Restoration of pore-forming activity in staphylococcal alpha-hemolysin by targeted covalent modification.
In previous studies, the replacement of His35 in the pore-forming protein alpha-hemolysin (alpha HL) with Leu, Ile, Pro, Arg, Ser, Thr or Cys yielded inactive polypeptides. Here, we show that modification of the inactive single-cysteine mutant alpha HL-H35C with iodoacetamide, to form H35CamC, generates significant pore-forming activity. The closely related polypeptides H35N and H35Q have, respectively, essentially no activity and greatly reduced activity. The modified residue in H35CamC, S-carboxamidomethyl-cysteine, mimicks histidine in volume, polarity and hydrogen bonding potential, but is unable to ionize. Unmodified H35C is defective in the final step of pore formation: the conversion of an inactive heptameric membrane-bound assembly intermediate to a structure containing open channels. It is this step in assembly that is ameliorated in H35CamC.
Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545, USA.
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Walker
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D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007460:Iodoacetamide / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D018272:Porins / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D011817:Rabbits; D013210:Staphylococcus / Q000378:metabolism*; D013696:Temperature
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D018272:Porins; C021091:staphylococcal alpha-toxin; D006639:Histidine; D003545:Cysteine; D007460:Iodoacetamide