TY - JOUR
T1 - Subunit stoichiometry of staphylococcal α-hemolysin in crystals and on membranes
T2 - A heptameric transmembrane pore
AU - Gouaux, J. Eric
AU - Braha, Orit
AU - Hobaugh, Michael R.
AU - Song, Langzhou
AU - Cheley, Stephen
AU - Shustak, Christopher
AU - Bayley, Hagan
PY - 1994/12/20
Y1 - 1994/12/20
N2 - Elucidation of the accurate subunit stoichiometry of oligomeric membrane proteins is fraught with complexities. The interpretations of chemical cross- linking, analytical ultracentrifugation, gel filtration, and low-resolution electron microscopy studies are often ambiguous. Staphylococcal α-hemolysin (αHL), a homooligomeric toxin that forms channels in cell membranes, was believed to possess six subunits arranged around a sixfold axis of symmetry. Here, we report that analysis of x-ray diffraction data and chemical modification experiments indicate that the αHL oligomer is a heptamer. Self- rotation functions calculated using x-ray diffraction data from single crystals of αHL oligomers show a sevenfold axis of rotational symmetry. The αHL pore formed on rabbit erythrocyte membranes was determined to be a heptamer by electrophoretic separation of αHL heteromers formed from subunits with the charge of wild-type αHL and subunits with additional negative charge generated by targeted chemical modification of a single- cysteine mutant. These data establish the heptameric oligomerization state of the αHL transmembrane pore both in three-dimensional crystals and on a biological membrane.
AB - Elucidation of the accurate subunit stoichiometry of oligomeric membrane proteins is fraught with complexities. The interpretations of chemical cross- linking, analytical ultracentrifugation, gel filtration, and low-resolution electron microscopy studies are often ambiguous. Staphylococcal α-hemolysin (αHL), a homooligomeric toxin that forms channels in cell membranes, was believed to possess six subunits arranged around a sixfold axis of symmetry. Here, we report that analysis of x-ray diffraction data and chemical modification experiments indicate that the αHL oligomer is a heptamer. Self- rotation functions calculated using x-ray diffraction data from single crystals of αHL oligomers show a sevenfold axis of rotational symmetry. The αHL pore formed on rabbit erythrocyte membranes was determined to be a heptamer by electrophoretic separation of αHL heteromers formed from subunits with the charge of wild-type αHL and subunits with additional negative charge generated by targeted chemical modification of a single- cysteine mutant. These data establish the heptameric oligomerization state of the αHL transmembrane pore both in three-dimensional crystals and on a biological membrane.
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U2 - 10.1073/pnas.91.26.12828
DO - 10.1073/pnas.91.26.12828
M3 - Article
C2 - 7809129
AN - SCOPUS:0028567173
SN - 0027-8424
VL - 91
SP - 12828
EP - 12831
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 26
ER -