STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

点击翻译按钮获取中文摘要

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, where expression of barstar counters the lethal effect of barnase expression. The structures of both proteins and their complex are known. 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 and preliminary binding studies have been done for several combinations. 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(C42,80A), 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 (e.g. H102K) which are poorly inhibited by wild type barstar. Several such barstar mutants have been found, with the strongest suppressors of barnase (H102K) all including the mutation Y30W. As the side chain of this tyrosine is buried within barstar, this suggests that the improved binding involves some structural rearrangement of surrounding residues. In vitro studies confirm that in vivo behavior correlates with strength of barnase-barstar binding. We have found that, for at least two different Streptomyces ribonucleases (barnase homologs), barstar is a good enough inhibitor to provide in vivo protection and thus allow the production of these enzymes in E. coli. In vitro binding studies and co-crystallization with barstar for structural studies are planned. Work elsewhere, in which the barnase gene becomes a tissue-specific killer when attached to eukaryotic promoters, has aroused considerable interest in its use in developmental studies and as the key to a variety of anti-viral strategies.