Experimental Characterization of the Denatured State Ensemble of Proteins

  • Jae-Hyun Cho
  • Daniel P. Raleigh
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 490)

Abstract

The traditional view of the denatured state ensemble of proteins is that it behaves as a classic random coil. This model has important implications for the analysis of protein stability, protein folding, and cooperativity; namely that the effects of mutations on the free energy of the denatured state ensemble can be ignored. This assumption, which is still routinely made, at least at the implicit level, greatly simplifies the analysis of such experiments. However it has long been recognized that the denatured state ensemble (DSE) of real proteins is often quite different from a random coil and can exhibit significant structural preferences. In some cases parts of the chain can even adopt relatively well-defined conformations, particularly under native conditions. Well-studied examples of DSE interactions include elements of hydrogen-bonded secondary structure, particularly helices or turns, as well hydrophobic clusters, hydrophobic aromatic clusters, and more recently interactions involving charged residues. Deviations from random-coil behavior are of practical importance if they influence protein folding, stability, or function, or if they compromise our analysis and interpretation of experiments. The existence of residual structure in the DSE naturally leads to the question of its role in protein folding and stability, and raises the possibility that some mutations could exert a significant part of their effect by altering the DSE. Much of our understanding of the interactions governing protein stability and the folding process have been generated by mutational studies; thus, a detailed understanding of the denatured state ensemble is critical.

Key words

Denatured state ensemble unfolded state protein stability protein folding protein engineering NMR protein design thermodynamics peptides random coil 

