Characterization of the photoconversion on reaction of the fluorescent protein kaede on the single-molecule level
Characterization of the photoconversion on reaction of the fluorescent protein kaede on the single-molecule level
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DOI：
10.1529/biophysj.105.061713
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发表时间：
2005-11-01
影响因子：
3.4
通讯作者：
Schwille, P
中科院分区：
文献类型：
--
作者：
Dittrich, PS;Schäfer, SP;Schwille, P
荧光蛋白现在被广泛用于荧光显微镜，作为任何感兴趣的蛋白质的遗传标签。最近，一种新的荧光蛋白Kaeed被引入，它在波长为350-410 nm的光激活后表现出从绿色到红色的不可逆色移，从而允许在光照前后对蛋白质进行特异性的细胞跟踪。在这项工作中，我们用基于单分子光谱的荧光技术研究了凯德的光转化反应动力学。通过荧光相关光谱，揭示了生色团的快速闪烁动力学。尽管这些动力学在亚毫秒的时间尺度上被发现依赖于绿色荧光凯德生色团的pH，但光转化的红色生色团的闪烁时间在很大的pH范围内是恒定的，但随着488 nm激发光的强度而变化。这些发现表明，光转化反应导致发色团及其封闭环境的全面重组。为了更详细地研究光转化，我们引入了一种新的实验装置，在微流控通道中进行单分子尺度的连续流动实验。在这里，我们可以看到。样品由半导体激光器(波长405 nm)聚焦而成。在波长为488 nm的激发下，将原始的和光转化的Kaeed蛋白区分开来。通过改变起始激光的流量和强度，我们得到了完全光转化的反应速率为38.6·S(-1)，这比生色团的内部动力学慢得多。没有荧光中间态被揭示出来。
Fluorescent proteins are now widely used in fluorescence microscopy as genetic tags to any protein of interest. Recently, a new fluorescent protein, Kaede, was introduced, which exhibits an irreversible color shift from green to red fluorescence after photoactivation with lambda = 350-410 nm and, thus, allows for specific cellular tracking of proteins before and after exposure to the illumination light. In this work, the dynamics of this photoconversion reaction of Kaede are studied by fluorescence techniques based on single-molecule spectroscopy. By fluorescence correlation spectroscopy, fast flickering dynamics of the chromophore group were revealed. Although these dynamics on a submillisecond timescale were found to be dependent on pH for the green fluorescent Kaede chromophore, the flickering timescale of the photoconverted red chromophore was constant over a large pH range but varied with intensity of the 488-nm excitation light. These findings suggest a comprehensive reorganization of the chromophore and its close environment caused by the photoconversion reaction. To study the photoconversion in more detail, we introduced a novel experimental arrangement to perform continuous flow experiments on a single-molecule scale in a microfluidic channel. Here, the reaction in the. owing sample was induced by the focused light of a diode laser (lambda = 405 nm). Original and photoconverted Kaede protein were differentiated by subsequent excitation at lambda = 488 nm. By variation of flow rate and intensity of the initiating laser we found a reaction rate of 38.6 s(-1) for the complete photoconversion, which is much slower than the internal dynamics of the chromophores. No fluorescent intermediate states could be revealed.