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Transient non-native secondary structures during the refolding of α-lactalbumin detected by infrared spectroscopy

Nature Structural Biology volume 7, pages 78–86 (2000)Cite this article

Abstract

Stopped-flow Fourier-transform infrared spectroscopy (SF-FTIR) was used to identify native as well as non-native secondary structures during the refolding of the calcium-binding protein α-lactalbumin. Infrared absorbance spectra were recorded in real time after a pH jump induced refolding of the protein. In the presence of calcium, the refolding is fast with concerted appearance of secondary structures; in its absence, folding is much slower and intricate, with transient formation and disappearance of non-native β-sheet. The possibility of detecting native as well as non-native structures at the same time is especially valuable in providing insight into the complexity of the refolding process of a protein.

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Figure 1: Molscript49 representation of the structure of bovine α-lactalbumin. The diagram is generated from the X-ray structure (1HFZ).
Figure 2: IR spectra of α-lactalbumin in the native states and the A-state.
Figure 3: Comparison between the A-state and the unfolded state in 8 M urea.
Figure 4: Refolding kinetics of α-lactalbumin from the A-state to the native holo- and apo-forms at 3 °C.
Figure 5: Kinetics of the absorbance changes during the refolding of α-lactalbumin at 3 °C.
Figure 6: Comparison between the refolding kinetics of the apo-form at 3 °C and 8 °C.
Figure 7: Effect of protein concentration on the refolding kinetics in D2O.

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References

  1. Levinthal, C. Are there pathways for protein folding? J. Chim. Phys. 65, 44–45 (1968).

    Article  Google Scholar 

  2. Plaxco, K.W. & Dobson, C.M. Time-resolved biophysical methods in the study of protein folding. Curr. Opin. Struct. Biol. 6, 630–636 (1996).

    Article  CAS  Google Scholar 

  3. Van Nuland, N.A.J., Forge, V. Balbach, J. & Dobson, C.M. Real-time NMR studies of protein folding. Acc. Chem. Res. 31, 773–778 (1998).

    Article  CAS  Google Scholar 

  4. Englander, S.W. & Mayne, L. Protein folding studied using hydrogen-exchange labeling and two dimensional NMR. Annu. Rev. Biophys. Biomol. Struct. 21, 243– 265 (1992).

    Article  CAS  Google Scholar 

  5. Ptitsyn, O.B. Structures of folding intermediates. Curr. Opin. Struct. Biol. 5, 74–78 (1995 ).

    Article  CAS  Google Scholar 

  6. Shortle, D.R. Structural analysis of non-native states of proteins by NMR. Curr. Opin. Struct. Biol. 6, 24–30 (1996).

    Article  CAS  Google Scholar 

  7. Dyson, H.J. & Wright, P.E. Equilibrium NMR studies of unfolded and partially folded proteins. Nature Struct. Biol. 5(Suppl), 499–503 ( 1998).

    Article  CAS  Google Scholar 

  8. Dill, K.A. & Chan, H.S. From Levinthal to pathways to funnels . Nature Struct. Biol. 4, 10– 19 (1997).

    Article  CAS  Google Scholar 

  9. Dobson, C.M., Sali, A. & Karplus, M. Protein folding: a perspective from theory and experiment . Angew. Chem. Int. Ed. 37, 868– 893 (1998).

    Article  Google Scholar 

  10. Shiraki, K., Nishikawa, K. & Goto, Y. Trifluoroethanol-induced stabilization of the α-helical structure of β-lactoglobulin: implication for non-hierarchical protein folding. J. Mol. Biol. 245, 180– 194 (1995).

    Article  CAS  Google Scholar 

  11. Schonbrunner, N., Wey, J., Engels, J., Georg, H. & Kiefhaber, T. Native-like β-structure in a trifluoroethanol-induced partially folded state of the all-β-sheet protein tendamistat. J. Mol. Biol. 260, 432–445 (1996).

