Genetic dissection of pancreatic trypsin inhibitor.
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.
Goldenberg
D P
DP
Department of Biology, University of Utah, Salt Lake City 84112.
Berger
J M
JM
Laheru
D A
DA
Wooden
S
S
Zhang
J X
JX
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004252:DNA Mutational Analysis; D004229:Dithiothreitol / Q000737:chemistry; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010179:Pancreas; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D014357:Trypsin; D004229:Dithiothreitol