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

Bioluminescence

Osamu Shimomura

Prasher’s two good idea

Expression of the fluorescent GFP

FP Family

The palette of mutated FPs

The Nobel Prize

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

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

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

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

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

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

2, Experiment research

First excited states Ground states Fluorescence Internal Conversion × Guess

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

Internal Conversion Conical intersection Coordinates ?

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.

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

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.

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

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

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

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

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

3,Theoretical calculation

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

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

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

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

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

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

4, Some new events

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

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

②

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

Liangxing Wu et. al J. AM. CHEM. SOC. 2008, 130, Compare quantum efficiency

Liangxing Wu et. al J. AM. CHEM. SOC. 2008, 130, ③

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

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

Thank you for your attention!