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. 1999 Dec;8(12):2765–2772. doi: 10.1110/ps.8.12.2765

Effect of hydrostatic pressure on unfolding of alpha-lactalbumin: volumetric equivalence of the molten globule and unfolded state.

Y Kobashigawa 1, M Sakurai 1, K Nitta 1
PMCID: PMC2144233  PMID: 10631994

Abstract

The effect of pressure on the unfolding of bovine alpha-lactalbumin was investigated by ultraviolet absorption methods. The change of molar volume associated with unfolding, deltaV, was measured in the presence or absence of guanidine hydrochloride at pH 7. The deltaV was estimated to be -63 cm3/mol in the absence of a chemical denaturant. While in the presence of guanidine hydrochloride (GuHCl), it was found that deltaV was -66 cm3/mol at 25 degrees C and was independent of the concentration of GuHCl, despite the fact that the molten globule fraction in the total unfolding product decreased with the increase of GuHCl concentration. The results indicate that the volume of alpha-lactalbumin only changes at the transition from a native to a molten globule state, and almost no volume change has been found during the transition from a molten globule to the unfolded state.

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Selected References

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  1. Alexandrescu A. T., Broadhurst R. W., Wormald C., Chyan C. L., Baum J., Dobson C. M. 1H-NMR assignments and local environments of aromatic residues in bovine, human and guinea pig variants of alpha-lactalbumin. Eur J Biochem. 1992 Dec 15;210(3):699–709. doi: 10.1111/j.1432-1033.1992.tb17471.x. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Anfinsen C. B. Principles that govern the folding of protein chains. Science. 1973 Jul 20;181(4096):223–230. doi: 10.1126/science.181.4096.223. [DOI] [PubMed] [Google Scholar]
  4. Barrick D., Baldwin R. L. Stein and Moore Award address. The molten globule intermediate of apomyoglobin and the process of protein folding. Protein Sci. 1993 Jun;2(6):869–876. doi: 10.1002/pro.5560020601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baum J., Dobson C. M., Evans P. A., Hanley C. Characterization of a partly folded protein by NMR methods: studies on the molten globule state of guinea pig alpha-lactalbumin. Biochemistry. 1989 Jan 10;28(1):7–13. doi: 10.1021/bi00427a002. [DOI] [PubMed] [Google Scholar]
  6. Brandts J. F., Oliveira R. J., Westort C. Thermodynamics of protein denaturation. Effect of pressu on the denaturation of ribonuclease A. Biochemistry. 1970 Feb 17;9(4):1038–1047. doi: 10.1021/bi00806a045. [DOI] [PubMed] [Google Scholar]
  7. Chalikian T. V., Breslauer K. J. Thermodynamic analysis of biomolecules: a volumetric approach. Curr Opin Struct Biol. 1998 Oct;8(5):657–664. doi: 10.1016/s0959-440x(98)80159-0. [DOI] [PubMed] [Google Scholar]
  8. Chalikian T. V., Gindikin V. S., Breslauer K. J. Volumetric characterizations of the native, molten globule and unfolded states of cytochrome c at acidic pH. J Mol Biol. 1995 Jul 7;250(2):291–306. doi: 10.1006/jmbi.1995.0377. [DOI] [PubMed] [Google Scholar]
  9. Chalikian T. V., Völker J., Anafi D., Breslauer K. J. The native and the heat-induced denatured states of alpha-chymotrypsinogen A: thermodynamic and spectroscopic studies. J Mol Biol. 1997 Nov 28;274(2):237–252. doi: 10.1006/jmbi.1997.1394. [DOI] [PubMed] [Google Scholar]
  10. Chaudhuri T. K., Horii K., Yoda T., Arai M., Nagata S., Terada T. P., Uchiyama H., Ikura T., Tsumoto K., Kataoka H. Effect of the extra n-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli. J Mol Biol. 1999 Jan 22;285(3):1179–1194. doi: 10.1006/jmbi.1998.2362. [DOI] [PubMed] [Google Scholar]
  11. Chyan C. L., Wormald C., Dobson C. M., Evans P. A., Baum J. Structure and stability of the molten globule state of guinea-pig alpha-lactalbumin: a hydrogen exchange study. Biochemistry. 1993 Jun 1;32(21):5681–5691. doi: 10.1021/bi00072a025. [DOI] [PubMed] [Google Scholar]
  12. Desmet J., Haezebrouck P., Van Cauwelaert F. Thermodynamic data on the binding of six M2(+)-ions to bovine, goat, and human alpha-lactalbumin. J Inorg Biochem. 1991 May 1;42(2):139–145. doi: 10.1016/0162-0134(91)80040-o. [DOI] [PubMed] [Google Scholar]
