INTERACTION OF THETA-TOXIN (PERFRINGOLYSIN-O), A CHOLESTEROL-BINDING CYTOLYSIN, WITH LIPOSOMAL MEMBRANES - CHANGE IN THE AROMATIC SIDE-CHAINS UPON BINDING AND INSERTION

TO understand the mechanism of membrane lysis by theta-toxin (perfring olysin O) from Clostridium perfringens, a cholesterol-binding, pore-fo rming cytolysin, we undertook a spectroscopic analysis of the structur al changes that occur during the lytic process using lipid vesicles. I n particular, the spectra were compared with those obtained using a mo dified theta-toxin, MC theta, that binds membrane cholesterol without forming oligomeric pores, thus bypassing the oligomerization step. The interaction of theta-toxin liposomes composed of cholesterol and phos phatidylcholine but not with cholesterol-free liposomes caused a remar kable increase in the intensity of the tryptophan fluorescence emissio n spectra and ellipticity changes in the near- and far-UV CD peaks. A CD peak shift from 292 to 300 nm was specific for theta-toxin, suggest ing oligomerization-specific changes occurring around tryptophan resid ues. Structural changes in the aromatic side chains were detected in t he near-UV CD and fluorescence spectra upon MC theta-liposome interact ion, although the far-UV CD spectra indicate that the beta-rich second ary structure of MC theta is well-conserved after membrane binding. Qu enching of the intrinsic tryptophan fluorescence of MC theta by bromin ated lecithin/cholesterol liposomes suggests that theta-toxin inserts at least partly into membranes in the absence of oligomerization. Thes e results indicate that regardless of oligomerization, the binding of theta-toxin to cholesterol induces partial membrane insertion and trig gers conformational changes accompanied by aromatic side chain rearran gement with retention of secondary structure. The spectral changes dep end on the cholesterol/toxin molar ratio and pH, with maxima at pH 5-7 , correlating with the optima for binding, suggesting that the cholest erol-induced insertion mechanism is distinct from the acid-induced one .