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
. 1998 Sep;7(9):1930–1938. doi: 10.1002/pro.5560070908

Electrostatic interactions in the acid denaturation of alpha-lactalbumin determined by NMR.

S Kim 1, J Baum 1
PMCID: PMC2144173  PMID: 9761473

Abstract

alpha-Lactalbumin (alpha-LA) undergoes a pH-dependent unfolding from the native state to a partially unfolded state (the molten globule state). To understand the role of electrostatic interactions in protein denaturation, NMR and CD pH titration experiments are performed on guinea pig alpha-LA. Variation of pH over the range of 7.0 to 2.0 simultaneously leads to the acid denaturation of the protein and the titration of individual ionizable groups. The pH titrations are interpreted in the context of these coupled events, and indicate that acid denaturation in alpha-LA is a cooperative event that is triggered by the protonation of two ionizable residues. Our NMR results suggest that the critical electrostatic interactions that contribute to the denaturation of alpha-LA are concentrated in the calcium binding region of the protein.

Full Text

The Full Text of this article is available as a PDF (5.1 MB).

Selected References

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

  1. Acharya K. R., Ren J. S., Stuart D. I., Phillips D. C., Fenna R. E. Crystal structure of human alpha-lactalbumin at 1.7 A resolution. J Mol Biol. 1991 Sep 20;221(2):571–581. doi: 10.1016/0022-2836(91)80073-4. [DOI] [PubMed] [Google Scholar]
  2. Acharya K. R., Stuart D. I., Phillips D. C., McKenzie H. A., Teahan C. G. Models of the three-dimensional structures of echidna, horse, and pigeon lysozymes: calcium-binding lysozymes and their relationship with alpha-lactalbumins. J Protein Chem. 1994 Aug;13(6):569–584. doi: 10.1007/BF01901539. [DOI] [PubMed] [Google Scholar]
  3. Acharya K. R., Stuart D. I., Walker N. P., Lewis M., Phillips D. C. Refined structure of baboon alpha-lactalbumin at 1.7 A resolution. Comparison with C-type lysozyme. J Mol Biol. 1989 Jul 5;208(1):99–127. doi: 10.1016/0022-2836(89)90091-0. [DOI] [PubMed] [Google Scholar]
  4. Alexandrescu A. T., Evans P. A., Pitkeathly M., Baum J., Dobson C. M. Structure and dynamics of the acid-denatured molten globule state of alpha-lactalbumin: a two-dimensional NMR study. Biochemistry. 1993 Feb 23;32(7):1707–1718. doi: 10.1021/bi00058a003. [DOI] [PubMed] [Google Scholar]
  5. Anderson D. E., Becktel W. J., Dahlquist F. W. pH-induced denaturation of proteins: a single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme. Biochemistry. 1990 Mar 6;29(9):2403–2408. doi: 10.1021/bi00461a025. [DOI] [PubMed] [Google Scholar]
  6. Anderson P. J., Brooks C. L., Berliner L. J. Functional identification of calcium binding residues in bovine alpha-lactalbumin. Biochemistry. 1997 Sep 30;36(39):11648–11654. doi: 10.1021/bi9709598. [DOI] [PubMed] [Google Scholar]
  7. Bartik K., Redfield C., Dobson C. M. Measurement of the individual pKa values of acidic residues of hen and turkey lysozymes by two-dimensional 1H NMR. Biophys J. 1994 Apr;66(4):1180–1184. doi: 10.1016/S0006-3495(94)80900-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cocco M. J., Kao Y. H., Phillips A. T., Lecomte J. T. Structural comparison of apomyoglobin and metaquomyoglobin: pH titration of histidines by NMR spectroscopy. Biochemistry. 1992 Jul 21;31(28):6481–6491. doi: 10.1021/bi00143a018. [DOI] [PubMed] [Google Scholar]
