Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Ligand binding and conformational motions in myoglobin

Nature volume 404, pages 205–208 (2000)Cite this article

Abstract

Myoglobin, a small globular haem protein that binds gaseous ligands such as O2, CO and NO reversibly at the haem iron, serves as a model for studying structural and dynamic aspects of protein reactions. Time-resolved spectroscopic measurements after photodissociation of the ligand revealed a complex ligand-binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein1,2,3. To observe the structural changes induced by ligand dissociation, we have carried out X-ray crystallographic investigations of carbon monoxy-myoglobin (MbCO mutant L29W) crystals illuminated below and above 180 K, complemented by time-resolved infrared spectroscopy of CO rebinding. Here we show that below 180 K photodissociated ligands migrate to specific sites within an internal cavity—the distal haem pocket—of an essentially immobilized, frozen protein, from where they subsequently rebind by thermally activated barrier crossing. Upon photodissociation above 180 K, ligands escape from the distal pocket, aided by protein fluctuations that transiently open exit channels. We recover most of the ligands in a cavity on the opposite side of the haem group.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: (2Fo-Fc)exp(iαc) electron density maps of the active site of L29W MbCO at 105 K, contoured at 1.5σ (standard deviation).
Figure 2: Root-mean-squared differences of the amino-acid positions (backbone average, N–Cα–C) between MbCO and the two structures created by photodissociation below 180 K (dashed line) and above 180 K (solid line), as a function of the sequence number.
Figure 3: (2Fo-Fc)exp(iαc) electron density map, contoured at 1.5σ, of the active site of L29W MbCO at 105 K after extended illumination above 180 K and stick model with colour code as in Fig. 1.
Figure 4: Flash photolysis kinetics of L29W MbCO below 180 K, measured in the infrared band of the bound CO at 1,945 cm-1.

Similar content being viewed by others

References

  1. Austin, R. H., Beeson, K. W., Eisenstein, L., Frauenfelder, H. & Gunsalus, I. C. Dynamics of ligand binding to myoglobin. Biochemistry 14, 5355– 5373 (1975).

    Article  CAS  Google Scholar 

  2. Steinbach, P. J. et al. Ligand binding to heme proteins: connection between dynamics and function. Biochemistry 30, 3988– 4001 (1991).

    Article  CAS  Google Scholar 

  3. Olson, J. S. & Phillips, G. N. Jr. Kinetic pathways and barriers for ligand binding to myoglobin. J. Biol. Chem. 271, 17593–17596 (1996).

    Article  CAS  Google Scholar 

  4. Henry, E. R., Sommer, J. H., Hofrichter, J. & Eaton, W. A. Geminate recombination of carbon-monoxide to myoglobin. J. Mol. Biol. 166, 443–451 ( 1983).

    Article  CAS  Google Scholar 

  5. Huang, X. & Boxer, S. G. Discovery of new ligand binding pathways in myoglobin by random mutagenesis. Nature Struct. Biol. 1, 226–229 ( 1994).

    Article  CAS  Google Scholar 

  6. Quillin, M. L. et al. Structural and functional effects of apolar mutations of the distal valine in myoglobin. J. Mol. Biol. 245, 416–436 (1995).

    Article  CAS  Google Scholar 

  7. Scott, E. E. & Gibson, Q. H. Ligand migration in sperm whale myoglobin. Biochemistry 36, 11909– 11917 (1997).

    Article  CAS  Google Scholar 

  8. Parak, F., Knapp, E. W. & Kucheida, D. Protein dynamics: Mössbauer-spectroscopy on deoxymyoglobin crystals. J. Mol. Biol. 161, 177– 194 (1982).

    Article  CAS  Google Scholar 

  9. Doster, W., Cusack, S. & Petry, W. Dynamical transition of myoglobin revealed by inelastic neutron-scattering. Nature 337, 754– 756 (1989).

    Article  ADS  CAS  Google Scholar 

  10. Nienhaus, G. U., Heinzl, J., Huenges, E. & Parak, F. Protein crystal dynamics studied by time-resolved analysis of X-ray diffuse scattering. Nature 338, 665–666 ( 1989).

    Article  ADS  CAS  Google Scholar 

  11. Rasmussen, B. F., Stock, A. M., Ringe, D. & Petsko, G. A. Crystalline ribonuclease A loses function below the dynamical transition at 220 K. Nature 357, 423–424 ( 1992).

    Article  ADS  CAS  Google Scholar 

  12. Schlichting, I., Berendzen, J., Phillips, G. N. Jr & Sweet, R. M. Crystal structure of photolysed carbonmonoxy-myoglobin. Nature 371, 808–812 ( 1994).

    Article  ADS  CAS  Google Scholar 

  13. Teng, T. Y., Srajer, V. & Moffat, K. Photolysis-induced structural changes in single crystals of carbonmonoxy myoglobin at 40 K. Nature Struct. Biol. 1, 701–705 (1994).

    Article  CAS  Google Scholar 

  14. Hartmann, H. et al. X-ray structure determination of a metastable state of carbonmonoxy myoglobin after photodissociation. Proc. Natl Acad. Sci. USA 93, 7013–7016 (1996).

    Article  ADS  CAS  Google Scholar 

  15. Lim, M., Jackson, T. A. & Anfinrud, P. A. Ultrafast rotation and trapping of carbon monoxide dissociated from myoglobin. Nature Struct. Biol. 4, 209–214 (1997).

    Article  CAS  Google Scholar 

  16. Srajer, V. et al. Photolysis of the carbon-monoxide complex of myoglobin: nanosecond time-resolved crystallography. Science 274, 1726–1729 (1996).

    Article  ADS  CAS  Google Scholar 

  17. Vitkup, D., Petsko, G. A. & Karplus, M. A comparison between molecular dynamics and X-ray results for dissociated CO in myoglobin. Nature Struct. Biol. 4, 202–208 (1997).

    Article  CAS  Google Scholar 

  18. Nienhaus, G. U., Mourant, J. R. & Frauenfelder, H. Spectroscopic evidence for conformational relaxation in myoglobin. Proc. Natl Acad. Sci. USA 89, 2902–2906 (1992).

    Article  ADS  CAS  Google Scholar 

  19. Springer, B. A., Sligar S. G., Olson J. S. & Phillips, G. N. Jr. Mechanisms of ligand recognition in myoglobin. Chem. Rev. 94, 699–714 (1994).

    Article  CAS  Google Scholar 

  20. Tilton, R. F., Kuntz, I. D. & Petsko, G. A. Cavities in proteins: structure of a metmyoglobin-xenon complex solved to 1.9-Å. Biochemistry 23, 2849–2857 (1984).

    Article  CAS  Google Scholar 

  21. Hirota, S. et al. Perturbation of the Fe-O2 bond by nearby residues in heme pocket: Observation of vFe-O2 Raman bands for oxymyoglobin mutants. J. Am. Chem. Soc. 118, 7845– 7846 (1996).

    Article  CAS  Google Scholar 

  22. Schoenborn, B. P., Watson, H. C. & Kendrew, J. C. Binding of xenon to sperm whale myoglobin. Nature 207, 28–30 ( 1965).

    Article  ADS  CAS  Google Scholar 

  23. Elber, R. & Karplus, M. Enhanced sampling in molecular-dynamics: use of the time-dependent Hartree approximation for a simulation of carbon-monoxide diffusion through myoglobin. J. Am. Chem. Soc. 112, 9161–9175 (1990).

    Article  CAS  Google Scholar 

  24. Frauenfelder, H., Parak, F. & Young, R. D. Conformational substates in proteins. Annu. Rev. Biophys. Biophys. Chem. 17, 451– 479 (1988).

    Article  CAS  Google Scholar 

  25. Nienhaus, G. U. & Young, R. D. Protein dynamics. Encyclopedia Appl. Phys. 15, 163– 184 (1996).

    Google Scholar 

  26. Alben, J. O. et al. Infrared spectroscopy of photodissociated carboxymyoglobin at low temperatures. Proc. Natl Acad. Sci. USA 79, 3744–3748 (1982).

    Article  ADS  CAS  Google Scholar 

  27. Nienhaus, G. U., Mourant, J. R., Chu, K. & Frauenfelder, H. Ligand binding to heme proteins: the effect of light on ligand binding in myoglobin. Biochemistry 33, 13413–13430 (1994).

    Article  CAS  Google Scholar 

  28. Springer, B. A. & Sligar, S. G. High-level expression of sperm whale myoglobin in Escherichia coli. Proc. Natl Acad. Sci. USA 84, 8961–8965 (1987).

    Article  ADS  CAS  Google Scholar 

  29. Bailey, S. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 ( 1994).

    Article  Google Scholar 

  30. Brünger, A. T. X-PLOR Version 3.1. (Yale Univ. Press, New Haven, CT, 1992 ).

  31. Read, R. J. Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A 42, 140 –149 (1986).

    Article  Google Scholar 

Download references

Acknowledgements

We thank J. S. Olson for providing the L29W plasmid, K. Nienhaus for the sample preparation, and E. Haustein for expert assistance with the kinetic experiments. This work was supported by DFG Sonderforschungsbereich 533 and Graduiertenkolleg 328, Stiftung Volkswagenwerk and the University of Ulm.

Author information

Authors and Affiliations

  1. Fakultät für Physik E17, Technische Universität München, Garching , D-85747, Germany

    Andreas Ostermann & Fritz G. Parak

  2. Department of Biophysics, University of Ulm, Ulm, D-89069, Germany

    Robert Waschipky & G. Ulrich Nienhaus

  3. Department of Physics, University of Illinois, Urbana, 61801-3080, Illinois, USA

    G. Ulrich Nienhaus

Authors
  1. Andreas Ostermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert Waschipky
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fritz G. Parak
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G. Ulrich Nienhaus
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ostermann, A., Waschipky, R., Parak, F. et al. Ligand binding and conformational motions in myoglobin. Nature 404, 205–208 (2000). https://doi.org/10.1038/35004622

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35004622

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing