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Characterization of the oligomeric states of wild type and mutant AraC

机译：野生型和突变AraC的低聚状态的表征

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摘要

AraC regulates transcription of the Escherichia coli arabinose operon, binding tandem DNA half-sites in the presence of arabinose and widely spaced half-sites in the absence of arabinose. In the structure of the AraC N-terminal dimerization domain with bound arabinose, the protein dimerizes via an antiparallel coiled-coil interface. The absence of bound ligand opens a second, beta-barrel interaction interface that also mediates interactions between unliganded AraC dimers in the crystal. The larger buried surface area of the beta-barrel interface, as compared with the coiled-coil interface, raised the possibility that protein-protein interactions mediated by the beta-barrel might play a role in ligand-mediated modulation of AraC DNA binding activity. For the crystallographically observed beta-barrel interaction to play a role in the cell, dimerization via this interface in the absence of arabinose would be predicted to be at least as energetically favorable as dimerization via the coiled-coil interface. In the study presented here, we use analytical ultracentrifugation to determine the oligomeric state of the AraC dimerization domain in the presence and absence of arabinose. Dimerization of the unliganded protein via the beta-barrel interface in the absence of interactions mediated by the coiled-coil interface is assayed using a mutant AraC protein with a disrupted coiled-coil interface. The results of these studies indicate that dimerization via the beta-barrel interface is substantially weaker than dimerization via the coiled-coil interface, indicating that the crystallographically observed beta-barrel interaction is not relevant to in vivo function. [References: 21]
机译：AraC调节大肠杆菌阿拉伯糖操纵子的转录，在存在阿拉伯糖的情况下结合串联的DNA半位点，在不存在阿拉伯糖的情况下结合宽间隔的半位点。在具有结合的阿拉伯糖的AraC N端二聚结构域的结构中，蛋白质通过反平行的卷曲螺旋界面二聚。缺乏结合配体会打开第二个β-桶相互作用界面，该界面也介导晶体中未配位的AraC二聚体之间的相互作用。与卷曲螺旋界面相比，β-桶界面的更大的埋入表面积增加了由β-桶介导的蛋白质-蛋白质相互作用可能在配体介导的AraC DNA结合活性调节中起作用的可能性。为了晶体学上观察到的β-桶相互作用在细胞中起作用，在没有阿拉伯糖的情况下通过该界面进行的二聚化预计至少与通过卷曲螺旋界面的二聚化在能量上有利。在这里提出的研究中，我们使用分析超速离心法确定存在和不存在阿拉伯糖的情况下AraC二聚化结构域的寡聚状态。在不存在由卷曲螺旋界面介导的相互作用的情况下，经由β-桶界面的未配体蛋白质的二聚化是使用具有破坏的卷曲螺旋界面的突变体AraC蛋白测定的。这些研究的结果表明，通过β-桶界面的二聚作用明显弱于通过盘绕线圈界面的二聚作用，这表明晶体学上观察到的β-桶相互作用与体内功能无关。 [参考：21]

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