RESTORATION OF PORE-FORMING ACTIVITY IN STAPHYLOCOCCAL ALPHA-HEMOLYSIN BY TARGETED COVALENT MODIFICATION

In previous studies, the replacement of His35 in the poreforming protein alpha-hemolysin (alpha HL) with Leu, Be, 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.