References

  1. 1.
    Whitten, S. T., Garcia-Moreno, E. B. (2000) pH dependence of stability of staphylococcal nuclease: Evidence of substantial electrostatic interactions in the denatured state. Biochemistry 39, 14292–14304.PubMedCrossRefGoogle Scholar
  2. 2.
    Shortle, D. (1996) The denatured state (the other half of the folding equation) and its role in protein stability. FASEB J 10, 27–34.PubMedGoogle Scholar
  3. 3.
    Millett, I. S., Doniach, S., Plaxco K. W. (2002) Toward a taxonomy of the denatured state: Small angle scattering studies of unfolded proteins. Adv Protein Chem 62, 241–262.PubMedCrossRefGoogle Scholar
  4. 4.
    Baldwin, R. L. (2002) A new perspective on unfolded proteins. Adv Protein Chem 62, 361–367.PubMedCrossRefGoogle Scholar
  5. 5.
    Choy, W. Y., Mulder, F. A. A., Crowhurst, K. A., et al. (2002) Distribution of molecular size within an unfolded state ensemble using small-angle X-ray scattering and pulse field gradient NMR techniques. J Mol Biol 316, 101–112.PubMedCrossRefGoogle Scholar
  6. 6.
    Cho, J. H., Sato, S., Raleigh, D. P. (2004) Thermodynamics and kinetics of non-native interactions in protein folding: a single point mutant significantly stabilizes the N-terminal domain of L9 by modulating non-native interactions in the denatured state. J Mol Biol 338, 827–837.PubMedCrossRefGoogle Scholar
  7. 7.
    Bowler, B. E. (2007) Thermodynamics of protein denatured states. Mol Biosyst 3, 88–99.PubMedCrossRefGoogle Scholar
  8. 8.
    Dill, K. A., Shortle, D. (1991) Denatured states of proteins. Annu Rev Biochem 60, 795–825.PubMedCrossRefGoogle Scholar
  9. 9.
    Sugase, K., Dyson, H. J., Wright, P. E. (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447, 1021–1024.PubMedCrossRefGoogle Scholar
  10. 10.
    Cho, J. H., Raleigh, D. P. (2005) Mutational analysis demonstrates that specific electrostatic interactions can play a key role in the denatured state ensemble of proteins. J Mol Biol 353, 174–185.PubMedCrossRefGoogle Scholar
  11. 11.
    Matthews, C. R. (1987) Effect of point mutations on the folding of globular proteins. Meth Enzymol 154, 498–511.PubMedCrossRefGoogle Scholar
  12. 12.
    Goldenberg, D. P., Frieden, R. W., Haack, J. A., et al. (1989) Mutational analysis of a protein folding pathway. Nature 338, 127–132.PubMedCrossRefGoogle Scholar
  13. 13.
    Matouschek, A., Kellis, J. T., Serrano, L., et al. (1989) Mapping the transition state and pathway of protein folding by protein engineering. Nature 340, 122–126.PubMedCrossRefGoogle Scholar
  14. 14.
    Fersht, A. R., Matouschek, A., Serrano, L. (1992) The folding of an enzyme: Theory of protein engineering analysis of stability and pathway of protein folding. J Mol Biol 224, 771–782.PubMedCrossRefGoogle Scholar
  15. 15.
    Cho, J. H., Raleigh, D. P. (2006) Denatured state effects and the origin of non-classical phi-values in protein folding. J Am Chem Soc 128, 16492–16493.Google Scholar
  16. 16.
    Neri, D., Billeter, M., Wider, G., Wuthrich, K. (1992) NMR determination of residual structure in a urea-denatured protein, the 434-repressor. Science 257, 1559–1563.PubMedCrossRefGoogle Scholar
  17. 17.
    Mok, Y. K., Kay, C. M., Kay, L. E., et al. (1999) NOE data demonstrating a compact unfolded state for an SH3 domain under non-denaturing conditions. J Mol Biol 289, 619–638.PubMedCrossRefGoogle Scholar
  18. 18.
    Mohana-Borges, R., Goto, N. K., Kroon, G. J. A., et al. (2004) Structural characterization of unfolded states of apomyoglobin using residual dipolar couplings. J Mol Biol 340, 1131–1142.PubMedCrossRefGoogle Scholar
  19. 19.
    Li, Y., Picart F., Raleigh, D. P. (2005) Direct characterization of the folded, unfolded and urea denatured states of the C-terminal domain of the ribosomal protein L9. J Mol Biol 349, 839–846.PubMedCrossRefGoogle Scholar
  20. 20.
    Pace, C. N., Laurents, D. V., Erickson, R. E. (1992) Urea denaturation of barnase: pH dependence and characterization of the unfolded state. Biochemistry 31, 2728–2734.PubMedCrossRefGoogle Scholar
  21. 21.
    Pace, C. N., Alston, R.W., Shaw, K.L. (2000) Charge–charge interactions influence the denatured state ensemble and contribute to protein stability. Protein Sci 9, 1395–1398.PubMedCrossRefGoogle Scholar
  22. 22.
    Shortle, D., Ackerman, M. S. (2001) Persistence of native-like topology in a denatured protein in 8 M urea. Science 293, 487–489.PubMedCrossRefGoogle Scholar
  23. 23.
    Kohn, J. E., Millett, I. S., Jacob, J., et al. (2004) Random-coil behavior and the dimensions of chemically unfolded proteins. Proc Natl Acad Sci USA 101, 12491–12496.Google Scholar
  24. 24.
