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. 1999 Jan;8(1):106–112. doi: 10.1110/ps.8.1.106

On the non-respect of the thermodynamic cycle by DsbA variants.

M Moutiez 1, T V Burova 1, T Haertlé 1, E Quéméneur 1
PMCID: PMC2144097  PMID: 10210189

Abstract

The mechanism of the disulfide-bond forming enzyme DsbA depends on the very low pKa of a cysteine residue in its active-site and on the relative instability of the oxidized enzyme compared to the reduced one. A thermodynamic cycle has been used to correlate its redox properties to the difference in the free energies of folding (deltadeltaGred/ox) of the oxidized and reduced forms. However, the relation was proved unsatisfied for a number of DsbA variants. In this study, we investigate the thermodynamic and redox properties of a highly destabilized variant DsbA(P151A) (substitution of cis-Pro151 by an alanine) by the means of intrinsic tryptophan fluorescence and by high-sensitivity differential scanning calorimetry (HS-DSC). When the value of deltadeltaGred/ox obtained fluorimetrically for DsbA(P151A) does not correlate with the value expected from its redox potential, the value of deltadeltaGred/ox provided by HS-DSC are in perfect agreement with the predicted thermodynamic cycle for both wild-type and variant. HS-DSC data indicate that oxidized wild-type enzyme and the reduced forms of both wild-type and variant unfold according to a two-state mechanism. Oxidized DsbA(P151A) shows a deviation from two-state behavior that implies the loss of interdomain cooperativity in DsbA caused by Pro151 substitution. The presence of chaotrope in fluorimetric measurements could facilitate domain uncoupling so that the fluorescence probe (Trp76) does not reflect the whole unfolding process of DsbA(P151A) anymore. Thus, theoretical thermodynamic cycle is respected when an appropriate method is applied to DsbA unfolding under conditions in which protein domains still conserve their cooperativity.

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Selected References

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

  1. Bardwell J. C. Building bridges: disulphide bond formation in the cell. Mol Microbiol. 1994 Oct;14(2):199–205. doi: 10.1111/j.1365-2958.1994.tb01281.x. [DOI] [PubMed] [Google Scholar]
  2. Bromberg S., Dill K. A. Side-chain entropy and packing in proteins. Protein Sci. 1994 Jul;3(7):997–1009. doi: 10.1002/pro.5560030702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bulaj G., Kortemme T., Goldenberg D. P. Ionization-reactivity relationships for cysteine thiols in polypeptides. Biochemistry. 1998 Jun 23;37(25):8965–8972. doi: 10.1021/bi973101r. [DOI] [PubMed] [Google Scholar]
  4. Charbonnier J. B., Belin P., Moutiez M., Stura E. A., Quéméneur E. On the role of the cis-proline residue in the active site of DsbA. Protein Sci. 1999 Jan;8(1):96–105. doi: 10.1110/ps.8.1.96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chivers P. T., Prehoda K. E., Raines R. T. The CXXC motif: a rheostat in the active site. Biochemistry. 1997 Apr 8;36(14):4061–4066. doi: 10.1021/bi9628580. [DOI] [PubMed] [Google Scholar]
  6. Couprie J., Remerowski M. L., Bailleul A., Courçon M., Gilles N., Quéméneur E., Jamin N. Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy. Protein Sci. 1998 Oct;7(10):2065–2080. doi: 10.1002/pro.5560071003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Evans P. A., Dobson C. M., Kautz R. A., Hatfull G., Fox R. O. Proline isomerism in staphylococcal nuclease characterized by NMR and site-directed mutagenesis. Nature. 1987 Sep 17;329(6136):266–268. doi: 10.1038/329266a0. [DOI] [PubMed] [Google Scholar]
  8. Freedman R. B., Hirst T. R., Tuite M. F. Protein disulphide isomerase: building bridges in protein folding. Trends Biochem Sci. 1994 Aug;19(8):331–336. doi: 10.1016/0968-0004(94)90072-8. [DOI] [PubMed] [Google Scholar]
  9. Freire E., Murphy K. P., Sanchez-Ruiz J. M., Galisteo M. L., Privalov P. L. The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase. Biochemistry. 1992 Jan 14;31(1):250–256. doi: 10.1021/bi00116a034. [DOI] [PubMed] [Google Scholar]
  10. Freire E. Thermodynamics of partly folded intermediates in proteins. Annu Rev Biophys Biomol Struct. 1995;24:141–165. doi: 10.1146/annurev.bb.24.060195.001041. [DOI] [PubMed] [Google Scholar]
  11. Gleason F. K. Mutation of conserved residues in Escherichia coli thioredoxin: effects on stability and function. Protein Sci. 1992 May;1(5):609–616. doi: 10.1002/pro.5560010507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grauschopf U., Winther J. R., Korber P., Zander T., Dallinger P., Bardwell J. C. Why is DsbA such an oxidizing disulfide catalyst? Cell. 1995 Dec 15;83(6):947–955. doi: 10.1016/0092-8674(95)90210-4. [DOI] [PubMed] [Google Scholar]
  13. Guddat L. W., Bardwell J. C., Zander T., Martin J. L. The uncharged surface features surrounding the active site of Escherichia coli DsbA are conserved and are implicated in peptide binding. Protein Sci. 1997 Jun;6(6):1148–1156. doi: 10.1002/pro.5560060603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gómez J., Hilser V. J., Xie D., Freire E. The heat capacity of proteins. Proteins. 1995 Aug;22(4):404–412. doi: 10.1002/prot.340220410. [DOI] [PubMed] [Google Scholar]
  15. Hennecke J., Spleiss C., Glockshuber R. Influence of acidic residues and the kink in the active-site helix on the properties of the disulfide oxidoreductase DsbA. J Biol Chem. 1997 Jan 3;272(1):189–195. doi: 10.1074/jbc.272.1.189. [DOI] [PubMed] [Google Scholar]
  16. Hodel A., Rice L. M., Simonson T., Fox R. O., Brünger A. T. Proline cis-trans isomerization in staphylococcal nuclease: multi-substrate free energy perturbation calculations. Protein Sci. 1995 Apr;4(4):636–654. doi: 10.1002/pro.5560040405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jacobi A., Huber-Wunderlich M., Hennecke J., Glockshuber R. Elimination of all charged residues in the vicinity of the active-site helix of the disulfide oxidoreductase DsbA. Influence of electrostatic interactions on stability and redox properties. J Biol Chem. 1997 Aug 29;272(35):21692–21699. doi: 10.1074/jbc.272.35.21692. [DOI] [PubMed] [Google Scholar]
  18. Kelley R. F., Richards F. M. Replacement of proline-76 with alanine eliminates the slowest kinetic phase in thioredoxin folding. Biochemistry. 1987 Oct 20;26(21):6765–6774. doi: 10.1021/bi00395a028. [DOI] [PubMed] [Google Scholar]
  19. Kellis J. T., Jr, Nyberg K., Fersht A. R. Energetics of complementary side-chain packing in a protein hydrophobic core. Biochemistry. 1989 May 30;28(11):4914–4922. doi: 10.1021/bi00437a058. [DOI] [PubMed] [Google Scholar]
  20. Kortemme T., Darby N. J., Creighton T. E. Electrostatic interactions in the active site of the N-terminal thioredoxin-like domain of protein disulfide isomerase. Biochemistry. 1996 Nov 19;35(46):14503–14511. doi: 10.1021/bi9617724. [DOI] [PubMed] [Google Scholar]
  21. Lundström J., Krause G., Holmgren A. A Pro to His mutation in active site of thioredoxin increases its disulfide-isomerase activity 10-fold. New refolding systems for reduced or randomly oxidized ribonuclease. J Biol Chem. 1992 May 5;267(13):9047–9052. [PubMed] [Google Scholar]
  22. Makhatadze G. I., Privalov P. L. Energetics of protein structure. Adv Protein Chem. 1995;47:307–425. doi: 10.1016/s0065-3233(08)60548-3. [DOI] [PubMed] [Google Scholar]
  23. Martin J. L., Bardwell J. C., Kuriyan J. Crystal structure of the DsbA protein required for disulphide bond formation in vivo. Nature. 1993 Sep 30;365(6445):464–468. doi: 10.1038/365464a0. [DOI] [PubMed] [Google Scholar]
  24. Matthews B. W., Nicholson H., Becktel W. J. Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6663–6667. doi: 10.1073/pnas.84.19.6663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mayr L. M., Landt O., Hahn U., Schmid F. X. Stability and folding kinetics of ribonuclease T1 are strongly altered by the replacement of cis-proline 39 with alanine. J Mol Biol. 1993 Jun 5;231(3):897–912. doi: 10.1006/jmbi.1993.1336. [DOI] [PubMed] [Google Scholar]
  26. Myers J. K., Pace C. N., Scholtz J. M. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 1995 Oct;4(10):2138–2148. doi: 10.1002/pro.5560041020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nozaki Y. The preparation of guanidine hydrochloride. Methods Enzymol. 1972;26:43–50. doi: 10.1016/s0076-6879(72)26005-0. [DOI] [PubMed] [Google Scholar]
  28. Pace C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986;131:266–280. doi: 10.1016/0076-6879(86)31045-0. [DOI] [PubMed] [Google Scholar]
  29. Privalov P. L. Intermediate states in protein folding. J Mol Biol. 1996 May 24;258(5):707–725. doi: 10.1006/jmbi.1996.0280. [DOI] [PubMed] [Google Scholar]
  30. Privalov P. L. Stability of proteins: small globular proteins. Adv Protein Chem. 1979;33:167–241. doi: 10.1016/s0065-3233(08)60460-x. [DOI] [PubMed] [Google Scholar]
  31. Richards F. M., Lim W. A. An analysis of packing in the protein folding problem. Q Rev Biophys. 1993 Nov;26(4):423–498. doi: 10.1017/s0033583500002845. [DOI] [PubMed] [Google Scholar]
  32. Schultz D. A., Baldwin R. L. Cis proline mutants of ribonuclease A. I. Thermal stability. Protein Sci. 1992 Jul;1(7):910–916. doi: 10.1002/pro.5560010709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tweedy N. B., Nair S. K., Paterno S. A., Fierke C. A., Christianson D. W. Structure and energetics of a non-proline cis-peptidyl linkage in a proline-202-->alanine carbonic anhydrase II variant. Biochemistry. 1993 Oct 19;32(41):10944–10949. doi: 10.1021/bi00092a003. [DOI] [PubMed] [Google Scholar]
  34. Wunderlich M., Glockshuber R. Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli. Protein Sci. 1993 May;2(5):717–726. doi: 10.1002/pro.5560020503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wunderlich M., Jaenicke R., Glockshuber R. The redox properties of protein disulfide isomerase (DsbA) of Escherichia coli result from a tense conformation of its oxidized form. J Mol Biol. 1993 Oct 20;233(4):559–566. doi: 10.1006/jmbi.1993.1535. [DOI] [PubMed] [Google Scholar]
  36. Zapun A., Bardwell J. C., Creighton T. E. The reactive and destabilizing disulfide bond of DsbA, a protein required for protein disulfide bond formation in vivo. Biochemistry. 1993 May 18;32(19):5083–5092. doi: 10.1021/bi00070a016. [DOI] [PubMed] [Google Scholar]
  37. Zapun A., Cooper L., Creighton T. E. Replacement of the active-site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo. Biochemistry. 1994 Feb 22;33(7):1907–1914. doi: 10.1021/bi00173a038. [DOI] [PubMed] [Google Scholar]

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