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.
|Original language||English (US)|
|Number of pages||4|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Dec 20 1994|
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