Bortoleto R K, de Oliveira A H, Ruller R, Arni R K, Ward R J
Department of Physics, IBILCE/UNESP, São José do Rio Preto, SP, Brazil.
Arch Biochem Biophys. 1998 Mar 1;351(1):47-52. doi: 10.1006/abbi.1997.0550.
The interaction of alpha-hemolysin (also called alpha-toxin) from Staphylococcus aureus with mixed egg-yolk phosphatidylcholine/cholesterol liposomes has been investigated using the intrinsic tryptophan fluorescence emission (ITFE) signal. The ITFE intensity of alpha-hemolysin, which was obtained using a novel purification protocol, showed a triphasic increase on incubation with liposomes at low protein/lipid ratios. The first, rapid phase results in an increase in ITFE of 10%, which reflects rapid conformation changes in the alpha-hemolysin on association with the liposome membrane. The second phase of the ITFE increase is associated with a red shift from 334 to 339 nm in the maximum emission wavelength, suggesting the transition to a partially unfolded intermediate in the oligomerization process. The third phase of the ITFE intensity change demonstrates a temporal correlation with the appearance of SDS-stable oligomers. The results demonstrate the feasibility of identification of intermediate protein conformations in complex membrane-associated processes by manipulation of the liposomal membrane composition.
利用内在色氨酸荧光发射（ITFE）信号，研究了金黄色葡萄球菌的α-溶血素（也称为α-毒素）与混合蛋黄磷脂酰胆碱/胆固醇脂质体的相互作用。采用一种新颖的纯化方案获得的α-溶血素的ITFE强度，在低蛋白/脂质比下与脂质体孵育时呈现出三相增加。第一个快速阶段导致ITFE增加10%，这反映了α-溶血素与脂质体膜结合时的快速构象变化。ITFE增加的第二阶段与最大发射波长从334nm红移至339nm相关，表明在寡聚化过程中转变为部分展开的中间体。ITFE强度变化的第三阶段与SDS稳定寡聚体的出现呈现出时间相关性。结果表明，通过操纵脂质体膜组成来鉴定复杂膜相关过程中中间蛋白质构象是可行的。
