Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1996 Jul;5(7):1282–1289.

Thermal-induced unfolding domains in aldolase identified by amide hydrogen exchange and mass spectrometry.

Z Zhang 1, D L Smith 1
PMCID: PMC2143457  PMID: 8819161

Abstract

Amide hydrogen exchange has been measured in short segments of intact rabbit muscle aldolase at temperatures of 14-50 degrees C by the protein fragmentation/mass spectrometry method (Zhang Z, Smith DL, 1993, Protein Sci 2:522-531). Deuterium levels in some segments did not change over the temperature range of the measurements, whereas deuterium levels in other segments increased rapidly with temperature. These results demonstrate that the equilibrium constant for local unfolding, Kunf, of some segments increases with temperature in the low temperature range (14-30 degrees C) of this study. Aldolase begins to lose activity at temperatures above 40 degrees C. In the 40-50 degrees C temperature range, Kunf is greater than 10(-4) in some regions and less than 10(-6) in other regions. This wide range of regional stability in the temperature range where aldolase begins to denature is interpreted in terms of cooperative unfolding/folding domains. Regions of highest stability were located along the hydrophobic subunit binding surface. It is proposed that hydrogen exchange might be used to identify unfolding domains in multidomain proteins whose thermodynamic properties have been determined by differential scanning calorimetry.

Full Text

The Full Text of this article is available as a PDF (5.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bai Y., Milne J. S., Mayne L., Englander S. W. Primary structure effects on peptide group hydrogen exchange. Proteins. 1993 Sep;17(1):75–86. doi: 10.1002/prot.340170110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bai Y., Milne J. S., Mayne L., Englander S. W. Protein stability parameters measured by hydrogen exchange. Proteins. 1994 Sep;20(1):4–14. doi: 10.1002/prot.340200103. [DOI] [PubMed] [Google Scholar]
  3. Bai Y., Sosnick T. R., Mayne L., Englander S. W. Protein folding intermediates: native-state hydrogen exchange. Science. 1995 Jul 14;269(5221):192–197. doi: 10.1126/science.7618079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beernink P. T., Tolan D. R. Subunit interface mutants of rabbit muscle aldolase form active dimers. Protein Sci. 1994 Sep;3(9):1383–1391. doi: 10.1002/pro.5560030904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  6. Buck M., Radford S. E., Dobson C. M. Amide hydrogen exchange in a highly denatured state. Hen egg-white lysozyme in urea. J Mol Biol. 1994 Apr 1;237(3):247–254. doi: 10.1006/jmbi.1994.1228. [DOI] [PubMed] [Google Scholar]
  7. Elöve G. A., Bhuyan A. K., Roder H. Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands. Biochemistry. 1994 Jun 7;33(22):6925–6935. doi: 10.1021/bi00188a023. [DOI] [PubMed] [Google Scholar]
  8. Englander J. J., Calhoun D. B., Englander S. W. Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry. Anal Biochem. 1979 Jan 15;92(2):517–524. doi: 10.1016/0003-2697(79)90693-6. [DOI] [PubMed] [Google Scholar]
  9. Englander S. W., Englander J. J., McKinnie R. E., Ackers G. K., Turner G. J., Westrick J. A., Gill S. J. Hydrogen exchange measurement of the free energy of structural and allosteric change in hemoglobin. Science. 1992 Jun 19;256(5064):1684–1687. doi: 10.1126/science.256.5064.1684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gamblin S. J., Cooper B., Millar J. R., Davies G. J., Littlechild J. A., Watson H. C. The crystal structure of human muscle aldolase at 3.0 A resolution. FEBS Lett. 1990 Mar 26;262(2):282–286. doi: 10.1016/0014-5793(90)80211-z. [DOI] [PubMed] [Google Scholar]
  11. HVIDT A. A DISCUSSION OF THE PH DEPENDENCE OF THE HYDROGEN-DEUTERIUM EXCHANGE OF PROTEINS. C R Trav Lab Carlsberg. 1964;34:299–317. [PubMed] [Google Scholar]
  12. Heyduk T., Kochman M. Temperature-induced conformational transition in rabbit muscle aldolase studied by temperature dependence of sulfhydryl reactivity. Eur J Biochem. 1985 Sep 2;151(2):337–343. doi: 10.1111/j.1432-1033.1985.tb09106.x. [DOI] [PubMed] [Google Scholar]
  13. Hvidt A., Nielsen S. O. Hydrogen exchange in proteins. Adv Protein Chem. 1966;21:287–386. doi: 10.1016/s0065-3233(08)60129-1. [DOI] [PubMed] [Google Scholar]
  14. Jollès P., Berthou J., Lifchitz A., Clochard A., Saint-Blancard J. Contribution to the study in solution and solid state of the rabbit aldolase temperature dependence. FEBS Lett. 1980 Jul 11;116(1):48–50. doi: 10.1016/0014-5793(80)80526-6. [DOI] [PubMed] [Google Scholar]
  15. Kim K. S., Fuchs J. A., Woodward C. K. Hydrogen exchange identifies native-state motional domains important in protein folding. Biochemistry. 1993 Sep 21;32(37):9600–9608. doi: 10.1021/bi00088a012. [DOI] [PubMed] [Google Scholar]
  16. Lehrer G. M., Barker R. Conformational changes in rabbit muscle aldolase. Kinetic studies. Biochemistry. 1970 Mar 31;9(7):1533–1540. doi: 10.1021/bi00809a009. [DOI] [PubMed] [Google Scholar]
  17. Lehrer G. M., Barker R. Conformational changes in rabbit muscle aldolase. Ultraviolet spectroscopic studies. Biochemistry. 1971 Apr 27;10(9):1705–1714. doi: 10.1021/bi00785a031. [DOI] [PubMed] [Google Scholar]
  18. Loh S. N., Prehoda K. E., Wang J., Markley J. L. Hydrogen exchange in unligated and ligated staphylococcal nuclease. Biochemistry. 1993 Oct 19;32(41):11022–11028. doi: 10.1021/bi00092a011. [DOI] [PubMed] [Google Scholar]
  19. Louie G., Tran T., Englander J. J., Englander S. W. Allosteric energy at the hemoglobin beta chain C terminus studied by hydrogen exchange. J Mol Biol. 1988 Jun 20;201(4):755–764. doi: 10.1016/0022-2836(88)90472-x. [DOI] [PubMed] [Google Scholar]
  20. Marmorino J. L., Auld D. S., Betz S. F., Doyle D. F., Young G. B., Pielak G. J. Amide proton exchange rates of oxidized and reduced Saccharomyces cerevisiae iso-1-cytochrome c. Protein Sci. 1993 Nov;2(11):1966–1974. doi: 10.1002/pro.5560021118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Miller D. W., Dill K. A. A statistical mechanical model for hydrogen exchange in globular proteins. Protein Sci. 1995 Sep;4(9):1860–1873. doi: 10.1002/pro.5560040921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Privalov P. L. Thermodynamic problems of protein structure. Annu Rev Biophys Biophys Chem. 1989;18:47–69. doi: 10.1146/annurev.bb.18.060189.000403. [DOI] [PubMed] [Google Scholar]
  23. Ray J., Englander S. W. Allosteric sensitivity in hemoglobin at the alpha-subunit N-terminus studied by hydrogen exchange. Biochemistry. 1986 May 20;25(10):3000–3007. doi: 10.1021/bi00358a040. [DOI] [PubMed] [Google Scholar]
  24. Robertson A. D., Baldwin R. L. Hydrogen exchange in thermally denatured ribonuclease A. Biochemistry. 1991 Oct 15;30(41):9907–9914. doi: 10.1021/bi00105a014. [DOI] [PubMed] [Google Scholar]
  25. Roder H., Wagner G., Wüthrich K. Amide proton exchange in proteins by EX1 kinetics: studies of the basic pancreatic trypsin inhibitor at variable p2H and temperature. Biochemistry. 1985 Dec 3;24(25):7396–7407. doi: 10.1021/bi00346a055. [DOI] [PubMed] [Google Scholar]
  26. Roder H., Wagner G., Wüthrich K. Individual amide proton exchange rates in thermally unfolded basic pancreatic trypsin inhibitor. Biochemistry. 1985 Dec 3;24(25):7407–7411. doi: 10.1021/bi00346a056. [DOI] [PubMed] [Google Scholar]
  27. Sygusch J., Beaudry D., Allaire M. Molecular architecture of rabbit skeletal muscle aldolase at 2.7-A resolution. Proc Natl Acad Sci U S A. 1987 Nov;84(22):7846–7850. doi: 10.1073/pnas.84.22.7846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sygusch J., Boulet H., Beaudry D. Structure of rabbit muscle aldolase at low resolution. J Biol Chem. 1985 Dec 5;260(28):15286–15290. [PubMed] [Google Scholar]
  29. Vimard C., Orsini G., Goldberg M. E. Renaturation of acid-denatured rabbit muscle aldolase. Existence and properties of a stable monomeric intermediate. Eur J Biochem. 1975 Feb 21;51(2):521–527. doi: 10.1111/j.1432-1033.1975.tb03952.x. [DOI] [PubMed] [Google Scholar]
  30. Woodward C., Simon I., Tüchsen E. Hydrogen exchange and the dynamic structure of proteins. Mol Cell Biochem. 1982 Oct 29;48(3):135–160. doi: 10.1007/BF00421225. [DOI] [PubMed] [Google Scholar]
  31. Zhang Z., Post C. B., Smith D. L. Amide hydrogen exchange determined by mass spectrometry: application to rabbit muscle aldolase. Biochemistry. 1996 Jan 23;35(3):779–791. doi: 10.1021/bi952227q. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES