New insights into the photophysics of DsRed by multiparameter spectroscopy on single proteins.
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.
Institute of Physical and Theoretical Chemistry, University of Tuebingen, Tuebingen, Germany.
Frank
F
Schleifenbaum
Christian
C
Blum
Kirstin
K
Elgass
Vinod
V
Subramaniam
Alfred J
AJ
Meixner
D031541:Fluorescence Resonance Energy Transfer; D008164:Luminescent Proteins / Q000737:chemistry*; D010777:Photochemistry; D055585:Physical Phenomena; D010825:Physics; D011485:Protein Binding
D008164:Luminescent Proteins; C413663:fluorescent protein 583