Single-molecule chemistry studies with engineered alpha-hemolysin pores

Engineered protein nanopores can be used to investigate a wide range of dynamic processes in real time and at the single-molecule level, for example covalent bond making and breaking or the interaction of ligands with their cognate binding sites. The detection of such processes is accomplished by monitoring the current carried by ions through the pore in an applied potential, which is modulated as molecules of interest interact with engineered binding sites within the pore. In contrast to ensemble measurements, where the behaviour of individual molecules is obscured by averaging, single-channel recordings can identify short-lived intermediates and rare reaction pathways, thereby adding to our understanding of fundamental processes in chemistry and biology. The goal of my thesis work was to engineer alpha-hemolysin (αHL) pores to gain insight into such processes.
Chapter 1 provides an overview of common techniques used to study single- molecule processes, in particular single channel recordings. General techniques to engineer ion channels and pores are presented, followed by examples of how the alpha-HL pore has been engineered to monitor dynamic processes at the single- molecule level.
Chapter 2 describes how alpha-HL pores can be chemically modifeed with a tridentate "half-chelator" ligand. Single channel recordings show that this modifeed pore can be used to determine rates of chelation and the stability of divalent metal ion complexes. The modifeed pore can also be used as a stochastic sensor for the detection of different divalent metal ions in solution.
Chapter 3 investigates the chelate-cooperativity between two half-chelator ligands installed in close proximity in the alpha-HL pore, as they form a full complex with a single Zn2+ ion. The single channel recordings reveal a two step process, in which the Zn2+ ion must fiferst bind to one of the two half-chelators, before the second one completes the complex. The rate constants for all the major steps of the process are determined and the extent of cooperativity between the half-chelators is quantifeed.
Chapter 4 demonstrates that genetically encoded subunit dimers of alpha-HL can be used to control the subunit arrangement in the heptameric pore. Although techniques exist to prepare heteroheptameric pores, pores containing more than one type of modifeed subunit are not commonly used because it is impossible to distinguish between the permutations of the pore. By using subunit dimers, heptamers in which two defefined subunits are adjacent to each other can be formed, which increases the range of structures that can be obtained from engineered protein nanopores.
Chapter 5 explores the possibility of following the nuclease activity of a metal complex in the alpha-HL pore at the single-molecule level. The Rh(III) complex [Rh(bpy)2phzi]2+ binds strongly to CC mismatches in dsDNA, and on activation with UV light promotes the cleavage of one of the two strands. To follow this reaction by single channel recording, a piece of dsDNA with the bound Rh-complex was immobilised in the HL pore and the single current changes under UV irradiation were monitored. The preliminary data indicate that the rate of the photocleavage reaction can be measured.

Keywords: Protein Engineering
Advisor: Bayley, H