Sided functions of an arginine-agmatine antiporter oriented in liposomes.
Sided functions of an arginine-agmatine antiporter oriented in liposomes.
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DOI：
10.1021/bi201897t
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发表时间：
2012-02-28
期刊：
影响因子：
2.9
通讯作者：
Miler, Christopher
中科院分区：
文献类型：
--
作者：
Tsai, Ming-Feng;Fang, Yiling;Miler, Christopher
依赖精氨酸的极端耐酸系统帮助肠道细菌在恶劣的胃环境中生存。在这个多蛋白系统的中心是精氨酸-agmatine反转运蛋白AdiC。为了维持细胞质pH值，AdiC进口精氨酸并出口其脱羧产物agmatine，导致每次周转净挤出一个“虚质子”。AdiC在重组脂质体中的随机取向给量化其转运机制带来了障碍。为了克服这个问题，我们在底物结合位点附近引入了一个突变S26C。该突变体表现出与野生型蛋白相似的底物识别和ph依赖活性，但在与硫醇试剂反应时完全失去功能。然后，膜外MTSES试剂可以用作清洁侧抑制剂，以沉默那些细胞外部分从脂质体外侧突出的S26C-AdiC蛋白。或者，膜透性MTSEA和膜透性还原试剂TCEP可以一起使用，以相反的方向抑制蛋白质。这种方法能够以侧面的方式对AdiC进行稳态动力学分析。精氨酸和精氨酸在蛋白质的两侧具有相似的Michaelis-Menten参数，而细胞外侧比细胞质侧更有效地选择精氨酸而不是精氨酸酰胺，精氨酸酰胺是精氨酸的羧酸质子化形式的模拟物。此外，AdiC的两侧具有不同的ph敏感性。当细胞外侧酸化时，AdiC的活性在pH为4时达到平台，而细胞质侧的最佳pH为5.5，进一步的酸化抑制了运输。这种定向系统可以比以前更精确地分析adic介导的底物运输，并可以与酸胁迫下细菌膜的情况进行比较。
The arginine-dependent extreme acid resistance system helps enteric bacteria survive the harsh gastric environment. At the center of this multi-protein system is an arginine-agmatine antiporter, AdiC. To maintain cytoplasmic pH, AdiC imports arginine and exports its decarboxylated product agmatine, resulting in a net extrusion of one “virtual proton” in each turnover. The random orientation of AdiC in reconstituted liposomes throws up an obstacle to quantifying its transport mechanism. To overcome this problem, we introduced a mutation, S26C, near the substrate-binding site. This mutant exhibits similar substrate recognition and pH-dependent activity as wild-type protein, but loses function completely upon reaction with thiol reagents. The membrane-impermeant MTSES reagent can then be used as a cleanly sided inhibitor to silence those S26C-AdiC proteins whose extracellular portion projects from the external side of the liposome. Alternatively, the membrane-permeant MTSEA and membrane-impermeant reducing reagent, TCEP, can be used together to inhibit proteins in the opposite orientation. This approach enables steady-state kinetic analysis of AdiC in a sided fashion. Arginine and agmatine have similar Michaelis-Menten parameters for both sides of the protein, while the extracellular side selects arginine over argininamide, a mimic of the carboxylate-protonated form of arginine, more effectively than does the cytoplasmic side. Moreover, the two sides of AdiC have different pH-sensitivities. AdiC activity increases to a plateau at pH 4 as the extracellular side is acidified, while the cytoplasmic side shows an optimal pH of 5.5, with further acidification inhibiting transport. This oriented system allows more precise analysis of AdiC-mediated substrate transport than has been previously available and permits comparison to the situation experienced by the bacterial membrane under acid stress.
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影响因子：
3.2
作者：
Castanie-Cornet, MP;Penfound, TA;Foster, JW
通讯作者：
Foster, JW
DOI：
10.1073/pnas.1018081108
发表时间：
2011-03-08
影响因子：
11.1
作者：
Kowalczyk, Lukasz;Ratera, Merce;Palacin, Manuel
通讯作者：
Palacin, Manuel
影响因子：
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作者：
通讯作者：
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影响因子：
3.2
作者：
Gong, S;Richard, H;Foster, JW
通讯作者：
Foster, JW
影响因子：
64.8
作者：
Gao, Xiang;Zhou, Lijun;Shi, Yigong
通讯作者：
Shi, Yigong