STUDY OF A 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 ribonuclease occurring 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 and x- ray structures of both are known as well as the NMR solution structure of barnase. 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. Close to a hundred directed mutations in each protein have been produced. 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. The two Cys residues of barstar can both be replaced by Ala without loss of activity (in vitro or in vivo, but with some reduction in the strength of the bond) 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. Several methods have been developed for measuring the relative and absolute strength of the bond between barnase and barstar for various combinations of wild-type and mutant proteins. For the wild-type proteins the dissociation coefficient is on the order of 10 to the -13. 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 developmental studies and is the key to a variety of anti-viral strategies.