Reconstitution of KCNE1 into Lipid Bilayers: Comparing the Structural, Dynamic, and Activity Differences in Micelle and Vesicle Environments
Reconstitution of KCNE1 into Lipid Bilayers: Comparing the Structural, Dynamic, and Activity Differences in Micelle and Vesicle Environments
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DOI：
10.1021/bi2009294
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发表时间：
2011-12-20
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：
Coey, Aaron T.;Sahu, Indra D.;Lorigan, Gary A.
KCNE1 (minK) 存在于人类心脏和耳蜗中，是一种跨膜蛋白，可调节电压门控钾 KCNQ1 通道。虽然 KCNE1 此前已成为溶血磷脂去污剂胶束中广泛结构研究的主题，但关键观察结果尚未在脂质双层中得到证实和完善。在本研究中，开发了一种将 KCNE1 重建为由 1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC）和 1-棕榈酰-2-油酰-sn-甘油-3-磷酸（1'-rac-甘油）（钠盐）（POPG）组成的脂质双层囊泡的可靠方法。将蛋白脂质体显微注射到表达人 KCNQ1 (K(V)7.1) 电压门控钾通道的爪蟾卵母细胞中，导致通道的天然调节。圆二色光谱表明，在脂质囊泡中重建的蛋白质的 KCNE1 螺旋度百分比显着高于先前描述的 1.0% 1-肉豆蔻酰基-2-羟基-sn-甘油-3-磷酸（1'-rac-甘油）（钠盐）（LMPG）胶束中的结构。采用SDSL电子顺磁共振波谱技术探测POPC/POPG囊泡和LMPG胶束中KCNE1的Ser28、Phe54、Phe57、Leu59和Ser64的局部结构和环境。自旋标记的 KCNE1 半胱氨酸突变体 Phe54、Phe57、Leu59 和 Ser64 被发现位于 POPC/POPG 囊泡内部，而 Ser28 被发现位于膜外部。 Ser64 在囊泡中被证明是不可与水接触的，但在 LMPG 胶束溶液中却是可与水接触的。这些结果表明，KCNE1 胶束衍生结构的关键成分延伸到了脂质双层中该蛋白质的结构，但也证明需要使用源自双层重构蛋白质的数据来完善该结构，以更准确地定义其天然结构。这项工作为未来的研究奠定了基础。
KCNE1 (minK), found in the human heart and cochlea, is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study, a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (POPG) was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (K(V)7.1) voltage-gated potassium channel led to nativelike modulation of the channel. Circular dichroism spectroscopy demonstrated that the percent helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles than for the previously described structure in 1.0% 1-myristoyl-2-hydroxy-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (LMPG) micelles. SDSL electron paramagnetic resonance spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu59, and Ser64 of KCNE1 in both POPC/POPG vesicles and LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water inaccessible in vesicles but found to be water accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers but also demonstrate the need to refine this structure using data derived from the bilayer-reconstituted protein to more accurately define its native structure. This work establishes the basis for such future studies.