Advertisement

Proteins in Motion

Resonance Raman Spectroscopy as a Probe of Functional Intermediates
  • Uri Samuni
  • Joel M. Friedman
Part of the Methods in Molecular Biology™ book series (MIMB, volume 305)

Abstract

Elucidating proteins function at a level that allows for intelligent design and manipulation is essential in realization of their potential role in biomedical and industrial applications. It has become increasingly apparent though, that probing structures and functionalities under equilibrium conditions is not sufficient. Rather, many aspects of protein behavior and reactivity are rooted in protein dynamics. Thus, there is a growing effort to probe intermediate structures that occur transiently during the course of a proteins function in particular linked to the binding or release of a ligand or substrate. However, studies following the sequence of conformational changes triggered by the binding of sub-strate/ligand and the concomitant change in functional properties are inherently difficult because often the diffusion times are of the order of conformational relaxation times. This chapter describes methodologies for generating resonance Raman spectra from transient forms of hemoglobin under conditions that allow for the systematic exploration of conformational relaxation and functionality. Special consideration is given to Raman compatible protocols based on sol-gel encapsulation that allow for the preparation, trapping and temporal tuning of nonequilibrium population generated from either the addition or the removal of ligands/substrates.

Key Words

Resonance Raman protein dynamics protein conformation ligand binding sol-gel hemoglobin. 

References

  1. 1.
    Spiro T. G. (1978) Resonance Raman spectra of hemoproteins. Methods Enzymol 54, 233–249.PubMedCrossRefGoogle Scholar
  2. 2.
    Spiro T. G. (1985) Resonance Raman spectroscopy as a probe of heme protein structure and dynamics. Adv. Protein Chem. 37, 111–159.PubMedCrossRefGoogle Scholar
  3. 3.
    Spiro T. G., Smulevich G., and Su C. (1990) Probing protein structure and dynamics with resonance Raman spectroscopy: cytochrome c peroxidase and hemoglobin. Biochemistry 29, 4497–4508.PubMedCrossRefGoogle Scholar
  4. 4.
    Spiro T. G. and Czernuszewicz R. S. (1995) Resonance Raman spectroscopy of metalloproteins. Methods Enzymol. 246, 416–460.PubMedCrossRefGoogle Scholar
  5. 5.
    Friedman J. M. (1994) Time-resolved resonance Raman spectroscopy as probe of structure, dynamics, and reactivity in hemoglobin. Methods Enzymol. 232, 205–231.PubMedCrossRefGoogle Scholar
  6. 6.
    Asher S. (1993) UV resonance Raman spectroscopy for analytical, physical and biophysical chemistry. Part 1. Anal. Chem. 65, 59A–66A.CrossRefGoogle Scholar
  7. 7.
    Austin J., Jordan T., and Spiro T. (1993) Ultraviolet resonance Raman studies of proteins and related model compounds. In Biomolecular Spectroscopy Part A (Clark R. J. H. and Hester R. E., eds.), John Wiley and Sons, New York, pp. 55–127.Google Scholar
  8. 8.
    Kitagawa T. (1988) The heme protein structure and the iron histidine stretching mode. In Biological Application of Raman Spectroscopy, Vol. III (Spiro T. G., ed.), John Wiley & Sons, New York, pp. 97–131.Google Scholar
  9. 9.
    Rousseau D. L. and Friedman J. M. (1988) Transient and cryogenic studies of photodissociated hemoglobin and myoglobin. In Biological Applications of Raman Spectroscopy, Vol. III (Spiro T. G., ed.), John Wiley & Sons, New York, pp. 133–215.Google Scholar
  10. 10.
    Friedman J. M., Scott T. W., Stepnoski R. A., Ikeda-Saito M., and Yonetani T. (1983) The iron-proximal histidine linkage and protein control of oxygen binding in hemoglobin. A transient Raman study. J. Biol. Chem. 258, 10,564–10,572.PubMedGoogle Scholar
  11. 11.
    Friedman J. M. (1985) Structure, dynamics, and reactivity in hemoglobin. Science 228, 1273–1280.PubMedCrossRefGoogle Scholar
  12. 12.
    Scott T. W. and Friedman J. M. (1984) Tertiary-structure relaxation in hemoglobin: a transient Raman study. J. Am. Chem. Soc. 106, 5677–5687.CrossRefGoogle Scholar
  13. 13.
    Avnir D., Braun S., Lev O., and Ottolenghi M. (1994) Enzymes and other proteins entrapped in sol-gel materials. Chem. Mater. 6, 1605–1614.CrossRefGoogle Scholar
  14. 14.
    Bettati S. and Mozzarelli A. (1997) T state hemoglobin binds oxygen noncooperatively with allosteric effects of protons, inositol hexaphosphate, and chloride. J. Biol. Chem. 272, 32,050–32,055.PubMedCrossRefGoogle Scholar
  15. 15.
    Bruno S., Bonaccio M., Bettati S., Rivetti C., Viappiani C., Abbruzzetti S., and Mozzarelli A. (2001) High and low oxygen affinity conformations of T state hemoglobin. Protein Sci. 10, 2401–2407.PubMedCrossRefGoogle Scholar
  16. 16.
    Dave B. C., Miller J. M., Dunn B., Valentine J. S., and Zink J. I. (1997) Encapsulation of proteins in bulk and thin film sol-gel matrices. J. Sol Gel Sci. Technol. 8, 629–634.Google Scholar
  17. 17.
    Das T. K., Khan I., Rousseau D. L., and Friedman J. M. (1999) Temperature dependent quaternary state relaxation in sol-gel encapsulated hemoglobin. Biospectroscopy 5, S64–S70.PubMedCrossRefGoogle Scholar
  18. 18.
    Ellerby L. M., Nishida C. R., Nishida F., Yamanaka S. A., Dunn B., Valentine J. S., and Zink J. I. (1992) Encapsulation of proteins in transparent porous silicate glasses prepared by the sol-gel method. Science 255, 1113–1115.PubMedCrossRefGoogle Scholar
  19. 19.
    Juszczak L. J. and Friedman J. M. (1999) UV resonance Raman spectra of ligand binding intermediates of sol-gel encapsulated hemoglobin. J. Biol. Chem. 274, 30,357–30,360.PubMedCrossRefGoogle Scholar
  20. 20.
    Khan I., Shannon C. F., Dantsker D., Friedman A. J., Perez-Gonzalezde-Apodaca J., and Friedman J. M. (2000) Sol-gel trapping of functional intermediates of hemoglobin: geminate and bimolecular recombination studies. Biochemistry 39, 16,099–16,109.PubMedCrossRefGoogle Scholar
  21. 21.
    Samuni U., Dantsker D., Khan I., Friedman A. J., Peterson E., and Friedman J. M. (2002) Spectroscopically and kinetically distinct conformational popula 300 tions of sol-gel encapsulated carbonmonoxy myoglobin: a comparison with hemoglobin. J. Biol. Chem. 25, 25.Google Scholar
  22. 22.
    Shibayama N. and Saigo S. (1995) Fixation of the quaternary structures of human adult haemoglobin by encapsulation in transparent porous silica gels. J. Mol. Biol. 251, 203–209.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Uri Samuni
    • 1
  • Joel M. Friedman
    • 1
  1. 1.Department of Physiology and BiophysicsAlbert Einstein College of MedicineBronx

Personalised recommendations