Walker B, Kasianowicz J, Krishnasastry M, Bayley H
Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Protein Eng. 1994 May;7(5):655-62. doi: 10.1093/protein/7.5.655.
Staphylococcal alpha-hemolysin, a pore-forming exotoxin, is a polypeptide of 293 amino acids that is secreted by Staphylococcus aureus as a water-soluble monomer. It assembles to form hexameric pores in lipid bilayers. Previous studies of pore formation have established the involvement of a central glycine-rich loop. Here, we show that when five consecutive histidine residues replace amino acids 130-134 at the midpoint of the loop, they provide a switch with which pore activity can be (i) turned off by micromolar concentrations of divalent zinc ions and (ii) turned back on with the chelating agent EDTA. Planar bilayer recordings show that Zn2+ and EDTA can act on open channels from either side of the bilayer and thus demonstrate that the central loop lines part of the conductive pathway. Our results suggest that genetically-engineered pore-forming proteins might make useful components of metal ion sensors.
葡萄球菌α-溶血素是一种形成孔道的外毒素，是由金黄色葡萄球菌分泌的一种含293个氨基酸的水溶性单体多肽。它能组装形成脂质双分子层中的六聚体孔道。以往对孔道形成的研究已证实富含甘氨酸的中央环参与其中。在此，我们表明，当五个连续的组氨酸残基取代环中点处的130 - 134位氨基酸时，它们提供了一个开关，通过该开关，孔道活性能够：（i）被微摩尔浓度的二价锌离子关闭；（ii）用螯合剂乙二胺四乙酸（EDTA）重新开启。平面双层记录显示，Zn2+和EDTA可从双层的任一侧作用于开放通道，从而证明中央环构成了传导途径的一部分。我们的结果表明，基因工程改造的形成孔道的蛋白质可能成为金属离子传感器的有用组件。
