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首页> 外文期刊>Biochemistry >Unfolding and Refolding in Vitro of a Tetrameric,alpha-Helical Membrane Protein:The Prokaryotic Potassium Channel KcsA
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Unfolding and Refolding in Vitro of a Tetrameric,alpha-Helical Membrane Protein:The Prokaryotic Potassium Channel KcsA

机译：在四聚体，α-螺旋膜蛋白的体外展开和重折叠：原核钾通道KCSA

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摘要

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 this is followed by the irreversible conversion of the unfolded monomers into a denatured protein form.
机译：2,2,2-三氟乙醇（TFE）有效地稳定了钾通道KCSA的其他高度稳定的四聚体结构，这是一种主要的α-螺旋膜蛋白[ValiyaveTil，Fi，Zhou，Y.和Mackinnon，R。（2002）生物化学41,10771-10777]。我们报告说，对洗涤剂溶液中增加浓度TFE浓度的蛋白质结构的影响包括两个连续的蛋白质浓度依赖性的合作转变。在第一种转变中，在较低的TFE浓度下发生，四聚体KCSA同时增加色氨酸残基暴露于溶剂，部分失去其二级结构，并解离其组成亚基。在这些条件下，TFE的简单稀释允许在洗涤剂溶液中高效地重折叠和蛋白质的蛋白质的重折叠。在重建成雾素巨型脂质体之后，重折叠蛋白质表现出Nativelike钾通道活性，如P atch-clamp方法。在较高的TFE浓度下发生的第二合作转变导致蛋白质的不可逆变性。这些结果在折叠的四聚蛋白和部分展开的单体之间的蛋白质和TFE浓度依赖性可逆平衡方面解释。亚基，其中折叠和低聚（或在平衡过程的另一方向上展开和解离）是似乎偶联的方法。较高的TFE浓度，然后将展开单体的不可逆转化为变性蛋白质形式。

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  • 来源
    《Biochemistry》 |2005年第43期|共9页
  • 作者

    Francisco N.Barrera; M.Lourdes Renart; M.Luisa Molina; Jose A.Poveda; Jose A.Encinar;

  • 作者单位

    Instituto de Biologia Molecular y Celular Universidad Miguel Hernandez Elche 03202 Alicante Spain and Instituto de Biocomputacion y Fisica de los Sistemas Complejos 50009 Zaragoza Spain;

    Instituto de Biologia Molecular y Celular Universidad Miguel Hernandez Elche 03202 Alicante Spain and Instituto de Biocomputacion y Fisica de los Sistemas Complejos 50009 Zaragoza Spain;

    Instituto de Biologia Molecular y Celular Universidad Miguel Hernandez Elche 03202 Alicante Spain and Instituto de Biocomputacion y Fisica de los Sistemas Complejos 50009 Zaragoza Spain;

    Instituto de Biologia Molecular y Celular Universidad Miguel Hernandez Elche 03202 Alicante Spain and Instituto de Biocomputacion y Fisica de los Sistemas Complejos 50009 Zaragoza Spain;

    Instituto de Biologia Molecular y Celular Universidad Miguel Hernandez Elche 03202 Alicante Spain and Instituto de Biocomputacion y Fisica de los Sistemas Complejos 50009 Zaragoza Spain;

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  • 正文语种 eng
  • 中图分类 生物化学;
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