IDENTIFICATION OF A MEMBRANE-SPANNING DOMAIN OF THE THIOL-ACTIVATED PORE-FORMING TOXIN CLOSTRIDIUM-PERFRINGENS PERFRINGOLYSIN-O - AN ALPHA-HELICAL TO BETA-SHEET TRANSITION IDENTIFIED BY FLUORESCENCE SPECTROSCOPY

Clostridium perfringens perfringolysin O (PFO or theta-toxin) is a cyt olytic toxin that binds to cholesterol-containing membranes and then s elf-associates to spontaneously form aqueous pores of varying size in the bilayer. In this study, a membrane-spanning domain has been identi fied in PFO by a combination of fluorescence spectroscopic methods usi ng the fluorescent dye N'-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylenediam ine (NBD) whose emission properties are sensitive to water. PFO was su bstituted with a single cysteine at most of the residues between amino acids K189 and N218, and then each cysteine was modified with NBD. Ea ch purified NBD-labeled PFO was then bound to membranes, and the probe 's environment was ascertained by measuring its fluorescence lifetime, emission intensity, and collisional quenching with either aqueous (io dide ions) or nonaqueous (nitroxide-labeled phospholipids) quenchers. Lifetime and intensity measurements revealed that the amino acid side chains in this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or a nonaqueous environment. This pattern indicates that this portion of the membrane-bound PFO spans the membr ane in an antiparallel beta-sheet conformation. The alternating exposu re of these residues to the hydrophobic interior of the bilayer was de monstrated by their susceptibility to quenching by nitroxide moieties attached to phospholipid acyl chains. Residues K189-N218 therefore for m a two-stranded, amphipathic beta-sheet in the membrane-bound PFO tha t creates a stable interface between the pore and the membrane. This s ame region packs as three short cr-helices in the soluble, monomeric f orm of PFO, and therefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a major structural transition i n which three alpha-helices unfold to form a membrane-spanning amphipa thic beta-sheet.