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. 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.

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Selected References

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  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]

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