STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

Diabetes, Digestive, Kidney Diseases

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Abstract

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 a 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, barnase at 1.5 Angstrom resolution. Crystal structures of several barnase-barstar pairs having complementary mutations in the interface, obtained by an in vivo selective technique, have been solved, providing insight into the mechanisms that determine the strength of the bond. Barstar also inhibits a group of RNases from Streptomyces strains. These enzymes are distantly related to barnase with a sequence identity of only 25%. Among the four such enzymes in hand, identities range from 40% to 70%. The structures of two, RNases Sa and St, are known from work on nonrecombinant material and a third, RNase Sa2, from our recombinant material. The structure of recombinant RNase Sa in complex with barstar has also been solved. Two barstar homologs from Streptomyces, for Sa2, have been cloned and expressed and since RNase St of S. erythreus is insufficiently inhibited by barstar, its homologous inhibitor is being actively pursued. The gene yrdF, identified in the the complete sequence of B. subtilis 168 as similar to that of barstar, has been cloned and, after several codon modifications, expressed in E. coli. Although there is no recognizable homolog of barnase in B. subtilis 168, yrdF is an effective inhibitor, in vitro and in vivo, of barnase. The dissociation constants (Kd) of the two Sa inhibitors complexed with barnase are 3x10exp-11 and 3x10exp-10, compared to no more than 10exp-12 for yrdH and 6x10exp-14 for barstar. A phage display system has been developed for selection of varieties or homologs of barstar that bind tightly to barnase or its mutants. Procedures have been developed for total synthesis of the barstar gene with randomization of selected residues and a multiplicity (the number of independently randomized sequences)on the order of 10exp9. We have screened a synthetic barstar library with a spatially compact eight residue portion of the hydrophobic core randomized. The possible residues in each substituted position being Leu, Ile, Val, Met or Phe. 200 unselected genes transferred to a plasmid barstar vector showed no indication of functional barstar, while 120 selected genes all appear to be functional in vivo, allowing transfomation of their host by a compatible plasmid carrying barnase. About half of the latter produce measurable barstar activity in vitro and half of these yield amounts comparable to the wild type gene. Thirty of the genes producing in vitro activity have been sequenced. No two are alike. The randomized codon at barstar position 74, Phe in the wild-type, is Phe in all the selected genes sequenced, indicating the structural importance of this residue. One product, with non-native residues in 6 positions and produced in high yield, has been provisionally chosen for structural studies. We are currently screening a barstar gene library in which all 22 residues of the hydrophilic core have been randomized.