  13. Desmet J., Tieghem E., Van Dael H., Van Cauwelaert F. Thermodynamics of Mn(2+)-binding to goat alpha-lactalbumin. Eur Biophys J. 1991;20(5):263–268. doi: 10.1007/BF00450561. [DOI] [PubMed] [Google Scholar]
  14. Desmet J., Van Dael H., Van Cauwelaert F., Nitta K., Sugai S. Comparison of the binding of Ca2+ and Mn2+ to bovine alpha-lactalbumin and equine lysozyme. J Inorg Biochem. 1989 Nov;37(3):185–191. doi: 10.1016/0162-0134(89)80041-8. [DOI] [PubMed] [Google Scholar]
  15. Dolgikh D. A., Abaturov L. V., Bolotina I. A., Brazhnikov E. V., Bychkova V. E., Gilmanshin R. I., Lebedev YuO, Semisotnov G. V., Tiktopulo E. I., Ptitsyn O. B. Compact state of a protein molecule with pronounced small-scale mobility: bovine alpha-lactalbumin. Eur Biophys J. 1985;13(2):109–121. doi: 10.1007/BF00256531. [DOI] [PubMed] [Google Scholar]
  16. Foygel K., Spector S., Chatterjee S., Kahn P. C. Volume changes of the molten globule transitions of horse heart ferricytochrome c: a thermodynamic cycle. Protein Sci. 1995 Jul;4(7):1426–1429. doi: 10.1002/pro.5560040717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Frye K. J., Perman C. S., Royer C. A. Testing the correlation between delta A and delta V of protein unfolding using m value mutants of staphylococcal nuclease. Biochemistry. 1996 Aug 6;35(31):10234–10239. doi: 10.1021/bi960693p. [DOI] [PubMed] [Google Scholar]
  18. Goto Y., Aimoto S. Anion and pH-dependent conformational transition of an amphiphilic polypeptide. J Mol Biol. 1991 Mar 20;218(2):387–396. doi: 10.1016/0022-2836(91)90720-q. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Griko Y. V., Privalov P. L. Thermodynamic puzzle of apomyoglobin unfolding. J Mol Biol. 1994 Jan 28;235(4):1318–1325. doi: 10.1006/jmbi.1994.1085. [DOI] [PubMed] [Google Scholar]
  21. Hawley S. A. Reversible pressure--temperature denaturation of chymotrypsinogen. Biochemistry. 1971 Jun 22;10(13):2436–2442. doi: 10.1021/bi00789a002. [DOI] [PubMed] [Google Scholar]
  22. Hiraoka Y., Sugai S. Equilibrium and kinetic study of sodium- and potassium-induced conformational changes of apo-alpha-lactalbumin. Int J Pept Protein Res. 1985 Sep;26(3):252–261. doi: 10.1111/j.1399-3011.1985.tb03203.x. [DOI] [PubMed] [Google Scholar]
  23. Ikeguchi M., Kuwajima K., Sugai S. Ca2+-induced alteration in the unfolding behavior of alpha-lactalbumin. J Biochem. 1986 Apr;99(4):1191–1201. doi: 10.1093/oxfordjournals.jbchem.a135582. [DOI] [PubMed] [Google Scholar]
  24. Inaka K., Kuroki R., Kikuchi M., Matsushima M. Crystal structures of the apo- and holomutant human lysozymes with an introduced Ca2+ binding site. J Biol Chem. 1991 Nov 5;266(31):20666–20671. [PubMed] [Google Scholar]
  25. KAUZMANN W. Some factors in the interpretation of protein denaturation. Adv Protein Chem. 1959;14:1–63. doi: 10.1016/s0065-3233(08)60608-7. [DOI] [PubMed] [Google Scholar]
  26. Kharakoz D. P., Bychkova V. E. Molten globule of human alpha-lactalbumin: hydration, density, and compressibility of the interior. Biochemistry. 1997 Feb 18;36(7):1882–1890. doi: 10.1021/bi960264r. [DOI] [PubMed] [Google Scholar]
  27. Kuroda Y., Kidokoro S., Wada A. Thermodynamic characterization of cytochrome c at low pH. Observation of the molten globule state and of the cold denaturation process. J Mol Biol. 1992 Feb 20;223(4):1139–1153. doi: 10.1016/0022-2836(92)90265-l. [DOI] [PubMed] [Google Scholar]
  28. Kuroki R., Nitta K., Yutani K. Thermodynamic changes in the binding of Ca2+ to a mutant human lysozyme (D86/92). Enthalpy-entropy compensation observed upon Ca2+ binding to proteins. J Biol Chem. 1992 Dec 5;267(34):24297–24301. [PubMed] [Google Scholar]
  29. Kuroki R., Taniyama Y., Seko C., Nakamura H., Kikuchi M., Ikehara M. Design and creation of a Ca2+ binding site in human lysozyme to enhance structural stability. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6903–6907. doi: 10.1073/pnas.86.18.6903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kuwajima K. A folding model of alpha-lactalbumin deduced from the three-state denaturation mechanism. J Mol Biol. 1977 Aug 5;114(2):241–258. doi: 10.1016/0022-2836(77)90208-x. [DOI] [PubMed] [Google Scholar]
