Comparing the structural dynamics of the human KCNE3 in reconstituted micelle and lipid bilayered vesicle environments
Comparing the structural dynamics of the human KCNE3 in reconstituted micelle and lipid bilayered vesicle environments
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DOI：
10.1016/j.bbamem.2022.183974
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发表时间：
2022-10-01
影响因子：
3.4
通讯作者：
Sahu, Indra D.
中科院分区：
文献类型：
--
作者：
Campbell, Conner;Faleel, Fathima Dhilhani Mohammed;Sahu, Indra D.
KCNE3是KCNE家族中的一种跨膜蛋白，调节包括KCNQ1在内的多种电压门控性钾通道的功能和转运。KCNE3的结构研究以前已经在各种模型膜模拟物中进行过。然而，重要的是要评估所使用的膜模拟物对观察到的构象和动力学的影响。在这项研究中，我们优化了一种将KCNE3重组为POPC/POPG脂质双层囊泡的方法，用于电子顺磁共振(EPR)光谱。圆二色谱数据表明，KCNE3蛋白重组为脂质双层囊泡的规则二级结构程度明显高于DPC洗涤剂胶束。采用电子顺磁共振(EPR)和定点自旋标记(SDSL)相结合的方法，研究了KCNE3在DPC胶束和POPC/POPG脂质双层囊泡中S49C、M59C、L67C、V85C和S101C突变的结构动力学。我们的CW-EPR功率饱和数据表明，与DPC胶束结果相反，S74C位点埋在脂质双层膜内，而V85C位点位于膜外。这些结果表明，为了确定KCNE3的天然结构，需要使用在脂质双层囊泡中获得的数据来改进KCNE3的胶束结构。这项工作将为在更自然的膜环境中详细研究KCNE3的结构提供指导，并将脂双层结果与各向同性双胞结构和KCNQ1结合的冷冻-EM结构进行比较。
KCNE3 is a single transmembrane protein of the KCNE family that modulates the function and trafficking of several voltage-gated potassium channels, including KCNQ1. Structural studies of KCNE3 have been previously conducted in a wide range of model membrane mimics. However, it is important to assess the impact of the membrane mimics used on the observed conformation and dynamics. In this study, we have optimized a method for the reconstitution of the KCNE3 into POPC/POPG lipid bilayer vesicles for electron paramagnetic resonance (EPR) spectroscopy. Our CD spectroscopic data suggested that the degree of regular secondary structure for KCNE3 protein reconstituted into lipid bilayered vesicle is significantly higher than in DPC detergent micelles. Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) was used to probe the structural dynamics of S49C, M59C, L67C, V85C, and S101C mutations of KCNE3 in both DPC micelles and in POPC/POPG lipid bilayered vesicles. Our CW-EPR power saturation data suggested that the site S74C is buried inside the lipid bilayered membrane while the site V85C is located outside the membrane, in contrast to DPC micelle results. These results suggest that the KCNE3 micelle structures need to be refined using data obtained in the lipid bilayered vesicles in order to ascertain the native structure of KCNE3. This work will provide guidelines for detailed structural studies of KCNE3 in a more native membrane environment and comparing the lipid bilayer results to the isotropic bicelle structure and to the KCNQ1-bound cryo-EM structure.