A giant liposome for single-molecule observation of conformational changes in membrane proteins
A giant liposome for single-molecule observation of conformational changes in membrane proteins
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DOI：
10.1016/j.bbamem.2009.01.015
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发表时间：
2009-06-01
影响因子：
3.4
通讯作者：
Kinosita, Kazuhiko, Jr.
中科院分区：
文献类型：
--
作者：
Onoue, Yasuhiro;Suzuki, Toshiharu;Kinosita, Kazuhiko, Jr.
我们提出了一个实验系统，允许在单分子水平上的膜蛋白的构象变化的可视化。靶膜蛋白在巨大脂质体中重构，用于独立控制膜两侧的水性环境。为了直接观察构象变化，将靶蛋白的脂质体外位点结合到玻璃表面，并且将在显微镜下容易可见的探针（例如微米尺寸的塑料珠）连接到脂质体内侧的另一个位点。微小蛋白质分子的构象变化或角运动将表现为探针的可见运动。蛋白质在玻璃表面上的附着也固定了脂质体，大大方便了其操作，如探针注射。作为一个模型系统，我们重建ATP合酶（FOF 1）在脂质体几十μ m的大小，附着的蛋白质特异性的玻璃表面，并证明其ATP驱动的旋转膜通过运动的亚微米珠。(c)2009 Elsevier B.V.保留所有权利。
We present an experimental system that allows visualization of conformational changes in membrane proteins at the single-molecule level. The target membrane protein is reconstituted in a giant liposome for independent control of the aqueous environments on the two sides of the membrane. For direct observation of conformational changes, an extra-liposomal site(s) of the target protein is bound to a glass surface, and a probe that is easily visible under a microscope, such as a micron-sized plastic bead, is attached to another site on the intra-liposomal side. A conformational change, or an angular motion in the tiny protein molecule, would manifest as a visible motion of the probe. The attachment of the protein on the glass surface also immobilizes the liposome, greatly facilitating its manipulation such as the probe injection. As a model system, we reconstituted ATP synthase (FOF1) in liposomes tens of mu m in size, attached the protein specifically to a glass surface, and demonstrated its ATP-driven rotation in the membrane through the motion of a submicron bead. (c) 2009 Elsevier B.V. All rights reserved.