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Obligatory steps in protein folding and the conformational diversity of the transition state

Abstract

We have analyzed the existence of obligatory steps in the folding reaction of the α-spectrin SH3 domain by mutating Asp 48 (D48G), which is at position i+3 of an isolated two-residue type II' β-turn. Calorimetry and X-ray analysis show an entropic stabilizing effect resulting from local changes at the dihedral angles of the β-turn. Kinetic analysis of D48G shows that this β-turn is fully formed in the transition state, while there is no evidence of its formation in an isolated fragment. Introduction of several mutations in the D48G protein reveals that the local stabilization has not significantly altered the transition state ensemble. All these results, together with previous analysis of other α-spectrin and src SH3 mutants, indicate that: (i) in the folding reaction there could be obligatory steps which are not necessarily part of the folding nucleus; (ii) transition state ensembles in β-sheet proteins could be quite defined and conformationally restricted ('mechanic folding nucleus'); and (iii) transition state ensembles in some proteins could be evolutionarily conserved.

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Figure 1: Structure comparison between the wild type and D48G proteins.
Figure 2: Calorimetric traces at different pHs for the D48G protein.
Figure 3: Kinetic measurements of the unfolding and refolding reaction of α-spectrin SH3 domain.
Figure 4: Conformational shift plot of the CαH protons for the distal β-hairpin fragment.
Figure 5: Energetic analysis of the different mutations.
Figure 6: Schematic diagram for the folding reaction of the SH3 protein.
Figure 7: Schematic diagram showing three possible models for the transition state in protein folding.

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References

  1. Jackson, S.E. & Fersht, A.R. Folding of chymotrypsin inhibitor 2. 1. Evidence for a two-state transition. Biochemistry 30, 10428– 10435 (1991).

    Article  CAS  Google Scholar 

  2. Alexander, P., Orban, J. & Bryan, P. Kinetic analysis of folding and unfolding of the 56 amino acid IgG-binding domain of Streptococcal protein G. Biochemistry 31, 7243– 7248 (1992).

    Article  CAS  Google Scholar 

  3. Sosnick, T.R., Mayne, L., Hiller, R. & Englander, S.W. The barriers in protein folding. Nature Struct. Biol. 1, 149– 156 (1992).

    Article  Google Scholar 

  4. Viguera, A.R., Martinez, J.C., Filimonov, V.V., Mateo, P.L. & Serrano, L. Thermodynamic and kinetic analysis of the SH3 domain of spectrin shows a two-state folding transition. Biochemistry 33, 2142– 2150 ( 1994).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Huang, G.S. & Oas, T.G. Submillisecond folding of monomeric λ repressor. Proc. Natl. Acad. Sci. USA 92, 6878– 6882 (1995).

    Article  CAS  Google Scholar 

  7. 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 

  8. Kragelund, B.B., Robinson, C.V., Knudesn, J., Dobson, C.M. & Poulsen, F.M. Fast and one-step folding of closely and distantly related homologous proteins of a four-helix bundle family. J. Mol. Biol. 256, 187– 200 (1995).

    Article  Google Scholar 

  9. Kuhlman, B., Boice, J.A., Fairman, R. & Raleigh, D.P. Structure and stability of the N-terminal domain of the ribosomal protein L9: evidence for rapid two-state folding. Biochemistry 37, 1025– 1032 (1998).

    Article  CAS  Google Scholar 

  10. Plaxco, K.W. et al. The folding kinetics and thermodynamics of the Fyn-SH3 domain. Biochemistry 37, 2529– 2537 (1998).

    Article  CAS  Google Scholar 

  11. Plaxco, K.W., Spitzfaden, C., Campbell, I.D. & Dobson, C.M. Rapid refolding of a proline-rich all beta-sheet fibronectin type III module. Proc. Natl. Acad. Sci. USA 93, 10703– 10706 (1996).

    Article  CAS  Google Scholar 

  12. Fersht, A .R. Characterizing transition states in protein folding: an essential step in the puzzle. Curr. Opin. Struct. Biol. 5, 79– 84 (1995).

    Article  CAS  Google Scholar 

  13. Munoz, V. & Serrano, L. Local vs non-local interactions in protein folding and stability. An experimentalist point of view. Folding & Design 1, R71– R77. (1996).

    Article  CAS  Google Scholar 

  14. Dill, K.A. & Sun Chan, H. From levinthal to pathways to funnels . Nature Struct. Biol. 4, 10– 19 (1998).

    Article  Google Scholar 

  15. Onuchic, J.N., Socci, N.D., Luthey-Schulten, Z. & Wolynes, P.G. Protein folding funnels: the nature of the transition state ensemble. Folding & Design 1, 441– 450 (1996).

    Article  CAS  Google Scholar 

  16. Lopez-Hernandez, E. & Serrano, L. Structure of the transition state for folding of the 129aa protein, CheY, resembles that of a smaller protein, CI-2. Folding & Design 1, 43– 55 (1996).

