Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 Jul;75(1):471–476. doi: 10.1016/S0006-3495(98)77535-6

Pressure-induced subunit dissociation and unfolding of dimeric beta-lactoglobulin.

V L Valente-Mesquita 1, M M Botelho 1, S T Ferreira 1
PMCID: PMC1299720  PMID: 9649408

Abstract

Effects of hydrostatic pressure on dimeric beta-lactoglobulin A (beta-Lg) were investigated. Application of pressures of up to 3.5 kbar induced a significant red shift ( approximately 11 nm) and a 60% increase in intrinsic fluorescence emission of beta-Lg. These changes were very similar to those induced by guanidine hydrochloride, which caused subunit dissociation and unfolding of beta-Lg. A large hysteresis in the recovery of fluorescence parameters was observed upon decompression of beta-Lg. Pressure-induced dissociation and unfolding were not fully reversible, because of the formation of a nonnative intersubunit disulfide bond that hampered correct refolding of the dimer. Comparison between pressure dissociation/unfolding at 3 degrees C and 23 degrees C revealed a marked destabilization of beta-Lg at low temperature. The stability of beta-Lg toward pressure was significantly enhanced by 1 M NaCl, but not by glycerol (up to 20% v/v). These observations suggest that salt stabilization was not related to a general cosolvent effect, but may reflect charge screening. Interestingly, pressure-induced dissociation/unfolding was completely independent of beta-Lg concentration, in apparent violation of the law of mass action. Possible causes for this anomalous behavior are discussed.

Full Text

The Full Text of this article is available as a PDF (77.3 KB).

Selected References

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

  1. Aymard P., Durand D., Nicolai T. The effect of temperature and ionic strength on the dimerisation of beta-lactoglobulin. Int J Biol Macromol. 1996 Oct;19(3):213–221. doi: 10.1016/0141-8130(96)01130-0. [DOI] [PubMed] [Google Scholar]
  2. Brownlow S., Morais Cabral J. H., Cooper R., Flower D. R., Yewdall S. J., Polikarpov I., North A. C., Sawyer L. Bovine beta-lactoglobulin at 1.8 A resolution--still an enigmatic lipocalin. Structure. 1997 Apr 15;5(4):481–495. doi: 10.1016/s0969-2126(97)00205-0. [DOI] [PubMed] [Google Scholar]
  3. Dufour E., Genot C., Haertlé T. beta-Lactoglobulin binding properties during its folding changes studied by fluorescence spectroscopy. Biochim Biophys Acta. 1994 Mar 16;1205(1):105–112. doi: 10.1016/0167-4838(94)90098-1. [DOI] [PubMed] [Google Scholar]
  4. Erijman L., Lorimer G. H., Weber G. Reversible dissociation and conformational stability of dimeric ribulose bisphosphate carboxylase. Biochemistry. 1993 May 18;32(19):5187–5195. doi: 10.1021/bi00070a030. [DOI] [PubMed] [Google Scholar]
  5. Erijman L., Weber G. Oligomeric protein associations: transition from stochastic to deterministic equilibrium. Biochemistry. 1991 Feb 12;30(6):1595–1599. doi: 10.1021/bi00220a022. [DOI] [PubMed] [Google Scholar]
  6. Griko YuV, Kutyshenko V. P. Differences in the processes of beta-lactoglobulin cold and heat denaturations. Biophys J. 1994 Jul;67(1):356–363. doi: 10.1016/S0006-3495(94)80488-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Griko Y. V., Privalov P. L. Calorimetric study of the heat and cold denaturation of beta-lactoglobulin. Biochemistry. 1992 Sep 22;31(37):8810–8815. doi: 10.1021/bi00152a017. [DOI] [PubMed] [Google Scholar]
  8. Griko Y. V., Privalov P. L., Venyaminov S. Y., Kutyshenko V. P. Thermodynamic study of the apomyoglobin structure. J Mol Biol. 1988 Jul 5;202(1):127–138. doi: 10.1016/0022-2836(88)90525-6. [DOI] [PubMed] [Google Scholar]
  9. Gross M., Jaenicke R. Proteins under pressure. The influence of high hydrostatic pressure on structure, function and assembly of proteins and protein complexes. Eur J Biochem. 1994 Apr 15;221(2):617–630. doi: 10.1111/j.1432-1033.1994.tb18774.x. [DOI] [PubMed] [Google Scholar]
  10. Kella N. K., Kinsella J. E. Structural stability of beta-lactoglobulin in the presence of kosmotropic salts. A kinetic and thermodynamic study. Int J Pept Protein Res. 1988 Nov;32(5):396–405. doi: 10.1111/j.1399-3011.1988.tb01274.x. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Narayan M., Berliner L. J. Fatty acids and retinoids bind independently and simultaneously to beta-lactoglobulin. Biochemistry. 1997 Feb 18;36(7):1906–1911. doi: 10.1021/bi9621526. [DOI] [PubMed] [Google Scholar]
  13. Paladini A. A., Jr, Weber G. Pressure-induced reversible dissociation of enolase. Biochemistry. 1981 Apr 28;20(9):2587–2593. doi: 10.1021/bi00512a034. [DOI] [PubMed] [Google Scholar]
  14. Pedrosa C., Ferreira S. T. Deterministic pressure-induced dissociation of vicilin, the 7S storage globulin from pea seeds: effects of pH and cosolvents on oligomer stability. Biochemistry. 1994 Apr 5;33(13):4046–4055. doi: 10.1021/bi00179a033. [DOI] [PubMed] [Google Scholar]
  15. Privalov P. L., Griko YuV, Venyaminov SYu, Kutyshenko V. P. Cold denaturation of myoglobin. J Mol Biol. 1986 Aug 5;190(3):487–498. doi: 10.1016/0022-2836(86)90017-3. [DOI] [PubMed] [Google Scholar]
  16. Rietveld A. W., Ferreira S. T. Deterministic pressure dissociation and unfolding of triose phosphate isomerase: persistent heterogeneity of a protein dimer. Biochemistry. 1996 Jun 18;35(24):7743–7751. doi: 10.1021/bi952118b. [DOI] [PubMed] [Google Scholar]
  17. Rietveld A. W., Ferreira S. T. Kinetics and energetics of subunit dissociation/unfolding of TIM: the importance of oligomerization for conformational persistence and chemical stability of proteins. Biochemistry. 1998 Jan 20;37(3):933–937. doi: 10.1021/bi9721593. [DOI] [PubMed] [Google Scholar]
  18. Ruan K., Weber G. Dissociation of yeast hexokinase by hydrostatic pressure. Biochemistry. 1988 May 3;27(9):3295–3301. doi: 10.1021/bi00409a026. [DOI] [PubMed] [Google Scholar]
  19. Silva J. L., Miles E. W., Weber G. Pressure dissociation and conformational drift of the beta dimer of tryptophan synthase. Biochemistry. 1986 Sep 23;25(19):5780–5786. doi: 10.1021/bi00367a065. [DOI] [PubMed] [Google Scholar]
  20. Silva J. L., Weber G. Pressure stability of proteins. Annu Rev Phys Chem. 1993;44:89–113. doi: 10.1146/annurev.pc.44.100193.000513. [DOI] [PubMed] [Google Scholar]
  21. Subramaniam V., Steel D. G., Gafni A. In vitro renaturation of bovine beta-lactoglobulin A leads to a biologically active but incompletely refolded state. Protein Sci. 1996 Oct;5(10):2089–2094. doi: 10.1002/pro.5560051015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Tanaka N., Tsurui Y., Kobayashi I., Kunugi S. Modification of the single unpaired sulfhydryl group of beta-lactoglobulin under high pressure and the role of intermolecular S-S exchange in the pressure denaturation [single SH of beta-lactoglobulin and pressure denaturation]. Int J Biol Macromol. 1996 Jul;19(1):63–68. doi: 10.1016/0141-8130(96)01102-6. [DOI] [PubMed] [Google Scholar]
  24. Timasheff S. N. The control of protein stability and association by weak interactions with water: how do solvents affect these processes? Annu Rev Biophys Biomol Struct. 1993;22:67–97. doi: 10.1146/annurev.bb.22.060193.000435. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES