Surface labeling of key residues during assembly of the transmembrane pore formed by staphylococcal alpha-hemolysin.
Structural changes in staphylococcal alpha-hemolysin (alpha HL) 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 alpha HL 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 approximately 1000 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.
Krishnasastry
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M
Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Walker
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B
Braha
O
O
Bayley
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H
D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004912:Erythrocytes; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011817:Rabbits; D013211:Staphylococcus aureus / Q000378:metabolism*; D013267:Stilbenes
C090885:4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonate; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D013267:Stilbenes; C021091:staphylococcal alpha-toxin; D006639:Histidine; D003545:Cysteine