Functional Characterization of Escherichia coli MsbA
Functional Characterization of Escherichia coli MsbA
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大肠杆菌 MsbA 的功能表征
DOI：
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发表时间：
2008
影响因子：
4.8
通讯作者：
F. Sharom
中科院分区：
文献类型：
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作者：
Paul D. W. Eckford;F. Sharom
大肠杆菌MSBA蛋白是三磷酸腺苷结合盒超家族中一个65 kDa的成员。它被认为是一种依赖于三磷酸腺苷的脂类转位酶，将脂类A从细胞膜的内叶运输到外叶。分离到具有较高ATPase活性的MSBA，发现其在洗涤剂溶液中为均二聚体。蛋白ATPase活性被钒酸盐抑制，并表现出不同的刺激和抑制模式。对蛋白质的内源性色氨酸荧光进行了表征，并用丙烯酰胺对其进行了动态猝灭，结果表明脂质A的结合发生了构象变化。荧光猝灭用于表征MSBA与核苷酸和各种可能的底物(包括脂质、类脂化合物和药物)的相互作用。MSBA对∼2 mM的三磷酸腺苷有明显的亲和力，也能结合非水解性三磷酸腺苷类似物和荧光三磷酸腺苷衍生物。推测底物脂质A与蛋白质的相互作用亲和力为6.4μm。已知为ABC多药转运蛋白底物的药物也与MSBA相互作用，亲和力在0.2 5~5 0μm之间。本研究首次使用荧光方法估计核苷酸和假定转运底物的MSBA结合亲和力。
The Escherichia coli MsbA protein is a 65-kDa member of the ATP-binding cassette superfamily. It is thought to function as an ATP-dependent lipid translocase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. MsbA with high ATPase activity was isolated and found to be homodimeric in detergent solution. The protein ATPase activity was inhibited by vanadate and showed variable patterns of stimulation and inhibition by lipid A and other compounds. The intrinsic tryptophan fluorescence of the protein was characterized, and dynamic quenching using acrylamide showed that a conformational change took place on binding of lipid A. Fluorescence quenching was used to characterize the interactions of MsbA with nucleotides and various putative substrates, including lipids, lipid-like compounds, and drugs. MsbA had an apparent binding affinity for ATP of ∼2 mm and also bound nonhydrolyzable ATP analogs and fluorescent ATP derivatives. The putative substrate lipid A interacted with the protein with an affinity of 6.4 μm. Drugs that are known to be substrates for ABC multidrug transporters also interacted with MsbA with affinities in the range 0.25–50 μm. This study represents the first use of fluorescence approaches to estimate MsbA binding affinities for nucleotides and putative transport substrates.
影响因子：
16
作者：
Smith, PC;Karpowich, N;Hunt, JF
通讯作者：
Hunt, JF