Conversion of the ATP synthase subunit c into a hydrophilic pore
Kulik, Daryna 1988-
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.
DegreeMaster of Science (M.Sc.)
SupervisorDmitriev, Oleg; Lee, Jeremy
CommitteeMoore, Stanley; Leung, Adelaine; Wu, Yuliang
Copyright DateSeptember 2016
Nanopore, nanopore analysis, ATP synthase, c-ring, subunit c, single-molecule analysis