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Tryptophan fluorescence as a reporter for structural changes in photoactive yellow protein elicited by photo-activation

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Abstract

Light-activation of photoactive yellow protein (PYP) is followed by a series of dynamical transitions in the structure of the protein. Tryptophan fluorescence is well-suited as a tool to study selected aspects of these. Using site-directed mutagenesis eight ‘single-tryptophan’ mutants of PYP were made by replacement of either a tyrosine, phenylalanine or histidine residue by tryptophan, while simultaneously eliminating the endogenous W119. Surprisingly, only three of these eight mutants turn out to emit measurable tryptophan fluorescence: F6W/W119F, F96W/W119F and H108W/W119F. Significantly, all three show altered tryptophan fluorescence upon formation of the pB state. As F96 is located very close to the chromophore, the F96W/W119F mutant protein is particularly suitable for further studies on the dynamical changes of the polarity in the chromophore-binding pocket of PYP. Furthermore, WT PYP can be photo-activated by a UV photon via the highly conserved W119 and subsequent Förster resonance energy transfer. Placing a unique tryptophan residue elsewhere in the protein shows that position 119 is favoured for UV-activation of PYP.

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References

  1. B. L. Taylor, I. B. Zhulin, Microbiol. Mol. Biol. Rev., 1999, 63, 479–506.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. J. L. Pellequer, K. A. Wager-Smith, S. A. Kay, E. D. Getzoff, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 5884–5890.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. A. S. Halavaty, K. Moffat, Biochemistry, 2007, 46, 14001–14009.

    Article  CAS  PubMed  Google Scholar 

  4. A. Pandini, L. Bonati, Protein Eng., Des. Sel., 2005, 18, 127–137.

    Article  CAS  Google Scholar 

  5. J. Vreede, M. A. van der Horst, K. J. Hellingwerf, W. Crielaard, D. M. F. van Aalten, J. Biol. Chem., 2003, 278, 18434–18439.

    Article  CAS  PubMed  Google Scholar 

  6. T. E. Meyer, Biochim. Biophys. Acta, 1985, 806, 175–183.

    Article  CAS  PubMed  Google Scholar 

  7. W. W. Sprenger, W. D. Hoff, J. P. Armitage, K. J. Hellingwerf, J. Bacteriol., 1993, 175, 3096–3104.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. J. S. Khan, Y. Imamoto, Y. Yamazaki, M. Kataoka, F. Tokunaga, M. Terazima, Anal. Chem., 2005, 77, 6625–6629.

    Article  CAS  PubMed  Google Scholar 

  9. M. Baca, G. E. O. Borgstahl, M. Boissinot, P. M. Burke, D. R. Williams, K. A. Slater, E. D. Getzoff, Biochemistry, 1994, 33, 14369–14377.

    Article  CAS  PubMed  Google Scholar 

  10. W. D. Hoff, P. Dux, K. Hard, B. Devreese, I. M. Nugterenroodzant, W. Crielaard, R. Boelens, R. Kaptein, J. Vanbeeumen, K. J. Hellingwerf, Biochemistry, 1994, 33, 13959–13962.

    Article  CAS  PubMed  Google Scholar 

  11. J. J. Vanbeeumen, B. V. Devreese, S. M. Vanbun, W. D. Hoff, K. J. Hellingwerf, T. E. Meyer, D. E. McRee, M. A. Cusanovich, Protein Sci., 1993, 2, 1114–1125.

    Article  CAS  Google Scholar 

  12. M. Kim, R. A. Mathies, W. D. Hoff, K. J. Hellingwerf, Biochemistry, 1995, 34, 12669–12672.

    Article  CAS  PubMed  Google Scholar 

  13. G. E. O. Borgstahl, D. R. Williams, E. D. Getzoff, Biochemistry, 1995, 34, 6278–6287.

    Article  CAS  PubMed  Google Scholar 

  14. P. Dux, G. Rubinstenn, G. W. Vuister, R. Boelens, F. A. A. Mulder, K. Hard, W. D. Hoff, A. R. Kroon, W. Crielaard, K. J. Hellingwerf, R. Kaptein, Biochemistry, 1998, 37, 12689–12699.

    Article  CAS  PubMed  Google Scholar 

  15. T. Gensch, C. C. Gradinaru, I. H. M. van Stokkum, J. Hendriks, K. J. Hellingwerf, R. van Grondelle, Chem. Phys. Lett., 2002, 356, 347–354.

    Article  CAS  Google Scholar 

  16. R. Kort, H. Vonk, X. Xu, W. D. Hoff, W. Crielaard, K. J. Hellingwerf, FEBS Lett., 1996, 382, 73–78.

    Article  CAS  PubMed  Google Scholar 

  17. M. Unno, M. Kumauchi, J. Sasaki, F. Tokunaga, S. Yamauchi, Biochemistry, 2002, 41, 5668–5674.

    Article  CAS  PubMed  Google Scholar 

  18. L. van Wilderen, M. A. Van der Horst, I. H. M. van Stokkum, K. J. Hellingwerf, R. van Grondelle, M. L. Groot, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 15050–15055.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Z. Ren, B. Perman, V. Srajer, T. Y. Teng, C. Pradervand, D. Bourgeois, F. Schotte, T. Ursby, R. Kort, M. Wulff, K. Moffat, Biochemistry, 2001, 40, 13788–13801.

    Article  CAS  PubMed  Google Scholar 

  20. E. F. Chen, T. Gensch, A. B. Gross, J. Hendriks, K. J. Hellingwerf, D. S. Kliger, Biochemistry, 2003, 42, 2062–2071.

    Article  CAS  PubMed  Google Scholar 

  21. K. Takeshita, Y. Imamoto, M. Kataoka, K. Mihara, F. Tokunaga, M. Terazima, Biophys. J., 2002, 83, 1567–1577.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. J. Hendriks, W. D. Hoff, W. Crielaard, K. J. Hellingwerf, J. Biol. Chem., 1999, 274, 17655–17660.

    Article  CAS  PubMed  Google Scholar 

  23. A. H. Xie, L. Kelemen, J. Hendriks, B. J. White, K. J. Hellingwerf, W. D. Hoff, Biochemistry, 2001, 40, 1510–1517.

    Article  CAS  PubMed  Google Scholar 

  24. T. E. Meyer, G. Tollin, J. H. Hazzard, M. A. Cusanovich, Biophys. J., 1989, 56, 559–564.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. J. Hendriks, I. H. M. van Stokkum, K. J. Hellingwerf, Biophys. J., 2003, 84, 1180–1191.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. W. D. Hoff, I. H. M. Vanstokkum, H. J. Vanramesdonk, M. E. Vanbrederode, A. M. Brouwer, J. C. Fitch, T. E. Meyer, R. Vangrondelle, K. J. Hellingwerf, Biophys. J., 1994, 67, 1691–1705.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. C. J. Craven, N. M. Derix, J. Hendriks, R. Boelens, K. J. Hellingwerf, R. Kaptein, Biochemistry, 2000, 39, 14392–14399.

    Article  CAS  PubMed  Google Scholar 

  28. W. D. Hoff, A. Xie, I. H. M. Van Stokkum, X. J. Tang, J. Gural, A. R. Kroon, K. J. Hellingwerf, Biochemistry, 1999, 38, 1009–1017.

    Article  CAS  PubMed  Google Scholar 

  29. G. Rubinstenn, G. W. Vuister, F. A. A. Mulder, P. E. Dux, R. Boelens, K. J. Hellingwerf, R. Kaptein, Nat. Struct. Biol., 1998, 5, 568–570.

    Article  CAS  PubMed  Google Scholar 

  30. Y. Imamoto, H. Kamikubo, M. Harigai, N. Shimizu, M. Kataoka, Biochemistry, 2002, 41, 13595–13601.

    Article  CAS  PubMed  Google Scholar 

  31. N. Shimizu, Y. Imamoto, M. Harigai, H. Kamikubo, Y. Yamazaki, M. Kataoka, J. Biol. Chem., 2006, 281, 4318–4325.

    Article  CAS  PubMed  Google Scholar 

  32. J. Hendriks, T. Gensch, L. Hviid, M. A. van der Horst, K. J. Hellingwerf, J. J. van Thor, Biophys. J., 2002, 82, 1632–1643.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. M. A. van der Horst, I. H. van Stokkum, W. Crielaard, K. J. Hellingwerf, FEBS Lett., 2001, 497, 26–30.

    Article  PubMed  Google Scholar 

  34. H. Ihee, S. Rajagopal, V. Srajer, R. Pahl, S. Anderson, M. Schmidt, F. Schotte, P. A. Anfinrud, M. Wulff, K. Moffat, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 7145–7150.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. M. Unno, M. Kumauchi, N. Hamada, F. Tokunaga, S. Yamauchi, J. Biol. Chem., 2004, 279, 23855–23858.

