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 ribonucleases 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 to probe the barnase-barstar interaction. Both proteins can now be obtained from recombinant genes in E. coli with yields of 100 mg\l or better. Co-expression of barstar is necessary to counter the lethal effect of barnase expression. X-ray structures of both proteins and their complex are known as well as the NMR solution structure of barnase. A fast and relatively precise assay based on a fluorogenic substrate has allowed us to use barnase-barstar titration curves and competition between active and inactive barnase mutants for barstar to study the kinetics and stability of complex formation. Solution of the structure of the complex has confirmed the involvement of several residues identified as such by protein engineering. One or more mutations of all of the barstar residues in direct contact with barnase in the complex, as well as several such for barnase, have been prepared. Determination of the Gibbs free energy for all combinations of such mutants in complex will isolate the energy contributions of different portions of the interface. Barstar A (barstar(Cys42,80Ala)), Which binds barnase almost as well as the wild type, is being used for much of this work. A system has been devised to select, in vivo, barstar mutants with improved binding to barnase mutants which are poorly inhibited by wild type barstar. Several such barstar mutants have been found. Recent work, elsewhere, in which the barnase gene is attached to a eukaryotic promoter in order to kill the tissue in which the promoter is expressed (in the first instance to produce male sterility in plants) has aroused considerable interest in its use in developmental studies and is the key to a variety of anti-viral strategies.