    Fitzkee, N. C., Rose, G. D. (2004) Reassessing random-coil statistics in unfolded proteins. Proc Natl Acad Sci USA 101, 12497–12502.Google Scholar
  25. 25.
    Dyson, H. J., Wright, P. E. (2004) Unfolded proteins and protein folding studied by NMR. Chem Rev 104, 3607–3622.PubMedCrossRefGoogle Scholar
  26. 26.
    Dyson, J. H., Wright, P. E. (2002) Insights into the structure and dynamics of unfolded proteins from nuclear magnetic resonance. Advan Protein Chem 62, 311–340.CrossRefGoogle Scholar
  27. 27.
    Tollinger, M., Croehurst, K. A., Kay, L. E., et al. (2003) Site-specific contributions to the pH dependence of protein stability. Proc Natl Acad Sci USA 100, 4545–4550.PubMedCrossRefGoogle Scholar
  28. 28.
    Marsh, J. A., Singh, V. K., Jia, Z., et al. (2006) Sensitivity of secondary structure propensities to sequence differences between alpha- and gamma-synuclein: Implications for fibrillation. Protein Sci 15, 2795–2804.PubMedCrossRefGoogle Scholar
  29. 29.
    Gillespie, J. R., Shortle, D. (1997) Characterization of long-range structure in the denatured state of staphylococcal nuclease. 1. Paramagnetic relaxation enhancement by nitroxide spin labels. J Mol Biol 268, 158–169.PubMedCrossRefGoogle Scholar
  30. 30.
    Grey, M. J., Tang, Y., Alexov, E., et al. (2006) Characterizing a partially folded intermediate of the villin headpiece domain under non-denaturing conditions: contribution of His41 to the pH-dependent stability of the N-terminal subdomain. J Mol Biol 355, 1078–1094.PubMedCrossRefGoogle Scholar
  31. 31.
    Korzhnev, D. M., Religa, T. L., Lundstroem, P., et al. (2007) The folding pathway of an FF domain: Characterization of an on-pathway intermediate state under folding conditions by N-15, C-13(alpha) and C-13-methyl relaxation dispersion and 1H/2H H-exchange NMR spectroscopy. J Mol Biol 372, 497–512.PubMedCrossRefGoogle Scholar
  32. 32.
    Zhang, O. W., Kay, L. E., Shortle, D., et al. (1997) Comprehensive NOE characterization of a partially folded large fragment of staphylococal nuclease Δ 131 Δ, using NMR methods with improved resolution. J Mol Biol 272, 9–20.PubMedCrossRefGoogle Scholar
  33. 33.
    Religa, T. L., Markson, J. S., Mayor, U., et al. (2005) Solution structure of a protein denatured state and folding intermediate. Nature 437, 1053–1056.PubMedCrossRefGoogle Scholar
  34. 34.
    Yang, A. S., Honig, B. (1993) On the pH dependence of protein stability. J Mol Biol 231, 459–474.PubMedCrossRefGoogle Scholar
  35. 35.
    Tanford, C. (1970) Potein denaturation. Part C. Theoretical models for the mechanism of denaturation. Advan Protein Chem 24, 1–95.CrossRefGoogle Scholar
  36. 36.
    Guzman-Casado, M., Parody-Morreale, A., Robic, S., et al. (2003) Energetic evidence for formation of a pH dependent hydrophobic cluster in the denatured state of Thermus thermophilus ribonuclease H. J Mol Biol 329, 731–743.PubMedCrossRefGoogle Scholar
  37. 37.
    Rohl, C. A., Scholtz, J. M., York, E. J., et al.. (1992) Kinetics of amide proton exchange in helical peptides of varying chain length. Interpretation by the Lifson–Roig equation. Biochemistry 31, 1263–1269.PubMedCrossRefGoogle Scholar
  38. 38.
    Marmorino, J. L., Auld, D., Betz, S. F., et al. (1993) Amide proton exchange rates of oxidized and reduced Saccharomyces cerevisiae iso-1-cytochrome c. Protein Sci 2, 1966–1974.PubMedCrossRefGoogle Scholar
  39. 39.
    Mori, S., van Zijl, P. C., Shortle, D. (1997) Measurement of water-amide proton exchange rates in the denatured state of staphylococcal nuclease by a magnetization transfer technique. Proteins 28, 325–332.PubMedCrossRefGoogle Scholar
  40. 40.
    Matsumura, M., Matthews, B.W. (1991) Stabilization of functional proteins by introduction of multiple disulfide bonds. Meth Enzymol 202, 336–356.PubMedCrossRefGoogle Scholar
  41. 41.
    Betz, S. F. (1993) Disulfide bonds and the stability of globular proteins. Protein Sci 10, 1551–1558.CrossRefGoogle Scholar
  42. 42.
    Anil, B., Song, B., Tang, Y., et al. (2004) Exploiting the right side of the Ramachandran plot: substitution of glycines by D-alanine can significantly increase protein stability. J Am Chem Soc 126, 13194–13195.CrossRefGoogle Scholar
  43. 43.
    Anil, B., Craig-Schapiro, R., Raleigh, D. P. (2006) Design of a hyperstable protein by rational consideration of unfolded state interactions. J Am Chem Soc 128, 3144–3145.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Jae-Hyun Cho
    • 1
  • Daniel P. Raleigh
    • 2
  1. 1.Department of Biochemistry and Molecular BiophysicsColumbia UniversityNew YorkUSA
  2. 2.Department of ChemistryStony Brook UniversityStony BrookUSA

Personalised recommendations