CONTRIBUTION OF TRYPTOPHAN RESIDUES TO THE STRUCTURAL-CHANGES IN PERFRINGOLYSIN-O DURING INTERACTION WITH LIPOSOMAL MEMBRANES

Perfringolysin O (theta-toxin) is a cholesterol-binding and pore-formi ng toxin that shares with other thiol-activated cytolysins a highly co nserved sequence, ECTGLAWEWWR (residues 430-440), near the C-terminus. To understand the membrane-insertion and pore-forming mechanisms of t he toxin, we evaluated the contribution of each Trp to the toxin confo rmation during its interaction with liposomal membranes. Circular dich roism (CD) spectra of Trp mutant toxins indicated that only Trp436 has a significant effect on the secondary structure, and that Trp436, Trp 438, and Trp439 make large contributions to near-UV CD spectra. Quench ing the intrinsic Trp fluorescence of the wild-type and mutant toxins with brominated lecithin/cholesterol liposomes revealed that Trp438 an d probably Trp436, but not Trp439, contributes to toxin insertion into the liposomal membrane. Near-UV CD spectra of the membrane-associated mutant toxins indicated that both Trp438 and Trp439 are required for the CD peak shift from 292 to 300 nm, a signal related to theta-toxin oligomerization and/or pore formation, suggesting a conformational cha nge around Trp438 and Trp439 in these processes.