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Increasing Enzyme Stability

  • C. Nick Pace
Part of the Industry-University Cooperative Chemistry Program Symposia book series (IUCC)

Abstract

Enzymes are active only when their polypeptide chain is folded into a unique globular conformation with a functional active site. In most naturally occurring enzymes, this globular conformation is only 5 to 10 kcal/mole more stable than unfolded, biologically inactive conformations (1). The conformational stability of a protein is defined as the free energy change for the reaction:
$$ \textup{folded}\;\textup{(native)}\;<----->\;\textup{unfolded\;(denatured)} $$
(1)
under ambient conditions, such as water at 25°C. The most convenient methods of estimating the conformational stability of a protein are urea (or guanidine hydrochloride (GdnHCl)) unfolding curves and thermal unfolding curves. Estimates of the conformational stability based on urea unfolding curves are designated ΔG(H2O), and estimates from thermal unfolding curves are designated ΔG(25°C). In the first part of this article, we describe how to measure ΔG(H2O) and ΔG(25°C).

Keywords

Disulfide Bond Free Energy Change Conformational Stability Guanidine Hydrochloride Bovine Pancreatic Trypsin Inhibitor 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    C.N. Pace, Conformational Stability of Globular Proteins, Trends Biochem. Sci. 15:14 (1990).PubMedCrossRefGoogle Scholar
  2. 2.
    C. Mitchinson, and J.A. Wells, Protein Engineering of Disulfide Bonds in Subtilisin BPN, Biochemistry 28:4807 (1989).PubMedCrossRefGoogle Scholar
  3. 3.
    M.W. Pantoliano, M. Whitlow, J.F. Wood, S.W. Dodd, K.D. Hardman, M.L. Rollence, and P.N. Bryan, Large Increases in General Stability for Subtilisin BPN through Incremental Changes in the Free Energy of Unfolding, Biochemistry 28: 7205 (1989).PubMedCrossRefGoogle Scholar
  4. 4.
    L. Regan, and W.F. DeGrado, Characterization of a Helical Protein Designed from First Principles, Science 241:976 (1988).PubMedCrossRefGoogle Scholar
  5. 5.
    C.N. Pace, B.A. Shirley, and J.A. Thomson, Measuring the Conformational Stability of a Protein, in “Protein Structure: a practical approach” T.E. Creighton, ed., pp. 311, IRL Press, Oxford.Google Scholar
  6. 6.
    J.A. Thomson, B.A. Shirley, G.R. Grimsley, and C.N. Pace, Conformational Stability and Mechanism of Folding of Ribonuclease T1, J. Biol. Chem. 264:11614 (1989).PubMedGoogle Scholar
  7. 7.
    M.M. Santoro, and D.W. Bolen, Unfolding Free Energy Changes Determined by the Linear Extrapolation Method. 1. Unfolding of Phenylmethanesulfonyl α-Chymotryspsin Using Different Denaturants, Biochemistry 27:8063 (1988).PubMedCrossRefGoogle Scholar
  8. 8.
    C.N. Pace, D.V. Laurents, and J.A. Thomson, PH Dependence of the Urea and Guanidine Hydrochloride Denaturation of Ribonuclease A and Ribonuclease T1, Biochemistry 29: in press.Google Scholar
  9. 9.
    W.M. Jackson, and J.F. Brandts, Thermodynamics of Protein Denaturation. A Calorimetric Study of the Reversible Denaturation of Chymostypsinogen and Conclusions Regarding the Accuracy of the Two-State Approximation, Biochemistry 9:2294 (1970).PubMedCrossRefGoogle Scholar
  10. 10.
    C.N. Pace, and C. Tanford, Thermodynamics of the Unfolding of β-Lactoglobulin in Aqueous Urea Solutions between 5 and 55°C, Biochemistry 7:198 (1968).PubMedCrossRefGoogle Scholar
  11. 11.
    P.L. Privalov, Stability of Proteins, Adv. Prot. Chem. 33:167 (1979).CrossRefGoogle Scholar
  12. 12.
    W.J. Becktel, and J.A. Schellman, Protein Stability Curves, Biopolvmers 26:1859 (1987).CrossRefGoogle Scholar
  13. 13.
    C.N. Pace, and D.V. Laurents, A New Method for Determining the Heat Capacity Change for Protein Folding, Biochemistry 28:2520 (1989).PubMedCrossRefGoogle Scholar
  14. 14.
    B. Chen., and J.A. Schellman, Low-Temperature Unfolding of a Mutant of Phage T4 Lysozyme. 1. Equilibrium Studies, Biochemistry 28:685 (1989).PubMedCrossRefGoogle Scholar
  15. 15.
    P.L. Privalov, Y. Gricko, S.Y. Venyaminov, and V.P. Kutyshenko Cold Denaturation of Myoglobin, J. Mol. Biol. 190:487 (1986).PubMedCrossRefGoogle Scholar
  16. 16.
    R.L. Baldwin, Temperature Dependence of the Hydrophobic Interaction, Proc. Nat. Acad. Sci. 83:8069 (1986).PubMedCrossRefGoogle Scholar
  17. 17.
    P.L. Privalov, and S.J. Gill, Stability of Protein Structure and Hydrophobic Interaction, Adv. Prot. Chem. 39:191 (1988).CrossRefGoogle Scholar
  18. 18.
    R.S. Spolar, J.-H. Ha, and T.M. Record, Hydrophobie Effect in Protein Folding and Other Noncovalent Processes Involving Proteins, Proc. Nat. Acad. Sci. 86:8382 (1989).PubMedCrossRefGoogle Scholar
  19. 19.
    D.J. States, T.E. Creighton, C.M. Dobson, and M. Karplus, Conformations of Intermediates in the Folding of the Pancreatic Trypsin Inhibitor, J-Mol. Biol. 195:731 (1987).PubMedCrossRefGoogle Scholar
  20. 20.
    R.E. Johnson, P. Adams, and J.A. Rupley, Thermodynamics of Protein Cross-Links, Biochemistry 17:1479 (1978).PubMedCrossRefGoogle Scholar
  21. 21.
    S.H. Lin, Y. Konishi, M.E. Denton, and H.A. Scheraga, Influence of an Extrinsic Cross-Link on the Folding Pathway of Ribonuclease A. Conformational and Thermodynamic Analysis of Cross-Linked (Lysine 7 — Lysine 41) Ribonuclease A, Biochemistry 23:5504 (1984).PubMedCrossRefGoogle Scholar
  22. 22.
    Y. Goto, M. Tsunenaga, Y. Kawata, and K. Hamaguchi, Conformation of the Constant Fragment of the Immunoglobulin Light Chain: Effect of Cleavage of the Polypeptide Chain and the Disulfide Bond, J. Biochem. 101:319 (1987).PubMedGoogle Scholar
  23. 23.
    C.N. Pace, G.R. Grimsley, J.A. Thomson, and B.J. Barnett, Conformational Stability of Ribonuclease Tl with Zero, One, and Two Intact Disulfide Bonds, J. Biol. Chem. 263: 11820 (1988).PubMedGoogle Scholar
  24. M. Matsumura, G. Signor, and B.W. Matthews, Substantial Increase in Protein Stability from Multiple Disulfide Bonds, Nature. 342:291 (1989).PubMedCrossRefGoogle Scholar
  25. 25.
    C.N. Pace, and G.R. Grimsley, Ribonuclease T1 is Stabilized by Cation and Anion Binding, Biochemistry 27:3242 (1988).PubMedCrossRefGoogle Scholar
  26. 26.
    M. Mitani, Y. Harushima, K. Kuwajima, M. Ikeguchi, M. Sugai, and S. Sugai, Innocuous Character of EDTA as Metal-Ion Buffers in Studying Ca2+ Binding by a-Lactalbumin, J. Biol. Chem 261:8824 (1986).PubMedGoogle Scholar
  27. 27.
    A.A. Pakula, and R.T. Sauer, Amino Acid Substitutions that Increase the Thermal Stability of the lambda Cro Protein, Proteins: Struc. Func. Gen. 5: 202 (1989).CrossRefGoogle Scholar
  28. 28.
    G. Das, D.R. Hickey, D. McLendon, G. McLendon, and F. Sherman, Dramatic Thermostabilization of Yeast Iso-1-Cytochrome C by an Asparagine → Isoleucine Replacement at Position 57, Proc. Nat. Acad. Sci. 86: 496 (1989).PubMedCrossRefGoogle Scholar
  29. 29.
    J.T. Kellis, K. Nyberg, and A.R. Fersht, Contribution of Hydrophobic Interactions to Protein Stability, Biochemistry 28:4914 (1988).CrossRefGoogle Scholar
  30. 30.
    K. Yutani, K. Ogasahara, T. Tsujita, and Y. Sugino, Dependence of Conformational Stability on Hydrophobicity of the Amino Acid Residue in a Series of Variant Proteins Substituted at a Unique Position of Tryptophan Synthase α Subunit, Proc. Nat. Acad. Sci. 84:4441 (1987).PubMedCrossRefGoogle Scholar
  31. 31.
    M. Matsumura, W.J. Becktel, M. Levitt, and B.W. Matthews, Stabilization of phage T4 Lysozyme by Engineered Disulfide Bonds, Proc. Nat. Acad. Sci. 86:6562 (1989).PubMedCrossRefGoogle Scholar
  32. 32.
    C.N. Pace, and T. McGrath, Substrate Stabilization of Lysozyme to Thermal and Guanidine Hydrochloride Denaturation, J. Biol. Chem. 255: 3862 (1980).PubMedGoogle Scholar
  33. 33.
    D. Kostrewa, H.-W. Choe, U. Heinemann, and W. Saenger, Crystal Structure of Guanosine-Free Ribonuclease T1 Complexed with Vanadate (V), Suggests Conformational Change upon Substrate Binding, Biochemistry 28:7592 (1989).PubMedCrossRefGoogle Scholar
  34. 34.
    K.R. Acharya, D.I. Stuart, N.P.C. Walker, M. Lewis, and D.C. Phillips, Refined Structure of Baboon a-Lactalbumin at 1.7A Resolution, J. Mol. Biol. 208:99 (1989).PubMedCrossRefGoogle Scholar
  35. 35.
    M.W. Pantoliano, M. Whitlow, J.F Wood, M.L. Rollence, B.C. Finzel, G.L. Gilliand, T.L. Poulus, and P.N. Bryan, The Engineering of Binding Affinity at Metal Binding Sites for the Stabilization of Proteins: Subtilisin as a Test Case, Biochemlatrv 27: 8311 (1988).CrossRefGoogle Scholar
  36. 36.
    R. Kuroki, Y. Taniyama, C. Seko, H. Nakmura, M. Kikuchi, and M. Ikehara, Design and Creation of a Ca2+ Binding Site in Human Lysozyme to Enhance Structural Stability, Proc. Nat. Acad. Sci. 86:6903 (1989).PubMedCrossRefGoogle Scholar
  37. 37.
    A.L. Swain, R.H. Kretsinger, and E.L. Amma, Restrained Least Squares Refinement of Native (Calcium) and Cadmium-substituted Carp Parvalbumin Using X-ray Crystallographic Data at 1.6Å Resolution, J_ Biol. Chem. 264:16620 (1989).PubMedGoogle Scholar
  38. 38.
    M. Fujinaga, T.J. Delbaere, G.D. Brayer, and M.N.G. James, Refined Structure of a-Lytic Protease at 1.7A Resolution, J. Mol. Biol. 183:479 (1985).CrossRefGoogle Scholar
  39. 39.
    J.W. Pflugrath, and F.A. Quiocho, Sulphate Sequestered in the Sulphate-Binding Protein of Salmonella Typhimurium is bound Solely by Hydrogen Bonds, Nature 314:257 (1985).PubMedCrossRefGoogle Scholar
  40. 40.
    T. Alber, Mutational Effects on Protein Stability, Ann. Rev. Biochem. 58:765 (1989).PubMedCrossRefGoogle Scholar
  41. 41.
    B.A. Shirley, P. Stanssens, J. Steyaert, and C.N. Pace, Conformational Stability and Activity of Ribonuclease T1 and Mutants: Gin 25 → Lys, Glu 58 → Ala, and the Double Mutant, J. Biol. Chem. 264:11621 (1989).PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1990

Authors and Affiliations

  • C. Nick Pace
    • 1
  1. 1.Biochemistry DepartmentTexas A&M UniversityCollege StationUSA

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