Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 196-245). Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 196-245). Also available in print.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

EmrE from Escherichia coli is a member of the small multidrug resistance protein family that oligomerizes to export hydrophobic cationic antimicrobials by utilizing the proton motive force. We studied the helix-helix interactions of the four transmembrane (TM) segments of EmrE to determine how this protein might assemble into its oligomeric forms. Using a combination of biochemical and biophysical techniques, we assessed the oligomerization propensities of Lys-tagged EmrE TM peptides in membrane-mimetic environments. Our results established that each of the TMs of EmrE display detergent-sensitive self-association, but in particular, TM2 had the greatest dimerization capability that was not completely abolished even by scrambling the native sequence. Mutations made to TM2 in full-length EmrE also revealed that efflux-defective mutations are located on one face of the helix. These findings reveal another potential oligomerization site for EmrE - and perhaps SMRs - and may provide a target for development of novel efflux-inhibitors.

No description available.

Plants within the Caryophyllaceae, and certain other families, produce cyclic peptides (CPs) which generally consist of 5–12 proteinogenic amino acids. Until recently, very little was known about the biosynthesis of CPs in the Caryophyllaceae. Recently, in the Covello lab, two enzymes in Saponaria vaccaria were found to be involved in the processing of ribosome-derived linear precursors, giving rise to cyclic peptides. Thus, oligopeptidase 1 (OLP1) and peptide cyclase 1 (PCY1) are involved in the biosynthesis of segetalin A (a six-membered CP) from a 32 amino acid linear peptide precursor called presegetalin A1. PCY1 carries out the unusual cyclization reaction to form mature segetalin A from a linear intermediate. The purified recombinant PCY1, the first cloned plant enzyme whose function is peptide cyclization, was identified as a homologue of a prolyl-oligopeptidase from the S9 serine protease family. In principle, PCY1 performs an intra-molecular transpeptidation reaction to produce a CP. A homology-based structural model of PCY1 suggests that it has two domains, a catalytic α/β hydrolase domain and an unusual β- propeller domain. In an effort to define the substrate specificity of PCY1, a wide variety of synthetic peptide precursors were tested in assays and the results are discussed.

EmrE from Escherichia coli is a member of the small multidrug resistance protein family that oligomerizes to export hydrophobic cationic antimicrobials by utilizing the proton motive force. We studied the helix-helix interactions of the four transmembrane (TM) segments of EmrE to determine how this protein might assemble into its oligomeric forms. Using a combination of biochemical and biophysical techniques, we assessed the oligomerization propensities of Lys-tagged EmrE TM peptides in membrane-mimetic environments. Our results established that each of the TMs of EmrE display detergent-sensitive self-association, but in particular, TM2 had the greatest dimerization capability that was not completely abolished even by scrambling the native sequence. Mutations made to TM2 in full-length EmrE also revealed that efflux-defective mutations are located on one face of the helix. These findings reveal another potential oligomerization site for EmrE - and perhaps SMRs - and may provide a target for development of novel efflux-inhibitors.

No description available.