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Entropic effects of disulphide bonds on protein stability

Nature Structural Biology volume 1, pages 434–438 (1994)Cite this article

Abstract

To measure the thermodynamic consequences of the reduction in the number of polypeptide-chain conformations that accompanies protein folding, we developed a method called loop permutation analysis. In this approach, the stabilizing contributions of three engineered disulphide bonds were compared in extended and circularly permutated mutants of phage T4 lysozyme. The observed differences in disulphide contributions, although qualitatively consistent with theoretical estimates, were not solely proportional to the differences in loop length. These findings suggest that in addition to the length of the chain, the polypeptide sequence may influence the energetic consequences of conformational restrictions.

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References

  1. Dill, K.A. Theory for the folding and stability of globular proteins., Biochemistry 24, 1501–1509 (1985).

    Article  CAS  PubMed  Google Scholar 

  2. Flory, P. Theory of elastic mechanisms in fibrous proteins. J. Am. chem. Soc. 28, 5222–5235 (1956).

    Article  Google Scholar 

  3. Schellman, J.A. The stability of hydrogen-bonded peptide structures in aqueous solution. C.r. Trav. Lab. Carlsberg Ser. Chim 29, 230–259 (1955).

    CAS  Google Scholar 

  4. Poland, D.C. & Scheraga, H.A. Statistical mechanics of noncovalent bonds in polyamino acids. VIII Covalent loops in proteins. Biopolymers 3, 379–399 (1965).

    Article  CAS  Google Scholar 

  5. Lin, S.H., Konishi, Y., Denton, M.E. & Scheraga, H.A. Influence of an extrinsic cross-link on the folding pathway of ribonuclease A. Conformational and thermodynamic analysis of cross-linked (Lysine7-Lysine41) ribonuclease. Biochemistry 23, 5504–5512 (1984).

    Article  CAS  PubMed  Google Scholar 

  6. Pace, C.N., Grimsley, G.R., Thomson, J.A. & Barnett, B.J. Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds. J. biol. Chem. 263, 11820–11825 (1988).

    CAS  PubMed  Google Scholar 

  7. Chan, H.S. & Dill, K.A. Intrachain loops in polymers: Effects of excluded volume. J. chem. Phys. 90, 492–509 (1989).

    Article  CAS  Google Scholar 

  8. Chan, H.S. & Dill, K.A. The effects of internal constraints on the configurations of chain molecules. J. chem. Phys. 92, 3118–3135 (1990).

    Article  CAS  Google Scholar 

  9. Cooper, A., Eyles, S.J., Radford, S.E. & Dobson, C.M. Thermodynamic consequences of the removal of a disulphide bridge from hen lysozyme. J. molec. Biol. 225, 939–943 (1992).

    Article  CAS  PubMed  Google Scholar 

  10. Betz, S.F. Disulfide bonds and the stability of globular proteins. Prot. Sci. 2, 1551–1558 (1993).

    Article  CAS  Google Scholar 

  11. Doig, A.D. & Williams, D.H. Is the hydrophobic effect stabilizing or destabilizing in proteins? The contribution of disulphide bonds to protein stability. J. molec. Biol. 217, 389–398 (1991).

    Article  CAS  PubMed  Google Scholar 

  12. Creighton, T.E. An empirical approach to protein conformation stability and flexibility. Biopolymers 22, 49–59 (1983).

    Article  CAS  PubMed  Google Scholar 

  13. Goldenberg, D.P. Dissecting the roles of individual interactions in protein stability: Lessons from a circularized protein. J. cell. Biochem. 29, 321–335 (1985).

    Article  CAS  PubMed  Google Scholar 

  14. Creighton, T.E. On the relevance of non-random polypeptide conformations for protein folding. Biophys. Chem. 31, 155–162 (1988).

    Article  CAS  PubMed  Google Scholar 

  15. Zhang, T., Bertelson, E., Benvegnu, N. & Alber, T. Circular permutation of T4 lysozyme. Biochemistry 32, 12311–12318 (1993).

    Article  CAS  PubMed  Google Scholar 

  16. Matsumura, M., Becktel, W.J., Levitt, M. & Matthews, B.W. Stabilization of phage T4 lysozyme by engineered disulfide bonds. Proc. natn. Acad. Sci. U.S.A. 86, 6562–6566 (1989).

    Article  CAS  Google Scholar 

  17. Matsumura, M., Signor, G. & Matthews, B.W. Substantial increase of protein stability by multiple disulfide bonds. Nature 342, 291–293 (1989).

    Article  CAS  PubMed  Google Scholar 

  18. Pjura, P.E., Matsumura, M., Wozniak, J.A. & Matthews, B.W. Structure of a thermostable disulfide-bridge mutant of phage T4 lysozyme shows that an engineered cross-link in flexible region does not increase the rigidity of the folded protein. Biochemistry 29, 2592–2598 (1990).

