Ultrafast Studies of the     Photophysics of Cis and Trans    States of the Green Fluorescent                Protein Chrom...
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Ultrafast Studies of the Photophysics of Cis and Trans States of the Green Fluorescent Protein Chromophore

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  1. 1. Ultrafast Studies of the Photophysics of Cis and Trans States of the Green Fluorescent Protein ChromophoreKiri Addison,† Jamie Conyard,† Tara Dixon,† PhilipC. 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 StatesJ. Phys. Chem. Lett. 2012, 3, 2298 -2302 1
  2. 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 American Chemical Society
  3. 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. 3American Chemical Society Kao, Y., et al., PNAS, 2012. 109 3220-25.
  4. 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. 4American Chemical Society Voliani, et al. J. Phys. Chem. B. 2008, 112, 10714-22.
  5. 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 5American Chemical Society Joo et al. Optics Exp. 2008,16, 20742
  6. 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 80Normalised 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 American Chemical Society
  7. 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) Proprietary and Confidential 7 American Chemical Society
  8. 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 American Chemical Society Kao. PNAS. 2012, 109, 3220-25.
  9. 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.Proprietary and Confidential 9American Chemical Society

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