42 hetero-oligomers of intramolecular H-bonding. These hetero-oligomers displayed enhanced toxicity to PC12 cells compared to individual peptides and induced greater calcium release from lipid vesicles than unmodified Ab. These results indicate that Ab and AbpE mutually inhibit fibrillogenesis and stabilize hetero-oligomers of enhanced cytotoxicity, possibly through a membrane permeabilization mechanism. Collectively, our findings lead to a new concept that Ab/AbpE hetero-oligomers, not just Ab or AbpE oligomers, are the main cytotoxic species in AD
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Date Issued: 2016
Identifier: CFE0006108, ucf:51195
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006108

Title: Modulation of cholera toxin structure and function by host proteins.
Creator: Burress, Helen, Teter, Kenneth, Self, William, Zervos, Antonis, Tatulian, Suren, University of Central Florida
Abstract / Description: Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the...
Show moreCholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared (FTIR) spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and its refolding of CTA1 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance (SPR) experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allowed disordered CTA1 to attain an active state and did not prevent further stimulation of toxin activity by ADP-ribosylation factor 6, a host cofactor for CTA1. This activity is consistent with its role as a chaperone that refolds endogenous cytosolic proteins as part of a foldosome complex consisting of Hsp90, Hop, Hsp40, p23, and Hsc70. A role for Hsc70 in CT intoxication has not yet been established. Here, biophysical, biochemical, and cell-based assays demonstrate Hsp90 and Hsc70 play overlapping roles in the processing of CTA1. Using SPR we determined that Hsp90 and Hsc70 could bind independently to CTA1 at distinct locations with high affinity, even in the absence of the Hop linker. Studies using isotope-edited FTIR spectroscopy found that, like Hsp90, Hsc70 induces a gain-of-structure in unfolded CTA1. The interaction between CTA1 and Hsc70 is essential for intoxication, as an RNAi-induced loss of the Hsc70 protein generates a toxin-resistant phenotype. Further analysis using isotope-edited FTIR spectroscopy demonstrated that the addition of both Hsc70 and Hsp90 to unfolded CTA1 produced a gain-of-structure above that of the individual chaperones. Our data suggest that CTA1 translocation involves a ratchet mechanism which couples the Hsp90-mediated refolding of CTA1 with extraction from the ER. The subsequent binding of Hsc70 further refolds CTA1 in a manner not previously observed in foldosome complex formation. The interaction of CTA1 with these chaperones is essential to intoxication and this work elucidates details of the intoxication process not previously known.
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Date Issued: 2014
Identifier: CFE0005310, ucf:50511
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005310

Title: In Vitro Characterization of Unmodified and Pyroglutamylated Alzheimer's Amyloid beta peptide.
Creator: Matos, Jason, Tatulian, Suren, Teter, Kenneth, Davidson, Victor, University of Central Florida
Abstract / Description: Plaques of amyloid ? peptide (A?) are a hallmark trait of Alzheimer's disease (AD). However, the precise role of A? aggregates is not well understood. Recent studies have identified that naturally occurring N-terminal truncation and pyroglutamylation of A? significantly increases its neurotoxicity by an unknown mechanism. Content of pyroglutamylated A? (pE-A?) in AD brains has been shown to reach up to 50% of total A?. Modified pE-A? co-aggregates with A? by a seeding mechanism and forms...
Show morePlaques of amyloid ? peptide (A?) are a hallmark trait of Alzheimer's disease (AD). However, the precise role of A? aggregates is not well understood. Recent studies have identified that naturally occurring N-terminal truncation and pyroglutamylation of A? significantly increases its neurotoxicity by an unknown mechanism. Content of pyroglutamylated A? (pE-A?) in AD brains has been shown to reach up to 50% of total A?. Modified pE-A? co-aggregates with A? by a seeding mechanism and forms structurally distinct and highly toxic oligomers. We studied structural transitions of the full-length A?1-42, its pyroglutamylated form A?pE3-42, their 9:1 (A?1-42/A?pE3-42) and 1:1 molar combinations. Transmission electron microscopy was used to directly visualize the fibrils of the samples in a buffer mimicking physiological environment. Atomic force microscopy measurements were done to determine rate of second nucleation events in fibrils. Thioflavin-T fluorescence indicated that low ionic strength suppressed the aggregation of A?pE3-42 but promoted that of A?1-42, suggesting different paths of fibrillogenesis of unmodified A? and pE-A?. Interestingly, A?pE3-42 at only 10% significantly facilitated the fibrillization of A?1-42 at near physiological ionic strength but had little effect at low salt. Circular dichroism and Fourier transform infrared (FTIR) spectroscopy were used to characterize the structural transitions during fibrillogenesis. In aqueous buffer, both unmodified A? and pE-A? peptides adopted parallel intermolecular ?-structure. Interestingly, A?pE3-42 contained lower ?-sheet content than 13C-A?1-42, while retaining significantly larger fractions of ?-helical and turn structures. Structural details of A? and pE-A? combinations were unveiled by isotope-edited FTIR spectroscopy, using 13C-labeled A?1-42 and unlabeled A?pE3-42. When exposed to environmental humidity, A?pE3-42 not only maintained an increased fraction of ?-helix but also was able to reverse 13C-A?1-42 ?-sheet structure. These data provide a novel structural mechanism for pE-A? hypertoxicity; pE-A? undergoes fasternucleation due to its increased hydrophobicity, thus promoting formation of smaller, hypertoxic oligomers of partial ?-helical structure.
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Date Issued: 2014
Identifier: CFE0005378, ucf:50465
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005378

Title: Resistive Pulse study of Vesicles and Liposomes.
Creator: Lin, Yuqing, Chow, Lee, Schulte, Alfons, Tatulian, Suren, Yuan, Jiann-Shiun, University of Central Florida
Abstract / Description: In this work, the properties of the liposomes, the artificially created vesicles by various methods, are explored by a resistive pulse method using micropipettes. The fact that vesicles are fundamental in the wide range of functionalities they fulfill as organelles strengthen the desire of understanding the properties of them. The motivation of this work comes from the significant roles that liposomes play in the development of targeted drug delivery systems. Among other significant variables...
Show moreIn this work, the properties of the liposomes, the artificially created vesicles by various methods, are explored by a resistive pulse method using micropipettes. The fact that vesicles are fundamental in the wide range of functionalities they fulfill as organelles strengthen the desire of understanding the properties of them. The motivation of this work comes from the significant roles that liposomes play in the development of targeted drug delivery systems. Among other significant variables, the size of liposomes is found to be one of the dominating parameters in liposome based drug delivery, and the correlation between liposome size and delivery efficiency is discussed. To help improving the size evaluation ability, a few mainstream methods for liposome size detection and measurements are reviewed. As a reliable and accessible alternative method for liposomes detection, the resistive pulse method is introduced and the measurement on liposomes size change upon pH gradient was performed using this method. With our current liposome composition, we found the size increases as environmental pH increases. Further investigation is performed with vesicular pH=6, 7, and 8, respectively. Lastly, the stability of the small unilamellar vesicles (SUV) was studied via resistive pulse method, by monitoring the size change of 50nm liposomes as function of time. A significant size change in freshly prepared 50nm liposomes is recorded. This information will provide invaluable knowledge for targeting tumor with tight tissues, where small size liposomes are needed.
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Date Issued: 2015
Identifier: CFE0005826, ucf:50933
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005826

Title: Two-Component Covalent Inhibitors (TCCI) of the Human Immunodeficiency Virus Reverse Transcriptase (HIV-RT).
Creator: Ledezma, Carlos, Kolpashchikov, Dmitry, Yestrebsky, Cherie, Hernandez, Florencio, Zhai, Lei, Tatulian, Suren, University of Central Florida
Abstract / Description: The traditional design of nucleoside reverse transcriptase inhibitors (NRTI's) involves the synthesis of chain-terminated nucleoside analogs. HIV-RT has relatively low fidelity which facilitates mutations that confer resistance towards NRTI's, also, drug promiscuity from NRTI's result in various side-effects that lead to poor patient adherence to treatment. We designed and tested two-component covalent inhibitors against HIV-RT. Our inhibitor design results in higher specificity due to its...
Show moreThe traditional design of nucleoside reverse transcriptase inhibitors (NRTI's) involves the synthesis of chain-terminated nucleoside analogs. HIV-RT has relatively low fidelity which facilitates mutations that confer resistance towards NRTI's, also, drug promiscuity from NRTI's result in various side-effects that lead to poor patient adherence to treatment. We designed and tested two-component covalent inhibitors against HIV-RT. Our inhibitor design results in higher specificity due to its binary approach, which has previously been used in biosensing applications, where both components are necessary for therapeutic effect, and lower chances for mutagenesis because of its inhibitory action. The TCCI approach results in up to 93% inhibition of HIV-RT Furthermore, our inhibitor design is highly modular and can be adjusted towards the therapeutic targeting of other biopolymers.
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Date Issued: 2017
Identifier: CFE0006893, ucf:51712
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006893

