CORRECT OLIGOMERIZATION IS A PREREQUISITE FOR INSERTION OF THE CENTRAL MOLECULAR DOMAIN OF STAPHYLOCOCCAL ALPHA-TOXIN INTO THE LIPID BILAYER

Staphylococcal alpha-toxin is a primarily hydrophilic molecule that bi nds as a monomer to target membranes and then aggregates to form amphi philic oligomers that represent water-filled transmembrane channels. C urrent evidence indicates that a region located in the center of the m olecule inserts deeply into the bilayer. In the present study, we soug ht to determine whether membrane insertion was triggered by the oligom erization process, and whether insertion correlated with pore formatio n. Double mutants of alpha-toxin were prepared in which His-35 was rep laced by Arg, and cysteine residues were introduced at positions 69, 1 30 and 186. Substitution of His-35 with Arg rendered the toxin molecul es incapable of proper oligomerization, so that they remained in nonly tic form after binding to membranes. The sulfhydryl groups were labell ed with the polarity-sensitive fluorescent dye acrylodan. Functionally intact, single mutant toxins containing only the cysteine residues we re utilized as controls. Measurements of the fluorescence emission spe ctrum of acrylodan were performed for the active and inactive alpha-to xin mutants in free solution and in membrane-bound form. The collectiv e results demonstrate that proper oligomerization is required for memb rane insertion of the central region in the alpha-toxin molecule, and that lack of insertion correlates with absence of pore formation.