Dynamic transitions of the transmembrane domain of diphtheria toxin: disulfide trapping and fluorescence proximity studies.
Dynamic transitions of the transmembrane domain of diphtheria toxin: disulfide trapping and fluorescence proximity studies.
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白喉毒素跨膜结构域的动态转变：二硫键捕获和荧光邻近研究。
DOI：
10.1021/bi00203a022
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Collier,RJ
中科院分区：
文献类型：
--
作者：
Zhan,H;Choe,S;Huynh,PD;Finkelstein,A;Eisenberg,D;Collier,RJ
1994年7月19日收到的修订版Mandalpt ®摘要：白喉毒素催化结构域跨内体膜至胞质区室的易位取决于毒素T（跨膜）结构域在膜中的低pH触发插入。T结构域，由九个α-螺旋排列在三层，克隆和表达作为一个离散的蛋白质在大肠杆菌中，和突变体形式的制备和表征。为了研究在各种条件下的三层的相对运动，我们产生了两种突变形式的域，每个包含一个人工intramoleculardisulfide桥连接埋非极性发夹（TH 8-TH 9）的其他两个层之一。这两个二硫化物抑制暴露的域的非极性区域在溶液中，在低pH值，2-p-toluidinylnaphthalene-6-sulfonate结合，并阻止其能力，形成人工双层通道。桥梁的减少消除了这些影响。还原形式的突变蛋白与芘基马来酰亚胺（一种荧光探针）反应，以监测层的分离。强激基缔合物带中看到的两种突变体在中性pH值在pH值为5时不减少，表明保留在溶液中的总构象在酸性条件下。在pH 5时加入磷脂囊泡，而在pH7.5时不加入，淬灭了荧光，反映了T结构域与双层相互作用时TH 8-TH 9发夹与其他层的物理分离。结果表明：（i）分离的T结构域的构象与在整个毒素中看到的非常相似，（ii）TH 8-TH 9发夹结构域与结构域的其他两层分离是膜插入的必要步骤，（iii）这种分离是通过结构域在酸性条件下与膜接触而触发的。
Revised Manuscript Received July 19, 1994® abstract: Translocation of the catalytic domain of diphtheria toxin across the endosomal membrane to the cytosolic compartment depends on low-pH-triggered insertion of the toxin’s T (transmembrane) domain into the membrane. The T domain, consisting of nine a-helices arranged in three layers, was cloned and expressed as a discrete protein in Escherichia coli, and mutant forms were prepared and characterized. To investigate the relative movements of the three layers under various conditions, we generated two mutant forms of the domain, each containing an artificial intramoleculardisulfide bridge linking the buried apolar hairpin (TH8-TH9) to one of the other two layers. Both disulfides inhibited exposure of the domain’s apolar regions in solution at low pH, as determined by 2-p-toluidinylnaphthalene-6-sulfonate binding, and blocked its ability to form channels in artificial bilayers. Reduction of the bridges abolished these effects. Reduced forms of the mutant proteins were reacted with pyrenylmaleimide, a fluorescent probe, to monitor separation of the layers. Strong excimer bands seen in both mutants at neutral pH were undiminished at pH 5, indicating the retention of gross conformation in solution under acidic conditions. The addition of phospholipid vesicles at pH 5, but not at pH 7.5, quenchedexcimer fluorescence, reflecting the physical separation of the TH8-TH9 hairpin from the other layers upon the T domain’s interactionwith the bilayer. The results indicatethat (i) the conformation of the isolated T domain closely resembles that seen in the whole toxin,(ii) the TH8-TH9 hairpin separates from both of the other layers of the domain as an essential step of membrane insertion, and (iii) this separation is triggered by contact of the domain with the membrane under acidic conditions.