This presentation has been moved. To view this presentation, please visit http://pubs.acs.org/iapps/liveslides/pages/index.htm?mscNo=jz3008408 Ultrafast Studies of the Photophysics of Cis and Trans States of the Green Fluorescent Protein Chromophore

1. Ultrafast Studies of the
Photophysics of Cis and Trans
States of the Green Fluorescent
Protein Chromophore
Kiri Addison,† Jamie Conyard,† Tara Dixon,† Philip
C. Bulman Page,† Kyril M. Solntsev,‡ and Stephen
R. Meech*,†
† School of Chemistry, University of East Anglia, Norwich Research
Park, Norwich, NR4 7TJ, United Kingdom.
‡ School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic
Drive, Atlanta, Georgia 30332-0400, United States
J. Phys. Chem. Lett. 2012, 3, 2298 -2302 1

2. Fluorescent Proteins
 Fluorescent proteins are used in a wide range of applications including ultra
resolution bio imaging, cell labelling and studying protein interactions.
Image from gfp.conncoll.edu
 Most FPs contain a chromophore with a common core structure (HBDI).
 Minor modifications to the chromophore structure or to its surroundings can
cause dramatic changes in optical behaviour.
 To aid the design of new FPs, a better understanding of the photophysics
of the chromophore is necessary.
Proprietary and Confidential Tsien, R., Nobel Lecture, 2009. 48 5612-26. 2
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3. Dronpa
 Dronpa is a photoactivatable fluorescent protein.
 It can be switched between bright and dark states by irradiation with 405 nm
and 488 nm light.
Image from http://www.olympusfluoview.com/applications/opticalhighlighters.html
 In the bright state the chromophore adopts the cis conformation.
 In the dark state the chromophore adopts the trans conformation.
How important is isomerisation?
Proprietary and Confidential Habuchi, S., et al., PNAS, 2005. 102 9511-6. 3
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Kao, Y., et al., PNAS, 2012. 109 3220-25.

4. Cis – Trans HBDI
 Irradiation with UV light creates a metastable trans population.
 Up to 40% of the initial cis population can be converted.
 Isomerisation causes small changes in the steady state absorption
and emission spectra. 0.3
dark
5 minutes irradiation
5 hours irradiation
0.2
Absorption
Dark
0.1
0.0
300 350 400 450 500
Wavelength (nm)
Irradiated 700000
cis
cis + trans
600000
500000
Intensity (cps)
400000
300000
200000
100000
0
400 450 500 550 600 650
Wavelength (nm)
Proprietary and Confidential Dong et al. J. Am. Chem. Soc. 2007, 129, 10084-85. 4
American Chemical Society
Voliani, et al. J. Phys. Chem. B. 2008, 112, 10714-22.

5. Ultrafast Fluorescence Upconversion
1.0 HBDI in methanol
Raman scattering
from heptane at 475nm
Normlaised Intensity
 Probes the excited state dynamics. 0.5
47 fs
 Time resolution of better than 50 fs.
0.0
-0.1 0.0 0.1 0.2 0.3 0.4
Time [ps]
Proprietary and Confidential Heisler et al. J. Phys. Chem. B 2009, 113, 1623 5
American Chemical Society
Joo et al. Optics Exp. 2008,16, 20742

6. Time Resolved Fluorescence of Cis – Trans
HBDI
 The cis and cis + trans decays are experimentally indistinguishable.
 Must have similar excited state potential energy surfaces.
 Supported by calculations.
1
Neutral cis 100
Neutral cis + trans
90
Anionic cis
Anionic cis + trans 80
Normalised Emission
0.1 70
Energy [kcal/mol]
60
50
40
0.01 30
Cis S0
20 Cis S1
Trans S0
10
Trans S1
0
-90 -75 -60 -45 -30 -15 0 15 30 45 60 75 90
Torsional Angle [deg]
0 1 2 3 4 5
Dealy time (ps)
Proprietary and Confidential Olsen & smith. J. Am. Chem. Soc. 2008, 130, 8677-89. 6
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7. Simulated Decays
 The cis decay was fitted with a bi-exponential function.
 The time constants were varied to simulate a trans population with a
different lifetime.
 If the cis and trans lifetimes were to differ by more than 20%, this
should be observable in these data.
1
cis measured
cis + trans measured
cis fit
c
 20%
Normalised emission
0.1
0.01
1E-3
0 1 2 3 4
Delay time (ps)
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8. Significance
 The trans state cannot be a dark state because there is not a significant
decrease in the steady state fluorescence.
 Cis – trans isomerisation cannot be solely responsible for the dramatic
photoswitching behaviour of Dronpa.
 Photoswitching also involves the movement of residues surrounding the
chromophore, changes in the hydrogen bonding network and flexibility of
the chromophore.
Dronpa on (green) and off (blue) structures from Andresen. PNAS. 2007, 104, 13005 -09.
 Differential protein-chromophore interactions could dramatically alter the
photophysics.
Proprietary and Confidential 8
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Kao. PNAS. 2012, 109, 3220-25.

9. Conclusions
 The fluorescence decays of cis HBDI and
of a mixture of cis and trans HBDI are
experimentally indistinguishable.
 Photoisomerization of the chromophore
cannot be solely responsible for the
photoswitching of GFP like PAFPs.
 This study highlights the importance of the
protein environment in modulating the
photophysics of fluorescent proteins.
Acknowledgments
K.A and J.C thanks UEA for the award of
studentships. This work was supported by EPSRC
(EP/H02715) for S.RM. and by NSF (CHE-1213047)
to K.M.S. The authors thank Anthony Baldridge for
the HBDI synthesis.
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