RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

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Two proteins, barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens, and barstar, its intracellular inhibitor, are used as a model system for the study of protein folding and protein-protein interactions. Barnase is one of an homologous group of ribonucleases occuring in both prokaryotes and eukaryotes. Recombinant DNA techniques are being applied with three major aims: (1) to facilitate production of wild type and mutant proteins; (2) to examine the structural and control sequences of the genes; and (3) to make specific changes in the sequences to test theories of folding and probe the barnase- barstar interaction. The lethal effect of the cloned wild type barnase gene can be repressed by expression of the barstar gene on the same plasmid. E. coli plasmid vectors have been devised for both proteins and both can now be obtained essentially pure in 100 mg quantities. DNA and amino acid sequences are known for both and the x-ray structure of barnase has been refined to 2.0 A. The structures of both proteins in solution are being studied by 2-D NMR and x-ray work on both barstar and the barnase-barstar complex are under way. A synthetic fluorescent substrate has been used to study hydrolysis kinetics and to look at the kinetics and stability of the barnase-barstar interaction for native and mutant proteins. A number of variations in the sequence of each protein (80 in barnase, 60 in barstar) have been obtained by oligonucleotide-directed mutagenesis. Some of these were aimed at specific questions but most are part of a survey of the protein surfaces designed to locate their areas of interaction and residues on both have been identified as being so involved. Our finding that the two Cys residues of barstar can both be replaced by Ala without loss of activity or yield will greatly simplify future studies of barstar folding. Such replacement of either or both of the Cys residues reduces the stability of barstar only to that of the wild-type measured in the presence of mercaptoethanol or DTT. A method has been developed for measuring the relative strength of the bond between barnase and barstar for various combinations of wild-type and mutant proteins. Recent work, elsewhere, in which the barnase gene was attached to a eukaryotic promoter in order to kill the tissue in which that promoter is expressed (in the first instance to produce male sterility in plants) has aroused considerable interest in its possible use in development studies and as the key to a variety of anti-viral strategies.