Nanoscience Center, Department of Biological and Environmental Science, University of Jyväskylä , P.O. Box 35, 40014 Jyväskylä, Finland.
J Phys Chem B. 2013 Sep 26;117(38):11049-57. doi: 10.1021/jp312061b. Epub 2013 Apr 3.
Fluorescent proteins are versatile tools for molecular imaging. In this study, we report a detailed analysis of the absorption and fluorescence properties of the chromophore-binding domain from Deinococcus radiodurans and its D207H mutant. Using single photon counting and transient absorption techniques, the average excited state lifetime of both studied systems was about 370 ps. The D207H mutation slightly changed the excited state decay profile but did not have a considerable effect on the average decay time of the system or the shape of the absorption and emission spectra of the biliverdin chromophore. We confirmed that the fluorescence properties of both samples are very similar in vivo and in vitro. However, we found that the paraformaldehyde fixation of the Escherichia coli cells containing the recombinant phytochrome protein significantly changed the fluorescence properties of the chromophore-binding domain. The biliverdin fluorescence was diminished almost completely, and the fluorescence originated only from the protoporphyrin molecules. Our results emphasize that the effect of protoporphyrin IXa should not be ignored in the fluorescence experiments with phytochrome systems while designing better red fluorescence markers for cellular imaging.
荧光蛋白是分子成像的多功能工具。在这项研究中，我们报告了对来自耐辐射球菌的生色团结合域及其 D207H 突变体的吸收和荧光性质的详细分析。使用单光子计数和瞬态吸收技术，研究的两种系统的平均激发态寿命约为 370 ps。D207H 突变略微改变了激发态衰减曲线，但对系统的平均衰减时间或胆红素生色团的吸收和发射光谱的形状没有显著影响。我们证实，两种样品的荧光性质在体内和体外非常相似。然而，我们发现含有重组光敏色素蛋白的大肠杆菌细胞的多聚甲醛固定显著改变了生色团结合域的荧光性质。胆红素荧光几乎完全消失，荧光仅源自原卟啉分子。我们的结果强调，在设计用于细胞成像的更好的红色荧光标记物时，在光敏色素系统的荧光实验中不应忽视原卟啉 IXa 的影响。
