De Angelis D A, Miesenböck G, Zemelman B V, Rothman J E
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 251, New York, NY 10021, USA.
Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12312-6. doi: 10.1073/pnas.95.21.12312.
We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 vs. 475 nm result. These spectral changes provide a sensitive ratiometric index of the extent of self-association that can be exploited to quantitatively image homo-oligomerization or clustering processes of GFP-tagged proteins in vivo. The method, which we term proximity imaging (PRIM), complements fluorescence resonance energy transfer between a blue fluorescent protein donor and a GFP acceptor, a powerful method for imaging proximity relationships between different proteins. However, unlike fluorescence resonance energy transfer (which is a spectral interaction), PRIM depends on direct contact between two GFP modules, which can lead to structural perturbations and concomitant spectral changes within a module. Moreover, the precise spatial arrangement of the GFP molecules within a given dimer determines the magnitude and direction of the spectral change. We have used PRIM to detect FK1012-induced dimerization of GFP fused to FK506-binding protein and clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces.
我们报告了一项意外发现，该发现扩展了由绿色荧光蛋白（GFP）或其衍生物所执行的令人印象深刻的报告功能目录。当两个GFP分子靠近时，在395nm与475nm激发下发射的绿色荧光的相对强度会发生变化。这些光谱变化提供了一个敏感的比例指数，可用于衡量自缔合程度，从而能够在体内对GFP标记蛋白的同源寡聚化或聚集过程进行定量成像。我们将该方法称为邻近成像（PRIM），它补充了蓝色荧光蛋白供体与GFP受体之间的荧光共振能量转移，后者是一种用于成像不同蛋白之间邻近关系的强大方法。然而，与荧光共振能量转移（这是一种光谱相互作用）不同，PRIM依赖于两个GFP模块之间的直接接触，这可能会导致模块内的结构扰动和伴随的光谱变化。此外，给定二聚体内GFP分子的精确空间排列决定了光谱变化的幅度和方向。我们已使用PRIM检测FK1012诱导的与FK506结合蛋白融合的GFP的二聚化以及糖基磷脂酰肌醇锚定的GFP在细胞表面的聚集。