Title: Theoretical-Experimental Study of the Two-Photon Circular Dichroism of Helicenes and Aromatic Amino Acids in the UV Region: From the Structure-Property Relationship to the Final Implementation.
Creator: Vesga Prada, Yuly Katherine, Hernandez, Florencio, Huo, Qun, Chumbimuni Torres, Karin, Zou, Shengli, Tatulian, Suren, University of Central Florida
Abstract / Description: Two-photon circular dichroism (TPCD) has been recognized for its exceptional spectroscopic ability for the structural and conformational analysis of chiral systems due to its high sensitivity to small peptide structural distortions. In 2008, Hernandez and co-workers demonstrated TPCD experimentally by the development of the Double L-scan technique. Since then, we have been working on a systematic theoretical-experimental study of chiral molecules using TPCD. In this dissertation, I present my...
Show moreTwo-photon circular dichroism (TPCD) has been recognized for its exceptional spectroscopic ability for the structural and conformational analysis of chiral systems due to its high sensitivity to small peptide structural distortions. In 2008, Hernandez and co-workers demonstrated TPCD experimentally by the development of the Double L-scan technique. Since then, we have been working on a systematic theoretical-experimental study of chiral molecules using TPCD. In this dissertation, I present my contribution to the continuation to the study of the structure-property relationship of TPCD in molecules with axial chirality in solution, as well as the implementation of the TPCD measurements in the near- and far-UV regions. Employing a theoretical-experimental approach I will discuss: 1) the effect of the pulse width of the excitation source on the TPCD spectra of biaryl derivatives, 2) the theoretical study of the TPCD signal in the far-UV on molecular structures simulating aromatic amino acid residues in proteins with secondary structures, and 3) the pros and cons of the implementation of the FUV-TPCD spectrometer. The outcomes of my research reveal the potential of TPCD for the conformational analysis of relatively complex molecular systems such as peptides in the far-UV region, an area never accessed before. Additionally, we exposed the applicability of TPCD as a complimentary method to standard electronic circular dichroism (ECD) for the study of complex structures. Finally, I demonstrate for the very first time experimental evidence of TPCD in the near- to Far-UV region.
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Date Issued: 2016
Identifier: CFE0006514, ucf:51375
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006514

