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Acceleration of the folding of acylphosphatase by stabilization of local secondary structure

Abstract

The addition of trifluoroethanol or hexafluoroisopropanol converts the apparent two-state folding of acylphosphatase, a small α/β protein, into a multistate mechanism where secondary structure accumulates significantly in the denatured state before folding to the native state. This results in a marked acceleration of folding as revealed by following the intrinsic fluorescence and circular dichroism changes upon folding. The folding rate is at a maximum when the secondary-structure content of the denatured state corresponds to that of the native state, while further stabilization of secondary structure decreases the folding rate. These findings indicate that stabilization of intermediate structure can either enhance or retard folding depending on its nature and content of native-like interactions.

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Figure 1: Equilibrium conformational changes induced by fluoroalcohols.
Figure 2: TFE and HFIP concentration dependence of the natural logarithm of the observed folding rate constant for muscle (,) and CT (,) AcP.
Figure 3: Folding rate constant of CT AcP, expressed as its natural logarithm, versus urea concentration.
Figure 4: Folding of CT AcP monitored by 222 nm far-UV CD.
Figure 5: Relationship between secondary-structure content of the denatured state and folding rate.

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References

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

    Article  CAS  Google Scholar 

  2. 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 

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

    Article  CAS  Google Scholar 

  4. Roder, H. & Colon, W. Kinetic role of early intermediates in protein folding. Curr. Opin. Struct. Biol. 7, 15–28 (1997).

    Article  CAS  Google Scholar 

  5. Khorasanizadeh, S., Peters, I.D. & Roder, H. Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues. Nat. Struct. Biol. 3, 193–205 (1996).

    Article  CAS  Google Scholar 

  6. Lopez-Hernandez, E., Cronet, P., Serrano, L. & Munoz, V. Folding kinetics of Che Y mutants with enhanced native alpha-helix propensities. J. Mol. Biol. 266, 610–620 (1997).

    Article  CAS  Google Scholar 

  7. Khorasanizadeh, S., Peters, I.D., Butt, T.R. & Roder, H. Folding and stability of a tryptophan-containing mutant of ubiquitin. Biochemistry 32, 7054–7063 (1993).

    Article  CAS  Google Scholar 

  8. Kragelund, B.B., Robinson, C.V., Knudsen, J., Dobson, C.M. & Poulsen, F.M. Folding of a four-helix bundle: studies of acyl-coenzyme A binding protein. Biochemistry 34, 7217–7224 (1995).

    Article  CAS  Google Scholar 

  9. Villegas, V. et al. Evidence for a two-state transition in the folding process of the activation domain of human procarboxypeptidase A2. Biochemistry 34, 15105–15110 ( 1995).

    Article  CAS  Google Scholar 

  10. Schindler, T., Herrler, M., Marahiel, M.A. & Schmid, F.X. Extremely rapid protein folding in the absence of intermediates. Nat. Struct. Biol. 2, 663–673 (1995).

    Article  CAS  Google Scholar 

  11. Huang, G.S. & Oas, T.G. Structure and stability of monomeric λ-repressor: NMR evidence for two-state folding. Biochemistry 34 , 3884–3892 (1995).

    Article  CAS  Google Scholar 

  12. Sosnick, T.R., Mayne, L. & Englander, S.W. Molecular collapse: the rate-limiting step in two-state cytochrome c folding. Proteins 24, 413– 426 (1996).

    Article  CAS  Google Scholar 

  13. Stefani, M., Taddei, N. & Ramponi, G. Insights into acylphosphatase structure and catalytic mechanism. Cell. Mol. Life Sci. 53, 141– 151 (1997).

    Article  CAS  Google Scholar 

  14. Saudek, V. et al. Identification and description of beta-structure in horse muscle acylphosphatase by nuclear magnetic resonance spectroscopy. J. Mol. Biol. 207, 405–415 (1989).

    Article  CAS  Google Scholar 

  15. Pastore, A., Saudek, V., Ramponi, G. & Williams, R.J.P. Three-dimensional structure of acylphosphatase. Refinement and structure analysis. J. Mol. Biol. 224, 427–440 (1992).

    Article  CAS  Google Scholar 

  16. Thunnissen, M.M.G.M., Taddei, N., Liguri, G., Ramponi, G. & Nordlund, P. Crystal structure of common type acylphosphatase from bovine testis. Structure 5, 69– 79 (1997).

    Article  CAS  Google Scholar 

  17. van Nuland, N.A.J., Chiti, F., Taddei, N., Raugei, G., Ramponi, G. & Dobson, C.M. Slow folding of muscle acylphosphatase in the absence of intermediates. J. Mol. Biol. 283, 883–891 ( 1998).

    Article  CAS  Google Scholar 

  18. Taddei, N. et al. Thermodynamics and kinetics of folding of common-type acylphosphatase: comparison to the highly homologous muscle isoenzyme. Biochemistry 38, 2135–2142 ( 1999).

    Article  CAS  Google Scholar 

  19. Buck, M., Radford, S.E. & Dobson, C.M. A partially folded state of HEWL in TFE: structural characterisation and implications for protein folding. Biochemistry 32, 669–678 ( 1993).

    Article  CAS  Google Scholar 

  20. Thomas, P.D., & Dill, D.A. Local and nonlocal interactions in global proteins and mechanism of alcohol denaturation. Protein Sci. 2, 2050–2065 ( 1993).

    Article  CAS  Google Scholar 

  21. 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 

  22. Lu, H., Buck, M., Radford, S.E. & Dobson, C.M. Acceleration of the folding of hen lysozyme by trifluoroethanol. J. Mol. Biol. 265, 112–117 ( 1997).

    Article  CAS  Google Scholar 

  23. Chiti, F., et al. Structural characterisation of the transition state for folding of muscle acylphosphatase. J. Mol. Biol. 283, 893–903 (1998).

    Article  CAS  Google Scholar 

  24. Yang, J.T., Wu, C.C. & Martinez, H.M. Calculation of protein conformation from circular dichroism. Methods. Enzymol. 130, 208– 269 (1986).

    Article  CAS  Google Scholar 

  25. Alexandrescu, A.T., Ng, Y-L. & Dobson, C.M. Characterization of a trifluoroethanol-induced partially folded state of α-lactalbumin. J. Mol. Biol. 235, 587–599 (1994).

    Article  CAS  Google Scholar 

  26. 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 34, 13219– 13232 (1995).

    Article  CAS  Google Scholar 

  27. Muñoz, V. & Serrano, L. Elucidating the folding problem of helical peptides using empirical parameters. Nat. Struct. Biol. 1, 399–409 (1994).

    Article  Google Scholar 

  28. Muñoz, V. & Serrano, L. Elucidating the folding problem of helical peptides using empirical parameters. II. Helix macrodipole effects and rational modification of the helical content of natural peptides. J. Mol. Biol. 245, 275– 296 (1994).

    Article  Google Scholar 

  29. Rost, B. & Sander, C. Combining evolutionary information and neural networks to predict protein secondary structure. Proteins 19, 55–72 ( 1994).

    Article  CAS  Google Scholar 

  30. Rost, B., Sander, C. & Schineider, R. PHD—an automatic mail server for protein secondary structure prediction. CABIOS 10, 53– 60 (1994).

    CAS  PubMed  Google Scholar 

  31. Cammers-Goodwin, A. et al. Mechanism of stabilization of helical conformations of polypeptides by water containing trifluoroethanol. J. Am. Chem. Soc. 118, 3082–3090 (1996).

    Article  CAS  Google Scholar 

  32. Hirota, N., Mizuno, K. & Goto, Y. Cooperative α-helix formation of β-lactoglobulin and melittin by hexafluoroisopropanol. Protein Sci. 6, 416–421 (1997).

    Article  CAS  Google Scholar 

  33. Matouschek, A., Kellis, J.T., Serrano, L., Bycroft, M. & Fersht, A.R. Transient folding intermediates characterized by protein engineering. Nature 346, 440–445 (1990).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. Schulman, B.A. & Kim, P.S. Proline scanning mutagenesis of a molten globule reveals non-cooperative formation of a protein's overall topology. Nat. Struct. Biol. 3, 682–687 (1996).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  37. Plaxco, K.W., Simons, K.T. & Baker, D. Contact order, transition state placement and the folding kinetics of single domain proteins. J. Mol. Biol., 277, 985–994 (1998).

    Article  CAS  Google Scholar 

  38. Modesti, A. et al. Expression, purification and characterisation of acylphosphatase muscular isoenzyme as fusion protein with glutathione S-transferase. Protein Expr. Purif. 6, 799– 805 (1995).

    Article  CAS  Google Scholar 

  39. Manavalan, P. & Johnson, W.C. Variable selection method improves the prediction of protein secondary structure from circular-dichroism spectra. Anal. Biochem. 167, 76– 85 (1987).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to L. Serrano and M. Buck for useful discussions. F.C. was supported by a grant from the European Community. D.H. was supported by JSPS Postdoctoral Fellowships for Research Abroad. This is a contribution from the Oxford Centre for Molecular Sciences, which is funded by BBSRC, EPSRC and MRC. The work has also been supported by funds from the Italian CNR (Target Project Biotechnology), from MURST (Project Structural Biology) and from the European Community (Biotechnology Unit). The research of C.M.D. is supported in part by an International Research Scholars award from the Howard Hughes Medical Institute and by The Wellcome Trust.

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Correspondence to Christopher M. Dobson.

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Chiti, F., Taddei, N., Webster, P. et al. Acceleration of the folding of acylphosphatase by stabilization of local secondary structure. Nat Struct Mol Biol 6, 380–387 (1999). https://doi.org/10.1038/7616

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