Schleifenbaum Frank, Blum Christian, Elgass Kirstin, Subramaniam Vinod, Meixner Alfred J
Institute of Physical and Theoretical Chemistry, University of Tuebingen, Tuebingen, Germany.
J Phys Chem B. 2008 Jun 26;112(25):7669-74. doi: 10.1021/jp7114753. Epub 2008 Jun 4.
The red fluorescent protein from DsRed from Discosoma reef coral exhibits complex photophysics. One key reason for this is that DsRed forms obligate tetrameric units containing green and red emitting monomers in random composition. Experimental investigations have proven that these different chromophores within one tetramer are coupled by fluorescence resonance energy transfer (FRET) and that the observed strong red emission is due to a nonradiative energy transfer from the green to the red chromophore when the green chromophore is exclusively excited. Ensemble studies can only provide averaged data on statistical mixtures of tetramers with different compositions, since it is impossible to separate the tetramers into functional monomers containing only red or green emitting chromophores. We present here the results of DsRed multiparameter single molecule spectroscopy. By combination of spectral and time domain spectroscopy, we were able to isolate single tetramers containing only green chromophores and thus record the fluorescence lifetime of the green emitting species without interference from FRET to the red chromophore for the first time. The fluorescence lifetime for the green chromophore of DsRed is remarkably longer than for the green fluorescent protein, which is a chemical analogue to the green chromophore in DsRed. On the basis of our single protein experiments, we can derive a complete set of spectroscopic parameters to describe Forster energy transfer in the DsRed system without any further assumptions. Hence in combination with X-ray studies our data allow for an accurate quantitative description of the radiative and nonradiative relaxation processes in DsRed proteins.
来自盘状珊瑚礁珊瑚的红色荧光蛋白DsRed表现出复杂的光物理性质。造成这种情况的一个关键原因是，DsRed形成了包含随机组成的绿色和红色发射单体的 obligate 四聚体单元。实验研究已经证明，一个四聚体内的这些不同发色团通过荧光共振能量转移（FRET）耦合，并且观察到的强烈红色发射是由于当绿色发色团被单独激发时，从绿色发色团到红色发色团的非辐射能量转移。整体研究只能提供关于不同组成的四聚体统计混合物的平均数据，因为不可能将四聚体分离成仅包含红色或绿色发射发色团的功能性单体。我们在此展示了DsRed多参数单分子光谱学的结果。通过光谱和时域光谱学的结合，我们首次能够分离出仅包含绿色发色团的单个四聚体，从而记录绿色发射物种的荧光寿命，而不受从绿色发色团到红色发色团的FRET干扰。DsRed绿色发色团的荧光寿命明显长于绿色荧光蛋白，绿色荧光蛋白是DsRed中绿色发色团的化学类似物。基于我们的单蛋白实验，我们可以推导出一套完整的光谱参数，无需任何进一步假设即可描述DsRed系统中的福斯特能量转移。因此，结合X射线研究，我们的数据能够对DsRed蛋白中的辐射和非辐射弛豫过程进行准确的定量描述。