    Article  CAS  PubMed  Google Scholar 

  19. Matthews, B.W., Nicholson, H. & Becktel, W.J. Enhanced protein thermostability from site-directed mutation that decreases the entropy of unfolding. Proc. natn. Acad. Sci. U.S.A. 84, 6663–6667 (1987).

    Article  CAS  Google Scholar 

  20. Villafranca, J.E. et al. Directed mutagenesis of dihydrofolate reductase. Science 222, 782–788 (1983).

    Article  CAS  PubMed  Google Scholar 

  21. Perry, L.J. & Wetzel, R. Disulfide bond engineered into T4 lysozyme: stabilization of the protein toward thermal inactivation. Science 226, 555–557 (1984).

    Article  CAS  PubMed  Google Scholar 

  22. Ueda, T., Yamada, H., Hirata, M. & Imoto, T. An intramolecular cross-linkage of lysozyme. Formation of cross-links between lysine-1 and histidine-15 with bis(bromoacetamide) derivatives by a two-stage reaction procedure and properties of the resulting derivatives. Biochemistry 24, 6316–6322, (1985).

    Article  CAS  PubMed  Google Scholar 

  23. Sauer, R.T. et al. An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of λ repressor. Biochemistry 24, 5992–5998, (1984).

    Google Scholar 

  24. Wells, J.A. & Powers, D.B. In vivo formation and stability of engineered disulfide bonds in subtilisin. J. biol. Chem. 261, 6564–6570, (1986).

    CAS  PubMed  Google Scholar 

  25. Pantoliano, M.W. et al. Protein engineering of subtilisin BPN': Enhanced stabilization through the introduction of two cysteines to form a disulfide bond. Biochemistry 26, 2077–2082 (1987).

    Article  CAS  PubMed  Google Scholar 

  26. Villafranca, J.E., Howell, E.E., Oatley, S.J., Xuong, N.-h. & Kraut, J. An engineered disulfide bond on dihydrofolate reductase. Biochemistry 26, 2077–2082 (1987).

    Article  Google Scholar 

  27. Betz, S.F. & Pielak, G.J. Introduction of a disulfide bond into cytochrome c stabilizes a compact denatured state. Biochemistry 31, 12337–12344 (1992).

    Article  CAS  PubMed  Google Scholar 

  28. Eder, J. & Wilmanns, M. Protein engineering of a disulfide bond in a beta/alpha-barrel protein. Biochemistry 31, 4437–4444 (1992).

    Article  CAS  PubMed  Google Scholar 

  29. Goldenberg, D.P. & Creighton, T.E. Circular and circularly permuted forms of bovine pancreatic trypsin inhibitor. J. molec. Biol. 165, 407–413 (1983).

    Article  CAS  PubMed  Google Scholar 

  30. Luger, K., Hommel, U., Herold, M., Hofsteenge, J. & Kirschner, K. Correct folding of circularly permuted variants of a ba barrel enzyme in vivo. Science 243, 206–210 (1989).

    Article  CAS  PubMed  Google Scholar 

  31. Buchwalder, A., Szadkowski, H. & Kirschner, K. A fully active variant of dihydrofolate reductase with a circularly permuted sequence. Biochemistry 31, 1621–1630 (1992).

    Article  CAS  PubMed  Google Scholar 

  32. Yang, Y.R. & Schachman, H.K. Aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains. Proc. natn. Acad. Sci. U.S.A., (1993).

  33. Kunkel, T.A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. natn. Acad. Sci. U.S.A. 82, 488–492 (1985).

    Article  CAS  Google Scholar 

  34. Muchmore, D.C., McIntosh, L.P., Russell, C.B., Anderson, D.E. & Dahlquist, F.W. Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Meth. Enzymol. 177, 43–73 (1989).

    Google Scholar 

  35. Becktel, W.J. & Schellman, J.A. Protein stability curves. Biopolymers 26, 1859–1877 (1987).

    Article  CAS  PubMed  Google Scholar 

  36. Burington, R.S. & May, D.C. Handbook of Probability and Statistics with Tables, (McGraw-Hill, New York, 1967).

    Google Scholar 

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Author notes

  1. Eric Bertelsen

    Present address: Institute of Molecular Biology, University of Oregon, Eugene, OR, 97403, U.S.A.

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, U.S.A.

    Tao Zhang & Tom Alber

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Zhang, T., Bertelsen, E. & Alber, T. Entropic effects of disulphide bonds on protein stability. Nat Struct Mol Biol 1, 434–438 (1994). https://doi.org/10.1038/nsb0794-434

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