The mechanism of folding of a small, well-characterized protein, bovine pancreatic trypsin inhibitor (BPTI), will be studied by testing the effects of amino acid substitutions on the stabilities of folding intermediates and oh the stability and conformation of the native protein. this project is directed towards the long-term goal of understanding how the three-dimensional structures of globular proteins are specified by their amino acid sequences and how changes in the amino acid sequence may alter the properties of the native protein or the process of forming it. Many diseases involving defects in protein function may arise because of mutations that prevent proteins from acquiring the correct three-dimensional structures required to interact specifically with other molecules. Modified forms of BPTI will be produced using in vitro mutagenesis and recombinant DNA methods. A cloned gene coding for BPTI will be modified using synthetic oligonucleotides and used to direct the synthesis of mutant proteins in Escherichia coli. The disulfide-coupled folding reactions of the mutant proteins will be studied by chemically trapping and characterizing disulfide-bonded intermediates in folding and unfolding. To allow quantitative comparisons of he mutant and wild-type proteins, the relative stabilities of the native , unfolded and intermediate state will be measured, as well as their rates of interconversion. Some of the mutations to be studied ar site-directed changes designed to test the roles of specific residues and interactions in stabilizing the native protein and determining the pathway of folding. These site- directed changes are intended to disrupt interactions present in the native protein, including hydrogen bonds, salt bridges and packing interactions. Other mutants to be studied were identified amount randomly mutagenized clones using a genetic screen to isolate mutants with altered folding intermediate as well as in the native protein is expected to alter the stability of both species similarly. From the observed effects of the mutations on the relative stabilities of the different species and their kinetics of interconversion, the roles of he altered residues at the various stages of folding will be inferred.
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