Mel S F, Falick A M, Burlingame A L, Stroud R M
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Biochemistry. 1993 Sep 14;32(36):9473-9. doi: 10.1021/bi00087a027.
Channel-forming colicins exist in at least two different membrane-associated conformations: a voltage-independent closed-channel state and a voltage-dependent open-channel state. In a voltage-independent membrane-associated conformation, we find that two major regions of colicin Ia are protected from pepsin proteolysis after association with negatively charged membranes. In contrast, colicin Ia is rapidly and completely proteolyzed in the absence of membranes. The major protected region includes an electrophysiologically defined C-terminal channel-forming domain as well as 96 residues upstream of this region. Approximately 100 residues spanning Ala79- approximately Arg189 within the N-terminal domain are protected as well. The first N-terminal 76 residues of colicin Ia and a large region which includes much of the putative central receptor-binding domain are not protected from proteolysis. Both N- and C-termini of protected peptides have been identified using a combination of gel electrophoresis, N-terminal sequencing, and mass spectrometry, thereby defining specific residues that are located on the outside of the lipid bilayer. These data suggest a role for regions other than the electrophysiologically defined C-terminal channel-forming domain in membrane insertion and channel formation.
一种电压非依赖性的封闭通道状态和一种电压依赖性的开放通道状态。在电压非依赖性的与膜相关的构象中，我们发现与带负电荷的膜结合后，大肠杆菌素Ia的两个主要区域免受胃蛋白酶的蛋白水解作用。相比之下，在没有膜的情况下，大肠杆菌素Ia会迅速且完全被蛋白水解。主要的受保护区域包括一个电生理学定义的C端通道形成结构域以及该区域上游的96个残基。N端结构域内跨越Ala79至约Arg189的大约100个残基也受到保护。大肠杆菌素Ia的前76个N端残基以及包括大部分假定的中央受体结合结构域的一个大区域不受蛋白水解的影响。使用凝胶电泳、N端测序和质谱相结合的方法鉴定了受保护肽段的N端和C端，从而确定了位于脂质双层外部的特定残基。这些数据表明，除了电生理学定义的C端通道形成结构域之外的区域在膜插入和通道形成中发挥作用。
