Deactivation Mechanism of the Green Fluorescent Chromophore

Speaker: Junfeng LiAdvisor : 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, 13617

Temperature dependence of the fluorescence spectrum of p-HBDI

An important question

How does the protein convert such a poor fluorophore t

o 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

FluorescenceInternal

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 et

hanol (1.200 mPa.s) is 0.6ps while in ethylene glycol (2

5.66 mPa.s) it is 1.3ps; a twenty-fold increase in viscosi

ty causes only a two-fold increase in excited state lifeti

me.

Suggestion

This result suggests that the coordinate promoting IC

in HBDI is not very sensitive to solvent friction, so is u

nlikely to involve a large scale structural change disp

lacing significant volumes of solvent (volume conser

ving), such as a complete rotation about the exocycli

c double bond

Temperatures dependence

In isoviscosity analysis, the temperatures dependence

observed reflects the activation energy.

For HBDI this procedure leads to the conclusion that t

he 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

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, 4089-4096

Compare quantum efficiency

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

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!