Miller C E, Majewski J, Watkins E B, Kuhl T L
Manuel Lujan Neutron Scattering Center, Los Alamos National Laboratory, Los Alamos, New Mexico, USA.
Biophys J. 2008 Jul;95(2):629-40. doi: 10.1529/biophysj.107.120725. Epub 2008 Mar 21.
Cholera toxin is a highly efficient biotoxin, which is frequently used as a tool to investigate protein-membrane interactions and as a reporter for membrane rafts. Cholera toxin binds selectively to gangliosides with highest affinity to GM(1). However, the mechanism by which cholera toxin crosses the membrane remains unresolved. Using x-ray reflectivity and grazing incidence diffraction, we have been able to monitor the binding and penetration of cholera toxin into a model lipid monolayer containing the receptor GM(1) at the air-water interface. Very high toxin coverage was obtained allowing precise measurements of how toxin binding alters lipid packing. Grazing incidence x-ray diffraction revealed the coexistence of two monolayer phases after toxin binding. The first was identical to the monolayer before toxin binding. In regions where toxin was bound, a second membrane phase exhibited a decrease in order as evidenced by a larger area per molecule and tilt angle with concomitant thinning of the monolayer. These results demonstrate that cholera toxin binding induces the formation of structurally distinct, less ordered domains in gel phases. Furthermore, the largest decrease in lateral order to the monolayer occurred at low pH, supporting a low endosomal pH in the infection pathway. Surprisingly, at pH = 8 toxin penetration by the binding portion of the toxin, the B(5) pentamer, was also observed.
霍乱毒素是一种高效生物毒素，常被用作研究蛋白质 - 膜相互作用的工具以及膜筏的报告分子。霍乱毒素选择性地与神经节苷脂结合，对GM(1)具有最高亲和力。然而，霍乱毒素穿过膜的机制仍未解决。利用X射线反射率和掠入射衍射，我们能够监测霍乱毒素在气 - 水界面与含有受体GM(1)的模型脂质单层的结合和穿透情况。获得了非常高的毒素覆盖率，从而能够精确测量毒素结合如何改变脂质堆积。掠入射X射线衍射显示毒素结合后存在两个单层相。第一个与毒素结合前的单层相同。在毒素结合的区域，第二个膜相显示出有序性降低，表现为每个分子的面积和倾斜角增大，同时单层变薄。这些结果表明霍乱毒素结合诱导在凝胶相中形成结构上不同、有序性较低的结构域。此外，单层横向有序性的最大降低发生在低pH值时，这支持了感染途径中内体的低pH值。令人惊讶的是，在pH = 8时，也观察到毒素的结合部分B(5)五聚体对毒素的穿透作用。
