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首页> 外文期刊>Biochemistry >Characterization of the walker a motif of MsbA using site-directed spin-labeling electron paramagnetic resonance spectroscopy
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Characterization of the walker a motif of MsbA using site-directed spin-labeling electron paramagnetic resonance spectroscopy

机译：使用定点自旋标记电子顺磁共振波谱表征沃克MsbA的基序

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MsbA is an ABC transporter that transports lipid A across the inner membrane of Gram-negative bacteria such as Escherichia coli. Without functional MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes [Chang and Roth (2001) Science 293, 1793-1800]. Although a structure of MsbA is now available, several functionally important motifs common to ABC transporters are unresolved in the crystal structure. The Walker A domain, one of the ABC transporter consensus motifs that is directly involved in ATP binding, is located within a large unresolved region of the MsbA ATPase domain. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful technique for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions. MsbA reconstituted into lipid membranes has been evaluated by EPR spectroscopy, and it has been determined that the Walker A domain forms an cc-helical structure, which is consistent with the structure of this motif observed in other crystallized ABC transporters. In addition, the interaction of the Walker A residues with ATP before, during, and after hydrolysis was followed using SDSL EPR spectroscopy in order to identify the residues directly involved in substrate binding and hydrolysis.
机译：MsbA是一种ABC转运蛋白，可将脂质A跨革兰氏阴性细菌（如大肠杆菌）的内膜转运。在不存在功能性MsbA的情况下，细菌细胞会在其内膜中积聚有毒的脂质A。最近获得了MsbA的晶体结构，为膜中的功能动力学研究提供了极好的起点[Chang and Roth（2001）Science 293，1793-1800]。尽管现在可以使用MsbA的结构，但ABC转运蛋白共有的几个功能上重要的基序在晶体结构中尚未解析。 Walker A结构域是直接参与ATP结合的ABC转运蛋白共有基元之一，位于MsbA ATPase结构域的较大未解析区域内。定点自旋标记（SDSL）电子顺磁共振（EPR）光谱技术是一种强大的技术，不仅可以检测和跟踪由于动态相互作用而引起的局部结构变化，而且可以表征大型蛋白质结构内的局部区域。已通过EPR光谱法评估了重构为脂质膜的MsbA，并确定Walker A结构域形成cc螺旋结构，该结构与在其他结晶的ABC转运蛋白中观察到的该基序结构一致。此外，使用SDSL EPR光谱追踪水解之前，水解过程中和水解之后Walker A残基与ATP的相互作用，以鉴定直接参与底物结合和水解的残基。

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