  9. Dobson C. M. Protein folding. Solid evidence for molten globules. Curr Biol. 1994 Jul 1;4(7):636–640. doi: 10.1016/s0960-9822(00)00141-x. [DOI] [PubMed] [Google Scholar]
  10. Dolgikh D. A., Gilmanshin R. I., Brazhnikov E. V., Bychkova V. E., Semisotnov G. V., Venyaminov SYu, Ptitsyn O. B. Alpha-Lactalbumin: compact state with fluctuating tertiary structure? FEBS Lett. 1981 Dec 28;136(2):311–315. doi: 10.1016/0014-5793(81)80642-4. [DOI] [PubMed] [Google Scholar]
  11. Ewbank J. J., Creighton T. E. The molten globule protein conformation probed by disulphide bonds. Nature. 1991 Apr 11;350(6318):518–520. doi: 10.1038/350518a0. [DOI] [PubMed] [Google Scholar]
  12. Forman-Kay J. D., Clore G. M., Gronenborn A. M. Relationship between electrostatics and redox function in human thioredoxin: characterization of pH titration shifts using two-dimensional homo- and heteronuclear NMR. Biochemistry. 1992 Apr 7;31(13):3442–3452. doi: 10.1021/bi00128a019. [DOI] [PubMed] [Google Scholar]
  13. Griko Y. V., Freire E., Privalov G., van Dael H., Privalov P. L. The unfolding thermodynamics of c-type lysozymes: a calorimetric study of the heat denaturation of equine lysozyme. J Mol Biol. 1995 Sep 29;252(4):447–459. doi: 10.1006/jmbi.1995.0510. [DOI] [PubMed] [Google Scholar]
  14. Griko Y. V., Freire E., Privalov P. L. Energetics of the alpha-lactalbumin states: a calorimetric and statistical thermodynamic study. Biochemistry. 1994 Feb 22;33(7):1889–1899. doi: 10.1021/bi00173a036. [DOI] [PubMed] [Google Scholar]
  15. Haezebrouck P., De Baetselier A., Joniau M., Van Dael H., Rosenberg S., Hanssens I. Stability effects associated with the introduction of a partial and a complete Ca(2+)-binding site into human lysozyme. Protein Eng. 1993 Aug;6(6):643–649. doi: 10.1093/protein/6.6.643. [DOI] [PubMed] [Google Scholar]
  16. Hall L., Campbell P. N. Alpha-lactalbumin and related proteins: a versatile gene family with an interesting parentage. Essays Biochem. 1986;22:1–26. [PubMed] [Google Scholar]
  17. Haruyama H., Qian Y. Q., Wüthrich K. Static and transient hydrogen-bonding interactions in recombinant desulfatohirudin studied by 1H nuclear magnetic resonance measurements of amide proton exchange rates and pH-dependent chemical shifts. Biochemistry. 1989 May 16;28(10):4312–4317. doi: 10.1021/bi00436a028. [DOI] [PubMed] [Google Scholar]
  18. Kim S., Baum J., Anderson S. Production of correctly folded recombinant [13C, 15N]-enriched guinea pig [Val90]-alpha-lactalbumin. Protein Eng. 1997 Apr;10(4):455–462. doi: 10.1093/protein/10.4.455. [DOI] [PubMed] [Google Scholar]
  19. Kita N., Kuwajima K., Nitta K., Sugai S. Equilbrium and kinetics of the unfolding of alpha-lactalbumin by guanidine hydrochloride (II). Biochim Biophys Acta. 1976 Mar 18;427(1):350–358. doi: 10.1016/0005-2795(76)90310-x. [DOI] [PubMed] [Google Scholar]
  20. Kronman M. J., Sinha S. K., Brew K. Characteristics of the binding of Ca2+ and other divalent metal ions to bovine alpha-lactalbumin. J Biol Chem. 1981 Aug 25;256(16):8582–8587. [PubMed] [Google Scholar]
  21. Kuhlman B., Boice J. A., Wu W. J., Fairman R., Raleigh D. P. Calcium binding peptides from alpha-lactalbumin: implications for protein folding and stability. Biochemistry. 1997 Apr 15;36(15):4607–4615. doi: 10.1021/bi962901j. [DOI] [PubMed] [Google Scholar]
  22. Kuwajima K., Nitta K., Sugai S. Intramolecular perturbation of tryptophans induced by the protonation of ionizable groups in goat alpha-lactalbumin. Biochim Biophys Acta. 1980 Jun 26;623(2):389–401. doi: 10.1016/0005-2795(80)90268-8. [DOI] [PubMed] [Google Scholar]
  23. Kuwajima K., Ogawa Y., Sugai S. Role of the interaction between ionizable groups in the folding of bovine alpha-lactalbumin. J Biochem. 1981 Mar;89(3):759–770. doi: 10.1093/oxfordjournals.jbchem.a133256. [DOI] [PubMed] [Google Scholar]
  24. Kuwajima K. The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins. 1989;6(2):87–103. doi: 10.1002/prot.340060202. [DOI] [PubMed] [Google Scholar]
  25. Kuwajima K. The molten globule state of alpha-lactalbumin. FASEB J. 1996 Jan;10(1):102–109. doi: 10.1096/fasebj.10.1.8566530. [DOI] [PubMed] [Google Scholar]
  26. McKenzie H. A., White F. H., Jr Lysozyme and alpha-lactalbumin: structure, function, and interrelationships. Adv Protein Chem. 1991;41:173–315. doi: 10.1016/s0065-3233(08)60198-9. [DOI] [PubMed] [Google Scholar]
  27. Nitta K., Tsuge H., Iwamoto H. Comparative study of the stability of the folding intermediates of the calcium-binding lysozymes. Int J Pept Protein Res. 1993 Feb;41(2):118–123. doi: 10.1111/j.1399-3011.1993.tb00121.x. [DOI] [PubMed] [Google Scholar]
  28. Oda Y., Yamazaki T., Nagayama K., Kanaya S., Kuroda Y., Nakamura H. Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR. Biochemistry. 1994 May 3;33(17):5275–5284. doi: 10.1021/bi00183a034. [DOI] [PubMed] [Google Scholar]
  29. Oliveberg M., Arcus V. L., Fersht A. R. pKA values of carboxyl groups in the native and denatured states of barnase: the pKA values of the denatured state are on average 0.4 units lower than those of model compounds. Biochemistry. 1995 Jul 25;34(29):9424–9433. doi: 10.1021/bi00029a018. [DOI] [PubMed] [Google Scholar]
  30. Oliveberg M., Vuilleumier S., Fersht A. R. Thermodynamic study of the acid denaturation of barnase and its dependence on ionic strength: evidence for residual electrostatic interactions in the acid/thermally denatured state. Biochemistry. 1994 Jul 26;33(29):8826–8832. doi: 10.1021/bi00195a026. [DOI] [PubMed] [Google Scholar]
  31. Pardon E., Haezebrouck P., De Baetselier A., Hooke S. D., Fancourt K. T., Desmet J., Dobson C. M., Van Dael H., Joniau M. A Ca(2+)-binding chimera of human lysozyme and bovine alpha-lactalbumin that can form a molten globule. J Biol Chem. 1995 May 5;270(18):10514–10524. doi: 10.1074/jbc.270.18.10514. [DOI] [PubMed] [Google Scholar]
  32. Peng Z. Y., Kim P. S. A protein dissection study of a molten globule. Biochemistry. 1994 Mar 1;33(8):2136–2141. doi: 10.1021/bi00174a021. [DOI] [PubMed] [Google Scholar]
  33. Permyakov E. A., Morozova L. A., Burstein E. A. Cation binding effects on the pH, thermal and urea denaturation transitions in alpha-lactalbumin. Biophys Chem. 1985 Jan;21(1):21–31. doi: 10.1016/0301-4622(85)85003-1. [DOI] [PubMed] [Google Scholar]
  34. Permyakov E. A., Yarmolenko V. V., Kalinichenko L. P., Morozova L. A., Burstein E. A. Calcium binding to alpha-lactalbumin: structural rearrangement and association constant evaluation by means of intrinsic protein fluorescence changes. Biochem Biophys Res Commun. 1981 May 15;100(1):191–197. doi: 10.1016/s0006-291x(81)80081-2. [DOI] [PubMed] [Google Scholar]
