Determination of Reaction Intermediate Structures in Heme Proteins

  • Kelvin Chu
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 364)

Abstract

Developments in structural biology and molecular biology have allowed increasingly detailed investigations of structure-function relationships. Although atomic-resolution structures of proteins are becoming more common, a growing number of structural studies have focused on the role played by dynamics and have sought to determine the structure of intermediates in protein reactions. These experiments have revealed the first atomic-level pictures of enzyme catalysis and the conformational motions required for biological function. This chapter uses the cryotrapping of reaction intermediates in horse heart myoglobin (Mb) to illustrate the methods utilized in determining the structures of reaction intermediates in protein systems. The techniques described here are applicable to a wide variety of heme proteins including Mb, hemoglobin, photosynthetic reaction centers, and cytochrome p450cam.

Key Words

Myoglobin cytochrome p450 heme proteins reaction intermediates cryotrapping 

References

  1. 1.
    Stoddard, B. L. (1998) New results using Laue diffraction and time-resolved crystallography. Curr. Opin. Struct. Biol. 8, 612–618.PubMedCrossRefGoogle Scholar
  2. 2.
    Ridder, I. S., Rozeboom, H. J., Kalk, K. H., and Dijkstra, B. W. (1999) Crystal structures of intermediates in the dehalogenation of haloalkanoates by L-2-haloacid dehalogenase. J. Biol. Chem. 274, 30,672–30,678.PubMedCrossRefGoogle Scholar
  3. 3.
    Pannifer, A. D., Flint, A. J., Tonks, N. K., and Barford, D. (1998) Visualization of the cysteinyl-phosphate intermediate of a protein-tyrosine phosphatase by x-ray crystallography. J. Biol. Chem. 273, 10,454–10,462.PubMedCrossRefGoogle Scholar
  4. 4.
    Burzlaff, N. I., Rutledge, P. J., Clifton, I. J., et al. (1999) The reaction cycle of isopenicillin N synthase observed by X-ray diffraction. Nature 401, 721–724.PubMedCrossRefGoogle Scholar
  5. 5.
    Ogle, J. M., Clifton, I. J., Rutledge, P. J., et al. (2001) Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction. Chem. Biol. 8, 1231–1237.PubMedCrossRefGoogle Scholar
  6. 6.
    Wilmot, C. M., Hajdu, J., McPherson, M. J., Knowles, P. F., and Phillips, S. E. (1999) Visualization of dioxygen bound to copper during enzyme catalysis. Science 286, 1724–1728.PubMedCrossRefGoogle Scholar
  7. 7.
    Murray, J. B., Szoke, H., Szoke, A., and Scott, W. G. (2000) Capture and visualization of a catalytic RNA enzyme-product complex using crystal lattice trapping and X-ray holographic reconstruction. Mol. Cell. 5, 279–287.PubMedCrossRefGoogle Scholar
  8. 8.
    Kuhlbrandt, W. (2000) Bacteriorhodopsin—the movie. Nature 406, 569–570.PubMedCrossRefGoogle Scholar
  9. 9.
    Luecke, H., Schobert, B., Richter, H. T., Cartailler, J. P., and Lanyi, J. K. (1999) Structural changes in bacteriorhodopsin during ion transport at 2 angstrom resolution. Science 286, 255–261.PubMedCrossRefGoogle Scholar
  10. 10.
    Luecke, H., Schobert, B., Richter, H. T., Cartailler, J. P., and Lanyi, J. K. (1999) Structure of bacteriorhodopsin at 1.55 A resolution. J. Mol. Biol. 291, 899–911.PubMedCrossRefGoogle Scholar
  11. 11.
    Edman, K., Nollert, P., Royant, A., et al. (1999) High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle. Nature 401, 822–826.PubMedCrossRefGoogle Scholar
  12. 12.
    Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2000) Helix deformation is coupled to vectorial proton transport in the photocycle of bacteriorhodopsin. Nature 406, 645–648.PubMedCrossRefGoogle Scholar
  13. 13.
    Sass, H. J., Buldt, G., Gessenich, R., et al. (2000) Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin. Nature 406, 649–653.PubMedCrossRefGoogle Scholar
  14. 14.
    Chu, K., Vojtchovsky, J., McMahon, B. H., Sweet, R. M., Berendzen, J., and Schlichting, I. (2000) Structure of a new ligand-binding intermediate in wildtype carbonmonoxymyoglobin. Nature 403, 921–923.PubMedCrossRefGoogle Scholar
  15. 15.
    Schlichting, I., Berendzen, J., Phillips, G. N., Jr., and Sweet, R. M. (1994) Crystal structure of photolyzed myoglobin. Nature 371, 808–812.PubMedCrossRefGoogle Scholar
  16. 16.
    Ostermann, A., Waschipky, R., Parak, F. G., and Nienhaus, G. U. (2000) Ligand binding and conformational motions in myoglobin. Nature 404, 205–208.PubMedCrossRefGoogle Scholar
  17. 17.
    Teng, T. Y., Srajer, V., and Moffat, K. (1994) Photolysis-induced structural changes in single crystals of carbonmonoxymyoglobin at 40K. Nat. Struct. Biol. 1, 701–705.PubMedCrossRefGoogle Scholar
  18. 18.
    Adachi, S., Park, S. Y., Tame, J. R., Shiro, Y., and Shibayama, N. (2003) Direct observation of photolysis-induced tertiary structural changes in hemoglobin. Proc. Natl. Acad. Sci. USA 100, 7039–7044.PubMedCrossRefGoogle Scholar
  19. 19.
    Stowell, M. H., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Light-induced structural changes in photosynthetic reaction center: implications for mechanism of electron-proton transfer. Science 276, 812–816.PubMedCrossRefGoogle Scholar
  20. 20.
    Schlichting, I., Berendzen, J., Chu, K., et al. (2000) The catalytic pathway of cytochrome P450cam at atomic resolution. Science 287, 1615–1622.PubMedCrossRefGoogle Scholar
  21. 21.
    Stoddard, B. L. (1999) Visualizing enzyme intermediates using fast diffraction and reaction trapping methods: isocitrate dehydrogenase. Biochem. Soc. Trans. 27, 42–48.PubMedGoogle Scholar
  22. 22.
    Scott, W.G. (1999) Biophysical and biochemical investigations of RNA catalysis in the hammerhead ribozyme. Q. Rev. Biophys. 32, 241–284.PubMedCrossRefGoogle Scholar
  23. 23.
    Genick, U. K., Borgstahl, G. E., Ng, K., et al. (1997) Structure of a protein photocycle intermediate by millisecond time-resolved crystallography. Science 275, 1471–1475.PubMedCrossRefGoogle Scholar
  24. 24.
    Genick, U. K., Soltis, S. M., Kuhn, P., Canestrelli, I. L., and Getzoff, E. D. (1998) Structure at 0.85 A resolution of an early protein photocycle intermediate. Nature 392, 206–209.PubMedCrossRefGoogle Scholar
  25. 25.
    Perman, B., Srajer, V., Ren, Z., et al. (1998) Energy transduction on the nanosecond time scale: early structural events in a xanthopsin photocycle. Science 279, 1946–1950.PubMedCrossRefGoogle Scholar
  26. 26.
    Subramaniam, S. and R. Henderson (2000) Molecular mechanism of vectorial proton translocation by bacteriorhodopsin. Nature 406, 653–657.PubMedCrossRefGoogle Scholar
  27. 27.
    Vonck, J. (2000) Structure of the bacteriorhodopsin mutant F219L N intermediate revealed by electron crystallography. Embo. J. 19, 2152–2160.PubMedCrossRefGoogle Scholar
  28. 28.
    Brunori, M., Vallone, B., Cutruzzola, F., et al. (2000) The role of cavities in protein dynamics: crystal structure of a photolytic intermediate of a mutant myoglobin. Proc. Natl. Acad. Sci. USA 97, 2058–2063.PubMedCrossRefGoogle Scholar
