Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Mar;8(3):625–634. doi: 10.1110/ps.8.3.625

Trifluoroethanol-induced conformational transitions of proteins: insights gained from the differences between alpha-lactalbumin and ribonuclease A.

K Gast 1, D Zirwer 1, M Müller-Frohne 1, G Damaschun 1
PMCID: PMC2144273  PMID: 10091665

Abstract

The trifluoroethanol (TFE)-induced structural changes of two proteins widely used in folding experiments, bovine alpha-lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: alpha-lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so-called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE-induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments.

Full Text

The Full Text of this article is available as a PDF (174.5 KB).

Selected References

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

  1. Alexandrescu A. T., Ng Y. L., Dobson C. M. Characterization of a trifluoroethanol-induced partially folded state of alpha-lactalbumin. J Mol Biol. 1994 Jan 14;235(2):587–599. doi: 10.1006/jmbi.1994.1015. [DOI] [PubMed] [Google Scholar]
  2. Arai M., Ikura T., Semisotnov G. V., Kihara H., Amemiya Y., Kuwajima K. Kinetic refolding of beta-lactoglobulin. Studies by synchrotron X-ray scattering, and circular dichroism, absorption and fluorescence spectroscopy. J Mol Biol. 1998 Jan 9;275(1):149–162. doi: 10.1006/jmbi.1997.1456. [DOI] [PubMed] [Google Scholar]
  3. Arai M., Kuwajima K. Rapid formation of a molten globule intermediate in refolding of alpha-lactalbumin. Fold Des. 1996;1(4):275–287. doi: 10.1016/s1359-0278(96)00041-7. [DOI] [PubMed] [Google Scholar]
  4. Balbach J., Forge V., van Nuland N. A., Winder S. L., Hore P. J., Dobson C. M. Following protein folding in real time using NMR spectroscopy. Nat Struct Biol. 1995 Oct;2(10):865–870. doi: 10.1038/nsb1095-865. [DOI] [PubMed] [Google Scholar]
  5. Bhattacharjya S., Balaram P. Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme. Protein Sci. 1997 May;6(5):1065–1073. doi: 10.1002/pro.5560060513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bodkin M. J., Goodfellow J. M. Hydrophobic solvation in aqueous trifluoroethanol solution. Biopolymers. 1996 Jul;39(1):43–50. doi: 10.1002/(SICI)1097-0282(199607)39:1%3C43::AID-BIP5%3E3.0.CO;2-V. [DOI] [PubMed] [Google Scholar]
  7. Buck M., Radford S. E., Dobson C. M. A partially folded state of hen egg white lysozyme in trifluoroethanol: structural characterization and implications for protein folding. Biochemistry. 1993 Jan 19;32(2):669–678. doi: 10.1021/bi00053a036. [DOI] [PubMed] [Google Scholar]
  8. Buck M., Schwalbe H., Dobson C. M. Characterization of conformational preferences in a partly folded protein by heteronuclear NMR spectroscopy: assignment and secondary structure analysis of hen egg-white lysozyme in trifluoroethanol. Biochemistry. 1995 Oct 10;34(40):13219–13232. doi: 10.1021/bi00040a038. [DOI] [PubMed] [Google Scholar]
  9. Bychkova V. E., Dujsekina A. E., Klenin S. I., Tiktopulo E. I., Uversky V. N., Ptitsyn O. B. Molten globule-like state of cytochrome c under conditions simulating those near the membrane surface. Biochemistry. 1996 May 14;35(19):6058–6063. doi: 10.1021/bi9522460. [DOI] [PubMed] [Google Scholar]
