The internal cavity of the staphylococcal alpha-hemolysin pore accommodates approximately 175 exogenous amino acid residues

The cavity within the cap domain of the transmembrane staphylococcal alpha-hemolysin (alphaHL) pore is roughly a sphere of diameter approximately 45 A (molecular surface volume approximately 39,500 A(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 alphaHL pore. Homomeric pores containing 25 or less residues in each loop (n or= 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, L(1)W(6) 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 approximately 175 amino acids. The findings suggest that small functional peptides or proteins might be assembled within the alphaHL pore.