Membrane Topology of the Colicin E1 Channel Using Genetically Encoded Fluorescence

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• 作者：Derek Ho ; Miguel R. Lugo ; Andrei L. Lomize ; Irina D. Pogozheva ; Suneel P. Singh ; Adrian L. Schwan ; A. Rod Merrill
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：June 7, 2011
• 年：2011
• 卷：50
• 期：22
• 页码：4830-4842
• 全文大小：1056K
• 年卷期：v.50,no.22(June 7, 2011)
• ISSN：1520-4995

文摘

The membrane topology of the colicin E1 channel domain was studied by fluorescence resonance energy transfer (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鈥?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鈥? 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鈥? 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鈥? 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.