Title: The Role of SOD1 Acetylation in Neurodegeneration.
Creator: Kaliszewski, Michael, Bossy-Wetzel, Ella, Estevez, Alvaro, Kim, Yoon-Seong, Tatulian, Suren, University of Central Florida
Abstract / Description: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons. Cu, Zn superoxide dismutase (SOD1), a cytoplasmic free radical scavenging enzyme, is mutated in familial ALS (fALS) and post-translational modification of the wild-type protein has been associated with sporadic ALS (sALS). Proteomic studies indicate that SOD1 is acetylated at Lys123; however, the role of this modification remains unknown. To investigate its function, we generated antibodies for...
Show moreAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons. Cu, Zn superoxide dismutase (SOD1), a cytoplasmic free radical scavenging enzyme, is mutated in familial ALS (fALS) and post-translational modification of the wild-type protein has been associated with sporadic ALS (sALS). Proteomic studies indicate that SOD1 is acetylated at Lys123; however, the role of this modification remains unknown. To investigate its function, we generated antibodies for Lys123-acetylated SOD1 (Ac-K123 SOD1). Sod1 deletion in Sod1-/- mice, K123 mutation, or preabsorption with Ac-K123 peptide suppressed immunoreactivity, confirming antibody specificity. In the normal central nervous system, Ac-K123 SOD1 maps to glutamatergic neurons of the cerebellar cortex, dentate gyrus, hippocampus, olfactory bulb, and retina. In cultured neurons, Ac-K123 SOD1 localized to defined regions of axons and dendrites. Previous studies have suggested a role for SOD1 in cell cycle regulation. Therefore, we tested the distribution of Ac-K123 SOD1 during the cell cycle of astrocytes. In G1 Ac-K123 SOD1 localized to the nucleus, in G0 to the primary cilium, in metaphase and anaphase to chromosomes, and in telophase to the midbody. The deacetylase HDAC6 and acetyl-transferase ?-TAT1 are associated with the primary cilium. Therefore, we tested whether they regulate reversible acetylation of SOD1. HDAC6 knockdown or pharmacological inhibition markedly increased, while HDAC6 overexpression decreased, SOD1 Lys123 acetylation. By contrast, SOD1 Lys123 acetylation was decreased by ?-TAT1 knockdown and increased by ?-TAT1 overexpression. These results suggest that HDAC6 and ?-TAT1 regulate SOD1 Lys123 acetylation. Next, we examined Lys123 acetylation in fALS SOD1 mutants. Remarkably, Lys123 acetylation was dramatically increased in fALS mutants including SOD1 A4V. The acetyl-Lys123 mimetic of wild-type SOD1 caused axonal transport deficits similar to those observed in SOD1 pathogenic mutants such as A4V. Interestingly, HDAC6 deacetylation or acetylation resistance by Lys123 mutation, abolished A4V protein misfolding, axonal transport defects, and neuronal cell death. These results suggest that Lys123 acetylation plays a key role in the neurotoxicity of fALS mutants and may have implications in sALS. Because Ac-K123 SOD1 maps to the primary cilium, we examined whether ciliogenesis is altered in fALS mutant SOD1 astrocytes. Strikingly, fALS mutants caused centriole and primary cilia proliferation with ciliary ectosome secretion. Notably, multiciliated ependymal cells in the brain ventricles and spinal cord central canal, which are critical for cerebral spinal fluid circulation, stained strongly for Ac-K123 SOD1. Thus, we speculate that ciliary ectosome shedding from ependymal cells accounts for the presence of misfolded SOD1 in the CSF in fALS and perhaps sALS. In summary, we identified SOD1 Lys123 acetylation as a novel mechanism underlying protein misfolding and neurodegeneration in ALS. Ac-K123 SOD1 may emerge as novel target for the diagnosis and treatment of ALS.
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Date Issued: 2016
Identifier: CFE0006467, ucf:51409
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006467

Bax (2)	+ -
Circular Dichroism (2)	+ -
amyloid beta (2)	+ -
apoptosis (2)	+ -
cholera (2)	+ -

cholera toxin (2)	+ -
fibrillogenesis (2)	+ -
solid-state NMR (2)	+ -
AB toxins (1)	+ -
AFM (1)	+ -
AFM-IR (1)	+ -
ATR-FTIR (1)	+ -
Alzheimer's (1)	+ -
Alzheimer's Disease (1)	+ -
Alzheimer's disease (1)	+ -
Amyloid beta (1)	+ -
Amyloid fibrillization (1)	+ -
Bioenergetics (1)	+ -
Brownian Dynamics (1)	+ -
CT20p (1)	+ -
CTA1 (1)	+ -
Cholera (1)	+ -
Covalent Inhibition (1)	+ -
DNA (1)	+ -
Fluorescence (1)	+ -
Fluorescence Spectroscopy (1)	+ -
Fourier transform infrared spectroscopy (1)	+ -
G protein (1)	+ -
HIV-RT (1)	+ -
Interdiscliplinary approach (1)	+ -

ETD Collection (21)	+ -
Honors Collection (1)	+ -

Tatulian, Suren (22)	+ -
University of Central Florida (22)	+ -
Teter, Kenneth (7)	+ -
Chen, Bo (4)	+ -
Schulte, Alfons (4)	+ -

Self, William (4)	+ -
Zervos, Antonis (4)	+ -
Cole, Alexander (3)	+ -
Davidson, Victor (3)	+ -
Hernandez, Florencio (2)	+ -
Jewett, Travis (2)	+ -
Khaled, Annette (2)	+ -
Bader, Carly (1)	+ -
Bhattacharya, Aniket (1)	+ -
Boohaker, Rebecca (1)	+ -
Bossy-Wetzel, Ella (1)	+ -
Boykin, Tommy (1)	+ -
Burress, Helen (1)	+ -
Campiglia, Andres (1)	+ -
Chow, Lee (1)	+ -
Chumbimuni Torres, Karin (1)	+ -
Dow, Brian (1)	+ -
Estevez, Alvaro (1)	+ -
Garg, Pranav (1)	+ -
Goldblatt, Gregory (1)	+ -
Guyette, Jessica (1)	+ -
Huang, Aiqun (1)	+ -
Huo, Qun (1)	+ -
Jeon, Jaekyun (1)	+ -
Jewett, Mollie (1)	+ -

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