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首页> 外文期刊>Biochemistry >Substrate-induced conformational changes of the perplasmic N-terminus of an outer-membrane transporter by site-directed spin labeling.
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Substrate-induced conformational changes of the perplasmic N-terminus of an outer-membrane transporter by site-directed spin labeling.

机译：底物诱导的外膜转运蛋白周向N末端的构象变化通过定点自旋标记。

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摘要

The structure and dynamics of the N-terminal and core regions of BtuB, an outer membrane vitamin B(12) transporter from Escherichia coli, were investigated by site-directed spin labeling. Cysteine mutants were generated by site-directed mutagenesis to place spin labels in the N-terminal region (residues 1-17), the core region (residues 25-30), and double labels into the Ton box (residues 6-12). BtuB mutants were expressed, spin labeled, purified, and reconstituted into phosphatidylcholine. In the presence of substrate (vitamin B(12)), EPR spectroscopy demonstrates that there is a conformational change in the Ton box similar to that seen previously for BtuB in intact outer membranes. The Ton box is positioned within the beta-barrel of BtuB in the absence of substrate (docked configuration) but becomes unfolded and increases its aqueous exposure upon substrate binding (undocked configuration). This conformational change and the similarity in the EPR spectra between reconstituted and native membranes indicate that BtuB is correctly folded and functional in the reconstituted system. The protein segment on the N-terminal side of the Ton box is highly mobile, and it becomes more mobile in the presence of substrate. Side chains in the region C-terminal to the Ton box also show increases in mobility with substrate addition, but position 16 appears to define a hinge point for this conformation change. EPR line shapes and relaxation data indicate that residues 25-30 form a beta-strand structure, which is analogous to the first beta-strand in the cores of the homologous iron transporters. When substrate binds to BtuB, this first beta-strand remains folded. The EPR spectra of double-nitroxide labels within the Ton box are broadened because of dipolar and collisional exchange interactions. The broadening pattern indicates that the Ton box is not helical but is in an extended or beta-strand structure.
机译：结构和动态的N末端和核心区域的BtuB，大肠杆菌的外膜维生素B（12）转运蛋白，通过定点旋转标记进行了调查。半胱氨酸突变体通过定点诱变产生，将自旋标记置于N末端区域（残基1-17），核心区域（残基25-30）和双标记放入Ton框（残基6-12）。表达，旋转标记，纯化BtuB突变体，然后将其重组为磷脂酰胆碱。在存在底物（维生素B（12））的情况下，EPR光谱表明，完整包装的外膜中Ton盒中的构象变化与BtuB先前所见相似。在没有底物的情况下，Ton盒位于BtuB的β桶内（对接构型），但在与底物结合时（未对接构型），Ton盒会展开并增加其水暴露量。重构膜和天然膜之间的这种构象变化和EPR谱的相似性表明BtuB在重构系统中正确折叠并起作用。 Ton盒N端侧的蛋白质片段具有高度的移动性，在存在底物的情况下，其移动性也更高。 Ton盒的C端区域中的侧链也随着底物的添加而显示出迁移率的增加，但位置16似乎为该构象变化定义了一个铰接点。 EPR线形和弛豫数据表明，残基25-30形成一个β链结构，类似于同源铁转运蛋白核心中的第一个β链。当底物与BtuB结合时，第一条β链保持折叠状态。由于偶极和碰撞交换相互作用，Ton盒内双硝基氧标记物的EPR谱变宽了。变宽的模式表示Ton框不是螺旋形的，而是呈扩展或β链结构。

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