Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions
Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions
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DOI：
10.3791/54640
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发表时间：
2016-10-01
影响因子：
1.2
通讯作者：
Sanchez-Puig, Nuria
中科院分区：
文献类型：
--
作者：
Gijsbers, Abril;Nishigaki, Takuya;Sanchez-Puig, Nuria
蛋白质-蛋白质相互作用在生物体的功能中起着至关重要的作用。一旦确定并验证了相互作用，就有必要在结构和机制层面上对其进行表征。为此目的，存在几种生物化学和生物物理方法。其中，荧光各向异性是当荧光团标记的蛋白质的荧光强度在蛋白质-蛋白质相互作用时保持恒定时特别使用的强大技术。在该技术中，荧光团标记的蛋白质用适当波长的垂直偏振光激发，该垂直偏振光根据荧光团与入射光束的相对取向选择性地激发荧光团的子集。所得发射还具有方向性，其在垂直和水平平面中的关系如下定义各向异性（r）：r =（I-VV-I-VH）/（I-VV +2I（VH）），其中I-VV和I-VH分别是垂直和水平分量的荧光强度。荧光各向异性对荧光团的旋转扩散敏感，即附着于蛋白质的荧光团的表观分子大小，其在蛋白质-蛋白质相互作用时改变。在本文中，使用荧光各向异性作为研究蛋白质-蛋白质相互作用的工具，以解决在Shwachman-Diamond综合征（SBDS）中突变的蛋白质与延伸因子样-1 GTf3（EFL 1）之间的结合为例。通常，用荧光团标记蛋白质是在蛋白质的巯基（半胱氨酸）或氨基（N-末端胺或赖氨酸）上进行的。然而，SBDS具有几个半胱氨酸和赖氨酸，不允许定点标记它。作为替代技术，染料4 '，5'-双（1，3，2-二硫阿索兰-2-基）荧光素用于特异性标记四半胱氨酸基序，Cys-Cys-Pro-Gly-Cys-Cys，基因工程改造的重组SBDS蛋白的C-末端。拟合的实验数据提供了定量和机制的信息，这些蛋白质之间的结合模式。
Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(I-VV-I-VH)/(I-VV+ 2I(VH)), where I-VV and I-VH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4', 5'-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.