MEMBRANE-PENETRATING DOMAIN OF STREPTOLYSIN-O IDENTIFIED BY CYSTEINE SCANNING MUTAGENESIS

Streptolysin O (SLO), a polypeptide of 571 amino acids, belongs to a f amily of highly homologous toxins that bind to cell membranes containi ng cholesterol and then polymerize to form large transmembrane pores, A conserved region close to tile C terminus contains the single cystei ne residue of SLO and has been implicated ill membrane binding, which has been the only clear assignment of function to a part of the sequen ce, We have used a cysteine-less active mutant of SLO to introduce sin gle cysteine residues at 19 positions distributed throughout the seque nce, The cysteines were derivatized with the polarity-sensitive fluoro phore acrylodan, and the fluorescence emission of the label was examin ed. at the different stages of SLO pore assembly. With several mutants , oligomerization on membranes was accompanied by emission blue-shifts , indicating movement of the label into a more hydrophobic environment , These effects were essentially confined to the range of amino acids 213-305. With oligomeric mutants L274C, S286C, and S305C, additional e nvironmental alterations were induced when different nondenaturing det ergents were used to dislodge the membrane lipids from the oligomers. The corresponding amino acid residues thus insert. into the lipid bila yer during pore formation, Conversely, the spectra of oligomeric mutan ts A213C and T245C were not affected by detergents, Devoid of contact with the lipid bilayer, these amino acid residues probably participate in the interaction of SLO molecules within the oligomer.