Hink M A, Griep R A, Borst J W, van Hoek A, Eppink M H, Schots A, Visser A J
MicroSpectroscopy Centre, Department of Biomolecular Sciences and the Laboratory for Monoclonal Antibodies, Wageningen University, 6703 HA Wageningen, The Netherlands.
J Biol Chem. 2000 Jun 9;275(23):17556-60. doi: 10.1074/jbc.M001348200.
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.
随着水母维多利亚多管发光水母绿色荧光蛋白（GFP）与内源性蛋白质的细胞内融合物在细胞生物学和生物化学中的应用越来越广泛，对其结构信息的需求也日益增加。我们研究了单独的GFP以及与针对革兰氏阴性菌外细胞壁脂多糖产生的单链抗体融合的GFP（简称为scFv-GFP）的动态特性。scFv部分具有功能，这在结合试验中得到了证实，该试验包括使用荧光相关光谱观察scFv-GFP与革兰氏阴性菌的结合以及一个含有吸附脂多糖抗原的表面等离子体共振细胞。我们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，无法解释整个蛋白质的球状旋转。只有假设两种融合蛋白之间存在快速铰链运动，才能解释这一结果。scFv-GFP的模拟结构支持了这一观察结果。
