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= 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 >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.
Jung
Yuni
Y
Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.
Cheley
Stephen
S
Braha
Orit
O
Bayley
Hagan
H
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013210:Staphylococcus / Q000201:enzymology; D013696:Temperature
D000596:Amino Acids; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; D021122:Protein Subunits; C021091:staphylococcal alpha-toxin; D012694:Serine; D005998:Glycine