Conversion of the ATP synthase subunit c into a hydrophilic pore
| dc.contributor.advisor | Dmitriev, Oleg | |
| dc.contributor.advisor | Lee, Jeremy | |
| dc.contributor.committeeMember | Moore, Stanley | |
| dc.contributor.committeeMember | Leung, Adelaine | |
| dc.contributor.committeeMember | Wu, Yuliang | |
| dc.creator | Kulik, Daryna 1988- | |
| dc.creator.orcid | 0000-0001-7306-4986 | |
| dc.date.accessioned | 2016-10-18T19:44:42Z | |
| dc.date.available | 2017-11-27T16:32:00Z | |
| dc.date.created | 2016-09 | |
| dc.date.issued | 2016-10-18 | |
| dc.date.submitted | September 2016 | |
| dc.date.updated | 2016-10-18T19:44:42Z | |
| dc.description.abstract | Nanopore analysis is a very promising technique for many applications, such as the study of intrinsically disordered proteins, folding and misfolding of proteins and DNA sequencing. Nanopore is a small pore in the lipid bilayer membrane, which can let solutes through. Nanopores of various diameters would be useful for different applications. Currently, the most widely used nanopore is alpha-hemolysin with an internal diameter of ~1.4 nm. This nanopore is mostly used to study unfolded protein molecules and single-stranded DNA. The limitations of this pore include inability to pass larger folded protein molecules and double-stranded DNA, as well as inability to effectively discriminate biopolymers from smaller molecules. Our goal was to design a nanopore alternative to alpha-hemolysin, which would be stable for a long time in the artificial membrane and be suitable for a wider spectrum of applications in nanopore analysis. We chose c-rings of ATP synthase from two bacterial species as scaffolds for a new type of a nanopore: the c-ring from E.coli, which consists of 10 c-subunits, and the c-ring from I. tartaricus, which consists of 11 c-subunits. The size and diameter of the c-ring differ from species to species, which makes it a versatile model for a novel nanopore. We modified each c-ring by introducing polar amino acid substitutions into its internal cavity to make the ring interior hydrophilic. We used the wild type c-subunit from E. coli as a control. Purified monomeric c-subunits were reconstituted into proteoliposomes and tested for nanopore formation. Both the wild type and the polar interior versions of the c-ring formed stable nanopores with similar electrical properties. Further studies are needed to investigate the ability of these nanopores to translocate biomolecules and their potential for use in biological assays. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | http://hdl.handle.net/10388/7535 | |
| dc.subject | Nanopore, nanopore analysis, ATP synthase, c-ring, subunit c, single-molecule analysis | |
| dc.title | Conversion of the ATP synthase subunit c into a hydrophilic pore | |
| dc.type | Thesis | |
| dc.type.material | text | |
| local.embargo.terms | 2017-10-18 | |
| thesis.degree.department | Biochemistry | |
| thesis.degree.discipline | Biochemistry | |
| thesis.degree.grantor | University of Saskatchewan | |
| thesis.degree.level | Masters | |
| thesis.degree.name | Master of Science (M.Sc.) |