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Conservation of rapid two-state folding in mesophilic, thermophilic and hyperthermophilic cold shock proteins

Nature Structural Biology volume 5, pages 229–235 (1998)Cite this article

Abstract

The cold shock protein CspB from Bacillus subtilis is only marginally stable, but it folds extremely fast in a simple N ⇌ U two-state reaction. The corresponding cold shock proteins from the thermophile Bacillus caldolyticus and the hyperthermophile Thermotoga maritima show strongly increased conformational stabilities, but unchanged very fast two-state refolding kinetics. The absence of intermediates in the folding of B. subtilis CspB is thus not a corollary of its low stability. Rather, two-state folding and an unusually native-like activated state of folding seem to be inherent properties of these small all-β proteins. There is no link between stability and folding rate, and numerous sequence positions exist which can be varied to modulate the stability without affecting the rate and mechanism of folding.

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References

  1. Kim, P.S. & Baldwin, R.L. Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. Annu. Rev. Biochem. 51, 459–489 (1982).

    Article  CAS  Google Scholar 

  2. Kim, P.S. & Baldwin, R.L. Intermediates in the folding reactions of small proteins. Annu. Rev. Biochem. 59, 631–660 (1990).

    Article  CAS  Google Scholar 

  3. Jaenicke, R. Folding and association of proteins. Prog. Biophys. molec. Biol. 49, 117–237 (1987).

    Article  CAS  Google Scholar 

  4. Kuwajima, K. The molten globule as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins Struct. Funct. Genet. 6, 87–103 (1989).

    Article  CAS  Google Scholar 

  5. Matthews, C.R. Pathways of Protein Folding. Annu. Rev. Biochem. 62, 653–683 (1993).

    Article  CAS  Google Scholar 

  6. Sosnick, T.R., Mayne, L., Hiller, R. & Englander, S.W. The barriers in protein folding. Nature Struct. Biol. 1, 149–156 (1994).

    Article  CAS  Google Scholar 

  7. Kiefhaber, T. Kinetic traps in lysozyme folding. Proc. Natl. Acad. Sci. USA 92, 9029–9033 (1995).

    Article  CAS  Google Scholar 

  8. Baldwin, R.L. The nature of protein folding pathways: The classical versus the new view. J. Biomolec. NMR 5, 103–109 (1995).

    Article  CAS  Google Scholar 

  9. Baldwin, R.L. On-pathway versus off-pathway folding intermediates. Folding & Design 1, R1–R8 (1996).

    Article  CAS  Google Scholar 

  10. Rothwarf, D.M. & Scheraga, H.A. Role of non-native aromatic and hydrophobic interactions in the folding of hen egg white lysozyme. Biochemistry 35, 13797–13807 (1996).

    Article  CAS  Google Scholar 

  11. Ikai, A. & Tanford, C. Kinetics of unfolding and refolding of proteins I. Mathematical analysis. J. Mol. Biol. 73, 145–163 (1973).

    Article  CAS  Google Scholar 

  12. Ikai, A., Fish, W.W. & Tanford, C. Kinetics of unfolding and refolding of proteins II. Results for cytochrome c. J. Mol. Biol. 73, 165–184 (1973).

    Article  CAS  Google Scholar 

  13. 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 

  14. Jackson, S.E. elMasry, N. & Fersht, A.R. Structure of the hydrophobic core in the transition state for folding of chymotrypsin inhibitor 2: a critical test of the protein engineering method of analysis. Biochemistry 32, 11270–1278 (1993).

    Article  CAS  Google Scholar 

  15. Schindler, T., Herrler, M., Marahiel, M.A. & Schmid, F.X. Extremely rapid folding in the absence of intermediates: the cold-shock protein from Bacillus subtilis. Nature Struct. Biol. 2, 663–673 (1995).

    Article  CAS  Google Scholar 

  16. Schindler, T. & Schmid, F.X. Thermodynamic properties of an extremely rapid protein folding reaction. Biochemistry 35, 16833–16842 (1996).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Burton, R.E., Huang, G.S., Daugherty, M.A., Fullbright, P.W. & Oas, T.G. Microsecond protein folding through a compact transition state. J. Mol. Biol. 263, 311–322 (1996).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Kragelund, B.B. et al. Fast and one-step folding of closely and distantly related homologous proteins of a four-helix bundle family. J. Mol. Biol. 256, 187–200 (1996).

    Article  CAS  Google Scholar 

  21. 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 32, 2142–2150 (1994).

    Article  Google Scholar 

  22. 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  Google Scholar 

  23. Bai, Y.W., Karimi, A., Dyson, H.J. & Wright, P.E. Absence of a stable intermediate on the folding pathway of protein A. Prot. Sci. 6, 1449–1457 (1997).

    Article  CAS  Google Scholar 

  24. Jones, P.G., van Bogelen, R.A. & Neidhardt, F.C. Induction of proteins in response to low temperature in Escherichia coli. J. Bacteriol. 169, 2092–2095 (1987).

    Article  CAS  Google Scholar 

  25. Willimsky, G., Bang, H., Fischer, G. & Marahiel, M.A. Characterization of cspB, a Bacillus subtilis inducible cold shock gene affecting cell viability at low temperature. J. Bacteriol. 174, 6326–6335 (1992).

