Site-directed fluorescence studies of a prokaryotic ClC antiporter
Site-directed fluorescence studies of a prokaryotic ClC antiporter
复制标题
DOI：
10.1021/bi0523815
复制
发表时间：
2006-06-06
期刊：
影响因子：
2.9
通讯作者：
Mindell, Joseph A.
中科院分区：
文献类型：
--
作者：
Bell, Susan P.;Curran, Patricia K.;Mindell, Joseph A.
ClC家族的通道和转运体具有多种生理功能。对它们的门控和运输机制的理解仍然不完整，对蛋白质构象变化的程度存在分歧。利用位点定向荧光标记，我们探测了原核细胞ClC-ec1与运输相关的结构重排。我们用环境敏感的荧光团AlexaFluor 488特异性标记了ClC-ec1 R螺旋中引入的几个位置的半胱氨酸，并证明标记的突变体表现出与野生型蛋白没有区别的H+/Cl-运输活性。在我们检查的每个位置，我们观察到在已知激活运输的相同pH范围内酸化时荧光的变化。荧光变化对Cl-浓度也很敏感；此外，如果荧光变化反映了输运所需的构象变化，则Cl-和H+依赖性是耦合的。总之，结果表明，荧光的变化报告了转运过程中蛋白质构象的变化。标记的转运体突变去除了对质子偶联氯离子转运至关重要的谷氨酸，保留了pH依赖的荧光变化，这表明多个残基赋予了pH依赖的转运机制。这些结果对ClC通道和转运体的转运和门控模型具有启示意义。
Channels and transporters of the ClC family serve a variety of physiological functions. Understanding of their gating and transport mechanisms remains incomplete, with disagreement over the extent of protein conformational change involved. Using site-directed fluorescence labeling, we probe ClC-ec1, a prokaryotic ClC, for transport-related structural rearrangements. We specifically label cysteines introduced at several positions in the R helix of ClC-ec1 with AlexaFluor 488, an environment-sensitive fluorophore, and demonstrate that the labeled mutants show H+/Cl- transport activity indistinguishable from that of the wild-type protein. At each position that we examined we observe fluorescence changes upon acidification over the same pH range that is known to activate transport. The fluorescence change is also sensitive to Cl- concentration; furthermore, the Cl- and H+ dependencies are coupled as would be expected if the fluorescence change reflected a conformational change required for transport. Together, the results suggest that the changes in fluorescence report protein conformational changes underlying the transport process. Labeled transporters mutated to remove a glutamate critical to proton-coupled chloride transport retain pH-dependent fluorescence changes, suggesting that multiple residues confer pH dependence on the transport mechanism. These results have implications for models of transport and gating in ClC channels and transporters.