Unfolding and refolding in vitro of a tetrameric, α-helical membrane protein:: The prokaryotic potassium channel KcsA
Unfolding and refolding in vitro of a tetrameric, α-helical membrane protein:: The prokaryotic potassium channel KcsA
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DOI：
10.1021/bi050845t
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发表时间：
2005-11-01
期刊：
影响因子：
2.9
通讯作者：
González-Ros, JM
中科院分区：
文献类型：
--
作者：
Barrera, FN;Renart, ML;González-Ros, JM
2，2，2-三氟乙醇（TFE）有效地使钾通道KcsA（一种主要为α-螺旋的膜蛋白）的原本高度稳定的四聚体结构不稳定[Valiyaveetil，F.一、Zhou，Y.，（1996年），中国科学院，和MacKinnon，R.（2002）Biochemistry 41，10771-10777]。在这里，我们报告说，在洗涤剂溶液中的TFE浓度增加的蛋白质结构的影响，包括两个连续的蛋白质浓度依赖性，合作过渡。在第一个这样的转变，发生在较低的TFE浓度，四聚体KcsA同时增加色氨酸残基的溶剂暴露，部分失去其二级结构，并解离成其组成亚基。在这些条件下，TFE的简单稀释允许蛋白质在洗涤剂溶液中的高效重折叠和四聚化。此外，重组成asolectin巨型脂质体后，再折叠的蛋白质表现出天然钾通道活性，如通过膜片钳方法评估的。相反，在较高的TFE浓度下发生的第二次合作转变导致蛋白质的不可逆变性。这些结果被解释在折叠的四聚体蛋白质和部分未折叠的单体亚基之间的蛋白质和TFE浓度依赖性可逆平衡，其中折叠和寡聚化（或在平衡过程的其他方向展开和解离）似乎耦合的过程。在较高的TFE浓度下
2,2,2-Trifluoroethanol (TFE) effectively destabilizes the otherwise highly stable tetrameric structure of the potassium channel KcsA, a predominantly alpha-helical membrane protein [Valiyaveetil, F. I., Zhou, Y., and MacKinnon, R. (2002) Biochemistry 41, 10771-10777]. Here, we report that the effects on the protein structure of increasing concentrations of TFE in detergent solution include two successive protein concentration-dependent, cooperative transitions. In the first of such transitions, occurring at lower TFE concentrations, the tetrameric KcsA simultaneously increases the exposure of tryptophan residues to the solvent, partly loses its secondary structure, and dissociates into its constituent subunits. Under these conditions, simple dilution of the TFE permits a highly efficient refolding and tetramerization of the protein in the detergent solution. Moreover, following reconstitution into asolectin giant liposomes, the refolded protein exhibits nativelike potassium channel activity, as assessed by patch-clamp methods. Conversely, the second cooperative transition occurring at higher TFE concentrations results in the irreversible denaturation of the protein. These results are interpreted in terms of a protein and TFE concentration-dependent reversible equilibrium between the folded tetrameric protein and partly unfolded monomeric subunits, in which folding and oligomerization (or unfolding and dissociation in the other direction of the equilibrium process) are seemingly coupled processes. At higher TFE concentrations