Two-dimensional 1H nuclear magnetic resonance study of the (5-55) single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor

Carlo P.M. van Mierlo*, Nigel J. Darby, David Neuhaus, Thomas E. Creighton

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

84 Citations (Scopus)

Abstract

An analogue of the bovine pancreatic trypsin inhibitor (BPTI) folding intermediate that contains only the disulphide bond between Cys5 and Cys55 has been prepared in Escherichia coli by protein engineering methods, with the other four Cys residues replaced by Ser. Two-dimensional 1H nuclear magnetic resonance studies of the analogue have resulted in essentially complete resonance assignments of the folded form of the protein. The folded protein has a compact conformation that is structurally very similar to that of native BPTI, although there are subtle differences and the folded conformation is not very stable. Approximately half of the protein molecules are unfolded at 3 °C, and this proportion increases at higher temperatures. The folded and unfolded conformations are in slow exchange. The conformational properties of the analogue can explain many aspects of the kinetic role that the normal (5-55) intermediate plays in the folding of BPTI.

Original languageEnglish
Pages (from-to)373-390
JournalJournal of Molecular Biology
Volume222
Issue number2
DOIs
Publication statusPublished - 20 Nov 1991

Keywords

  • bovine pancreatic trypsin inhibitor (BPTI)
  • disulphide bonds
  • folding intermediate
  • n.m.r.
  • protein folding

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