Topology of the outer membrane usher PapC determined by site-directed fluorescence labeling.
Topology of the outer membrane usher PapC determined by site-directed fluorescence labeling.
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通过定点荧光标记确定 PapC 的外膜拓扑结构。
DOI：
10.1074/jbc.m409192200
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Thanassi,DavidG
中科院分区：
文献类型：
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作者：
Henderson,NadineS;So,StephaneShuKin;Martin,Cheryl;Kulkarni,Ritwij;Thanassi,DavidG
与通过跨膜α-螺旋跨越脂质双层的典型膜蛋白相比，细菌外膜蛋白采用由反平行跨膜β-链组成的β-桶结构。外膜蛋白的拓扑结构很难用计算机算法准确预测，通常用于α-螺旋膜蛋白的拓扑结构映射方案对β-桶蛋白不起作用。我们在这里提出的拓扑结构的PapC usher，外膜蛋白所需的组装和分泌的P皮利的伴侣/usher途径在uropathicEscherichia coli。最初试图通过插入蛋白酶切割位点来绘制PapC拓扑结构的尝试在很大程度上是不成功的，这是由于在大多数位点缺乏切割并且需要破坏外膜以鉴定周质位点。因此，我们采用了一种定点荧光标记技术，允许在完整的细菌中使用小分子探针的外膜蛋白的拓扑映射。使用这种方法，我们证明了PapC具有编码多达32个跨膜β链的潜力。基于实验证据，我们提出引导器由26条β链组成的N-末端β-桶结构域组成，并且不同的C-末端结构域不插入膜中，而是位于N-末端β-桶的腔内，类似于由外膜铁-铁载体摄取蛋白编码的塞子结构域。
In contrast to typical membrane proteins that span the lipid bilayer via transmembrane α-helices, bacterial outer membrane proteins adopt a β-barrel architecture composed of antiparallel transmembrane β-strands. The topology of outer membrane proteins is difficult to predict accurately using computer algorithms, and topology mapping protocols commonly used for α-helical membrane proteins do not work for β-barrel proteins. We present here the topology of the PapC usher, an outer membrane protein required for assembly and secretion of P pili by the chaperone/usher pathway in uropathogenicEscherichia coli. An initial attempt to map PapC topology by insertion of protease cleavage sites was largely unsuccessful due to lack of cleavage at most sites and the requirement to disrupt the outer membrane to identify periplasmic sites. We therefore adapted a site-directed fluorescence labeling technique to permit topology mapping of outer membrane proteins using small molecule probes in intact bacteria. Using this method, we demonstrated that PapC has the potential to encode up to 32 transmembrane β-strands. Based on experimental evidence, we propose that the usher consists of an N-terminal β-barrel domain comprised of 26 β-strands and that a distinct C-terminal domain is not inserted into the membrane but is located instead within the lumen of the N-terminal β-barrel similar to the plug domains encoded by the outer membrane iron-siderophore uptake proteins.