Surface labeling of key residues during assembly of the transmembrane pore formed by staphylococcal α‐hemolysin

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Abstract
Structural changes in staphylococcal α‐hemolysin (αHL) that occur during oligomerization and pore formation on membranes have been examined by using a simple gel‐shift assay to determine the rate of modification of key single‐cysteine mutants with the hydrophilic sulfhydryl reagent, 4‐acetamido‐4′‐((iodoacetyl)amino)stilbene‐2,2′‐disulfonate (IASD). The central glycine‐rich loop of αHL lines the lumen of the transmembrane channel. A residue in the loop remains accessible to IASD after assembly, in keeping with the ability of the pore to pass molecules of 1̃000 Da. By contrast, residues near the N‐terminus, which are critical for pore function, become deeply buried during oligomerization, while a residue at the extreme C‐terminus increases in reactivity after assembly, consistent with a location in the part of the pore that projects from the surface of the lipid bilayer.