  10. Conio G., Patrone E., Brighetti S. The effect of aliphatic alcohols on the helix-coil transition of poly-L-ornithine and poly-L-glutamic acid. J Biol Chem. 1970 Jul 10;245(13):3335–3340. [PubMed] [Google Scholar]
  11. Cort J. R., Andersen N. H. Formation of a molten-globule-like state of myoglobin in aqueous hexafluoroisopropanol. Biochem Biophys Res Commun. 1997 Apr 28;233(3):687–691. doi: 10.1006/bbrc.1997.6524. [DOI] [PubMed] [Google Scholar]
  12. Damaschun G., Damaschun H., Gast K., Misselwitz R., Müller J. J., Pfeil W., Zirwer D. Cold denaturation-induced conformational changes in phosphoglycerate kinase from yeast. Biochemistry. 1993 Aug 3;32(30):7739–7746. doi: 10.1021/bi00081a019. [DOI] [PubMed] [Google Scholar]
  13. Fontana A., Zambonin M., De Filippis V., Bosco M., Polverino de Laureto P. Limited proteolysis of cytochrome c in trifluoroethanol. FEBS Lett. 1995 Apr 10;362(3):266–270. doi: 10.1016/0014-5793(95)00237-4. [DOI] [PubMed] [Google Scholar]
  14. Gast K., Chaffotte A. F., Zirwer D., Guillou Y., Mueller-Frohne M., Cadieux C., Hodges M., Damaschun G., Goldberg M. E. Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain. Protein Sci. 1997 Dec;6(12):2578–2588. doi: 10.1002/pro.5560061210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gast K., Damaschun G., Misselwitz R., Zirwer D. Application of dynamic light scattering to studies of protein folding kinetics. Eur Biophys J. 1992;21(5):357–362. doi: 10.1007/BF00188349. [DOI] [PubMed] [Google Scholar]
  16. Gast K., Zirwer D., Müller-Frohne M., Damaschun G. Compactness of the kinetic molten globule of bovine alpha-lactalbumin: a dynamic light scattering study. Protein Sci. 1998 Sep;7(9):2004–2011. doi: 10.1002/pro.5560070917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hamada D., Segawa S., Goto Y. Non-native alpha-helical intermediate in the refolding of beta-lactoglobulin, a predominantly beta-sheet protein. Nat Struct Biol. 1996 Oct;3(10):868–873. doi: 10.1038/nsb1096-868. [DOI] [PubMed] [Google Scholar]
  18. Hirota N., Mizuno K., Goto Y. Group additive contributions to the alcohol-induced alpha-helix formation of melittin: implication for the mechanism of the alcohol effects on proteins. J Mol Biol. 1998 Jan 16;275(2):365–378. doi: 10.1006/jmbi.1997.1468. [DOI] [PubMed] [Google Scholar]
  19. Hoshino M., Hagihara Y., Hamada D., Kataoka M., Goto Y. Trifluoroethanol-induced conformational transition of hen egg-white lysozyme studied by small-angle X-ray scattering. FEBS Lett. 1997 Oct 13;416(1):72–76. doi: 10.1016/s0014-5793(97)01172-1. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Jasanoff A., Fersht A. R. Quantitative determination of helical propensities from trifluoroethanol titration curves. Biochemistry. 1994 Mar 1;33(8):2129–2135. doi: 10.1021/bi00174a020. [DOI] [PubMed] [Google Scholar]
  22. Johnson W. C., Jr Protein secondary structure and circular dichroism: a practical guide. Proteins. 1990;7(3):205–214. doi: 10.1002/prot.340070302. [DOI] [PubMed] [Google Scholar]
  23. KRONMAN M. J., ANDREOTTI R. E. INTER- AND INTRAMOLECULAR INTERACTIONS OF ALPHA-LACTALBUMIN. I. THE APPARENT HETEROGENEITY AT ACID PH. Biochemistry. 1964 Aug;3:1145–1151. doi: 10.1021/bi00896a024. [DOI] [PubMed] [Google Scholar]
  24. Kamatari Y. O., Konno T., Kataoka M., Akasaka K. The methanol-induced globular and expanded denatured states of cytochrome c: a study by CD fluorescence, NMR and small-angle X-ray scattering. J Mol Biol. 1996 Jun 14;259(3):512–523. doi: 10.1006/jmbi.1996.0336. [DOI] [PubMed] [Google Scholar]
