Department of Biochemistry and Molecular Biology, Center for Integrative Science, University of Chicago, Chicago, IL 60637, USA.
Biochemistry. 2012 Oct 16;51(41):8132-42. doi: 10.1021/bi300980q. Epub 2012 Oct 5.
The voltage-sensing domain (VSD) is the common scaffold responsible for the functional behavior of voltage-gated ion channels, voltage sensitive enzymes, and proton channels. Because of the position of the voltage dependence of the available VSD structures, at present, they all represent the activated state of the sensor. Yet in the absence of a consensus resting state structure, the mechanistic details of voltage sensing remain controversial. The voltage dependence of the VSD from Ci-VSP (Ci-VSD) is dramatically right shifted, so that at 0 mV it presumably populates the putative resting state. Appropriate biochemical methods are an essential prerequisite for generating sufficient amounts of Ci-VSD protein for high-resolution structural studies. Here, we present a simple and robust protocol for the expression of eukaryotic Ci-VSD in Escherichia coli at milligram levels. The protein is pure, homogeneous, monodisperse, and well-folded after solubilization in Anzergent 3-14 at the analyzed concentration (~0.3 mg/mL). Ci-VSD can be reconstituted into liposomes of various compositions, and initial site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopic measurements indicate its first transmembrane segment folds into an α-helix, in agreement with the homologous region of other VSDs. On the basis of our results and enhanced relaxation EPR spectroscopy measurement, Ci-VSD reconstitutes essentially randomly in proteoliposomes, precluding straightforward application of transmembrane voltages in combination with spectroscopic methods. Nevertheless, these results represent an initial step that makes the resting state of a VSD accessible to a variety of biophysical and structural approaches, including X-ray crystallography, spectroscopic methods, and electrophysiology in lipid bilayers.
电压感应域（VSD）是负责电压门控离子通道、电压敏感酶和质子通道功能行为的常见支架。由于可用 VSD 结构的电压依赖性的位置，目前它们都代表传感器的激活状态。然而，在缺乏共识的静止状态结构的情况下，电压感应的机制细节仍然存在争议。Ci-VSP（Ci-VSD）的 VSD 的电压依赖性显著右移，因此在 0 mV 时，它可能占据假定的静止状态。适当的生化方法是产生足够量 Ci-VSD 蛋白进行高分辨率结构研究的必要前提。在这里，我们提出了一种简单而稳健的方案，用于在大肠杆菌中以毫克级水平表达真核 Ci-VSD。该蛋白质是纯的、均一的、单分散的，并且在 Anzergent 3-14 中溶解后折叠良好，在分析浓度下（约 0.3 mg/mL）。Ci-VSD 可以重组到各种组成的脂质体中，并且初始的定点自旋标记和电子顺磁共振（EPR）光谱测量表明其第一个跨膜片段折叠成α-螺旋，与其他 VSD 的同源区域一致。基于我们的结果和增强的弛豫 EPR 光谱测量，Ci-VSD 在蛋白脂质体中基本上随机重组，排除了在与光谱方法结合的同时施加跨膜电压的直接应用。然而，这些结果代表了一个初始步骤，使 VSD 的静止状态可以通过多种生物物理和结构方法来研究，包括 X 射线晶体学、光谱方法和脂质双层中的电生理学。
