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dc.creatorKrasniqi, Besniken_US
dc.date.accessioned2014-01-21T19:01:30Z
dc.date.available2014-01-21T19:01:30Z
dc.date.created2013-11en_US
dc.date.issued2013-11-28en_US
dc.date.submittedNovember 2013en_US
dc.identifier.urihttp://hdl.handle.net/10388/ETD-2013-11-1280en_US
dc.description.abstractIn recent years the application of single-molecule techniques to probe biomolecules and intermolecular interactions at single-molecule resolution has expanded rapidly. Here, I investigate a series of peptides and proteins in an attempt to gain a better understanding of nanopore sensing as a single-molecule technique. The analysis of retro, inversed, and retro-inversed isomers of glucagon and α-helical Fmoc-D2A10K2 peptide showed that nanopore sensing utilizing a wild-type α-hemolysin pore can distinguish between all four isomers while circular dichroism can only distinguish between chiral isomers, but not between directional isomers. The investigation of a series of proteins of different chemical and physical properties revealed important information about nanopore analysis of proteins. Contrary to some reports in the literature, all proteins analysed here induced large blockade events. The frequency of total events and the proportion of large blockade events were significantly reduced in tris(hydroxymethyl)aminomethane or 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid buffers and were only restored by the addition of ethylenediaminetetraacetic acid or the use of phosphate buffer, both of which can sequester metal ions. Furthermore, the results obtained with the proteins in the presence of ligands demonstrated that transient or partial unfolding of proteins can be detected by nanopore analysis confirming the usefulness of this technique for conformational studies or for protein/ligand interactions. Interestingly, while the blockade current histograms were different for each protein there was no obvious correlation between the properties of the proteins and the blockade current histograms. In an attempt to identify whether the large blockade events were translocation or intercalation, both an indirect and a direct approach were taken. The indirect approach which relies on the effect of voltage on the interaction of the molecule with the pore provided no conclusive answer to the question of protein translocation through the α-hemolysin pore. In contrast, the direct approach in which ribonuclease A is added to the cis side of the pore and then the trans side is tested for enzyme activity showed that ribonuclease A doesn't translocate through the α-hemolysin pore.en_US
dc.language.isoengen_US
dc.subjectnanoporeen_US
dc.subjectnanopore sensingen_US
dc.subjectsolid-state poresen_US
dc.subjectalpha-hemolysinen_US
dc.subjectisomersen_US
dc.subjectzeta potentialen_US
dc.subjectmetal ion bindingen_US
dc.subjectprotein ligand interactionsen_US
dc.subjectRibonuclease Aen_US
dc.titleNanopore Sensing Of Peptides And Proteinsen_US
thesis.degree.departmentBiochemistryen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophy (Ph.D.)en_US
dc.type.materialtexten_US
dc.type.genreThesisen_US
dc.contributor.committeeMemberLee, Jeremy S.en_US
dc.contributor.committeeMemberMoore, Stanleyen_US
dc.contributor.committeeMemberHoward, Peteren_US
dc.contributor.committeeMemberNapper, Scotten_US


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