Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104, United States.
Langmuir. 2012 Jul 17;28(28):10504-20. doi: 10.1021/la301219z. Epub 2012 Jun 29.
The voltage-sensor domain (VSD) is a modular four-helix bundle component that confers voltage sensitivity to voltage-gated cation channels in biological membranes. Despite extensive biophysical studies and the recent availability of X-ray crystal structures for a few voltage-gated potassium (Kv) channels and a voltage-gate sodium (Nav) channel, a complete understanding of the cooperative mechanism of electromechanical coupling, interconverting the closed-to-open states (i.e., nonconducting to cation conducting) remains undetermined. Moreover, the function of these domains is highly dependent on the physical-chemical properties of the surrounding lipid membrane environment. The basis for this work was provided by a recent structural study of the VSD from a prokaryotic Kv-channel vectorially oriented within a single phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) membrane investigated by X-ray interferometry at the solid/moist He (or solid/vapor) and solid/liquid interfaces, thus achieving partial to full hydration, respectively (Gupta et al. Phys. Rev. E2011, 84, 031911-1-15). Here, we utilize neutron interferometry to characterize this system in substantially greater structural detail at the submolecular level, due to its inherent advantages arising from solvent contrast variation coupled with the deuteration of selected submolecular membrane components, especially important for the membrane at the solid/liquid interface. We demonstrate the unique vectorial orientation of the VSD and the retention of its molecular conformation manifest in the asymmetric profile structure of the protein within the profile structure of this single bilayer membrane system. We definitively characterize the asymmetric phospholipid bilayer solvating the lateral surfaces of the VSD protein within the membrane. The profile structures of both the VSD protein and phospholipid bilayer depend upon the hydration state of the membrane. We also determine the distribution of water and exchangeable hydrogen throughout the profile structure of both the VSD itself and the VSD:POPC membrane. These two experimentally determined water and exchangeable hydrogen distribution profiles are in good agreement with molecular dynamics simulations of the VSD protein vectorially oriented within a fully hydrated POPC bilayer membrane, supporting the existence of the VSD's water pore. This approach was extended to the full-length Kv-channel (KvAP) at a solid/liquid interface, providing the separate profile structures of the KvAP protein and the POPC bilayer within the reconstituted KvAP:POPC membrane.
电压传感器域（VSD）是一个模块化的四螺旋束组件，它使生物膜中的电压门控阳离子通道具有电压敏感性。尽管进行了广泛的生物物理研究，并且最近可获得一些电压门控钾（Kv）通道和电压门控钠（Nav）通道的 X 射线晶体结构，但对机电耦合的协同机制，即从关闭状态到开放状态（即，不导电到阳离子导电）的相互转换的完整理解仍未确定。此外，这些结构域的功能高度依赖于周围脂质膜环境的物理化学性质。这项工作的基础是最近对一个原核 Kv 通道的 VSD 的结构研究，该通道在单个磷脂（1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC））膜中定向排列，通过 X 射线干涉法在固体/潮湿的 He（或固体/蒸气）和固体/液体界面处进行研究，从而分别实现部分至完全水合（Gupta 等人，Phys. Rev. E2011，84，031911-1-15）。在这里，我们利用中子干涉测量法在亚分子水平上对该系统进行了更高结构细节的表征，这是由于溶剂对比度变化与选定亚分子膜成分的氘化相结合所带来的固有优势，对于固体/液体界面处的膜尤为重要。我们证明了 VSD 的独特定向和其分子构象的保留，这表现在该单双层膜系统的蛋白轮廓结构中的蛋白不对称轮廓结构中。我们明确地描绘了在膜内的 VSD 蛋白的侧向表面上溶剂化的不对称磷脂双层的结构。VSD 蛋白和磷脂双层的轮廓结构都取决于膜的水合状态。我们还确定了 VSD 本身和 VSD：POPC 膜的轮廓结构中水和可交换氢的分布。这两个实验确定的水和可交换氢的分布曲线与 VSD 蛋白在完全水合的 POPC 双层膜中定向的分子动力学模拟结果非常吻合，支持 VSD 的水通道的存在。该方法扩展到固体/液体界面处的全长 Kv 通道（KvAP），提供了重组的 KvAP：POPC 膜中 KvAP 蛋白和 POPC 双层的单独轮廓结构。
