Topology of the ExbB protein in the cytoplasmic membrane of Escherichia coli.
Topology of the ExbB protein in the cytoplasmic membrane of Escherichia coli.
复制标题
大肠杆菌细胞质膜中 ExbB 蛋白的拓扑结构。
DOI：
10.1016/s0021-9258(18)53424-4
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Volkmar BraunS
中科院分区：
文献类型：
--
作者：
K. Kampfenkel;Volkmar BraunS
ExbB蛋白与ExbD和TonB蛋白一起参与穿过大肠杆菌外膜的能量偶联转运。为了理解这个不寻常的过程，需要确定ExbB的亚细胞位置及其跨膜排列。使用ExbB-β-内酰胺酶融合蛋白作为报告的周质与细胞质的融合位点的位置，和访问的ExbB在原生质球和细胞裂解液氨肽酶K，胰蛋白酶，蛋白酶K，我们到达了一个模型的ExbB拓扑结构在细胞质膜。从周质中的N末端开始，ExbB含有三个跨膜区段（残基16-39、128-155、162-194），一个小的周质环和细胞质中的两个大部分。研究的18种融合蛋白中的两种，ExbB 34-β-内酰胺酶和ExbB 41-β-内酰胺酶，赋予高氨苄青霉素抗性。蛋白酶实验显示，在一个反向跨膜取向的分子的百分比分别高，低。这两种蛋白质都缺乏正电荷在细胞质膜的内侧，决定跨膜段的方向。
The ExbB protein together with the ExbD and TonB proteins is involved in energy-coupled transport across the outer membrane of Escherichia coli. To understand this unusual process it is required to determine the subcellular location of ExbB and its transmembrane arrangement. Using ExbB-beta-lactamase fusion proteins as reporters for a periplasmic versus a cytoplasmic location of the fusion sites, and accessibility of ExbB in spheroplasts and cell lysates to aminopeptidase K, trypsin, and proteinase K, we arrived at a model of ExbB topology in the cytoplasmic membrane. Starting with the N terminus in the periplasm ExbB contains three transmembrane segments (residues 16-39, 128-155, 162-194) a small periplasmic loop and two large portions in the cytoplasm. Two of the 18 fusion proteins studied, ExbB34-beta-lactamase and ExbB41-beta-lactamase, conferred a high ampicillin resistance. Protease experiments revealed a high respectively low percentage of the molecules in a reverse transmembrane orientation. Both proteins were lacking positive charges at the inner side of the cytoplasmic membrane which determine the orientation of transmembrane segments.