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Volume-conserving transcis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography

Abstract

Trans-to-cis isomerization, the key reaction in photoactive proteins, usually cannot occur through the standard one-bond-flip mechanism. Owing to spatial constraints imposed by a protein environment, isomerization probably proceeds through a volume-conserving mechanism in which highly choreographed atomic motions are expected, the details of which have not yet been observed directly. Here we employ time-resolved X-ray crystallography to visualize structurally the isomerization of the p-coumaric acid chromophore in photoactive yellow protein with a time resolution of 100 ps and a spatial resolution of 1.6 Å. The structure of the earliest intermediate (IT) resembles a highly strained transition state in which the torsion angle is located halfway between the trans- and cis-isomers. The reaction trajectory of IT bifurcates into two structurally distinct cis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue through E46Q mutation, which switches off the bicycle-pedal pathway.

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Figure 1: Isomerization mechanisms and overview of the PYP.
Figure 2: Time-resolved electron-density maps of pCA in the chromophore binding pocket.
Figure 3: Time-independent intermediates for WT-PYP and E46Q-PYP recovered from the SVD analysis of time-dependent difference electron-density maps.
Figure 4: Structures of pCA intermediates, reaction pathways and kinetics.
Figure 5: HT and BP pathways and a comparison of experimental and theoretical IT structures.

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References

  1. 1

    Liu, R. S. & Asato, A. E. The primary process of vision and the structure of bathorhodopsin: a mechanism for photoisomerization of polyenes. Proc. Natl Acad. Sci. USA 82, 259–263 (1985).

    CAS  Article  Google Scholar 

  2. 2

    Warshel, A. & Barboy, N. Energy storage and reaction pathways in the first step of the vision process. J. Am. Chem. Soc. 104, 1469–1476 (1982).

    CAS  Article  Google Scholar 

  3. 3

    Muller, A. M., Lochbrunner, S., Schmid, W. E. & Fuss, W. Low-temperature photochemistry of previtamin D: a hula-twist isomerization of a triene. Angew. Chem. Int. Ed. 37, 505–507 (1998).

    CAS  Article  Google Scholar 

  4. 4

    Liu, R. S., Yang, L. Y. & Liu, J. Mechanisms of photoisomerization of polyenes in confined media: from organic glasses to protein binding cavities. Photochem. Photobiol. 83, 2–10 (2007).

    CAS  PubMed  Google Scholar 

  5. 5

    Imamoto, Y., Kataoka, M. & Liu, R. S. Mechanistic pathways for the photoisomerization reaction of the anchored, tethered chromophore of the photoactive yellow protein and its mutants. Photochem. Photobiol. 76, 584–589 (2002).

    CAS  Article  Google Scholar 

  6. 6

    Andruniow, T., Ferre, N. & Olivucci, M. Structure, initial excited-state relaxation, and energy storage of rhodopsin resolved at the multiconfigurational perturbation theory level. Proc. Natl Acad. Sci. USA 101, 17908–17913 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Frutos, L. M., Andruniow, T., Santoro, F., Ferre, N. & Olivucci, M. Tracking the excited-state time evolution of the visual pigment with multiconfigurational quantum chemistry. Proc. Natl Acad. Sci. USA 104, 7764–7769 (2007).

    CAS  Article  Google Scholar 

  8. 8

    Schapiro, I., Weingart, O. & Buss, V. Bicycle-pedal isomerization in a rhodopsin chromophore model. J. Am. Chem. Soc. 131, 16–17 (2009).

    CAS  Article  Google Scholar 

  9. 9

    Xie, A., Hoff, W. D., Kroon, A. R. & Hellingwerf, K. J. Glu46 donates a proton to the 4-hydroxycinnamate anion chromophore during the photocycle of photoactive yellow protein. Biochemistry 35, 14671–14678 (1996).

    CAS  Article  Google Scholar 

  10. 10

    van Wilderen, L. J. et al. Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein. Proc. Natl Acad. Sci. USA 103, 15050–15055 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Groot, M. L. et al. Initial steps of signal generation in photoactive yellow protein revealed with femtosecond mid-infrared spectroscopy. Biochemistry 42, 10054–10059 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Heyne, K. et al. Structural evolution of the chromophore in the primary stages of trans/cis isomerization in photoactive yellow protein. J. Am. Chem. Soc. 127, 18100–18106 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Genick, U. K., Soltis, S. M., Kuhn, P., Canestrelli, I. L. & Getzoff, E. D. Structure at 0.85 Å resolution of an early protein photocycle intermediate. Nature 392, 206–209 (1998).

    CAS  Article  Google Scholar 

  14. 14

    Kort, R., Hellingwerf, K. J. & Ravelli, R. B. Initial events in the photocycle of photoactive yellow protein. J. Biol. Chem. 279, 26417–26424 (2004).

    CAS  Article  Google Scholar 

  15. 15

    Anderson, S., Srajer, V. & Moffat, K. Structural heterogeneity of cryotrapped intermediates in the bacterial blue light photoreceptor, photoactive yellow protein. Photochem. Photobiol. 80, 7–14 (2004).

    CAS  Article  Google Scholar 

  16. 16

    Ren, Z. et al. A molecular movie at 1.8 Å resolution displays the photocycle of photoactive yellow protein, a eubacterial blue-light receptor, from nanoseconds to seconds. Biochemistry 40, 13788–13801 (2001).

    CAS  Article  Google Scholar 

  17. 17

    Ihee, H. et al. Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds. Proc. Natl Acad. Sci. USA 102, 7145–7150 (2005).

    CAS  Article  Google Scholar 

  18. 18

    Andresen, M. et al. Structure and mechanism of the reversible photoswitch of a fluorescent protein. Proc. Natl Acad. Sci. USA 102, 13070–13074 (2005).

    CAS  Article  Google Scholar 

  19. 19

    Sprenger, W. W., Hoff, W. D., Armitage, J. P. & Hellingwerf, K. J. The eubacterium Ectothiorhodospira halophila is negatively phototactic, with a wavelength dependence that fits the absorption spectrum of the photoactive yellow protein. J. Bacteriol. 175, 3096–3104 (1993).

    CAS  Article  Google Scholar 

  20. 20

    Borgstahl, G. E., Williams, D. R. & Getzoff, E. D. 1.4 Å structure of photoactive yellow protein, a cytosolic photoreceptor: unusual fold, active site, and chromophore. Biochemistry 34, 6278–6287 (1995).

    CAS  Article  Google Scholar 

  21. 21

    Ujj, L. et al. New photocycle intermediates in the photoactive yellow protein from Ectothiorhodospira halophila: picosecond transient absorption spectroscopy. Biophys. J. 75, 406–412 (1998).

    CAS  Article  Google Scholar 

  22. 22

    Hoff, W. D. et al. Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila. Biophys. J. 67, 1691–1705 (1994).

    CAS  Article  Google Scholar 

  23. 23

    Brudler, R., Rammelsberg, R., Woo, T. T., Getzoff, E. D. & Gerwert, K. Structure of the I1 early intermediate of photoactive yellow protein by FTIR spectroscopy. Nature Struct. Biol. 8, 265–270 (2001).

    CAS  Article  Google Scholar 

  24. 24

    Imamoto, Y. et al. Low-temperature Fourier transform infrared spectroscopy of photoactive yellow protein. Biochemistry 40, 8997–9004 (2001).

    CAS  Article  Google Scholar 

  25. 25

    Devanathan, S. et al. Femtosecond spectroscopic observations of initial intermediates in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila. Biophys. J. 77, 1017–1023 (1999).

    CAS  Article  Google Scholar 

  26. 26

    Unno, M., Kumauchi, M., Hamada, N., Tokunaga, F. & Yamauchi, S. Resonance Raman evidence for two conformations involved in the L intermediate of photoactive yellow protein. J. Biol. Chem. 279, 23855–23858 (2004).

    CAS  Article  Google Scholar 

  27. 27

    Takeshita, K., Imamoto, Y., Kataoka, M., Tokunaga, F. & Terazima, M. Themodynamic and transport properties of intermediate states of the photocyclic reaction of photoactive yellow protein. Biochemistry 41, 3037–3048 (2002).

    CAS  Article  Google Scholar 

  28. 28

    Schmidt, M. et al. Protein kinetics: structures of intermediates and reaction mechanism from time-resolved X-ray data. Proc. Natl Acad. Sci. USA 101, 4799–4804 (2004).

    CAS  Article  Google Scholar 

  29. 29

    Rajagopal, S. et al. A structural pathway for signaling in the E46Q mutant of photoactive yellow protein. Structure 13, 55–63 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Schotte, F. et al. Watching a protein as it functions with 150-ps time-resolved X-ray crystallography. Science 300, 1944–1947 (2003).

    CAS  Article  Google Scholar 

  31. 31

    Schmidt, M., Rajagopal, S., Ren, Z. & Moffat, K. Application of singular value decomposition to the analysis of time-resolved macromolecular X-ray data. Biophys. J. 84, 2112–2129 (2003).

    CAS  Article  Google Scholar 

  32. 32

    Tripathi, S., Srajer, V., Purwar, N., Henning, R. & Schmidt, M. pH dependence of the photoactive yellow protein photocycle investigated by time-resolved crystallography. Biophys. J. 102, 325–332 (2012).

    CAS  Article  Google Scholar 

  33. 33

    Kim, T. W. et al. Protein structural dynamics of photoactive yellow protein in solution revealed by pump-probe X-ray solution scattering. J. Am. Chem. Soc. 134, 3145–3153 (2012).

    CAS  Article  Google Scholar 

  34. 34

    Liu, R. S. & Hammond, G. S. The case of medium-dependent dual mechanisms for photoisomerization: one-bond-flip and hula-twist. Proc. Natl Acad. Sci. USA 97, 11153–11158 (2000).

    CAS  Article  Google Scholar 

  35. 35

    Imamoto, Y., Kataoka, M. & Tokunaga, F. Photoreaction cycle of photoactive yellow protein from Ectothiorhodospira halophila studied by low-temperature spectroscopy. Biochemistry 35, 14047–14053 (1996).