  25. Kiefhaber T., Baldwin R. L. Kinetics of hydrogen bond breakage in the process of unfolding of ribonuclease A measured by pulsed hydrogen exchange. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2657–2661. doi: 10.1073/pnas.92.7.2657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kuprin S., Gräslund A., Ehrenberg A., Koch M. H. Nonideality of water-hexafluoropropanol mixtures as studied by X-ray small angle scattering. Biochem Biophys Res Commun. 1995 Dec 26;217(3):1151–1156. doi: 10.1006/bbrc.1995.2889. [DOI] [PubMed] [Google Scholar]
  27. Kuwajima K., Hiraoka Y., Ikeguchi M., Sugai S. Comparison of the transient folding intermediates in lysozyme and alpha-lactalbumin. Biochemistry. 1985 Feb 12;24(4):874–881. doi: 10.1021/bi00325a010. [DOI] [PubMed] [Google Scholar]
  28. Kuwajima K., Mitani M., Sugai S. Characterization of the critical state in protein folding. Effects of guanidine hydrochloride and specific Ca2+ binding on the folding kinetics of alpha-lactalbumin. J Mol Biol. 1989 Apr 5;206(3):547–561. doi: 10.1016/0022-2836(89)90500-7. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Kuwajima K., Yamaya H., Sugai S. The burst-phase intermediate in the refolding of beta-lactoglobulin studied by stopped-flow circular dichroism and absorption spectroscopy. J Mol Biol. 1996 Dec 13;264(4):806–822. doi: 10.1006/jmbi.1996.0678. [DOI] [PubMed] [Google Scholar]
  31. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  32. Luo P., Baldwin R. L. Mechanism of helix induction by trifluoroethanol: a framework for extrapolating the helix-forming properties of peptides from trifluoroethanol/water mixtures back to water. Biochemistry. 1997 Jul 8;36(27):8413–8421. doi: 10.1021/bi9707133. [DOI] [PubMed] [Google Scholar]
  33. Makhatadze G. I., Privalov P. L. Protein interactions with urea and guanidinium chloride. A calorimetric study. J Mol Biol. 1992 Jul 20;226(2):491–505. doi: 10.1016/0022-2836(92)90963-k. [DOI] [PubMed] [Google Scholar]
  34. Monera O. D., Kay C. M., Hodges R. S. Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions. Protein Sci. 1994 Nov;3(11):1984–1991. doi: 10.1002/pro.5560031110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Muñoz V., Serrano L. Local versus nonlocal interactions in protein folding and stability--an experimentalist's point of view. Fold Des. 1996;1(4):R71–R77. doi: 10.1016/S1359-0278(96)00036-3. [DOI] [PubMed] [Google Scholar]
  36. Myers J. K., Pace C. N., Scholtz J. M. Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T1. Protein Sci. 1998 Feb;7(2):383–388. doi: 10.1002/pro.5560070219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nelson J. W., Kallenbach N. R. Stabilization of the ribonuclease S-peptide alpha-helix by trifluoroethanol. Proteins. 1986 Nov;1(3):211–217. doi: 10.1002/prot.340010303. [DOI] [PubMed] [Google Scholar]
  38. Nöppert A., Gast K., Müller-Frohne M., Zirwer D., Damaschun G. Reduced-denatured ribonuclease A is not in a compact state. FEBS Lett. 1996 Feb 12;380(1-2):179–182. doi: 10.1016/0014-5793(96)00048-8. [DOI] [PubMed] [Google Scholar]
  39. Nöppert A., Gast K., Zirwer D., Damaschun G. Initial hydrophobic collapse is not necessary for folding RNase A. Fold Des. 1998;3(3):213–221. doi: 10.1016/S1359-0278(98)00029-7. [DOI] [PubMed] [Google Scholar]
  40. Pace C. N., Laurents D. V., Thomson J. A. pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1. Biochemistry. 1990 Mar 13;29(10):2564–2572. doi: 10.1021/bi00462a019. [DOI] [PubMed] [Google Scholar]
  41. Polverino de Laureto P., De Filippis V., Di Bello M., Zambonin M., Fontana A. Probing the molten globule state of alpha-lactalbumin by limited proteolysis. Biochemistry. 1995 Oct 3;34(39):12596–12604. doi: 10.1021/bi00039a015. [DOI] [PubMed] [Google Scholar]
  42. Polverino de Laureto P., Scaramella E., De Filippis V., Bruix M., Rico M., Fontana A. Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol. Protein Sci. 1997 Apr;6(4):860–872. doi: 10.1002/pro.5560060413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ptitsyn O. B. Molten globule and protein folding. Adv Protein Chem. 1995;47:83–229. doi: 10.1016/s0065-3233(08)60546-x. [DOI] [PubMed] [Google Scholar]
  44. Rajan R., Balaram P. A model for the interaction of trifluoroethanol with peptides and proteins. Int J Pept Protein Res. 1996 Oct;48(4):328–336. doi: 10.1111/j.1399-3011.1996.tb00849.x. [DOI] [PubMed] [Google Scholar]
  45. Schmid F. X. Mechanism of folding of ribonuclease A. Slow refolding is a sequential reaction via structural intermediates. Biochemistry. 1983 Sep 27;22(20):4690–4696. doi: 10.1021/bi00289a013. [DOI] [PubMed] [Google Scholar]
  46. Schönbrunner N., Wey J., Engels J., Georg H., Kiefhaber T. Native-like beta-structure in a trifluoroethanol-induced partially folded state of the all-beta-sheet protein tendamistat. J Mol Biol. 1996 Jul 19;260(3):432–445. doi: 10.1006/jmbi.1996.0412. [DOI] [PubMed] [Google Scholar]
  47. Shiraki K., Nishikawa K., Goto Y. Trifluoroethanol-induced stabilization of the alpha-helical structure of beta-lactoglobulin: implication for non-hierarchical protein folding. J Mol Biol. 1995 Jan 13;245(2):180–194. doi: 10.1006/jmbi.1994.0015. [DOI] [PubMed] [Google Scholar]
  48. Sivaraman T., Kumar T. K., Yu C. Destabilisation of native tertiary structural interactions is linked to helix-induction by 2,2,2-trifluoroethanol in proteins. Int J Biol Macromol. 1996 Dec;19(4):235–239. doi: 10.1016/s0141-8130(96)01132-4. [DOI] [PubMed] [Google Scholar]
  49. Storrs R. W., Truckses D., Wemmer D. E. Helix propagation in trifluoroethanol solutions. Biopolymers. 1992 Dec;32(12):1695–1702. doi: 10.1002/bip.360321211. [DOI] [PubMed] [Google Scholar]
  50. Thomas P. D., Dill K. A. Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation. Protein Sci. 1993 Dec;2(12):2050–2065. doi: 10.1002/pro.5560021206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Udgaonkar J. B., Baldwin R. L. Early folding intermediate of ribonuclease A. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8197–8201. doi: 10.1073/pnas.87.21.8197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Udgaonkar J. B., Baldwin R. L. NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A. Nature. 1988 Oct 20;335(6192):694–699. doi: 10.1038/335694a0. [DOI] [PubMed] [Google Scholar]
  53. Udgaonkar J. B., Baldwin R. L. Nature of the early folding intermediate of ribonuclease A. Biochemistry. 1995 Mar 28;34(12):4088–4096. doi: 10.1021/bi00012a027. [DOI] [PubMed] [Google Scholar]

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

RESOURCES