  35. Pike A. C., Brew K., Acharya K. R. Crystal structures of guinea-pig, goat and bovine alpha-lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in lactose synthase. Structure. 1996 Jun 15;4(6):691–703. doi: 10.1016/s0969-2126(96)00075-5. [DOI] [PubMed] [Google Scholar]
  36. Ptitsyn O. B., Pain R. H., Semisotnov G. V., Zerovnik E., Razgulyaev O. I. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 1990 Mar 12;262(1):20–24. doi: 10.1016/0014-5793(90)80143-7. [DOI] [PubMed] [Google Scholar]
  37. Ptitsyn O. B. Structures of folding intermediates. Curr Opin Struct Biol. 1995 Feb;5(1):74–78. doi: 10.1016/0959-440x(95)80011-o. [DOI] [PubMed] [Google Scholar]
  38. Qin J., Clore G. M., Gronenborn A. M. Ionization equilibria for side-chain carboxyl groups in oxidized and reduced human thioredoxin and in the complex with its target peptide from the transcription factor NF kappa B. Biochemistry. 1996 Jan 9;35(1):7–13. doi: 10.1021/bi952299h. [DOI] [PubMed] [Google Scholar]
  39. Rao K. R., Brew K. Calcium regulates folding and disulfide-bond formation in alpha-lactalbumin. Biochem Biophys Res Commun. 1989 Sep 29;163(3):1390–1396. doi: 10.1016/0006-291x(89)91133-9. [DOI] [PubMed] [Google Scholar]
  40. Schulman B. A., Redfield C., Peng Z. Y., Dobson C. M., Kim P. S. Different subdomains are most protected from hydrogen exchange in the molten globule and native states of human alpha-lactalbumin. J Mol Biol. 1995 Nov 10;253(5):651–657. doi: 10.1006/jmbi.1995.0579. [DOI] [PubMed] [Google Scholar]
  41. Segawa T., Sugai S. Interactions of divalent metal ions with bovine, human, and goat alpha-lactalbumins. J Biochem. 1983 May;93(5):1321–1328. doi: 10.1093/oxfordjournals.jbchem.a134266. [DOI] [PubMed] [Google Scholar]
  42. Sommers P. B., Kronman M. J. Comparative fluorescence properties of bovine, goat, human and guinea pig alpha lactalbumin. Characterization of the environments of individual tryptophan residues in partially folded conformers. Biophys Chem. 1980 Apr;11(2):217–232. doi: 10.1016/0301-4622(80)80024-x. [DOI] [PubMed] [Google Scholar]
  43. Sugai S., Ikeguchi M. Conformational comparison between alpha-lactalbumin and lysozyme. Adv Biophys. 1994;30:37–84. doi: 10.1016/0065-227x(94)90010-8. [DOI] [PubMed] [Google Scholar]
  44. Szyperski T., Antuch W., Schick M., Betz A., Stone S. R., Wüthrich K. Transient hydrogen bonds identified on the surface of the NMR solution structure of Hirudin. Biochemistry. 1994 Aug 9;33(31):9303–9310. doi: 10.1021/bi00197a034. [DOI] [PubMed] [Google Scholar]
  45. Tanford C. Protein denaturation. C. Theoretical models for the mechanism of denaturation. Adv Protein Chem. 1970;24:1–95. [PubMed] [Google Scholar]
  46. Tanford C. Protein denaturation. Adv Protein Chem. 1968;23:121–282. doi: 10.1016/s0065-3233(08)60401-5. [DOI] [PubMed] [Google Scholar]
  47. Wu L. C., Peng Z. Y., Kim P. S. Bipartite structure of the alpha-lactalbumin molten globule. Nat Struct Biol. 1995 Apr;2(4):281–286. doi: 10.1038/nsb0495-281. [DOI] [PubMed] [Google Scholar]
  48. Wu L. C., Schulman B. A., Peng Z. Y., Kim P. S. Disulfide determinants of calcium-induced packing in alpha-lactalbumin. Biochemistry. 1996 Jan 23;35(3):859–863. doi: 10.1021/bi951408p. [DOI] [PubMed] [Google Scholar]
  49. Yang A. S., Honig B. On the pH dependence of protein stability. J Mol Biol. 1993 May 20;231(2):459–474. doi: 10.1006/jmbi.1993.1294. [DOI] [PubMed] [Google Scholar]

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

RESOURCES