    CAS  Article  Google Scholar 

  36. 36

    Imamoto, Y., Kataoka, M., Tokunaga, F., Asahi, T. & Masuhara, H. Primary photoreaction of photoactive yellow protein studied by subpicosecond–nanosecond spectroscopy. Biochemistry 40, 6047–6052 (2001).

    CAS  Article  Google Scholar 

  37. 37

    Ryan, W. L., Gordon, D. J. & Levy, D. H. Gas-phase photochemistry of the photoactive yellow protein chromophore trans-p-coumaric acid. J. Am. Chem. Soc. 124, 6194–6201 (2002).

    CAS  Article  Google Scholar 

  38. 38

    Espagne, A., Paik, D. H., Changenet-Barret, P., Martin, M. M. & Zewail, A. H. Ultrafast photoisomerization of photoactive yellow protein chromophore analogues in solution: influence of the protonation state. ChemPhysChem 7, 1717–1726 (2006).

    CAS  Article  Google Scholar 

  39. 39

    Sugishima, M. et al. Structure of photoactive yellow protein (PYP) E46Q mutant at 1.2 Å resolution suggests how Glu46 controls the spectroscopic and kinetic characteristics of PYP. Acta Crystallogr. D60, 2305–2309 (2004).

    CAS  Google Scholar 

  40. 40

    Schotte, F. et al. Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography. Proc. Natl Acad. Sci. USA 109, 19256–19261 (2012).

    CAS  Article  Google Scholar 

  41. 41

    Premvardhan, L. L., van der Horst, M. A., Hellingwerf, K. J. & van Grondelle, R. Stark spectroscopy on photoactive yellow protein, E46Q, and a nonisomerizing derivative, probes photo-induced charge motion. Biophys. J. 84, 3226–3239 (2003).

    CAS  Article  Google Scholar 

  42. 42

    Groenhof, G. et al. Photoactivation of the photoactive yellow protein: why photon absorption triggers a trans-to-cis isomerization of the chromophore in the protein. J. Am. Chem. Soc. 126, 4228–4233 (2004).

    CAS  Article  Google Scholar 

  43. 43

    Espagne, A. et al. Ultrafast light-induced response of photoactive yellow protein chromophore analogues. Photochem. Photobiol. Sci. 6, 780–787 (2007).

    CAS  Article  Google Scholar 

  44. 44

    Henry, E. R. The use of matrix methods in the modeling of spectroscopic data sets. Biophys. J. 72, 652–673 (1997).

    CAS  Article  Google Scholar 

  45. 45

    Boggio-Pasqua, M., Robb, M. A. & Groenhof, G. Hydrogen bonding controls excited-state decay of the photoactive yellow protein chromophore. J. Am. Chem. Soc. 131, 13580–13581 (2009).

    CAS  Article  Google Scholar 

  46. 46

    Ren, Z. & Moffat, K. Quantitative analysis of synchrotron Laue diffraction patterns in macromolecular crystallography. J. Appl. Crystallogr. 28, 461–481 (1995).

    CAS  Article  Google Scholar 

  47. 47

    Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).

    CAS  Article  Google Scholar 

  48. 48

    McRee, D. E. XtalView/Xfit – a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999).

    CAS  Article  Google Scholar 

  49. 49

    Devanathan, S., Lin, S., Cusanovich, M. A., Woodbury, N. & Tollin, G. Early intermediates in the photocycle of the Glu46Gln mutant of photoactive yellow protein: femtosecond spectroscopy. Biophys. J. 79, 2132–2137 (2000).

    CAS  Article  Google Scholar 

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Acknowledgements

We acknowledge extensive support from M. Wulff of beamline ID09 at ESRF during data collection there. We thank M. Wulff, P. Anfinrud, F. Schotte and H. S. Cho for their earlier contributions to this research. This work was supported by the Research Center Program (CA1201) of Institute for Basic Science (IBS) in Korea and by Creative Research Initiatives (Center for Time-Resolved Diffraction) of MEST/NRF of Korea. M.S. is supported by National Science Foundation grants 0952643 (Career) and 0843459. K.M. is supported by National Institutes of Health (NIH) grant GM036452. Use of the BioCARS Sector 14 at the APS was supported by NIH National Institute of General Medical Sciences grant P41GM103543. The time-resolved set up at Sector 14 was funded in part through collaboration with P. Anfinrud (NIH/NIDDK) through the Intramural Research Program of the NIDDK. Use of the APS was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under Contract No. DE-AC02-06CH11357.

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H.I. designed the study, and Y.O.J. and H.I. purified, crystallized, collected and analysed the X-ray data. V.S. helped with the analysis of the X-ray data. Y.O.J., J.H.L. and M.S. performed the kinetic analysis of time-dependent data. J.K. performed the DFT calculations. Y.O.J., K.M. and H.I. co-wrote the paper. All the authors discussed the results and commented on the manuscript.

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Correspondence to Hyotcherl Ihee.

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Jung, Y., Lee, J., Kim, J. et al. Volume-conserving transcis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography. Nature Chem 5, 212–220 (2013). https://doi.org/10.1038/nchem.1565

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