bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles.
bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles.
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DOI：
10.1085/jgp.201511448
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发表时间：
2016-01
期刊：
影响因子：
--
通讯作者：
Jiang QX
中科院分区：
文献类型：
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作者：
Zheng H;Lee S;Llaguno MC;Jiang QX
KvAP通过C-末端的His-Tag种子结合到珠子上，形成具有单向通道取向的支撑双层，用于功能研究。已经开发出融合或巨大囊泡、平面脂质双层、液滴膜系统和平面支撑膜来结合膜蛋白，用于对这些蛋白质或双层性质的电学和生物物理分析。然而，将包括离子通道在内的膜蛋白整合到重组膜系统中仍然是困难的，因为重组膜系统可以很容易地控制操作尺寸、膜蛋白的掺入方向和膜的脂组成。在这里，利用一种新开发的化学工程方法，我们报告了一种珠状支撑的单层膜(BSUM)系统，该系统可以很好地控制膜的尺寸、蛋白质取向和脂质组成。我们的新系统使用特定的配体来促进膜蛋白单向结合到脂质双层中。冷冻电子显微镜成像证实了bSUM的单层性质。在不同直径的bSUM中，来自电压门控离子通道的电子记录证明了新系统的多功能性。以KvAP为模型体系，我们发现，与其他体外膜系统相比，bSUM具有以下优点：(A)大部分通道以受控方式定向；(B)通道介导脂质双层的形成；(C)每个微球上只有一个双层膜；(D)脂组成可控，bSUM大小也可在0.2-20微米范围内实验控制；(E)通道活性可通过平面电极膜片钳记录；(F)bSUM在平面电极上的电压钳位速度快(0.2-0.5ms)，适合于研究具有快速门控动力学的离子通道。我们的观察表明，化学工程的bSUM为研究不同脂质组成的膜中的脂-蛋白质相互作用提供了一个新的平台，并可能用于其他应用，如靶向递送和单分子成像。
KvAP conjugated to beads via a C-terminal His-tag seeds formation of a supported bilayer with unidirectional channel orientation for functional studies. Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.