Zakharov S D, Cramer W A
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
Biochimie. 2002 May-Jun;84(5-6):465-75. doi: 10.1016/s0300-9084(02)01453-0.
The formation of integral membrane voltage-gated ion channels by the initially soluble C-terminal channel polypeptide (CP) of the pore-forming colicins is a fruitful area for studies on membrane protein import. The dependence of CP import on specific membrane parameters can be better understood using liposomes and planar membranes of defined lipid composition. The membrane surface and interfacial layer provide special conditions for the transition of a pore-forming colicin from the soluble to the integral membrane state. The colicin E1 CP is arranged in the membrane interfacial layer as a conformationally mobile helical array that is extended far more in the two dimensions parallel to the membrane surface than in the third dimension perpendicular to it. The alpha-helical content of CP(E1) increases by approximately 30% upon binding to the membrane. The sequence of kinetically distinguishable events in the CP(E1)-membrane interaction is binding, unfolding to a subtended area of 4200 A(2), helix extension, and insertion, the last three events overlapping in their time course ( approximately 10 s(-1)). The extension into two dimensions and the interaction with the membrane surface may explain the reversible denaturation and refolding of secondary structure that occurs after boiling of the CP-membrane complex. Although DSC showed the presence of helix-helix interactions in the membrane-bound state, the change in secondary structure and the extended surface area argue against a molten-globule intermediate in the CP-membrane interaction. However, the surface-bound state is mobile, as surface conformational mobility is a necessary prerequisite for insertion of CP trans-membrane helices into the bilayer. The requirement for this surface protein mobility, described by "thermal melting" FRET experiments, may provide the explanation for the precipitous decrease in the voltage-gated CP channel formation at high values of surface potential of planar bilayer membranes. Thus, the membrane interfacial layer, with the CP backbone situated near the acyl chain carbonyls, provides a favorable environment for the structure changes necessary for the transition from the soluble to the membrane-inserted state.
成孔性大肠杆菌素最初可溶的C端通道多肽（CP）形成整合膜电压门控离子通道，这是膜蛋白导入研究的一个富有成果的领域。使用具有特定脂质组成的脂质体和平面膜，可以更好地理解CP导入对特定膜参数的依赖性。膜表面和界面层为成孔性大肠杆菌素从可溶状态转变为整合膜状态提供了特殊条件。大肠杆菌素E1 CP以构象可移动的螺旋阵列形式排列在膜界面层中，在平行于膜表面的二维方向上比垂直于膜表面的第三维方向上延伸得更远。CP（E1）与膜结合后，其α-螺旋含量增加约30%。CP（E1）与膜相互作用中动力学上可区分的事件顺序为结合、展开至4200 Ų的张角面积、螺旋延伸和插入，后三个事件在时间进程上重叠（约10 s⁻¹）。在二维方向上的延伸以及与膜表面的相互作用可能解释了CP - 膜复合物煮沸后发生的二级结构的可逆变性和重折叠。尽管差示扫描量热法（DSC）显示在膜结合状态下存在螺旋 - 螺旋相互作用，但二级结构的变化和扩展的表面积表明CP与膜相互作用中不存在熔球中间体。然而，表面结合状态是可移动的，因为表面构象流动性是CP跨膜螺旋插入双层膜的必要前提。通过“热熔解”荧光共振能量转移（FRET）实验描述的这种表面蛋白流动性的要求，可能解释了在平面双层膜表面电位高值时电压门控CP通道形成的急剧下降。因此，CP主链位于酰基链羰基附近的膜界面层为从可溶状态转变为膜插入状态所需的结构变化提供了有利环境。
