Photophysics of DsRed, a red fluorescent protein, from the ensemble to the single-molecule level
Photophysics of DsRed, a red fluorescent protein, from the ensemble to the single-molecule level
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DOI：
10.1021/jp010116x
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发表时间：
2001-05-31
影响因子：
3.3
通讯作者：
Moerner, WE
中科院分区：
文献类型：
--
作者：
Lounis, B;Deich, J;Moerner, WE
DsRed是从珊瑚的Discosoma属中克隆的四聚体荧光蛋白，已经显示出作为用于体内蛋白标记的绿色荧光蛋白（GFP）突变体的更长波长的替代物的前景。重组蛋白的散装和单分子研究表明，DsRed发色团显示出高稳定性，对光漂白相比，GFP突变体。斯塔克调制光谱证实，电子结构的DsRed发色团是类似的GFP。然而，DsRed的四聚体性质导致亚基间能量转移，如固定化时分子的异常低的荧光各向异性（0.23 +/-0.02）所证明的。该值与基于初步晶体学信息的能量转移速率估计值大致一致。荧光发射漂白率线性的应用激发强度，这意味着在532 nm的泵浦过程中发射的停止是光驱动的，并与四聚体结构一致，几个光漂白“步骤”观察到个别复合物。由于在漂白之前会发射更多的光子，因此这项研究表明，只要四聚体在生理学研究中不是一个问题，DsRed可能比一些基于GFP的标记技术更上级。
DsRed, a tetrameric fluorescent protein cloned from the Discosoma genus of coral, has shown promise as a longer-wavelength substitute for green fluorescent protein (GFP) mutants for in vivo protein labeling. Bulk and single-molecule studies of the recombinant protein revealed that the DsRed chromophore shows high stability against photobleaching as compared to GFP mutants. Stark modulation spectra confirm that the electronic structure of the DsRed chromophore is similar to that of GFP. However, the tetrameric nature of DsRed leads to intersubunit energy transfer, as evidenced by the molecule's unusually low fluorescence anisotropy when immobilized (0.23 +/- 0.02). This value is approximately consistent with an estimate of the energy transfer rates based on preliminary crystallographic information. The fluorescence emission bleaches at a rate linear in the applied excitation intensity, implying that the cessation of emission during pumping at 532 nm is light-driven and, consistent with the tetrameric structure, several photobleaching "steps" were observed for individual complexes. Because more photons are emitted before bleaching, this study suggests that DsRed may be superior to some GFP-based labeling technologies as long as tetramerization is not an issue in physiological studies.