  31. Kuwajima K., Harushima Y., Sugai S. Influence of Ca2+ binding on the structure and stability of bovine alpha-lactalbumin studied by circular dichroism and nuclear magnetic resonance spectra. Int J Pept Protein Res. 1986 Jan;27(1):18–27. doi: 10.1111/j.1399-3011.1986.tb02761.x. [DOI] [PubMed] [Google Scholar]
  32. Kuwajima K., Nitta K., Yoneyama M., Sugai S. Three-state denaturation of alpha-lactalbumin by guanidine hydrochloride. J Mol Biol. 1976 Sep 15;106(2):359–373. doi: 10.1016/0022-2836(76)90091-7. [DOI] [PubMed] [Google Scholar]
  33. Lin T. Y. Lactose synthetase. Modification of carboxyl groups in alpha-lactalbumin. Biochemistry. 1970 Feb 17;9(4):984–995. doi: 10.1021/bi00806a038. [DOI] [PubMed] [Google Scholar]
  34. Livingstone J. R., Spolar R. S., Record M. T., Jr Contribution to the thermodynamics of protein folding from the reduction in water-accessible nonpolar surface area. Biochemistry. 1991 Apr 30;30(17):4237–4244. doi: 10.1021/bi00231a019. [DOI] [PubMed] [Google Scholar]
  35. Makhatadze G. I., Privalov P. L. Contribution of hydration to protein folding thermodynamics. I. The enthalpy of hydration. J Mol Biol. 1993 Jul 20;232(2):639–659. doi: 10.1006/jmbi.1993.1416. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Makino S., Noguchi H. Volume and sound velocity changes associated with coil- transition of poly(S-carboxymethyl L-cysteine) in aqueous solution. Biopolymers. 1971;10(7):1253–1260. doi: 10.1002/bip.360100714. [DOI] [PubMed] [Google Scholar]
  38. Mitani M., Harushima Y., Kuwajima K., Ikeguchi M., Sugai S. Innocuous character of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and EDTA as metal-ion buffers in studying Ca2+ binding by alpha-lactalbumin. J Biol Chem. 1986 Jul 5;261(19):8824–8829. [PubMed] [Google Scholar]
  39. Moerschell R. P., Hosokawa Y., Tsunasawa S., Sherman F. The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo. Processing of altered iso-1-cytochromes c created by oligonucleotide transformation. J Biol Chem. 1990 Nov 15;265(32):19638–19643. [PubMed] [Google Scholar]
  40. Nozaka M., Kuwajima K., Nitta K., Sugai S. Detection and characterization of the intermediate on the folding pathway of human alpha-lactalbumin. Biochemistry. 1978 Sep 5;17(18):3753–3758. doi: 10.1021/bi00611a013. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Privalov P. L., Gill S. J. Stability of protein structure and hydrophobic interaction. Adv Protein Chem. 1988;39:191–234. doi: 10.1016/s0065-3233(08)60377-0. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Sali A., Shakhnovich E., Karplus M. How does a protein fold? Nature. 1994 May 19;369(6477):248–251. doi: 10.1038/369248a0. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Semisotnov G. V., Rodionova N. A., Kutyshenko V. P., Ebert B., Blanck J., Ptitsyn O. B. Sequential mechanism of refolding of carbonic anhydrase B. FEBS Lett. 1987 Nov 16;224(1):9–13. doi: 10.1016/0014-5793(87)80412-x. [DOI] [PubMed] [Google Scholar]
  49. Semisotnov G. V., Rodionova N. A., Razgulyaev O. I., Uversky V. N., Gripas' A. F., Gilmanshin R. I. Study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers. 1991 Jan;31(1):119–128. doi: 10.1002/bip.360310111. [DOI] [PubMed] [Google Scholar]
  50. Spolar R. S., Livingstone J. R., Record M. T., Jr Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water. Biochemistry. 1992 Apr 28;31(16):3947–3955. doi: 10.1021/bi00131a009. [DOI] [PubMed] [Google Scholar]
  51. Sugai S., Yashiro H., Nitta K. Equilibrium and kinetics of the unfolding of alpha-lactalbumin by guanidine hydrochloride. Biochim Biophys Acta. 1973 Nov 11;328(1):35–41. doi: 10.1016/0005-2795(73)90327-9. [DOI] [PubMed] [Google Scholar]
  52. Tanaka N., Kunugi S. Effect of pressure on the deuterium exchange reaction of alpha-lactalbumin and beta-lactoglobulin. Int J Biol Macromol. 1996 Feb;18(1-2):33–39. doi: 10.1016/0141-8130(95)01053-x. [DOI] [PubMed] [Google Scholar]
  53. Vidugiris G. J., Royer C. A. Determination of the volume changes for pressure-induced transitions of apomyoglobin between the native, molten globule, and unfolded states. Biophys J. 1998 Jul;75(1):463–470. doi: 10.1016/S0006-3495(98)77534-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zipp A., Kauzmann W. Pressure denaturation of metmyoglobin. Biochemistry. 1973 Oct 9;12(21):4217–4228. doi: 10.1021/bi00745a028. [DOI] [PubMed] [Google Scholar]

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