Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles
Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles
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DOI：
10.1529/biophysj.104.053413
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发表时间：
2005-02-01
影响因子：
3.4
通讯作者：
Poolman, B
中科院分区：
文献类型：
--
作者：
Doeven, MK;Folgering, JHA;Poolman, B
GUV已被广泛用于研究脂质流动性，膜动力学和脂质结构域（筏）的形成，使用单分子技术，如荧光相关光谱。在这些类型的模型膜的膜蛋白动力学的报告是远远不够先进的，由于蛋白质纳入GUV在功能状态的困难。我们已经使用蔗糖来防止四种不同的膜蛋白（复合物）在GUV形成过程的脱水步骤期间失活。蔗糖的量进行了优化，使蛋白质保留100%的生物活性，并获得了许多蛋白-GUV。虽然GUV可以通过由中性和阴离子脂质组成的脂质混合物的水合作用形成，但中性脂质形成GUV需要交流电电场。通过共聚焦成像、荧光相关光谱、膜片钳测量和生物化学技术，确定了GUV中ATP结合盒转运系统、离子连接转运体和机械敏感通道的分布、横向流动性和功能。此外，我们表明，蔗糖减慢荧光脂质类似物的横向流动性，可能是由于氢键与脂质头基，导致更大的复合物的流动性降低。
GUVs have been widely used for studies on lipid mobility, membrane dynamics and lipid domain ( raft) formation, using single molecule techniques like fluorescence correlation spectroscopy. Reports on membrane protein dynamics in these types of model membranes are by far less advanced due to the difficulty of incorporating proteins into GUVs in a functional state. We have used sucrose to prevent four distinct membrane protein(s) ( complexes) from inactivating during the dehydration step of the GUV-formation process. The amount of sucrose was optimized such that the proteins retained 100% biological activity, and many proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid mixtures composed of neutral and anionic lipids, an alternate current electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility, and function of an ATP-binding cassette transport system, an ion-linked transporter, and a mechanosensitive channel in GUVs were determined by confocal imaging, fluorescence correlation spectroscopy, patch-clamp measurements, and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogs, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility.