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A third native one-disulphide intermediate in the folding of bovine pancreatic trypsin inhibitor

Nature Structural Biology volume 2, pages 674–679 (1995)Cite this article

Abstract

A previously unidentified intermediate has been detected in the early stages of the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI). The intermediate contains one disulphide bond between residues 14 and 38 and is denoted [14–38]. The 14–38 disulphide bond is also found in native BPTI. Although the other native one-disulphide intermediates, [30–51] and [5–55], are thermodynamically more stable, [14–38] can be populated substantially at the early stages of BPTI folding. Moreover, initial characterization of the kinetic properties of this intermediate strongly suggest that a substantial fraction of BPTI molecules fold by way of the [14–38] intermediate. Our results emphasize the importance of native-like tendencies in protein folding.

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References

  1. Creighton, T.E. Renaturation of the reduced bovine pancreatic trypsin inhibitor. J. molec. Biol. 87, 563–577 (1974).

    Article  CAS  Google Scholar 

  2. Creighton, T.E. Conformational restrictions on the pathway of folding and unfolding of BPTI. J. molec. Biol. 113, 275–293 (1977).

    Article  CAS  Google Scholar 

  3. Creighton, T.E. Energetics of folding and unfolding of pancreatic trypsin inhibitor. J. molec. Biol. 113, 295–312 (1977).

    Article  CAS  Google Scholar 

  4. Creighton, T.E. Effects of urea and guanidine. HCI on the folding and unfolding of pancreatic trypsin inhibitor. J. molec. Biol. 113, 313–328 (1977).

    Article  CAS  Google Scholar 

  5. Creighton, T.E. & Goldenberg, D.P. Kinetic role of a meta-stable native like two disulphide species in the folding transition of bovine pancreatic trypsin inhibitor. J. molec. Biol. 179, 497–526 (1984).

    Article  CAS  Google Scholar 

  6. Goldenberg, D.P. Kinetic analysis of the folding and unfolding of a mutant form of bovine pancreatic trypsin inhibitor lacking the cysteine-14 and -38 thiols. Biochemistry 27, 2481–2489 (1988).

    Article  CAS  Google Scholar 

  7. Weissman, J.S. & Kim, P.S. Reexamination of the folding of BPTI: predominance of native intermediates. Science 253, 1386–1393 (1991).

    Article  CAS  Google Scholar 

  8. Weissman, J.S. & Kim, P.S. Kinetic role of nonnative species in the folding of bovine pancreatic trypsin inhibitor. Proc. natn. Acad. Sci. U.S.A. 89, 9900–9904 (1992).

    Article  CAS  Google Scholar 

  9. Weissman, J.S. & Kim, P.S. The pro region of BPTI facilitates folding. Cell 71, 841–851 (1992).

    Article  CAS  Google Scholar 

  10. Weissman, J.S. & Kim, P.S. Efficient catalysis of disulphide bond rearrangements by protein disulphide isomerase. Nature 365, 185–188 (1993).

    Article  CAS  Google Scholar 

  11. Creighton, T.E. Reactivities of cysteine residues of the reduced pancreatic trypsin inhibitor. J. molec. Biol. 96, 777–782 (1975).

    Article  CAS  Google Scholar 

  12. Darby, N.J. & Creighton, T.E. Dissecting the disulphide-coupled folding pathway of bovine pancreatic trypsin inhibitor. J. molec. Biol. 232, 873–896 (1993).

    Article  CAS  Google Scholar 

  13. States, D.J., Creighton, T.E., Dobson, C.M. & Karplus, M. Conformations of intermediates in the folding of pancreatic trypsin inhibitor. J. molec. Biol. 195, 731–739 (1987).

    Article  CAS  Google Scholar 

  14. Oas, T.G. & Kim, P.S. A peptide model of a protein folding intermediate.Nature 336, 42–48 (1988).

    Article  CAS  Google Scholar 

  15. Staley, J.P. & Kim, P.S. Role of a subdomain in the folding of bovine pancreatic trypsin inhibitor. Nature 344, 685–688 (1990).

