Qiu X Q, Jakes K S, Kienker P K, Finkelstein A, Slatin S L
Department of Physiology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Gen Physiol. 1996 Mar;107(3):313-28. doi: 10.1085/jgp.107.3.313.
Colicin Ia, a bacterial protein toxin of 626 amino acid residues, forms voltage-dependent channels in planar lipid bilayer membranes. We have exploited the high affinity binding of streptavidin to biotin to map the topology of the channel-forming domain (roughly 175 residues of the COOH-terminal end) with respect to the membrane. That is, we have determined, for the channel's open and closed states, which parts of this domain are exposed to the aqueous solutions on either side of the membrane and which are inserted into the bilayer. This was done by biotinylating cysteine residues introduced by site-directed mutagenesis, and monitoring by electrophysiological methods the effect of streptavidin addition on channel behavior. We have identified a region of at least 68 residues that flips back and forth across the membrane in association with channel opening and closing. This identification was based on our observations that for mutants biotinylated in this region, streptavidin added to the cis (colicin-containing) compartment interfered with channel opening, and trans streptavidin interfered with channel closing. (If biotin was linked to the colicin by a disulfide bond, the effects of streptavidin on channel closing could be reversed by detaching the streptavidin-biotin complex from the colicin, using a water-soluble reducing agent. This showed that the cysteine sulfur, not just the biotin, is exposed to the trans solution). The upstream and downstream segments flanking the translocated region move into and out of the bilayer during channel opening and closing, forming two transmembrane segments. Surprisingly, if any of several residues near the upstream end of the translocated region is held on the cis side by streptavidin, the colicin still forms voltage-dependent channels, indicating that a part of the protein that normally is fully translocated across the membrane can become the upstream transmembrane segment. Evidently, the identity of the upstream transmembrane segment is not crucial to channel formation, and several open channel structures can exist.
大肠杆菌素Ia是一种由626个氨基酸残基组成的细菌蛋白毒素，能在平面脂质双分子层膜中形成电压依赖性通道。我们利用链霉亲和素与生物素的高亲和力结合来绘制通道形成结构域（大致为COOH末端的175个残基）相对于膜的拓扑结构。也就是说，我们已经确定了，对于通道的开放和关闭状态，该结构域的哪些部分暴露于膜两侧的水溶液中，哪些部分插入到双分子层中。这是通过对定点诱变引入的半胱氨酸残基进行生物素化，并用电生理方法监测添加链霉亲和素对通道行为的影响来完成的。我们确定了一个至少由68个残基组成的区域，该区域随着通道的开放和关闭在膜中来回翻转。这一确定是基于我们的观察结果，即对于在该区域进行生物素化的突变体，添加到顺式（含大肠杆菌素）隔室中的链霉亲和素会干扰通道开放，而反式链霉亲和素会干扰通道关闭。（如果生物素通过二硫键与大肠杆菌素相连，使用水溶性还原剂将链霉亲和素-生物素复合物从大肠杆菌素上分离，链霉亲和素对通道关闭的影响就可以逆转。这表明半胱氨酸硫，而不仅仅是生物素，暴露于反式溶液中）。易位区域两侧的上游和下游片段在通道开放和关闭期间进出双分子层，形成两个跨膜片段。令人惊讶的是，如果易位区域上游末端附近的几个残基中的任何一个被链霉亲和素固定在顺式一侧，大肠杆菌素仍然会形成电压依赖性通道，这表明通常完全跨膜转运的蛋白质的一部分可以成为上游跨膜片段。显然，上游跨膜片段的身份对通道形成并不关键，并且可以存在几种开放通道结构。
