Probing the Dynamics and Structural Topology of the Reconstituted Human KCNQ1 Voltage Sensor Domain (Q1-VSD) in Lipid Bilayers Using Electron Paramagnetic Resonance Spectroscopy
Probing the Dynamics and Structural Topology of the Reconstituted Human KCNQ1 Voltage Sensor Domain (Q1-VSD) in Lipid Bilayers Using Electron Paramagnetic Resonance Spectroscopy
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DOI：
10.1021/acs.biochem.8b01042
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发表时间：
2019-02-19
期刊：
影响因子：
2.9
通讯作者：
Lorigan, Gary A.
中科院分区：
文献类型：
--
作者：
Dixit, Gunjan;Sahu, Indra D.;Lorigan, Gary A.
KCNQ 1（Kv7.1或KvLQT 1）是一种钾离子通道蛋白，存在于心脏、耳朵和其他组织中。与KCNE 1辅助蛋白复合，在心脏动作电位的复极化阶段发挥作用。该通道的突变与几种疾病有关，包括先天性耳聋和长QT综合征。核磁共振（NMR）的结构研究在洗涤剂胶束和冷冻电镜结构的KCNQ 1从非洲爪蟾表明，电压传感器域（Q1-VSD）的通道有四个跨膜螺旋，S1-S4，整体结构与其他VSD相似。在这项研究中，我们描述了一种可靠的方法重建Q1-VSD到（POPC/POPG）脂质双层囊泡。利用定点自旋标记电子顺磁共振技术研究了Q1-VSD在POPC/POPG脂质双层囊泡中的结构动力学和拓扑学。探测几种突变体以确定它们相对于膜的位置和相应的浸没深度（以埃计）。采用P-31固态NMR光谱法，通过改变蛋白质：脂质摩尔比，研究了Q1-VSD掺入后双层囊泡的动力学，证实了蛋白质与双层囊泡的相互作用。圆二色光谱数据表明，Q1-VSD的α-螺旋含量是较高的蛋白质重组在囊泡比以前的研究中使用DPC洗涤剂胶束。这项研究提供了深入了解Q1-VSD在脂质双层环境中重建的结构拓扑和动力学，为更先进的结构和功能研究奠定了基础。
KCNQ1 (Kv7.1 or KvLQT1) is a potassium ion channel protein found in the heart, ear, and other tissues. In complex with the KCNE1 accessory protein, it plays a role during the repolarization phase of the cardiac action potential. Mutations in the channel have been associated with several diseases, including congenital deafness and long QT syndrome. Nuclear magnetic resonance (NMR) structural studies in detergent micelles and a cryo-electron microscopy structure of KCNQ1 from Xenopus laevis have shown that the voltage sensor domain (Q1-VSD) of the channel has four transmembrane helices, S1-S4, being overall structurally similar with other VSDs. In this study, we describe a reliable method for the reconstitution of Q1-VSD into (POPC/POPG) lipid bilayer vesicles. Site-directed spin labeling electron paramagnetic resonance spectroscopy was used to probe the structural dynamics and topology of several residues of Q1-VSD in POPC/POPG lipid bilayer vesicles. Several mutants were probed to determine their location and corresponding immersion depth (in angstroms) with respect to the membrane. The dynamics of the bilayer vesicles upon incorporation of Q1-VSD were studied using P-31 solid-state NMR spectroscopy by varying the protein:lipid molar ratios confirming the interaction of the protein with the bilayer vesicles. Circular dichroism spectroscopic data showed that the a-helical content of Q1-VSD is higher for the protein reconstituted in vesicles than in previous studies using DPC detergent micelles. This study provides insight into the structural topology and dynamics of Q1-VSD reconstituted in a lipid bilayer environment, forming the basis for more advanced structural and functional studies.