Assembly of a single protein pore

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.

Single-molecule techniques have made possible the study of biomolecule function without the limitations imposed by ensemble averaging. For example, single-molecule measurements have revealed that the F1-ATPase rotates in discrete 120 degree steps, and that structural dynamics of catalytic RNA are directly linked to changes in molecular kinetics. Such experiments exploit two of the unique properties of single-molecule observations: (1) To reveal sub-populations that would otherwise be undetectable in conventional bulk fluorescence measurements. (2) To 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. The behaviour of a biomolecule can be best understood by relating changes in its structure with changes in its function. To do this solely using single-molecule fluorescence is very difficult, and it is for this reason that motor proteins are widely studied; it is relatively easy to watch the movement of a molecule and hence observe its function directly. However for other proteins, alternative methods are required to probe biomolecule function. For membrane protein channels, there is an obvious measurable indicator of protein function, the ion current through the channel. We will construct an instrument capable of making simultaneous measurements of both the single-molecule fluorescence and ion current from a single fluorescently labelled membrane protein situated in an artificial bilayer. In this way the conformational changes and interactions of a membrane protein can be related to changes in its conduction properties. We will apply these techniques to a specific biological question: The assembly and insertion mechanism of the heptameric pore-forming protein, staphylococcal alpha-hemolysin (aHL). This combination of single-molecule fluorescence and electrical recording will allow this model to be tested: 1. Does the diffusional behaviour of aHL monomers change in response to the initial stages of pore assembly? 2. What is the mechanism of pore assembly? Does oligomerisation occurs via sequential monomer incorporation (monomer + monomer -> dimer + monomer -> trimer), or aggregation of larger intermediates (monomer + monomer -> dimer + dimer -> tetramer)? 3. Is the transition from pre-pore to active pore concomitant with changes in electrical activity, or does a delay exist between insertion of the beta-barrel portion of the complex and final electrical activity? Studying the detailed mechanism of aHL assembly will aid our understanding of the spontaneous insertion of beta-barrel membrane proteins, viral fusion, toxin action, and membrane protein biosynthesis. Understanding how this simple system works will help us tackle more complex membrane proteins.