Genetic dissection of pancreatic trypsin inhibitor

David P. Goldenberg, James M Berger, Daniel Laheru, Stagey Wooden, Jian Xin Zhang

Research output: Contribution to journalArticle

Abstract

In a previous study, a genetic screening procedure was used to identify variants of bovine pancreatic trypsin inhibitor that can fold to an active conformation but that are inactivated much more rapidly than the wild-type protein in the presence of dithiothreitol (DTT). The mechanisms by which 30 of these DTT-sensitive variants are inactivated have now been investigated. Some of the amino acid replacements cause rapid inactivation in the presence of DTT because the three disulfides of the native protein are reduced up to 300-fold faster than for the wild-type protein, leading to complete unfolding. Other substitutions, however, do not greatly increase the rate of complete reduction and unfolding but lead to accumulation of an inactive two-disulfide species. There is a striking correlation between the locations of the DTT-sensitive amino acid replacements in the three-dimensional structure of the protein and the mechanisms by which the variants are inactivated. All of the substitutions that cause rapid unfolding are clustered at one end of the folded protein, in the vicinity of the two disulfides that are reduced most slowly during unfolding of the wild-type protein, while substitutions of the other class are all located at the other end of the protein, near the trypsin binding site. These results indicate that the kinetic stability of native bovine pancreatic trypsin inhibitor and its ability to function as a protease inhibitor are largely influenced by residues in two distinguishable regions of the folded protein.

Original languageEnglish (US)
Pages (from-to)5083-5087
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume89
Issue number11
StatePublished - 1992
Externally publishedYes

Fingerprint

Trypsin Inhibitors
Dissection
Dithiothreitol
Disulfides
Proteins
Aprotinin
Amino Acids
Protein Unfolding
Genetic Testing
Protease Inhibitors
Trypsin
Binding Sites

Keywords

  • Bovine pancreatic trypsin inhibitor
  • Dithiothreitol-sensitive mutants
  • Protease inhibitors
  • Protein folding
  • Protein stability

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Genetic dissection of pancreatic trypsin inhibitor. / Goldenberg, David P.; Berger, James M; Laheru, Daniel; Wooden, Stagey; Zhang, Jian Xin.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 89, No. 11, 1992, p. 5083-5087.

Research output: Contribution to journalArticle

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