Osmolytes modulate conformational exchange in solvent-exposed regions of membrane proteins
Osmolytes modulate conformational exchange in solvent-exposed regions of membrane proteins
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DOI：
10.1002/pro.305
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发表时间：
2010-02-01
期刊：
影响因子：
8
通讯作者：
Cafiso, David S.
中科院分区：
文献类型：
--
作者：
Jimenez, Ricardo H. Flores;Do Cao, Marie-Ange;Cafiso, David S.
利用定点自旋标记技术（SDSL）研究了两种细菌外膜TonB依赖性转运蛋白BtuB和FecA的局部结构和构象交换。已知保护渗透剂，如聚乙二醇（PEG）调节BtuB的能量偶联基序（Ton盒）中的底物依赖性构象平衡。在这里，我们证明了一个片段，是N-末端的Ton盒在BtuB，是在有序和无序状态之间的构象交换有或没有基板。保护渗透剂改变了这种平衡，有利于更有序的折叠状态。然而，不暴露于溶剂的Ton盒的C-末端的BtuB区段对PEG不敏感。保护渗透剂还调节FecA的Ton盒中的构象平衡，其中较大分子量的PEG产生构象自由能的最大位移。这些数据表明，这些转运蛋白的溶剂暴露区域进行构象交换，这些转运蛋白的区域，参与蛋白质-蛋白质相互作用的样品多个构象substates。对溶质的敏感性为BtuB的两种高分辨率结构之间的差异提供了解释，这两种结构可能各自代表了蛋白质通常采样的状态子集中的一种构象。这项工作还说明了如何SDSL和渗透剂可用于表征和定量膜蛋白的构象平衡。
Site-directed spin labeling (SDSL) was used to investigate local structure and conformational exchange in two bacterial outer-membrane TonB-dependent transporters, BtuB and FecA. Protecting osmolytes, such as polyethylene glycols (PEGs) are known to modulate a substrate-dependent conformational equilibrium in the energy coupling motif (Ton box) of BtuB. Here, we demonstrate that a segment that is N-terminal to the Ton box in BtuB, is in conformational exchange between ordered and disordered states with or without substrate. Protecting osmolytes shift this equilibrium to favor the more ordered, folded state. However, a segment of BtuB that is C-terminal to the Ton box that is not solvent exposed is insensitive to PEGs. Protecting osmolytes also modulate a conformational equilibrium in the Ton box of FecA, with larger molecular weight PEGs producing the largest shifts in the conformational free energy. These data indicate that solvent-exposed regions of these transporters undergo conformational exchange and that regions of these transporters that are involved in protein-protein interactions sample multiple conformational substates. The sensitivity to solute provides an explanation for differences seen between two high-resolution structures of BtuB, which each likely represent one conformation from a subset of states that are normally sampled by the protein. This work also illustrates how SDSL and osmolytes may be used to characterize and quantitate conformational equilibria in membrane proteins.