Browsing by Subject "Peptide"
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Item Biosynthesis of sulfur containing heterocycles in natural products(2016-12) Gengler, Jon Peter; Liu, Hung-wen, 1952-This thesis is a comprehensive review of the biosynthesis of sulfur containing heterocycles in natural metabolites. The review focuses on sulfur incorporation and cyclization of the moieties, with a lesser examination of the role these heterocycles play in the chemistry of their compound's activity.Item Characterization and applications of affinity based surface modification of polypyrrole(2009-12) Nickels, Jonathan D.; Schmidt, Christine E.I present the characterization and applications of a technique to modify the surface of the conducting polymer, polypyrrole, via a novel, 12-amino acid peptide, THRTSTLDYFVI (T59). This peptide non-covalently binds to the chlorine-doped conducting polymer polypyrrole, allowing it to be used in tethering molecules to polypyrrole for uses such as a scaffold for the treatment of peripheral nerve injury or in surface coatings of neural recording electrodes. I have quantified the binding of this peptide as well as investigating the mechanism of the binding. The equilibrium constant of the binding interaction of PPyCl and the T59 peptide was found through a binding assay to be 92.6 nM, and the off rate was found to be approximately 2.49 s⁻¹, via AFM force spectroscopy. The maximum observed surface density of the peptide was 1.27 +/- 0.42 femtomoles/cm². Furthermore, my studies suggest that the eighth residue, aspartic acid, is the main contributor of the binding, by interacting with the partially positive charge on the backbone of polypyrrole. I have demonstrated practical applications of the technique in the successful modification of a PPyCl surface with the laminin fragment IKVAV, as well as the so-called stealth molecule poly(ethylene glycol) (PEG). A subcutaneous implant study was performed to confirm that the T59 peptide did not induce any significant reaction in vivo. Significantly, the conductivity of a PPyCl surface was unaffected by this surface modification technique.Item Development of matrix assisted laser desorption ionization-ion mobility-orthogonal time-of-flight mass spectrometry as a tool for proteomics(Texas A&M University, 2005-08-29) Ruotolo, Brandon ThomasSeparations coupled to mass spectrometry (MS) are widely used for large-scale protein identification in order to reduce the adverse effects of analyte ion suppression, increase the dynamic range, and as a deconvolution technique for complex datasets typical of cellular protein complements. In this work, matrix assisted laser desorption-ionization is coupled with ion mobility (IM) separation for the analysis of biological molecules. The utility of liquid-phase separations coupled to MS lies in the orthogonality of the two separation dimensions for all analytes. The data presented in this work illustrates that IM-MS relies on the correlation between separation dimensions for different classes (either structural or chemical) of analyte ions to obtain a useful separation. For example, for a series of peptide ions of increasing mass-to-charge (m/z) a plot drift time in the IM drift cell vs. m/z increases in a near-linear fashion, but DNA or lipids having similar m/z values will have very different IM drift time-m/z relationships, thus drift time vs. m/z can be used as a qualitative tool for compound class identification. In addition, IM-MS is applied to the analysis of large peptide datasets in order to determine the peak capacity of the method for bottom-up experiments in proteomics, and it is found that IM separation increases the peak capacity of an MS-only experiment by a factor of 5-10. The population density of the appearance area for peptides is further characterized in terms of the gas-phase structural propensities for tryptic peptide ions. It is found that a small percentage (~3%) of peptide sequences form extended (i.e., helical or β-sheet type) structures in the gas-phase, thus influencing the overall appearance area for peptide ions. Furthermore, the ability of IM-MS to screen for the presence of phosphopeptides is characterized, and it is found that post translationally modified peptides populate the bottom one-half to one-third of the total appearance area for peptide ions. In general, the data presented in this work indicates that IM-MS offers dynamic range and deconvolution capabilities comparable to liquid-phase separation techniques coupled to MS on a time scale (ms) that is fully compatible to current MS, including TOF-MS, technology.Item Development of Novel Cancer Immunotherapeutics Utilizing Cell-Targeting Peptides(2013-01-17) Diaz, Tracy Montie; Brown,Kathlynn Corrine, Ph.D.Cancer immunotherapy is an emerging treatment option that offers high tumor specificity and efficacy. Immune therapies for cancer can be divided into two main types: active and passive. Active therapy strives to achieve a long term protective immunity against a tumor antigen while passive therapy supplies exogenous immunological reagents for anti-tumor effector functions. Both immunotherapies can be improved by utilizing cell targeting peptides. Dendritic Cell Targeting Peptides: Cancer vaccines can elicit immune