    Article  CAS  Google Scholar 

  16. van Mierlo, C.P.M., Darby, N.J., Neuhaus, D. & Creighton, T.E. . Two-dimensional 1H nuclear magnetic resonance study of the [5-55] single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor. J. molec. Biol. 222, 373–390 (1991).

    Article  CAS  Google Scholar 

  17. Staley, J.P. & Kim, P.S. Complete folding of BPTI with only a single disulphide bond. Proc. natn. Acad. Sci. U.S.A. 89, 1519–1523 (1992).

    Article  CAS  Google Scholar 

  18. van Mierlo, C.P.M., Darby, N.J. & Creighton, T.E. . The partially folded conformation of the [30-51] intermediate in the disulphide folding pathway of bovine pancreatic trypsin inhibitor. Proc. natn. Acad. Sci. U.S.A. 89, 6775–6779 (1992).

    Article  CAS  Google Scholar 

  19. van Mierlo, C.P.M., Darby, N.J., Keeler, J., Neuhaus, D. & Creighton, T.E. . Partially folded conformation of the [30-51] intermediate in the disulphide folding pathway of bovine pancreatic trypsin inhibitor. J. molec. Biol. 229, 1125–1146 (1993).

    Article  CAS  Google Scholar 

  20. Staley, J.P. & Kim, P.S. Formation of a native-like subdomain in a partially folded intermediate of BPTI. Prot. Sci. 10, 1822–1832 (1994).

    Article  Google Scholar 

  21. Goto, Y. & Hamaguchi, K. Formation of the intrachain disulphide bond in the constant fragment of the immunoglobulin light chain. J. molec. Biol. 146, 321–340 (1981).

    Article  CAS  Google Scholar 

  22. Goldenberg, D.P. Native and non-native intermediates on the BPTI folding pathway. Trends biochem. Sci. 17, 257–261 (1992).

    Article  CAS  Google Scholar 

  23. Ferrer, M., Barany, G. & Woodward, C. Partially folded molten globule and molten coil states of bovine pancreatic trypsin inhibitor. Nature struct. Biology 2, 211–217 (1995).

    Article  CAS  Google Scholar 

  24. Deisenhofer, J. & Steigemann, W. Crystallographic refinement of the structure of bovine pancreatic trypsin inhibitor at 1.5Å resolution. Acta Crystallogr. B31, 238–250 (1975).

    Article  CAS  Google Scholar 

  25. Wlodawer, A., Walter, J., Huber, R. & Sjölin, L. Structure of bovine pancreatic trypsin inhibitor. J. molec. Biol. 180, 301–329 (1984).

    Article  CAS  Google Scholar 

  26. Berndt, K.D., Guntert, P., Orbons, L.P. & Wüthrich, K. Determination of a high-quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures. J. molec. Biol. 227, 757–775 (1992).

    Article  CAS  Google Scholar 

  27. Lee, B. & Richards, F.M. The interpretation of protein structures: estimation of static accessibility. J. molec. Biol. 55, 379–400 (1971).

    Article  CAS  Google Scholar 

  28. Marks, C.B., Naderi, H., Kosen, P.A., Kuntz, I. & Anderson, S. Mutants of bovine pancreatic trypsin inhibitor lacking cysteines 14 and 38 can fold properly. Science 235, 1370–1372 (1987).

    Article  CAS  Google Scholar 

  29. Zhang, J.-X. & Goldenberg, D.P. Amino acid replacement that eliminates kinetic traps in the folding pathway of pancreatic trypsin inhibitor. Biochemistry 32, 14075–14081 (1993).

    Article  CAS  Google Scholar 

  30. Creighton, T.E. Disulphide bonds as probes of protein folding pathways. Meth. Enzymol. 131, 83–107 (1986).

    Article  CAS  Google Scholar 

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Author notes

  1. Michal Dadlez

    Present address: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawinskiego 5A 02-106, Warszawa, Poland

Authors and Affiliations

  1. Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, Massachusetts, 02142, USA

    Michal Dadlez & Peter S. Kim

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  1. Michal Dadlez
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  2. Peter S. Kim
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Dadlez, M., Kim, P. A third native one-disulphide intermediate in the folding of bovine pancreatic trypsin inhibitor. Nat Struct Mol Biol 2, 674–679 (1995). https://doi.org/10.1038/nsb0895-674

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