A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy
A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy
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DOI：
10.1038/nbt1207
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发表时间：
2006-05-01
影响因子：
46.9
通讯作者：
Miyawaki, A
中科院分区：
文献类型：
--
作者：
Kogure, T;Karasawa, S;Miyawaki, A
双色荧光互相关光谱（FCCS）是一种很有前途的定量蛋白质-蛋白质相互作用的技术（1-5）。在该技术中，两种不同的荧光标记物在共同的测量体积内被同时激发和检测。然而，对准两个激光线和两个荧光团之间的发射交叉的困难使得该技术复杂。为了克服这些限制，我们开发了一种具有大斯托克斯位移的荧光蛋白。这种名为Keima的蛋白质分别在440 nm和620 nm处吸收和发射最大光。将单体形式的Keima与青色荧光蛋白相结合，允许使用单个458 nm激光线的双色FCCS和荧光蛋白发射的完全分离。这种FCCS方法能够灵敏地检测caspase-3的蛋白水解以及钙调素与钙调素依赖性酶的结合。此外，Keima和最大发射波长为570 nm的光谱变体可能有助于单波长激发的同时成像。
Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions(1-5). In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.