responses against tumor antigens. Antigen-pulsed in vitro matured dendritic cells (DCs) are used for higher efficacy. However, this method does not provide a significant therapeutic immune response. A more robust anti-tumor immune response could potentially be achieved through in vivo DC targeting of tumor antigens. Through phage-displayed peptide library panning protocol, four different DC-targeting phage clones were isolated. Of those, XS52.1 and XS52.3 bind specifically to the XS52 immature dendritic cells. The XS52.3 phage clone also binds bone-marrow dendritic cells (BMDCs) from Balb/c and C57BL/J6 mice. Each phage clone elicited heightened anti-phage antibody production in both mouse strains. Potential future studies will determine if these peptides can be used to target antigen to DCs for in vivo cancer vaccines. Peptide-Antibody Targeting: Monoclonal antibodies directed against tumor antigens have been successful in clinics, but problems remain with identifying and validating new targets. Modification of the antibody scaffold for distinct applications can also be problematic. Using our phage display panning protocol, we have identified ligands of high affinity and specificity against a panel of human non-small cell lung cancer (NSCLC) cell lines. Furthermore, these peptide-targeting ligands can be chemically synthesized and easily modified for different uses. In my studies, synthetic-peptide ligands have been used to redirect antibody targeting by using biotinylated-tetrameric peptides and anti-biotin antibodies. These results suggest that peptide-antibody conjugates utilizing isolated peptides can be used to redirect antibody targeting. This methodology would increase the antibody repertoire available for therapy.Item Identification of novel allosteric modulators of the glycine receptor using phage display technology(2011-08) Tipps, Megan Elizabeth; Mihic, S. John; Aldrich, Richard W.; Harris, Adron; Iverson, Brent L.; Zakon, Harold H.The glycine receptor (GlyR) is a ligand-gated ion channel and a member of the cys-loop receptor family. Like other members of this family, the GlyR is a target for many drugs of abuse, including alcohol. While the effects of alcohol on these receptors have been well-characterized, the contribution of each receptor subtype to the overall physiological and behavioral effects of alcohol use are unclear. This is partially due to the limited pharmacology of the GlyR, which limits the ability to isolate GlyR function within a complex system. One method for identifying compounds that bind to and modulate a given target is phage display. This approach uses bacteriophage to screen a large number of peptide sequences for affinity at a given target. We developed a phage selection protocol to identify peptides that bind to the GlyR. These peptides were then tested for functional effects at the GlyR using two-electrode voltage clamp physiology. We identified several peptides that were able to modulate GlyR function. Peptide D12-116 showed specificity for the GlyR over two closely related γ-aminobutyric acid (GABA) channels. In addition, this method is easily adapted for the selection of peptides that bind to any cell-expressed target, increasing the utility of phage display in the neurobiology field. Another shortcoming in GlyR pharmacology is the lack of modulators with specificity for a single GlyR subtype. We next adjusted our selection protocol to search for peptides that can distinguish between the different Gly R α subtypes. We identified several promising lead peptides that show subtype preference. Finally, we found that trifluoroacetic acid (TFA), a common peptide contaminant, also modulates GlyR function. This finding has important implications for both previously reported peptide modulators and the pharmacology of several volatile anesthetics, for which TFA is the major metaboliteItem Optimization of force fields for molecular dynamics(2014-12) Di Pierro, Michele; Elber, RonA technology for optimization of potential parameters from condensed phase simulations (POP) is discussed and illustrated. It is based on direct calculations of the derivatives of macroscopic observables with respect to the potential parameters. The derivatives are used in a local minimization scheme, comparing simulated and experimental data. In particular, we show that the Newton Trust-Region protocol allows for accurate and robust optimization. POP is illustrated for a toy problem of alanine dipeptide and is applied to folding of the peptide WAAAH. The helix fraction is highly sensitive to the potential parameters while the slope of the melting curve is not. The sensitivity variations make it difficult to satisfy both observations simultaneously. We conjecture that there is no set of parameters that reproduces experimental melting curves of short peptides that are modeled with the usual functional form of a force field. We then apply the newly developed technology to study the liquid mixture of tert-butanol and water. We are able to obtain, after 4 iterations, the correct phase behavior and accurately predict the value of the Kirkwood Buff (KB) integrals. We further illustrate that a potential that is determined solely by KB information, or the pair correlation function, is not necessarily unique.