Identification of a Membrane-Spanning Domain of the Thiol-Activated Pore-Forming Toxin Clostridium perfringens Perfringolysin O: An 
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- 作者：Laura A. Shepard ; Alejandro P. Heuck ; Brian D. Hamman ; Jamie Rossjohn ; Michael W. Parker ; Kathleen R. Ryan ; Arthur E. Johnson ; and Rodney K. Tweten
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：October 13, 1998
- 年：1998
- 卷：37
- 期：41
- 页码：14563 - 14574
- 全文大小：281K
- 年卷期：v.37,no.41(October 13, 1998)
- ISSN：1520-4995
文摘
Clostridium perfringens perfringolysin O (PFO or 
-toxin) is a cytolytic toxin that binds tocholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varyingsize in the bilayer. In this study, a membrane-spanning domain has been identified in PFO by a combinationof fluorescence spectroscopic methods using the fluorescent dye N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylenediamine (NBD) whose emission properties are sensitive to water.PFO was substituted with a single cysteine at most of the residues between amino acids K189 and N218,and then each cysteine was modified with NBD. Each purified NBD-labeled PFO was then bound tomembranes, and the probe's environment was ascertained by measuring its fluorescence lifetime, emissionintensity, and collisional quenching with either aqueous (iodide ions) or nonaqueous (nitroxide-labeledphospholipids) quenchers. Lifetime and intensity measurements revealed that the amino acid side chainsin this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or anonaqueous environment. This pattern indicates that this portion of the membrane-bound PFO spans themembrane in an antiparallel 
-sheet conformation. The alternating exposure of these residues to thehydrophobic interior of the bilayer was demonstrated by their susceptibility to quenching by nitroxidemoieties attached to phospholipid acyl chains. Residues K189-N218 therefore form a two-stranded,amphipathic -sheet in the membrane-bound PFO that creates a stable interface between the pore and themembrane. This same region packs as three short 
pha.gif" BORDER=0>-helices in the soluble, monomeric form of PFO, andtherefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a majorstructural transition in which three pha.gif" BORDER=0>-helices unfold to form a membrane-spanning amphipathic -sheet.
-toxin) is a cytolytic toxin that binds tocholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varyingsize in the bilayer. In this study, a membrane-spanning domain has been identified in PFO by a combinationof fluorescence spectroscopic methods using the fluorescent dye N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylenediamine (NBD) whose emission properties are sensitive to water.PFO was substituted with a single cysteine at most of the residues between amino acids K189 and N218,and then each cysteine was modified with NBD. Each purified NBD-labeled PFO was then bound tomembranes, and the probe's environment was ascertained by measuring its fluorescence lifetime, emissionintensity, and collisional quenching with either aqueous (iodide ions) or nonaqueous (nitroxide-labeledphospholipids) quenchers. Lifetime and intensity measurements revealed that the amino acid side chainsin this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or anonaqueous environment. This pattern indicates that this portion of the membrane-bound PFO spans themembrane in an antiparallel -sheet conformation. The alternating exposure of these residues to thehydrophobic interior of the bilayer was demonstrated by their susceptibility to quenching by nitroxidemoieties attached to phospholipid acyl chains. Residues K189-N218 therefore form a two-stranded,amphipathic -sheet in the membrane-bound PFO that creates a stable interface between the pore and themembrane. This same region packs as three short pha.gif" BORDER=0>-helices in the soluble, monomeric form of PFO, andtherefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a majorstructural transition in which three pha.gif" BORDER=0>-helices unfold to form a membrane-spanning amphipathic -sheet.