The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection
The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection
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DOI：
10.1073/pnas.251525598
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发表时间：
2001-12-04
影响因子：
11.1
通讯作者：
van Hulst, NF
中科院分区：
文献类型：
--
作者：
Garcia-Parajo, MF;Koopman, M;van Hulst, NF
最近的研究表明，新克隆的红色荧光蛋白DsRed从Discosoma属的巨大优势：明亮的红色荧光和高抗光漂白。然而，也已经清楚的是，蛋白质形成紧密堆积的四聚体，并且有迹象表明蛋白质不完全成熟，未成熟的绿色种类的比例未知。我们应用单分子方法来阐明DsRed中荧光发射的性质。实时荧光轨迹已获得偏振敏感检测。我们的研究结果表明，相同的单体之间的能量转移有效地发生与红色发光所产生的同样可能从任何发色单元。其中一个发色团的光解离会微弱地猝灭相邻发色团的发射。双色激发（在488和568 nm）单分子显微镜已被执行，以揭示每个四聚体内的红色与绿色物种的数量和分布。我们发现，86%的DsRed包含至少一个绿色物种的红色到绿色的比例为1.2-1.5。基于我们的发现，寡聚体抑制不仅有利于蛋白质融合，而且还将增加单个单体的荧光发射。
Recent studies on the newly cloned red fluorescence protein DsRed from the Discosoma genus have shown its tremendous advantages: bright red fluorescence and high resistance against photobleaching. However, it has also become clear that the protein forms closely packed tetramers, and there is indication for incomplete protein maturation with unknown proportion of immature green species. We have applied single-molecule methodology to elucidate the nature of the fluorescence emission in the DsRed. Real-time fluorescence trajectories have been acquired with polarization sensitive detection. Our results indicate that energy transfer between identical monomers occurs efficiently with red emission arising equally likely from any of the chromophoric units. Photodissociation of one of the chromophores weakly quenches the emission of adjacent ones. Dual color excitation (at 488 and 568 nm) single-molecule microscopy has been performed to reveal the number and distribution of red vs. green species within each tetramer. We find that 86% of the DsRed contain at least one green species with a red-to-green ratio of 1.2-1.5. On the basis of our findings, oligomer suppression would not only be advantageous for protein fusion but will also increase the fluorescence emission of individual monomers.