J. Biochem, 1998, Vol. 123, No. 6 1145-1155
© 1998 Japanese Biochemical Society
research-article |
Contribution of Tryptophan Residues to the Structural Changes in Perfringolysin O during Interaction with Liposomal Membranes
*Department of Enzyme Biochemistry, Tokyo Metropolitan Institute of Gerontology, Sakae-cho Itabashi-ku, Tokyo 173-0015;
+Toyama Medical and Pharmaceutical University Toyama 930-0152
1To whom correspondence should be addressed. Phone: +81-3-3964-3241 (Ext. 3068), Fax: +81-3-3579-4776, E-mail: megumi{at}tmig.or.jp
Perfringolysin O (
-toxin) is a cholesterol-binding and pore-forming toxin that shares with other thiol-activated cytolysins a highly conserved sequence, ECTGLAWEWWR (residues 430–440), near the C-terminus. To understand the membrane-insertion and pore-forming mechanisms of the toxin, we evaluated the contribution of each Trp to the toxin conformation during its interaction with liposomal membranes. Circular dichroism (CD) spectra of Trp mutant toxins indicated that only Trp436 has a significant effect on the secondary structure, and that Trp436, Trp438, and Trp439 make large contributions to near-UV CD spectra. Quenching the intrinsic Trp fluorescence of the wild-type and mutant toxins with brominated lecithin/cholesterol liposomes revealed that Trp438 and 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 -toxin oligomerization and/or pore formation, suggesting a conformational change around Trp438 and Trp439 in these processes.
2 Present address: Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113.
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