Structural Studies of Peptides that Influence the Pathogenicity of Bacterial Infections, and Investigation of Structure-Activity-Relationships of Antimicrobial Peptides with Application to Cancer Therapy.

  • Author / Creator
    Towle, Kaitlyn M.
  • Enterocin 7 is a two-component, leaderless bacteriocin comprised of an A and B peptide and produced by Enterococcus faecalis 710C. The secondary structure of enterocin 7B was investigated through circular dichroism. A high degree of α-helicity was discovered by circular dichroism, regardless of solvent. The solution structure of enterocin 7B was solved based on NMR spectroscopic data. The peptide was found to consist of three amphipathic α-helices, confirming the high degree of helicity predicted by circular dichroism. The overall structural fold in enterocin 7B was identified in other leaderless and circular bacteriocins and is a privileged motif in these classes of antimicrobial peptides. Subtilosin A is a circular, sactibiotic bacteriocin isolated from Bacillus subtillus JH642. This highly hydrophobic peptide of 34 amino acid residues contains three thioether bonds between the sulphur of cysteine side chains, at position 4, 7, and 13, and the α-carbon of partnering amino acids (Phe31, Thr28, and Phe22, respectively). A previous study of the solution structure of subtilosin A determined the stereochemistry of each α-carbon of Phe31, Thr28, and Phe22 to be D, D, and L, respectively. The all D energy minimization calculation was very close in energy to the reported D,D,L isomer, and therefore the crystal structure of this peptide is desirable. The solubility of subtilosin A was investigated; cyclodextrins were used as additives to increase the solubility of subtilosin A. A biotin bioconjugate of subtilosin A was synthesized to use as a non-covalent co-crystallization partner with streptavidin. A Meldrum’s acid derivative was synthesized to conjugate subtilosin A with carbonic anhydrase for use as a covalent co- crystallization partner. Efforts are underway to optimize the linkage between subtilosin A and carbonic anhydrase using the Meldrum’s acid derivative. Phenol-soluble modulins are virulence factors produced by a wide variety of staphylococcus bacteria. Of particular interest are the phenol-soluble modulins produced by the multi-drug resistant Staphylococcus aureus. Phenol-soluble modulins α1, and α3 were synthesized by solid phase peptide synthesis and phenol-soluble modulin β2 was isolated as a fusion protein with the small-ubiquitin like modifier protein that was subsequently cleaved. The secondary structure of these peptides was investigated using circular dichroism. Each peptide was found to be α-helical in a solution of 50 % trifluoroethanol: water. The solution structure of each peptide was solved using NMR spectroscopic data. Phenol-soluble modulins α1, and α3 were each found to be a single amphipathic α-helix and phenol-soluble modulin β2 was found to be comprised of three amphipathic α-helices that pack in such a way as to give a hydrophobic core and hydrophilic surface. The structure of phenol-soluble modulin α1 differed slightly from a predicted structure previously reported. Phenol-soluble modulin β2 was found to be primarily α- helical, despite the low values of α-helicity predicted by circular dichroism. Neopetrosiamide A and B are tricyclic peptides isolated from the marine sponge Neopetrosia spp. These peptides are potent metastasis inhibitors and they differ from one another only in the stereochemistry of the sulfoxide moiety of the oxidized methionine at position 24. Analogues of these peptides in which the methionine sulfoxide is replaced with various non- canonical and canonical amino acids revealed reduced or abolished activity, suggesting a mechanistic ‘hot-spot’ of this peptide. Efforts to reduce the synthetic steps required to form the three disulfide bridges by substituting the cysteine residues with hydrophobic residues were unsuccessful, indicating that the native neopetrosiamide is held tightly together by the three disulfides. Attempts to isolate and identify the biological receptor of neopetrosiamides through fluorescent labeling were unsuccessful. New folding studies using structure inducing solvents did not greatly improve the global oxidation of the cysteine residues to the correct disulfide connectivity. However, the use of chaotropic salts improved the global oxidation, resulting in a 1:1 mixture of desired neopetrosiamide with correct disulfide connectivity to undesired neopetrosiamide with incorrect disulfide connectivity.

  • Subjects / Keywords
  • Graduation date
    2017-11:Fall 2017
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Chemistry
  • Supervisor / co-supervisor and their department(s)
    • Vederas, John C. (Chemistry)
  • Examining committee members and their departments
    • Clive, Derrick L. J. (Chemistry)
    • Choi, Phillip Y.K. (Chemical and Materials Engineering)
    • Lowary, Todd L. (Chemistry)
    • Bowers, Albert A. (Pharmacy)