    Article  CAS  Google Scholar 

  12. Cordier-Ochsenbein, F. et al. Exploring the folding pathways of annexin I, a multidomain protein. I. Non-native structures stabilize the partially folded state of the isolated domain 2 of annexin I. J. Mol. Biol. 279 , 1163–1175 (1998).

    Article  CAS  Google Scholar 

  13. Guijarro, J.I., Jackson, M., Chaffotte, A.F., Delepierre, M., Mantsch, H.H. & Goldberg, M.E. Protein folding intermediates with rapidly exchangeable amide protons contain authentic hydrogen-bonded secondary structures. Biochemistry 34, 2998–3008 (1995).

    Article  CAS  Google Scholar 

  14. Hamada, D., Segawa, S. & Goto, Y. Non-native α-helical intermediate in the refolding of β-lactoglobulin, a predominantly β-sheet protein. Nature Struct. Biol. 3, 868–873 (1996).

    Article  CAS  Google Scholar 

  15. Arai, M., Ikura, T., Semisotnov, G.V., Kihara, H., Amemiya, Y. & Kuwajima, K. Kinetic Refolding of β-Lactoglobulin. Studies by Synchrotron X-ray Scattering, and Circular Dichroism, Absorption and Fluorescence Spectroscopy. J. Mol. Biol. 275, 149– 62 (1998).

    Article  CAS  Google Scholar 

  16. Surewicz, W.E., Mantsch, H.H. & Chapman, D. Determination of protein secondary structure by Fourier transform infrared spectroscopy: a critical assessment. Biochemistry 32, 389–394 ( 1993)

    Article  CAS  Google Scholar 

  17. Backmann, J., Fabian, H. & Naumann, D. Temperature-jump-induced refolding of ribonuclease A: a time-resolved FTIR spectroscopic study. FEBS. Lett. 364, 175–178 (1995).

    Article  CAS  Google Scholar 

  18. Troullier, A., Gerwert, K., & Dupont, Y. A time-resolved Fourier transformed infrared difference spectroscopy study of the sarcoplasmic reticulum Ca(2+)-ATPase: kinetics of the high-affinity calcium binding at low temperature. Biophys. J. 71, 2970–2983 ( 1996).

    Article  CAS  Google Scholar 

  19. Panick, G., Malessa, R., Winter, R., Rapp, G., Frye, K.J. & Royer, C.A. Structural characterization of the pressure-denatured state and unfolding/refolding kinetics of staphylococcal nuclease by synchrotron small-angle X-ray scattering and Fourier-transform infrared spectroscopy. J. Mol. Biol. 275, 389–402 (1998).

    Article  CAS  Google Scholar 

  20. Reinstadler, D., Fabian, H. & Naumann, D. New structural insights into the refolding of ribonuclease T1 as seen by time-resolved Fourier-transform infrared spectroscopy . Proteins 34, 303–316 (1999).

    Article  CAS  Google Scholar 

  21. Gerwert, K. Molecular reaction mechanisms of proteins as monitored by time-resolved FTIR spectroscopy. Curr. Opin. Struct. Biol. 3, 769–773 (1993).

    Article  CAS  Google Scholar 

  22. White, A.J., Drabble, K. & Wharton, C.W. A stopped-flow apparatus for infrared spectroscopy of aqueous solutions. Biochem. J. 306, 843 –849 (1995).

    Article  CAS  Google Scholar 

  23. Reinstadler, D., Fabian, H., Backmann, J. & Naumann, D. Refolding of thermally and urea-denatured ribonuclease A monitored by time-resolved FTIR spectroscopy . Biochemistry 35, 15822– 15830 (1996).

    Article  CAS  Google Scholar 

  24. Alexandrescu, A.T., Evans, P.A., Pitkeathly, M., Baum, J. & Dobson, C.M. Structure and dynamics of the acid-denatured molten globule state of α-lactalbumin: a two-dimensional NMR study. Biochemistry 32, 1707–1718 (1993).

    Article  CAS  Google Scholar 

  25. 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 α-lactalbumin . J. Mol. Biol. 253, 651– 657 (1995)

    Article  CAS  Google Scholar 

  26. Kuwajima, K. The molten globule state of α-lactalbumin. FASEB J. 10, 102–109 (1996).

    Article  CAS  Google Scholar 

  27. Kataoka, M., Kuwajima, K., Tokunaga, F. & Goto, Y. Structural characterization of the molten globule α-lactalbumin by solution X-ray scattering. Protein Sci. 6, 422– 430 (1997).

    Article  CAS  Google Scholar 

  28. Schulman, B.A., Kim, P.S., Dobson, C.M. & Redfield, C. A residue-specific NMR view of the non-cooperative unfolding of a molten globule. Nature Struct. Biol. 4, 630–634 (1997).

    Article  CAS  Google Scholar 

  29. Balbach, J., Forge, V., Lau, W.S., Jones, J.A., van Nuland, N.A.J. & Dobson, C.M. Detection of residue contacts in a protein folding intermediate. Proc. Natl. Acad. Sci. USA 94, 7182– 7185 (1997).

    Article  CAS  Google Scholar 

  30. Wu, L.C., Peng, Z.Y. & Kim, P.S. Bipartite structure of the α-lactalbumin molten globule. Nature Struct. Biol. 2, 281– 286 (1995).

    Article  CAS  Google Scholar 

  31. Chung, E.W. et al. Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR. Protein Sci. 6, 1316–1324 (1997).

    Article  CAS  Google Scholar 

  32. Wu, L.C., Schulman, B.A., Peng, Z.Y. & Kim, P.S. Disulfide determinants of calcium-induced packing in α-lactalbumin. Biochemistry 35, 859–863 (1996).

    Article  CAS  Google Scholar 

  33. 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 α-lactalbumin. J. Mol. Biol. 206, 547–61 (1989).

    Article  CAS  Google Scholar 

  34. Forge, V. et al. Rapid collapse and slow structural reorganisation during the refolding of bovine α-lactalbumin. J. Mol. Biol. 288, 673–688 (1999).

    Article  CAS  Google Scholar 

  35. Arai, M. & Kuwajima, K. Rapid formation of a molten globule intermediate in refolding of α-lactalbumin. Fold. Des. 1, 275–87 (1996).

    Article  CAS  Google Scholar 

  36. Balbach, J., Forge, V., van Nuland, N.A.J., Winder, S.L., Hore, P.J. & Dobson, C.M. Following protein folding in real time using NMR spectroscopy . Nature Struct. Biol. 2, 865– 870 (1995).

    Article  CAS  Google Scholar 

  37. Balbach, J., Forge, V., Lau, W.S., van Nuland, N.A.J., Brew, K. & Dobson, C.M. Protein folding monitored at individual residues during a two-dimensional NMR experiment. Science 274, 1161– 1163 (1996).

    Article  CAS  Google Scholar 

  38. Pike, A.C., Brew, K. & Acharya, K.R. Crystal structures of guinea-pig, goat and bovine α-lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in lactose synthase. Structure 4, 691–703 (1996).

    Article  CAS  Google Scholar 

  39. Kauppinen, J.K., Moffatt, D.J., Mantsch, H.H., & Cameron, D.G. Fourier self-deconvolution: a method for resolving intrinsically overlapped bands. Applied Spectroscopy 35, 271– 276 (1981).

    Article  CAS  Google Scholar 

  40. Kauppinen, J.K., Moffatt, D.J., Cameron, D.G. & Mantsch, H.H. Noise in Fourier self-deconvolution. Applied Optics 20, 1866–1879 (1981).

    Article  CAS  Google Scholar 

  41. Cameron, D.G. & Moffatt, D.J. A generalized approach to derivative spectroscopy. Applied Spectroscopy 41, 539 –544 (1987).

    Article  CAS  Google Scholar 

  42. Prestrelski, S.J., Byler, D.M. & Thompson, M.P. Effect of metal ion binding on the secondary structure of bovine α-lactalbumin as examined by infrared spectroscopy. Biochemistry 30, 8797–8804 (1991).

    Article  CAS  Google Scholar 

  43. Prestrelski, S.J., Byler, D.M. & Thompson, M.P. Infrared spectroscopic discrimination between α- and 310-helices in globular proteins. Int. J. Peptide Protein Res. 37, 508–512 (1991).

    Article  CAS  Google Scholar 

  44. Mizuguchi, M., Nara, M., Kawano, K. & Nitta, K. FT-IR study of the Ca2+-binding to bovine α-lactalbumin. FEBS Letters 417, 153–156 ( 1997).

    Article  CAS  Google Scholar 

  45. Casal, H.L., Kohler, U. & Mantsch, H.H. Structural and conformational changes of beta-lactoglobulin B: an infrared spectroscopic study of the effect of pH and temperature. Biochim. Biophys. Acta 957, 11–20 (1988).

    Article  CAS  Google Scholar 

  46. Alben, J.O. & Fiamingo, F.G. Fourier transform infrared spectroscopy . In Optical techniques in biological research (ed Rousseau, D.L.) 133–179 (Academic Press Inc, New York; 1984).

    Google Scholar 

  47. Fersht, A.R. Nucleation mechanisms in protein folding. Curr. Opin. Struct. Biol. 7, 3–9 (1997 ).

    Article  CAS  Google Scholar 

  48. Canet, D. et al. Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants. Biochemistry 38, 6419–6427 (1999).

    Article  CAS  Google Scholar 

  49. Kraulis, P.J. Molscript: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946 –950 (1991).

    Article  Google Scholar 

  50. Goormaghtigh, E., Cabiaux, V. & Ruysschaert, J.M. Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. Subcellular Biochemistry 23, 405–450 ( 1994).

    Article  CAS  Google Scholar 

  51. Miick, S.M., Martinez, G.V., Fiori, W.R., Todd, A.P. & Millhauser, G.L. Short alanine-based peptides may form 310-helices and not α-helices in aqueous solution . Nature 359, 653–655 (1992).

    Article  CAS  Google Scholar 

  52. Chirgadze, Y.N., Fedorov, O.V. & Trushina, N.P. Estimation of amino-acid residue side-chain absorption in the infrared spectra of protein solution in heavy water. Biopolymers 14, 679–694 ( 1975).

    Article  CAS  Google Scholar 

  53. Shaw, R.A., Perczel, A., Mantsch, H.H. & Fasman, G.D. Turns in small cyclic peptides -can infrared spectroscopy detect and discriminate amongst them? J. Mol. Struct. 324, 143– 150 (1994).

    Article  CAS  Google Scholar 

  54. Arrondo, J.L.R., Young, N.M. & Mantsch, H.H. The solution structure of concanavalin A probed by FT-IR spectroscopy. Biochim. Biophys. Acta 952, 261–268 (1988).

    Article  CAS  Google Scholar 

  55. Smail, A.A., Mantsch, H.H. & Wong, P.T. Aggregation of chymotrypsinogen: portrait by infrared spectroscopy. Biochim. Biophys. Acta 1121, 183–188 (1992).

    Article  Google Scholar 

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Acknowledgements

We thank F. Guillain and A. Sanson for careful reading of the manuscript, S. Crouzy for helping with Fig. 1, and E. Padros for the use of IR analysis software. A.T. was supported in part by a fellowship from Bio-Logic Co.

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  1. Département de Biologie Moléculaire et Structurale, Laboratoire de Biophysique Moléculaire et Cellulaire, URA CNRS 520, CEA-Grenoble, 17 av. des Martyrs, GRENOBLE, 38054 Cedex 09, France

    Agnès Troullier, Yves Dupont & Vincent Forge

  2. Robert Koch-Institut, Nordufer 20, Berlin, D-13353, Germany

    Diane Reinstädler & Dieter Naumann

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Correspondence to Vincent Forge.

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Troullier, A., Reinstädler, D., Dupont, Y. et al. Transient non-native secondary structures during the refolding of α-lactalbumin detected by infrared spectroscopy. Nat Struct Mol Biol 7, 78–86 (2000). https://doi.org/10.1038/71286

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