Determination of Real-Time Efflux Phenotypes in Escherichia coli AcrB Binding Pocket Phenylalanine Mutants Using a 1,2′-Dinaphthylamine Efflux Assay
Determination of Real-Time Efflux Phenotypes in Escherichia coli AcrB Binding Pocket Phenylalanine Mutants Using a 1,2′-Dinaphthylamine Efflux Assay
复制标题
DOI：
10.1371/journal.pone.0021196
复制
发表时间：
2011-06-15
期刊：
影响因子：
3.7
通讯作者：
Kern, Winfried V.
中科院分区：
文献类型：
--
作者：
Bohnert, Juergen A.;Schuster, Sabine;Kern, Winfried V.
为了评估苯丙氨酸残基在大肠杆菌外排泵蛋白AcrB中对底物转运的重要性，我们用新型近红外亲脂膜探针1，2 '-二萘胺（1，2'-DNA）对Phe-to-Ala结合口袋突变体进行实时外排测定。除F617 A外，所有突变均导致外排显著延迟。F610 A是影响最明显的点突变，其次是F628 A、F615 A、F136 A和F178 A。这是第一个研究，以证明单苯丙氨酸残基内的AcrB结合口袋的实时底物运输的重要性。
To evaluate the importance of phenylalanine residues for substrate transport in the Escherichia coli efflux pump protein AcrB, we subjected Phe-to-Ala binding pocket mutants to a real-time efflux assay with the novel near-infrared lipophilic membrane probe 1,2'-dinaphthylamine (1,2'-DNA). All mutations, with the exception of F617A, led to considerable retardation of efflux. F610A was the point mutation with the most pronounced impact, followed by F628A, F615A, F136A, and F178A. This is the first study to demonstrate the importance of single phenylalanine residues within the AcrB binding pocket for real-time substrate transport.