Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter.
Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter.
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DOI：
10.1016/j.bbrep.2018.10.006
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发表时间：
2018-12
影响因子：
2.7
通讯作者：
Wei Y
中科院分区：
文献类型：
--
作者：
Wang Z;Lu W;Rajapaksha P;Wilkop T;Cai Y;Wei Y
许多膜蛋白以低聚物或蛋白质复合体的形式存在并发挥作用。常规的分析方法包括用洗涤剂提取和增溶蛋白质，这可能会干扰它们的实际寡聚状态。AcrB是一种在大肠杆菌中的三聚体内膜多药转运蛋白。在以前的研究中，我们创造了一个突变体AcrBP223G，当从细胞膜中提取时，它的行为类似于单体。然而，AcrBP223G在细胞膜中的实际寡聚状态仍然不清楚，这使得对该突变影响功能的机制的解释变得更加复杂。在这里，我们使用了几种互补的方法来确定AcrBP223G在大肠杆菌细胞膜中的低聚状态。开发了两套定量荧光技术。为此，我们创建了荧光标记AcrB、AcrB-CFP和AcrB-YPet。用荧光共振能量转移(FRET)和光漂白后荧光恢复(FRAP)分别表征共表达的AcrB-CFP和AcrB-YPet之间的能量转移效率以及AcrB-YPet和AcrBP223G-YPet在活体细胞中的扩散系数。其次，我们在亚基间界面引入了半胱氨酸对，并使用受控氧化来探测亚基间的距离。所有研究的结果都集中在AcrBP223G作为三聚体存在于细胞膜中的结论，它在纯化步骤中解离。三聚体亲和力和结构的微小变化会导致AcrB活性的显著丧失。此外，在整个研究过程中，我们制定了方案并建立了基准值，这对进一步研究细胞膜中的膜蛋白相互作用很有用。AcrB功能对三聚体稳定性高度敏感。测定了AcrB三聚体在活细胞中的扩散系数。能量转移在CFP和YPet标记的AcrB三聚体中定量。洗涤剂提取可能会导致对膜蛋白复合体的错误结论。
Many membrane proteins exist and function as oligomers or protein complexes. Routine analytical methods involve extraction and solubilization of the proteins with detergents, which could disturb their actual oligomeric state. AcrB is a trimeric inner membrane multidrug transporter in E. coli. In previous studies, we created a mutant AcrBP223G, which behaves like a monomer when extracted from the cell membrane. However, the actual oligomeric state of AcrBP223G in cell membranes remained unclear, which complicated the interpretation of the mechanism by which the mutation affects function. Here we used several complementary methods to determine the oligomeric state of AcrBP223G in E. coli cell membranes. Two sets of quantitative fluorescent techniques were exploited. For these, we created fluorescent tagged AcrB, AcrB-CFP and AcrB-YPet. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) were employed to characterize independently the efficiency of energy transfer between co-expressed AcrB-CFP and AcrB-YPet, and the diffusion coefficient of AcrB-YPet and AcrBP223G-YPet in live E. coli cells. Second, we introduced Cys pairs at the inter-subunit interface and used controlled oxidation to probe inter-subunit distances. The results from all studies converge on the conclusion that AcrBP223G exists as a trimer in cell membranes, which dissociates during the purification steps. The small change in trimer affinity and structure leads to a significant loss of AcrB activity. In addition, throughout this study we developed protocols and established benchmark values, useful for further studies on membrane protein associations in cell membranes. AcrB function is highly sensitive to trimer stability. Diffusion coefficient was determined for AcrB trimer in live E. coli cells. Energy transfer was quantified in CFP and YPet tagged AcrB trimer. Detergent extraction may lead to wrong conclusion about membrane protein complex.
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影响因子：
2.5
作者：
Megias, Diego;Marrero, Raquel;Montoya, Maria C.
通讯作者：
Montoya, Maria C.
影响因子：
5.6
作者：
Lu, Wei;Chai, Qian;Wei, Yinan
通讯作者：
Wei, Yinan
影响因子：
15
作者：
Chen, Lirong;Novicky, Lawrence;Hristova, Kalina
通讯作者：
Hristova, Kalina
影响因子：
3.4
作者：
King, Christopher;Sarabipour, Sarvenaz;Hristova, Kalina
通讯作者：
Hristova, Kalina
DOI：
10.3390/s100605927
发表时间：
2010
期刊：
Sensors (Basel, Switzerland)
影响因子：
--
作者：
Rayan G;Guet JE;Taulier N;Pincet F;Urbach W
通讯作者：
Urbach W