Dmitriev, OlegLee, Jeremy2016-10-182017-11-272016-092016-10-18Septemberhttp://hdl.handle.net/10388/7535Nanopore 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.application/pdfNanopore, nanopore analysis, ATP synthase, c-ring, subunit c, single-molecule analysisThesis2016-10-18