High-level expression, refolding and probing the natural fold of the human voltage-dependent anion channel isoforms I and II
High-level expression, refolding and probing the natural fold of the human voltage-dependent anion channel isoforms I and II
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DOI：
10.1007/s00232-007-9038-8
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发表时间：
2007-04-01
影响因子：
2.4
通讯作者：
Zeth, Kornelius
中科院分区：
文献类型：
--
作者：
Engelhardt, Harald;Meins, Thomas;Zeth, Kornelius
电压依赖性阴离子通道（VDAC）是线粒体外膜中发现的主要蛋白质。该通道负责 ATP/ADP 的交换以及离子和其他小代谢物在膜上的易位。为了获得大量纯的且适当折叠的人VDAC用于功能和结构研究，在大肠杆菌中克隆了人亚型I和II（HVDAC1和HVDAC2）的基因。高水平表达导致包涵体形成。通过将变性蛋白质添加到两性离子或非离子去垢剂溶液中，这两种蛋白质都可以在体外重新折叠。开发了一种高效、快速的重折叠方案，每升细胞培养物产生超过 50 毫克的纯人 VDAC。通过傅里叶变换红外光谱法探测重折叠孔蛋白的天然和功能状态，以确定二级结构组成，并通过电生理学测量，证明 HVDAC1 的成孔活性。此外，还证明了 HVDAC1 与固定化 ATP 的结合。将嵌入洗涤剂胶束中的 HVDAC1 蛋白进行有限的蛋白水解，并结合基质辅助激光解吸电离质谱分析来识别蛋白质的胶束暴露区域，并开发改进的拓扑模型。我们的分析强烈表明存在一个 16 股反平行 β 桶，其中有一个大环和七个短环和转角。该蛋白质的初始结晶试验产生了衍射分辨率为 8 埃的晶体。
The voltage-dependent anion channel (VDAC) is the major protein found in the outer membrane of mitochondria. The channel is responsible for the exchange of ATP/ADP and the translocation of ions and other small metabolites over the membrane. In order to obtain large amounts of pure and suitably folded human VDAC for functional and structural studies, the genes of the human isoforms I and II (HVDAC1 and HVDAC2) were cloned in Escherichia coli. High-level expression led to inclusion body formation. Both proteins could be refolded in vitro by adding denatured protein to a solution of zwitterionic or nonionic detergents. A highly efficient and fast protocol for refolding was developed that yielded more than 50 mg of pure human VDACs per liter of cell culture. The native and functional state of the refolded porins was probed by Fourier transform infrared spectroscopy to determine the secondary structure composition and by electrophysiological measurements, demonstrating the pore-forming activity of HVDAC1. Furthermore, binding of HVDAC1 to immobilized ATP was demonstrated. Limited proteolysis of HVDAC1 protein embedded in detergent micelles in combination with matrix-assisted laser desorption ionization mass spectrometric analysis was applied to identify micelle-exposed regions of the protein and to develop an improved topology model. Our analysis strongly suggests a 16-stranded, antiparallel beta-barrel with one large and seven short loops and turns. Initial crystallization trials of the protein yielded crystals diffracting to 8 angstrom resolution.