Hemolysin Structure and Biology

a-Hemolysin is a 33.2 kDa protein toxin secreted by Staphylococcus aureus. It forms a multisubunit ring (four to eight identical subunits) on the surface of a cell or artificial bilayer and then inserts a beta-barrel channel across the target membrane.45,46 The x-ray crystal structure of the pore-forming heptamer was described in 1996 by Song and colleagues47 (Figure 11.3). The heptamer is about 10 nm long, and

FIGURE 11.1 Schematic of nucleic acid detection using a nanoscale pore. One end of a single molecule is captured in a nanometer-scale opening by an applied potential. Each strand impedes the ionic current as it is pulled through the opening (see Figure 11.2).

120 pA

120 pA

y—120 pA (open)

consists of a cap region and a transmembrane region. The cap region is outside of the lipid bilayer with a 2.6 nm aperture, leading into a hydrated vestibule about 5 nm deep and about 3.6 nm at its widest diameter. The vestibule terminates at a 1.5 nm limiting aperture defined by a ring of lysines (L147 of the monomeric protein), which leads into the 5 nm long transmembrane region of the protein. The inner diameter of the transmembrane channel ranges from 1.5 to 1.8 nm in diameter. Because of the relative robustness of this protein channel it has been used as a model ion channel to investigate a variety of characteristics, such as size of pore, ion sensitivity, ion selectivity, role of specific amino acids, and channel stability (for reviews, see Refs. [48-50]). The most stable (i.e., continuously open) form of the channel is the heptamer in high salt buffers (1 to 2 M KCl) at pH 7.5 to 8.5.41 First applied as a tool to investigate polymers in a confined space;41-43 the protein channel appeared to be an excellent candidate to examine DNA.39

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