  29. 29.
    Sjogren, T. and Hajdu, J. (2001) Structure of the bound dioxygen species in the cytochrome oxidase reaction of cytochrome cd1 nitrite reductase. J. Biol. Chem. 276, 13,072–13,076.PubMedCrossRefGoogle Scholar
  30. 30.
    Arndt, J. W., Gong, W., Zhong, X., et al. (2001) Insight into the catalytic mechanism of DNA polymerase beta: structures of intermediate complexes. Biochemistry 40, 5368–5375.PubMedCrossRefGoogle Scholar
  31. 31.
    Wilmouth, R. C., Edman, K., Neutze, R., et al. (2001) X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate. Nat. Struct. Biol. 8, 689–694.PubMedCrossRefGoogle Scholar
  32. 32.
    Kern, D., Volkman, B. F., Luginbuhl, P., Nohaile, M. J., Kustu, S., and Wemmer, D. E. (1999) Structure of a transiently phosphorylated switch in bacterial signal transduction. Nature 402, 894–898.PubMedCrossRefGoogle Scholar
  33. 33.
    Hadfield, A. and Hajdu, J. (1994) On the photochemical release of phosphate from 3,5-dinitrophenyl phosphate in a protein crystal. J. Mol. Biol. 236, 995–1000.PubMedCrossRefGoogle Scholar
  34. 34.
    Urayama, P., Phillips, G. N., and Gruner, S. M. (2002) Probing substates in sperm whale myoglobin using high-pressure crystallography. Structure 10, 51–60.PubMedCrossRefGoogle Scholar
  35. 35.
    Vojtechovsky, J., Chu, K., Berendzen, J., Sweet, R. M., and Schlichting, I. (1999) Crystal structures of myoglobin-ligand complexes at near-atomic resolution. Biophys. J. 77, 2153–2174.PubMedCrossRefGoogle Scholar
  36. 36.
    Schlichting, I. and Chu, K. (2000) Trapping intermediates in the crystal: ligand binding to myoglobin. Curr. Opin. Struct. Biol. 10, 744–752.PubMedCrossRefGoogle Scholar
  37. 37.
    Wilmot, C. M. and Pearson, A. R. (2002) Cryocrystallography of metalloprotein reaction intermediates. Curr. Opin. Chem. Biol. 6, 202–207.PubMedCrossRefGoogle Scholar
  38. 38.
    Schlichting, I. and Goody, R. S. (1997) Triggering Methods in crystallographic enzyme kinetics. Meth. Enzymol. 277, 467–490.PubMedCrossRefGoogle Scholar
  39. 39.
    Lim, M., Jackson, T. A., and Anfinrud, P. A. (1997) Ultrafast rotation and trapping of carbon monoxide dissociated from myoglobin. Nat. Struct. Biol. 4, 209–214.PubMedCrossRefGoogle Scholar
  40. 40.
    Chu, K., Ernst, R.M., Frauenfelder, H., Mourant, J. R., Nienhaus, G. U., and Philipp, R. (1995) Light-induced and thermal relaxation in a protein. Phys. Rev. Lett. 74, 2607–2610.PubMedCrossRefGoogle Scholar
  41. 41.
    Berendzen, J. and Braunstein, D. (1990) Temperature-derivative spectroscopy: a tool for protein dynamics. Proc. Natl. Acad. Sci. USA 87, 1–5.PubMedCrossRefGoogle Scholar
  42. 42.
    Petsko, G. A. and Ringe, D. (2000) Observation of unstable species in enzyme-catalyzed transformations using protein crystallography. Curr. Opin. Chem. Biol. 4, 89–94.PubMedCrossRefGoogle Scholar
  43. 43.
    Schlichting, I. (2000) Crystallographic structure determination of unstable species. Acc. Chem. Res. 33, 532–538.PubMedCrossRefGoogle Scholar
  44. 44.
    Ursby, T., Weik, M., Fioravanti, E., Delarue, M., Goeldner, M., and Bourgeois, D. (2002) Cryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals. Acta Crystallogr. D. Biol. Crystallogr. 58, 607–614.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Kelvin Chu
    • 1
  1. 1.Physics DepartmentUniversity of VermontBurlington

Personalised recommendations