Assembly of a single protein pore

点击翻译按钮获取中文摘要

Single-molecule fluorescence is a powerful technique for understanding the function of biomolecules. Studying biological systems at the level of individual molecules has many advantages, for example; we can reveal sub-populations that would otherwise be undetectable in conventional bulk measurements, and we can follow a single molecule as it undergoes a particular reaction or conformational change, enabling the observation of transient intermediate states that would otherwise be hidden. In order to relate changes in the structure of a biomolecule to changes in its function, techniques capable of monitoring both structure and function are required. For membrane protein channels, there is an obvious indicator of protein function, the flow of ions through the channel. Understanding the mechanisms that govern the behaviour of membrane proteins is very important. Membrane proteins are responsible for controlling many functions in the cell, including signalling and the transport of molecules across the cell membrane. However, due to their complexity, relatively little is known about their structure, interactions or behaviour. I propose to construct an instrument capable of making simultaneous measurements of both the single-molecule fluorescence and ion current from a fluorescently labelled membrane protein situated in an artificial bilayer. In this way, conformational changes within the membrane protein can be related to changes in its conduction. This technique will be tested by application to a specific biological problem: The assembly and insertion of the heptameric pore-forming protein, staphylococcal alpha-hemolysin (aHL). aHL is composed of 7 identical subunits, and when these 7 subunits combine they form a channel. This channel forms a beta-barrel structure. To understand how these subunits assemble, we will link them to fluorescent molecules. By simply counting the subunits as aHL forms we will be able to tell if the subunits come together one at a time, or in pairs, or in larger groups. By measuring the electrical current through a pore at the same time as we watch it form using fluorescent labels, we will be able to understand how the formation of a beta-barrel channel is related to the steps of pore assembly.