Protein nanopores as a platform for transmembrane nanodevices

Abstract

Nanopore sensing has seen vast development over the past four decades. The technique originally looked to use electrophysiological methods to study native protein channels. However, it is now possible to exploit these proteins for sensing applications. Herein, we explore methods for covalent and non-covalent modification of a biological nanopore to achieve new functionality. Chapter 1 summarises the history of nanopore technology; from its inception as a method for studying native channels, to its deployment in nanopore sensing. To achieve effective sensing, native proteins have undergone a broad range of chemical modification to achieve enhanced functionality. This chapter explores the amalgamation of biological and solid-state nanopores.

Chapter 2 seeks to the monitor the binding and catalytic turnover of substrates within a single cucurbituril molecule captured within a protein nanopore. Previous work has shown that cucurbiturils and cyclodextrins can transiently interact with an α-hemolysin channel. Capture of a single cucurbituril within a protein nanopore was achieved, and the dwell time of the binding events was optimised. Following this, it was demonstrated that observations of the catalysed Diels-Alder could be made at the single-molecule level. However, further optimisation of the resolution would be required to elucidate mechanistic information.

Chapter 3 presents methods for in situ chemical functionalisation of a biological nanopore. Here, the focus is upon the chemical modification of a wild-type protein thereby to circumventing the need for mutagenesis. Three target residues are discussed: lysine, methionine and tyrosine. Successful modification was achieved at both the lysine and methionine sites of α-hemolysin. While some provisional success was recorded with tyrosine, the modifications were not reproducible.

Chapter 4 introduces preliminary work towards the development of transmembrane molecular machines. This utilises the lysine modification discussed in Chapter 2 to covalently attach established synthetic molecular machines to the channel. Molecular switches, motors and pumps were all explored. Some success was achieved attaching the molecular machines to a protein channel. However, issues with pore stability limited the progress and true machine-like behaviour was not observed.