    Article  CAS  PubMed  Google Scholar 

  36. D. Hoersch, H. Otto, M. A. Cusanovich, M. P. Heyn, J. Phys. Chem. B, 2008, 112, 9118–9125.

    Article  CAS  PubMed  Google Scholar 

  37. M. Kamiya, S. Saito, I. Ohmine, J. Phys. Chem. B, 2007, 111, 2948–2956.

    Article  CAS  PubMed  Google Scholar 

  38. E. J. M. Leenders, J. VandeVondele, P. G. Bolhuis, E. J. Meijer, J. Phys. Chem. B, 2007, 111, 13591–13599.

    Article  CAS  PubMed  Google Scholar 

  39. M. A. van der Horst, I. H. M. van Stokkum, N. A. Dencher, K. J. Hellingwerf, Biochemistry, 2005, 44, 9160–9167.

    Article  PubMed  CAS  Google Scholar 

  40. A. D. Stahl, M. Hospes, K. Singhal, I. van Stokkum, R. van Grondelle, M. L. Groot, K. J. Hellingwerf, Biophys. J., 2011, 101, 1184–1192.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. S. Yamaguchi, H. Kamikubo, K. Kurihara, R. Kuroki, N. Niimura, N. Shimizu, Y. Yamazaki, M. Kataoka, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 440–444.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. J. Hendriks, M. Hospes and K. J. Hellingwerf, in CRC Handbook of Organic Photochemistry and Photobiology, CRC Press, 3rd edn, 2012, pp. 1137–1160.

    Google Scholar 

  43. J. M. Beechem, L. Brand, Annu. Rev. Biochem., 1985, 54, 43–71.

    Article  CAS  PubMed  Google Scholar 

  44. C. A. Royer, Chem. Rev., 2006, 106, 1769–1784.

    Article  CAS  PubMed  Google Scholar 

  45. T. Gensch, J. Hendriks, K. J. Hellingwerf, Photochem. Photobiol. Sci., 2004, 3, 531–536.

    Article  CAS  PubMed  Google Scholar 

  46. R. Kort, W. D. Hoff, M. VanWest, A. R. Kroon, S. M. Hoffer, K. H. Vlieg, W. Crielaard, J. J. VanBeeumen, K. J. Hellingwerf, EMBO J., 1996, 15, 3209–3218.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. R. W. Hendler, R. I. Shrager, J. Biochem. Biophys. Methods, 1994, 28, 1–33.

    Article  CAS  PubMed  Google Scholar 

  48. A. R. Kroon, W. D. Hoff, H. P. M. Fenneman, J. Gijzen, G. J. Koomen, J. W. Verhoeven, W. Crielaard, K. J. Hellingwerf, J. Biol. Chem., 1996, 271, 31949–31956.

    Article  CAS  PubMed  Google Scholar 

  49. T. E. Meyer, G. Tollin, T. P. Causgrove, P. Cheng, R. E. Blankenship, Biophys. J., 1991, 59, 988–991.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. B. C. Lee, P. A. Croonquist, W. D. Hoff, J. Biol. Chem., 2001, 276, 44481–44487.

    Article  CAS  PubMed  Google Scholar 

  51. R. Cordfunke, R. Kort, A. Pierik, B. Gobets, G. J. Koomen, J. W. Verhoeven, K. J. Hellingwerf, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 7396–7401.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. J. Hendriks, K. J. Hellingwerf, J. Biol. Chem., 2009, 284, 5277–5288.

    Article  CAS  PubMed  Google Scholar 

  53. Y. Imamoto, H. Koshimizu, K. Mihara, O. Hisatomi, T. Mizukami, K. Tsujimoto, M. Kataoka, F. Tokunaga, Biochemistry, 2001, 40, 4679–4685.

    Article  CAS  PubMed  Google Scholar 

  54. T. E. Meyer, S. Devanathan, T. Woo, E. D. Getzoff, G. Tollin, M. A. Cusanovich, Biochemistry, 2003, 42, 3319–3325.

    Article  CAS  PubMed  Google Scholar 

  55. K. Mihara, O. Hisatomi, Y. Imamoto, M. Kataoka, F. Tokunaga, J. Biochem., 1997, 121, 876–880.

    Article  CAS  PubMed  Google Scholar 

  56. R. Brudler, T. E. Meyer, U. K. Genick, S. Devanathan, T. T. Woo, D. P. Millar, K. Gerwert, M. A. Cusanovich, G. Tollin, E. D. Getzoff, Biochemistry, 2000, 39, 13478–13486.

    Article  CAS  PubMed  Google Scholar 

  57. W. D. Hoff, I. H. M. Van Stokkum, J. Gural, K. J. Hellingwerf, Biochim. Biophys. Acta, Bioenerg., 1997, 1322, 151–162.

    Article  CAS  Google Scholar 

  58. J. R. Lakowics, in Principles of Fluorescence Spectroscopy, Springer, 2006, pp. 529–575.

    Book  Google Scholar 

  59. E. C. Carroll, M. Hospes, C. Valladares, K. J. Hellingwerf, D. S. Larsen, Photochem. Photobiol. Sci., 2011, 10, 464–468.

    Article  CAS  PubMed  Google Scholar 

  60. H. Chosrowjan, S. Taniguchi, N. Mataga, M. Unno, S. Yamauchi, N. Hamada, M. Kumauchi, F. Tokunago, J. Phys. Chem. B, 2004, 108, 2686–2698.

    Article  CAS  Google Scholar 

  61. N. Mataga, H. Chosrowjan, Y. Shibata, Y. Imamoto, M. Kataoka, F. Tokunaga, Chem. Phys. Lett., 2002, 352, 220–225.

    Article  CAS  Google Scholar 

  62. N. Mataga, H. Chosrowjan, S. Taniguchi, J. Photochem. Photobiol., C: Photochem. Rev., 2004, 5, 155–168.

    Article  CAS  Google Scholar 

  63. X. Yu, D. M. Leitner, Chem. Phys. Lett., 2004, 391, 181–186.

    Article  CAS  Google Scholar 

  64. M. Harigai, M. Kataoka, Y. Imamoto, J. Am. Chem. Soc., 2006, 128, 10646–10647.

    Article  CAS  PubMed  Google Scholar 

  65. M. Hospes, J. H. Ippel, R. Boelens, K. J. Hellingwerf, J. Hendriks, J. Phys. Chem. B, 2012, 116(44), 13172–13182.

    Article  CAS  PubMed  Google Scholar 

  66. H. Kandori, T. Iwata, J. Hendriks, A. Maeda, K. J. Hellingwerf, Biochemistry, 2000, 39, 7902–7909.

    Article  CAS  PubMed  Google Scholar 

  67. Y. Imamoto, S. Tatsumi, M. Harigai, Y. Yamazaki, H. Kamikubo, M. Kataoka, Biophys. J., 2008, 94, 3620–3628.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. C. P. Joshi, H. Otto, D. Hoersch, T. E. Meyer, M. A. Cusanovich, M. P. Heyn, Biochemistry, 2009, 48, 9980–9993.

    Article  CAS  PubMed  Google Scholar 

  69. S. F. El-Mashtoly, M. Unno, M. Kumauchi, N. Hamada, K. Fujiwara, J. Sasaki, Y. Imamoto, M. Kataoka, F. Tokunaga, S. Yamauchi, Biochemistry, 2004, 43, 2279–2287.

    Article  CAS  PubMed  Google Scholar 

  70. T. Morishita, M. Harigai, Y. Yamazaki, H. Kamikubo, M. Kataoka, Y. Imamoto, Photochem. Photobiol., 2007, 83, 280–285.

    Article  CAS  PubMed  Google Scholar 

  71. D. Hoersch, H. Otto, M. A. Cusanovich, M. P. Heyn, Phys. Chem. Chem. Phys., 2009, 11, 5437–5444.

    Article  CAS  PubMed  Google Scholar 

  72. P. L. Ramachandran, J. E. Lovett, P. J. Carl, M. Cammarata, J. H. Lee, Y. O. Jung, H. Ihee, C. R. Timmel, J. J. van Thor, J. Am. Chem. Soc., 2011, 133, 9395–9404.

    Article  CAS  PubMed  Google Scholar 

  73. Y. Chen, M. D. Barkley, Biochemistry, 1998, 37, 9976–9982.

    Article  CAS  PubMed  Google Scholar 

  74. W. H. Qiu, T. P. Li, L. Y. Zhang, Y. Yang, Y. T. Kao, L. J. Wang, D. P. Zhong, Chem. Phys., 2008, 350, 154–164.

    Article  CAS  Google Scholar 

  75. H. Otto, D. Hoersch, T. E. Meyer, M. A. Cusanovich, M. P. Heyn, Biochemistry, 2005, 44, 16804–16816.

    Article  CAS  PubMed  Google Scholar 

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  1. Laboratory for Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1090 GE, Amsterdam, The Netherlands

    Marijke Hospes, Johnny Hendriks & Klaas J. Hellingwerf

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  1. Marijke Hospes
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† Electronic supplementary information (ESI) available: Experimental procedures and supplementary results. See DOI: 10.1039/c2pp25222h

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Hospes, M., Hendriks, J. & Hellingwerf, K.J. Tryptophan fluorescence as a reporter for structural changes in photoactive yellow protein elicited by photo-activation. Photochem Photobiol Sci 12, 479–488 (2013). https://doi.org/10.1039/c2pp25222h

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