A genetic screen to identify variants of bovine pancreatic trypsin inhibitor with altered folding energetics.
A genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild-type protein. Small cultures of Escherichia coli containing plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT). Under these conditions, unfolding and inactivation of the wild-type protein has a half-time of about 10 hours. Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate. Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated. The genes coding for 68 "DTT-sensitive" mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified. Most of the altered residues are largely buried in the core of the native wild-type structure and are highly conserved among proteins homologous to BPTI. These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated at most sites without completely preventing folding. Because this genetic screen is based on changes in folding energetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding and unfolding.
Coplen
L J
LJ
Department of Biology, University of Utah, Salt Lake City 84112.
Frieden
R W
RW
Goldenberg
D P
DP
D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000235:genetics*; D002642:Chick Embryo; D004229:Dithiothreitol / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics; D005819:Genetic Markers / Q000032:analysis; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation
D005819:Genetic Markers; D007611:Aprotinin; D004229:Dithiothreitol