    Article  CAS  Google Scholar 

  17. Itzhaki, L.S., Otzen, D.E. & Fersht, A.R. The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: Evidence for a nucleation-condensation mechanism for protein folding. J. Mol. Biol. 254, 260– 288 (1995).

    Article  CAS  Google Scholar 

  18. Viguera, A.R. Wilmanns, M. & Serrano, L. Different folding transition states could result in the some native structure . Nature Struct. Biol. 3, 874– 880 (1996).

    Article  CAS  Google Scholar 

  19. Abkevich, V.I., Gutin, A.M. & and Shakhnovich, E.I. . Specific nucleus as the transition state for protein folding: evidence from the lattice model. Biochemistry 33, 10026– 10036 (1994).

    Article  CAS  Google Scholar 

  20. Musacchio, A., M. E. M. Noble, M. E. M, Pautit, R. Wierenga, R. & Saraste, M. Crystal structure of a src-homology 3 (SH3) domain. Nature 359, 851– 855 (1992).

    Article  CAS  Google Scholar 

  21. Blanco, F.J., Ortiz, A.R. & Serrano, L. 1H and 15N-NMR assignment and solution structure of the SH3 domain of spectrin. Comparison with the crystal structure. J. Biomol. NMR. 9, 347– 357 (1997).

    Article  CAS  Google Scholar 

  22. Viguera, A.R., Blanco, F.J. & Serrano, L. The order of secondary structure elements does not determine the structure of a protein but does affect its folding kinetics. J. Mol. Biol. 247, 670– 681 ( 1995).

    CAS  PubMed  Google Scholar 

  23. Viguera, A.R. & Serrano, L. Loop length, intramolecular diffusion and protein folding. Nature Struct. Biol. 4, 939– 946 (1997).

    Article  CAS  Google Scholar 

  24. Perl, D. et al. Conservation of rapid two-state folding in mesophilic, thermophilic and hyperthermophilic cold shock proteins. Nature Struct. Biol. 5, 229– 235 (1998).

    Article  CAS  Google Scholar 

  25. Ramirez-Alvarado, M., Blanco, F.J., Niemann, H. & Serrano, L. Role of β-turns residues in β-hairpin formation and stability in designed peptides. J. Mol. Biol. 273, 898 – 912 (1997).

    Article  CAS  Google Scholar 

  26. Brunet, A.P. et al. The role of turns in the structure of an α-helical protein. Nature 363, 355– 358 ( 1993).

    Article  Google Scholar 

  27. Castagnoli, L., Vetriani, C. & Cesareni, C. Linking an easily detectable phenotype to the folding of a common structural motif. Selection of rare mutations that prevent the folding of Rop. J. Mol. Biol. 234, 378– 387 (1994).

    Article  Google Scholar 

  28. Ybe, J.A. & Hecht, M.H. Sequence replacements in the central β-turn of plastocyanin. Prot. Sci. 5, 814– 824 (1996).

    Article  CAS  Google Scholar 

  29. Hynes, T.R., Hodel, A. & Fox, R.O. Engineering alternative β-turn types in Staphylococcal nuclease. Biochemistry 33, 5021– 5030 (1994).

    Article  CAS  Google Scholar 

  30. Predki, P.F., Agrawal, V., Brunger, A.T. & Regan, L. Amino acid substitutions in a surface turn modulate protein stability. Nature Struct. Biol. 3, 54– 58 (1996 ).

    Article  CAS  Google Scholar 

  31. Zhou, H.X., Hoess, R.H. & deGrado, W.F. In vitro evolution of thermodynamically stable turns. Nature Struct. Biol. 3, 446– 451 (1996).

    Article  CAS  Google Scholar 

  32. Ohage, E.C., Grami, W., Walter, M.M., Steinbacher, S. & Steipe, B. β-turn propensities as paradigms for the analysis of structural motifs to engineer protein stability. Prot. Sci. 6, 233– 241 ( 1997).

    Article  CAS  Google Scholar 

  33. Kim, K., Ramanathan, R. & Frieden, C. Intestinal fatty acid binding protein: a specific residue in one turn appears to stabilize the native structure and be responsible for slow folding. Prot. Sci. 6, 364– 372 ( 1997).

    Article  CAS  Google Scholar 

  34. Gu H, Kim D, Baker D. Contrasting roles for symmetrically disposed beta-turns in the folding of a small protein . J. Mol. Biol. 274, 588– 596 (1997).

    Article  CAS  Google Scholar 

  35. Prieto, J., Wilmanns, M., Jimenez, M.A., Rico, M. & Serrano, L. Non-native interactions in protein folding and stability: Introducing a helical tendency in the all β-sheet α-spectrin SH3 domain. J. Mol. Biol. 268, 760– 778 (1997).

    Article  CAS  Google Scholar 

  36. Milla, M.E., Brown, B.M., Waldburger, C.D. & Sauer, R.T. P22 Arc repressor: transition state properties inferred from mutational effects on the rates of protein unfolding and refolding. Biochemistry 34 , 13914– 13919 (1995).

    Article  CAS  Google Scholar 

  37. Fersht, A.R., Itzhaki, L.S., elMasry, N., Matthews, J.M. & Otzen, D.E. . Single versus parallel pathways of protein folding and fractional formation of structure in the transition state. Proc. Natl. Acad. Sci. USA 91, 10426– 10429 (1994).

    Article  CAS  Google Scholar 

  38. Grantcharova, V.P., Riddle, D.S., Santiago, J.V. & Baker, D. Important role of hydrogen bonds in the structurally polarized transition sate for folding of the src SH3 domain. Nature Struct. Biol. 5, 714– 720 (1998).

    Article  CAS  Google Scholar 

  39. Musacchio, A., Wilmanns, M. & Saraste, M. Structure and function of the SH3 domains. Progr. Biophys. Molec. Biol. 61, 283– 297 ( 1994).

    Article  CAS  Google Scholar 

  40. Shakhnovich, E., Abkevich, V. & Ptitsyn, O. Conserved residues and the mechanism of protein folding . Nature 379, 96– 98 (1996).

    Article  CAS  Google Scholar 

  41. Plaxco, K.W. et al. The folding kinetics and thermodynamics of the Fyn-SH3 domain. Biochemistry 37, 2529– 2537 (1998).

    Article  CAS  Google Scholar 

  42. Burton, R.E., Huang, G.S., Daugherty, M.A., Calderone, T.F. & Oas, G.T. The energy landscape of a fast-folding protein mapped by Ala-Gly protein. Nature Struct. Biol. 4, 305– 310 (1998).

    Article  Google Scholar 

  43. Kunkel, T.A. Rapid and efficient site-directed mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488– 492 (1985).

    Article  CAS  Google Scholar 

  44. Gill S.C. & von Hippel. P.H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319– 326 (1989).

    Article  CAS  Google Scholar 

  45. Otwinowski,Z. & Minor,W. The HKL program suite. Unpublished programs.

  46. Navaza, J. AMORE : An automated package for molecular replacement. Acta Crystallogr. A 50, 157– 163 (1994).

    Article  Google Scholar 

  47. Jones, T.A., Zou, J.-Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A47, 110– 119 (1991).

    Article  CAS  Google Scholar 

  48. Brunger, A.T., Kuriyan, J. & Karplus, M. Crystallographic R factor refinement by molecular dynamics. Science 235, 458– 460 ( 1987).

    Article  CAS  Google Scholar 

  49. Lamzin, V.S. & Wilson, K.S. Automated refinement of protein models. Acta Crystallogr. A49, 129– 147 (1993).

    Google Scholar 

  50. Marion, D. & Wüthrich, K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurement of proton-proton spin-spin coupling constants. Biochem. Biophys. Res. Comm. 113, 967– 974 (1983).

    Article  CAS  Google Scholar 

  51. Aue, W.P., Bartholdi, E. & Ernst, R.R. Two dimensional spectroscopy.Application to nuclear magnetic resonance. J. Chem. Phys. 64, 2229– 2246 (1976).

    Article  CAS  Google Scholar 

  52. Piantini, U., Sørensen, O.W., Ernst, R.R. Multiple quantum filters for elucidating NMR coupling networks. J. Amer. Chem. Soc. 104, 6800– 6801 (1982).

    Article  CAS  Google Scholar 

  53. Kumar, A., Ernst, R.R. & Wüthrich, K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem. Biophys. Res. Comm. 95, 1– 6 (1980 ).

    Article  CAS  Google Scholar 

  54. Bax, A., & Davis, D.G. MLEV-17 based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. 65, 355– 360. (1985).

    CAS  Google Scholar 

  55. Wüthrich, K. In NMR of proteins and nucleic acids. (John Wiley & sons, New York; 1986).

    Book  Google Scholar 

  56. Johnson, C.M. & Fersht, A.R. Protein stability as a function of denaturant concentration: The thermal stability of barnase in the presence of urea. Biochemistry 34, 6795– 6804 (1995).

    Article  CAS  Google Scholar 

  57. Merutka, G., Dyson, H.J. & Wright, P.E. Random coil 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG. J. Biomol. NMR 5, 14– 24 (1995).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank I. Angrand for help with mutagenesis and molecular biology, G. Wallon and C. Vega for invaluable help with crystallographic techniques, and F. J. Blanco and M. Ramirez-Alvarado in NMR processing. We are also grateful to P. L. Mateo for the generous offer of calorimetric techniques. We are grateful to D. Baker for providing us with a copy of his manuscript regarding the analysis of the src SH3 domain prior to publication, as well as allowing us to discuss their unpublished results. J.C.M. is supported by a TMR fellowship from the EU. M.T.P. is supported by an EU biotechnology grant.

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Martinez, J., Pisabarro, M. & Serrano, L. Obligatory steps in protein folding and the conformational diversity of the transition state. Nat Struct Mol Biol 5, 721–729 (1998). https://doi.org/10.1038/1418

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