Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein
Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein
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DOI：
10.1074/jbc.m001348200
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发表时间：
2000-06-09
影响因子：
4.8
通讯作者：
Visser, AJWG
中科院分区：
文献类型：
--
作者：
Hink, MA;Griep, RA;Visser, AJWG
由于水母的绿色荧光蛋白(GFP)在细胞生物学和生物化学中的应用日益广泛，因此需要了解水母绿色荧光蛋白(GFP)与内源蛋白在细胞内融合的结构信息。我们研究了GFP单独的动态性质，并将其与抗革兰氏阴性菌外细胞壁脂多糖的单链抗体(简称scFv-GFP)融合，结合实验证明scFv部分具有功能，包括使用荧光相关光谱观察scFv-GFP与革兰氏阴性细菌的结合以及含有吸附的脂多糖抗原的表面血浆共振细胞。用时间分辨荧光各向异性研究了ScFv-GFP的旋转运动。然而，ScFv-GFP的旋转相关时间太短，不能解释整个蛋白质的球状旋转。这一结果只能通过假设两个融合蛋白之间的快速铰链运动来解释。ScFv-GFP的模型化结构支持这一观察结果。
Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry, We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of Gram-negative bacteria (abbreviated as scFv-GFP), The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to Gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation.