The internal cavity of the staphylococcal α-hemolysin pore accommodates ∼175 exogenous amino acid residuest

The cavity within the cap domain of the transmembrane staphylococcal alpha-hemolysin (alpha HL) pore is roughly a sphere of diameter similar to 45 angstrom (molecular surface volume similar to 39 500 angstrom(3)). We tested the ability of the cavity to accommodate exogenous polypeptide chains. Concatemerized Gly/Ser-containing sequences ("loops", L; number of repeats = n; number of residues = 10n + 5, n = 0-21) were inserted at a position located within the cavity of the fully assembled heptameric alpha HL pore. Homomeric pores containing 25 or less residues in each loop (n <= 2) could be assembled. The pores were protease-resistant, indicating that they had been formed correctly, and produced currents in planar lipid bilayers. The pores showed an up to 70% reduction in unitary conductance, depending on the length of the inserted loop. Protease-resistant heteromeric pores containing wild-type (W) and L subunits were also assembled: when n = 3, up to five L subunits were tolerated; when n = 4, three L subunits were tolerated; and when n = 5 or 6, two L subunits were tolerated. For n 7, only one L subunit was incorporated. As the inserted loop was lengthened, transient closures were observed in planar bilayer experiments with single pores. However, L1W6 pores with very long loops (n = 14 and 21) had unitary conductance values close to those Of W-7, suggesting that the loop is extruded through the opening in the cap of the pore into the external medium. Further analysis of bilayer recordings and electrophoretic migration patterns indicates that the upper capacity of the cavity is similar to 175 amino acids. The findings suggest that small functional peptides or proteins might be assembled within the alpha HL pore.