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首页> 外文期刊>Biochemistry >Membrane Topology of the Colicin E1 Channel Using Genetically Encoded Fluorescence
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Membrane Topology of the Colicin E1 Channel Using Genetically Encoded Fluorescence

机译：Colicin E1通道使用遗传编码的荧光的膜拓扑。

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The membrane topology of the colicin El channel domain was studied by fluorescence resonance energy ransfer (FRET). The FRET involved a genetically encoded fluorescent amino acid (coumarin) as the donor and a selectively labeled cysteine residue tethered with DABMI (4-(dimethylamino)phenylazophenyl-4'-maleimide) as the FRET acceptor. The fluorescent coumarin residue was incorporated into the protein via an orthogonal tRNA/aminoacyl-tRNA synthetase pair that allowed selective incorporation into any site within the colicin channel domain. Each variant harbored a stop (TAG) mutation for coumarin incorporation and a cysteine (TGT) mutation for DABMI attachment. Six interhelical distances within helices 1-6 were determined using FRET analysis for both the soluble and membrane-bound states. The FRET data showed large changes in the interhelical distances among helices 3-6 upon membrane association providing new insight into the membrane-bound structure of the channel domain. In general, the coumarin-DABMI FRET interhelical efficiencies decreased upon membrane binding, building upon the umbrella model for the colicin channel. A tentative model for the closed state of the channel domain was developed based on current and previously published FRET data. The model suggests circular arrangement of helices 1-7 in a clockwise direction from the extracellular side and membrane interfacial association of helices 1, 6, 7, and 10 around the central transmembrane hairpin formed by helices 8 and 9.
机译：通过荧光共振能量转移（FRET）研究了大肠菌素E1通道结构域的膜拓扑。 FRET涉及一个遗传编码的荧光氨基酸（香豆素）作为供体，一个选择性标记的半胱氨酸残基与DABMI（4-（二甲基氨基）苯基偶氮苯基-4'-马来酰亚胺）相连，作为FRET受体。荧光香豆素残基通过正交tRNA /氨酰基-tRNA合成酶对掺入蛋白质，从而可以选择性掺入大肠菌素通道域内的任何位点。每个变体都带有一个用于香豆素掺入的终止（TAG）突变和一个用于DABMI附着的半胱氨酸（TGT）突变。使用FRET分析确定了螺旋1-6内的六个螺旋间距，以了解可溶状态和膜结合状态。 FRET数据显示，在膜结合后，螺旋3-6之间的螺旋间距离发生了很大变化，从而提供了对通道结构域的膜结合结构的新见解。通常，基于大肠菌素通道的伞形模型，香豆素-DABMI FRET螺旋间效率在膜结合时降低。基于当前和先前发布的FRET数据，开发了用于通道域关闭状态的暂定模型。该模型建议从螺旋1、6、7和10的细胞外侧面和膜界面结合沿螺旋8-7形成的中心跨膜发夹沿顺时针方向排列螺旋1-7的圆形排列。

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