Stable self-assembly of a protein engineering scaffold on gold surfaces
Stable self-assembly of a protein engineering scaffold on gold surfaces
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DOI：
10.1110/ps.0206102
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发表时间：
2002-08-01
期刊：
影响因子：
8
通讯作者：
Lakey, JH
中科院分区：
文献类型：
--
作者：
Terrettaz, S;Ulrich, WP;Lakey, JH
大肠杆菌外膜蛋白OmpF是β-桶膜蛋白大家族中的一员。有些是孔形成蛋白，如OmpF，而另一些则是活性转运蛋白或酶。我们先前已经证明，毒素Colicin N的受体结合域(R-结构域)与OmpF高亲和力结合，在金电极上重组为锚定的脂双层。结合可以通过表面等离子体共振(SPR)和离子通道阻断(阻抗谱，IS)来测量。在这篇文章中，我们报道了一个突变体OmpF-E183C的使用，其中一个半胱氨酸被引入到一个短的周质转角上。OmpF-E183C直接结合到金表面，并通过从洗涤剂溶液中自组装创建高密度蛋白质层。当用β-硫醇对金表面进行处理，并在蛋白质固定化步骤后加入硫脂时，傅立叶变换红外光谱(FTIR)显示蛋白质保留了其富含β的结构。此外，我们还可以通过SPR和IS检测R结构域的结合，证实了金表面自组装膜蛋白单分子层的功能重构。由于这些β-桶蛋白是公认的蛋白质工程支架，该方法为从水溶液中简单地自组装蛋白质界面提供了一种通用方法。
The outer membrane protein OmpF from Escherichia coli is a member of a large family of beta-barrel membrane proteins. Some, like OmpF, are pore-forming proteins whilse others are active transporters or enzymes. We have previously shown that the receptor-binding domain (R-domain) of the toxin colicin N binds with high affinity to OmpF reconstituted into tethered lipid bilayers on gold electrodes. The binding can be measured by surface plasmon resonance (SPR) and ion channel blockage (impedance spectroscopy, IS). In this paper we report the use of a mutant OmpF-E183C in which a single cysteine had been introduced on a short periplasmic turn. OmpF-E183C binds directly to gold surfaces and creates high-density protein layers by self-assembly from detergent solution. When the gold surface is pretreated with beta-mercaptoethanol and thiolipids are added after the protein immobilisation step, the protein is shown, by Fourier transform infrared spectroscopy (FTIR), to retain its beta-rich structure. Furthermore, we could also measure R-domain binding by SPR and IS, confirming the functional reconstitution of a self-assembled membrane protein monolayer at the gold surface. Because these beta-barrel proteins are recognized protein engineering scaffolds, the method provides a generic method for the simple self-assembly of protein interfaces from aqueous solution.