Rapid Assembly of a Multimeric Membrane Protein Pore Observed by Single Molecule Fluorescence

Abstract

Pore-forming proteins play a vital role in the eukaryotic immune response, where immune surveillance can lead to the targeted-attack of infected cells and bacteria via pore generation. Conversely, many bacteria use similar protein pores to kill target cells and acquire nutrients from their host. However, the mechanisms by which many of these multimeric membrane proteins assemble to form pores are not well understood. We have observed the assembly of the staphylococcal pore-forming toxin α-hemolysin using single-molecule fluorescence imaging. Surprisingly, assembly from the monomer to the complete heptamer is extremely rapid, occurring in less than 5 ms. No lower-order oligomeric intermediates are detected. Monte Carlo simulation of our experiment shows that assembly is diffusion-limited, and pore formation is dependent on the stability of intermediate species. There are close similarities between bacterial pore-forming toxins, such as staphylococcal α-hemolysin, the anthrax protective antigen and the cholesterol-dependent cytolysins (CDCs), and their eukaryotic analogues, such as the complement pore - membrane attack complex and perforin domain (MACPF). The assembly mechanism we have observed for α-hemolysin provides a simple model that aids our understanding of these important pore-formers. To image rapidly diffusing α-hemolysin monomers and observe their assembly into higher-order oligomers requires bilayer longevity, high signal-to-noise, high time resolution and control of lipid composition. We have recently developed a new synthetic mimic of the cell membrane that fulfils these requirements; we form a Droplet Interface Bilayer by contact of two lipid monolayers between a nanolitre aqueous droplet and a hydrogel support immersed in a solution of phospholipid in hexadecane. To monitor the entire assembly process we introduced Cy3b-labeled α-hemolysin monomers into the droplet using a piezo-driven nanoinjector. The labeled α-hemolysin species were imaged on the bilayer using total internal reflection fluorescence (TIRF) microscopy.