Initial binding process of the membrane insertase YidC with its substrate Pf3 coat protein is reversible
Initial binding process of the membrane insertase YidC with its substrate Pf3 coat protein is reversible
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DOI：
10.1021/bi800116t
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发表时间：
2008-06-03
期刊：
影响因子：
2.9
通讯作者：
Kuhn, Andreas
中科院分区：
文献类型：
--
作者：
Gerken, Uwe;Erhardt, Dagmar;Kuhn, Andreas
用荧光光谱法研究了大肠杆菌内膜插入酶YidC与其底物Pf 3外壳蛋白的体外结合。纯化的YidC蛋白用脂质样去污剂正十二烷基磷酸胆碱溶解，并用1-苯胺基-萘-8-磺酸盐（ANS）非共价标记，而Pf 3外壳蛋白通过向缓冲液中加入10%（v/v）异丙醇保持在溶液中。Pf 3外壳蛋白的结合通过结合至YidC的ANS的荧光猝灭来分析。所有结合曲线在pH 9.0和5.0之间显示严格的双曲线形式，表明YidC与其底物之间的可逆和非合作结合。数据分析显示结合过程的解离常数K-D在1 μ M范围内。K-D值的pH曲线表明，Pf 3外壳蛋白的结合主要是由疏水相互作用。滴定实验为结合Pf 3外壳蛋白分子后插入酶的构象变化提供了强有力的证据。
The binding of the inner membrane insertase YidC from Escherichia coli to its substrate, the Pf3 coat protein, was examined in vitro by fluorescence spectroscopy. Purified YidC protein was solubilized with the lipid-like detergent n-dodecylphosphocholine and noncovalently labeled with 1-anilino-naphthalene-8-sulfonate (ANS), whereas the Pf3 coat protein was kept in solution by the addition of 10% (v/v) isopropanol to the buffer. The binding of Pf3 coat protein was analyzed by fluorescence quenching of ANS bound to YidC. All binding curves showed a strict hyperbolic form at pH values between 9.0 and 5.0, indicating a reversible and noncooperative binding between YidC and its substrate. Analysis of the data revealed a dissociation constant K-D for the binding process in the range of 1 mu M. The pH profile of the K-D values suggests that the binding of the Pf3 coat protein is dominated by hydrophobic interactions. The titration experiments provide strong evidence for a conformational change of the insertase upon binding a Pf3 coat protein molecule.