1. Deactivation Mechanism of the Green Fluorescent Chromophore Speaker: Junfeng Li Advisor : Zexing Cao 2010/4/2

2. Bioluminescence

3. Osamu Shimomura

4. Prasher’s two good idea

5. Expression of the fluorescent GFP

6. FP Family

7. The palette of mutated FPs

8. The Nobel Prize

9. Morise, H.; Shimomura, O.; et.al Biochemistry 1974, 13, 2656 Absorption and fluorescence spectrum of GFP

10. Fan Yang, Larry G. Moss et.al Nature biotechnology 1996, 14, 1246 Crystal structure of GFP

11. K.Brejc et.al PNAS USA 1997, 94, 2306

12. Satoshi Kojima, et.al. Tetrahedron Letters 1998, 39, 5239 p-HBDI showed weak fluorescence

13. Haruki niwa et,al Proc. Natl. Acad. Sci. USA 1996,93, 13617 Temperature dependence of the fluorescence spectrum of p-HBDI

14. An important question How does the protein convert such a poor fluorophore to the brightly fluorescent GFP? A clear answer to this question could assist in designing brighter GFP variants

15. 2, Experiment research

16. First excited states Ground states Fluorescence Internal Conversion × Guess

17. The dominant relaxation channel is internal conversion (IC) Stephen R. Meech et.al Chemical Physics Letters 2001, 346, 47 Ultrafast polarisation spectroscopy

18. Internal Conversion Conical intersection Coordinates ?

19. Exact information By making measurements of the excited state lifetime as a function of solvent viscosity it is possible to exact information about the nature of the coordinate promoting IC.

20. Viscosity experiment Andreas D. Kummer et.al J. Phys. Chem. B 2002, 106, 7554

21. Result The mean excited state lifetime for anionic HBDI in ethanol (1.200 mPa.s) is 0.6ps while in ethylene glycol (25.66 mPa.s) it is 1.3ps; a twenty-fold increase in viscosity causes only a two-fold increase in excited state lifetime.

22. Suggestion This result suggests that the coordinate promoting IC in HBDI is not very sensitive to solvent friction, so is unlikely to involve a large scale structural change displacing significant volumes of solvent (volume conserving), such as a complete rotation about the exocyclic double bond

23. Temperatures dependence In isoviscosity analysis, the temperatures dependence observed reflects the activation energy. For HBDI this procedure leads to the conclusion that the reaction is effectively barrierless SR Meech et. al J. Phys. Chem. A 2003, 107, 2616

24. Charge dependence The same rapid internal conversion was seen for neutral, cationic and anionic forms of HBDI. SR Meech et. al J. Phys. Chem. A 2003, 107, 2616

25. Distribution of conformers The decay is non-single exponential They proposed a distribution of conformers about the phenyl single bond, and were able to model the date suggesting that this mode specifically is involved in promoting radiationless. Dan Huppert et. al J. Phys. Chem. B 2006, 110, 4434

26. Summary Experiment A more detailed insight The volume of rotation The barrier height The mechanism of radiationless decay The formation of ground state isomers Theoretical calculations

27. 3,Theoretical calculation

28. Twisting about the imidazolinone double bond barrierless It did not lead to a crossing of ground and excited states for the anion IC is not charge dependent ≠ W.Weber et. al PNAS USA 1999, 96, 6177 Imidazolinone double bond

29. Single bond twist Crossing only occurred in the cation A fast internal conversion independent of charge state ≠ Single bond twist A.A. Voityuk et. al Chemical Physics Letters, 1998, 296, 6177

30. Hula twist Volume conserving Crossing via a significant energy barrier Barrierless IC mechanism ≠ Hula twist W.Weber et. al PNAS USA 1999, 96, 6177

31. A fast stretching coordinate, corresponding to reduced bond order Rotation about the phenolic single bond Temperature dependent The distribution about the phenyl single bond Massimo Olivucci et. al J. AM. CHEM. SOC. 2004, 126, 5452 Ab initio CASPT2//CASSCF relaxation path computations

32. Fernando Bernardi et. al J. AM. CHEM. SOC. 2005, 127, 3952 Conical intersection dynamics in solution

33. Population on S1 as a function of time T. J. Martinez et. al Faraday Discuss, 2004, 127, 149 Full multiple spawning (FMS) nonadiabatic wavepacket dynamics method CASSCF/CASPT2/AM1 ①

34. 4, Some new events

35. Jye-Shane Yang et. al Chem. Commun., 2008, 1344 The phenyl derivative

36. Jye-Shane Yang et. al Chem. Commun., 2008, 1344

37. ②

38. Liangxing Wu et. al J. AM. CHEM. SOC. 2008, 130, 4089 Lock ?

39. Liangxing Wu et. al J. AM. CHEM. SOC. 2008, 130, 4089-4096 Compare quantum efficiency

40. Liangxing Wu et. al J. AM. CHEM. SOC. 2008, 130, 4089-4096 ③

41. Jian Dong et. al J. AM. CHEM. SOC. 2009, 131, 662 Crystal phase p-HBDI

42. Jian Dong et. al J. AM. CHEM. SOC. 2009, 131, 662 ④

43. Thank you for your attention!