High Resolution Single-Molecule Enzyme Dynamics Using Nanopores

Abstract

DNA is a molecule that contains the genetic information of all living organisms. DNA provides the instructions that the cell uses to construct the proteins which carry out the complex functions required for life to thrive. Enzymes are a class of proteins that use chemical potentials to catalyze energetically unfavorable chemical reactions to perform tasks ranging from muscle contraction to DNA packaging. In this thesis I focus on a class of enzymes called `motor enzymes' which use the energy provided by ATP hydrolysis to produce directed motion along a molecular track, such as DNA or RNA, and perform mechanical tasks such as unwinding double-stranded nucleic acids or building double stranded nucleic acids from single-stranded nucleic acids. Classically, enzyme reactions were studied by biochemical methods that monitor a large number of reactions simultaneously. These methods are limited because enzymes operate near thermal energies, leading to asynchronous progression of the chemical reaction. In the past 30 years, methods to monitor the reactions of single enzyme molecules have provided numerous insights into the function of motor enzymes, but these techniques lack the resolution to provide full details of how these molecules transduce chemical energy into mechanical work. In this thesis I present the development of Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT), a method for monitoring the movement of single enzyme molecules on DNA at unprecedented spatiotemporal resolution using the biological nanopore MspA. In SPRNT, a single MspA protein pore (termed a `nanopore') in a phospholipid bilayer forms the only electrical connection between two salt solutions termed \textit{cis} and \textit{trans}. A voltage applied across the membrane causes an ion current to flow through the nanopore. Negatively charged single-stranded DNA complexed to a motor enzyme is attracted into the nanopore by the electric field. The DNA passes through the pore until the motor enzyme, which is too large to fit through the pore, comes to rest on the rim of MspA. The DNA bases in the pore reduce the ion current flowing through the pore depending on the bases therein. The motor enzyme then moves along the DNA, causing DNA to move through the pore, leading to a series of stochastic ion-current amplitudes which simultaneously provide measurements of the kinetics of the enzyme and the DNA sequence. This method leads to a higher spatiotemporal resolution than any other single-molecule technique. In this thesis I present my role in the development of SPRNT. In chapter 1 I introduce the relevant biomolecules and techniques used to examine them. In chapter 2 I discuss the development of SPRNT and quantify its spatiotemporal resolution. In chapters 3 and 4 I present the first enzyme dynamics studies done with SPRNT on the helicase Hel308, and use information from quantities that could not be measured previously to elucidate the precise details of Hel308 motion on DNA, and to determine the mechanism by which DNA modulates Hel308 translocation. Chapter 5 contains concluding remarks and a discussion of the future of SPRNT.