    Article  CAS  Google Scholar 

  26. Schindelin, H., Marahiel, M.A. & Heinemann, U. Universal nucleic acid-binding domain revealed by crystal structure of the B. subtilis major cold-shock protein. Nature 364, 164–168 (1993).

    Article  CAS  Google Scholar 

  27. Schnuchel, A. et al. Structure in solution of the major cold-shock protein from Bacillus subtilis. Nature 364, 169–171 (1993).

    Article  CAS  Google Scholar 

  28. Tanford, C., Aune, K.C. & Ikai, A. Kinetics of unfolding and refolding of proteins III. Results for lysozyme. J. Mol. Biol. 73, 185–197 (1973).

    Article  CAS  Google Scholar 

  29. 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 

  30. Fersht, A.R., Folding and Stability - The Pathway of Folding of Barnase. FEBS Lett. 325, 5–16 (1993).

    Article  CAS  Google Scholar 

  31. 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 

  32. 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. Nature Struct. Biol. 3, 193–205 (1996).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  34. Wildegger, G. & Kiefhaber, T. Three-state model for lysozyme folding: Triangular folding mechanism with an energetically trapped intermediate. J. Mol. Biol. 270, 294–304 (1997).

    Article  CAS  Google Scholar 

  35. Krieg, N.R. & Holt, J.G. Bergey's manual of systematic bacteriology. (Williams & Wilkins, Baltimore; 1986).

    Google Scholar 

  36. Huber, R. et al. Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90 °C. Arch. Microbiol. 144, 324–333 (1986).

    Article  CAS  Google Scholar 

  37. Onuchic, J.N., Wolynes, P.G., Luthey-Schulten, Z. & Socci, N.D. Toward an outline of the topography of a realistic protein- folding funnel. Proc. Natl. Acad. Sci. USA 92, 3626–3630 (1995).

    Article  CAS  Google Scholar 

  38. 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 

  39. Schönbrunner, N., Koller, K.P. & Kiefhaber, T. Folding of the disulfide-bonded beta-sheet protein tendamistat: Rapid two-state folding without hydrophobic collapse J. Mol. Biol. 268, 526–538 (1997).

    Article  Google Scholar 

  40. Prieto, J., Wilmanns, M., Jiménez, M.A., Rico, M. & Serrano, L. Non-native local interactions in protein folding and stability: introducing a helical tendency in the all-beta sheet alpha-spectrin SH3 domain. J. Mol. Biol. 268, 760–778 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  42. 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 

  43. Matouschek, A., Otzen, D.E., Itzhaki, L.S., Jackson, S.E. & Fersht, A.R. Movement of the position of the transition state in protein folding. Biochemistry 34, 13656–13662 (1995).

    Article  CAS  Google Scholar 

  44. Burton, R.E., Huang, G.S., Daugherty, M.A., Calderone, T.L. & Oas, T.G. The energy landscape of a fast-folding protein mapped by Ala-Gly Substitutions. Nature Struct. Biol. 4, 305–310 (1997).

    Article  CAS  Google Scholar 

  45. Laurents, D.V. & Baldwin, R.L. Protein folding: matching ‘theory’ and experiment. Biophys. J. in the press (1998).

  46. Mirny, L.A., Abkevich, V. & Shakhnovich, E.I. Universality and diversity of the protein folding scenarios: a comprehensive analysis with the aid of a lattice model. Folding & Design 1, 103–116 (1996).

    Article  CAS  Google Scholar 

  47. Sali, A., Shakhnovich, E. & Karplus, M. How does a protein fold? Nature 369, 248–251 (1994).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  49. Pace, C.N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Meth. Enz. 131, 266–280 (1986).

    Article  CAS  Google Scholar 

  50. Schindelin, H., Herrler, M., Willimsky, G., Marahiel, M.A. & Heinemann, U., Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB. Proteins Struct. Funct. Genet. 14, 120–124 (1992).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  52. Pace, C.N., Vajdos, F., Fee, L., Grimsley, G. & Gray, T. How to measure and predict the molar absorption coefficient of a protein. Prot. Sci. 4, 2411–2423 (1995).

    Article  CAS  Google Scholar 

  53. Santoro, M.M. & Bolen, D.W. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl α-chymotrypsin using different denaturants. Biochemistry 27, 8063–8068 (1988).

    Article  CAS  Google Scholar 

  54. Tonomura, B., Nakatani, H., Ohnishi, M., Yamaguchi-Ito, J. & Hiromi, K. Test reaction for a stopped-flow apparatus. Anal. Biochem. 84, 370–383 (1978).

    Article  CAS  Google Scholar 

  55. 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 

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Authors and Affiliations

  1. Laboratorium für Biochemie, Universität Bayreuth, D-95440, Bayreuth, Germany

    Dieter Perl, Thomas Schindler & Franz X. Schmid

  2. lnstitut für Biophysik und Physikalische Biochemie, Universität Regensburg, D-93040, Regensburg, Germany

    Christine Welker & Rainer Jaenicke

  3. Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Str., D-35043, Marburg, Germany

    Katja Schröder & Mohamed A. Marahiel

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  1. Dieter Perl
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Correspondence to Franz X. Schmid.

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Perl, D., Welker, C., Schindler, T. et al. Conservation of rapid two-state folding in mesophilic, thermophilic and hyperthermophilic cold shock proteins. Nat Struct Mol Biol 5, 229–235 (1998). https://doi.org/10.1038/nsb0398-229

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