Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The kinetic folding intermediate of ribonuclease H resembles the acid molten globule and partially unfolded molecules detected under native conditions

Nature Structural Biology volume 4, pages 298–304 (1997)Cite this article

An Erratum to this article was published on 01 June 1997

Abstract

Folding of ribonuclease HI from Escherichia coli populates a kinetic intermediate detectable by stopped-flow circular dichroism. Pulse labelling hydrogen exchange reveals that this intermediate consists of a structured core region of the protein, namely helices A and D and β-strand 4. This kinetic intermediate resembles both the acid molten globule of ribonuclease HI and rarely populated, partially unfolded forms detected under native conditions. These results indicate that the first portion of ribonuclease HI to fold is the most thermodynamically stable region of the native state, and that folding of this protein follows a hierarchical process.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

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. Matthews, C.R. Pathways of protein folding. Annu. Rev. Biochem. 62, 653–683 (1993).

    Article  CAS  Google Scholar 

  4. 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 

  5. 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 

  6. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Ptitsyn, O.B., Pain, R.H., Semisotnov, G.V., Zerovnik, E. & Razgulyaev, O.I. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 262, 20–24 (1990).

    Article  CAS  Google Scholar 

  10. Jennings, P.A. & Wright, P.E. Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science 262, 892–896 (1993).

    Article  CAS  Google Scholar 

  11. Arai, M. & Kuwajima, K. Rapid formation of a molten globule intermediate in refolding of α-lactalbumin. Folding & Design 1, 275–287 (1996).

    Article  CAS  Google Scholar 

  12. Bai, Y., Sosnick, T.R., Mayne, L. & Englander, S.W. Protein folding intermediates: native-state hydrogen exchange. Science 269, 192–197 (1995).

    Article  CAS  Google Scholar 

  13. Chamberlain, A.K., Handel, T.M. & Marqusee, S. Detection of rare partially folded molecules in equilibrium with the native conformation of RNase H. Nature Struct. Biol. 3, 782–787 (1996).

    Article  CAS  Google Scholar 

  14. Katayanagi, K. et al. Three-dimensional structure of ribonuclease H from E. coli. Nature 347, 306–309 (1990).

    Article  CAS  Google Scholar 

  15. Crouch, R.J. & Dirksen, M. Ribonucleases H. in Nucleases (ed. Linn, S.) 211–241 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1982).

    Google Scholar 

  16. Kanaya, S., Kimura, S., Katsuda, C. & Ikehara, M. Role of cysteine residues in ribonuclease H from Escherichia coli. Site-directed mutagenesis and chemical modification. Biochem. J. 271, 59–66 (1990).

    Article  CAS  Google Scholar 

  17. Yamasaki, K., Ogasahara, K., Yutani, K., Oobatake, M. & Kanaya, S. Folding pathway of Escherichia coli ribonuclease HI: a circular dichroism, fluorescence, and NMR study. Biochemistry 34, 16552–16562 (1995).

    Article  CAS  Google Scholar 

  18. Dabora, J.M. & Marqusee, S. Equilibrium unfolding of Escherichia coli ribonuclease H: characterization of a partially folded state. Protein Sci. 3, 1401–1408 (1994).

    Article  CAS  Google Scholar 

  19. Dabora, J. & Marqusee, S. Structure of the acid state of Escherichia coli ribonuclease H1. Biochemistry 35, 11951–11958 (1996).

    Article  CAS  Google Scholar 

  20. Kuwajima, K., Yamaya, H., Miwa, S., Sugai, S. & Nagamura, T. Rapid formation of secondary structure framework in protein folding studied by stopped-f low circular dichroism. FEBS Lett. 221, 115–118 (1987).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. 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 

  23. 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 

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

    Article  CAS  Google Scholar 

  25. Roder, H., Elove, G.A. & Englander, S.W. Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nature 335, 700–704 (1988).

    Article  CAS  Google Scholar 

  26. Udgaonkar, J.B. & Baldwin, R.L. NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A. Nature 335, 694–699 (1988).

    Article  CAS  Google Scholar 

  27. Elove, G.A. & Roder, H. Structure and stability of cytochrome c folding intermediates. in Protein Refolding (ed. Georgiou, G.) 51–63 (American Chemical Society, Washington, DC, 1991).

    Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Ptitsyn, O.B., Pain, R.H., Semisotnov, G.V., Zerovnik, E. & Razgulyaev, O.I. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 262, 20–24 (1990).

    Article  CAS  Google Scholar 

  30. Elove, G.A., Bhuyan, A.K. & Roder, H. Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands. Biochemistry 33, 6925–6935 (1994).

    Article  CAS  Google Scholar 

  31. Chen, H., Hughes, D.D., Chan, T.-A., Sedat, J.W. & Agard, D.A. IVE (lmage Visualization Environment): A software platform for all three-dimensional microscopy applications. J. Struct. Biol. 116, 56–60 (1996).

    Article  CAS  Google Scholar 

  32. Hvidt, A. & Nielsen, S.O. Hydrogen exchange in proteins. Adv. Prot. Chem. 21, 288–386 (1966).

    Google Scholar 

  33. Englander, S.W. & Kallenbach, N.R. Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q. Rev. Biophys. 16, 521–655 (1983).

    Article  CAS  Google Scholar 

  34. Bai, Y., Milne, J.S., Mayne, L. & Englander, S.W. Primary structure effects on peptide group hydrogen exchange. Proteins 17, 75–86 (1993).

    Article  CAS  Google Scholar 

  35. Connelly, G.P., Bai, Y., Jeng, M.F. & Englander, S.W. Isotope effects in peptide group hydrogen exchange. Proteins 17, 87–92 (1993).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, 229 Stanley Hall, Berkeley, Berkeley, CA, 94720, USA

    Tanya M. Raschke & Susan Marqusee

Authors
  1. Tanya M. Raschke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Susan Marqusee
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Raschke, T., Marqusee, S. The kinetic folding intermediate of ribonuclease H resembles the acid molten globule and partially unfolded molecules detected under native conditions. Nat Struct Mol Biol 4, 298–304 (1997). https://doi.org/10.1038/nsb0497-298

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb0497-298

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing