Protein engineering of homodimeric tyrosyl-tRNA synthetase to produce active heterodimers.
Heterodimers of tyrosyl-tRNA synthetase from Bacillus stearothermophilus have been produced by mutagenesis at the subunit interface. Oppositely charged groups have been engineered into the subunits so that they can form a complementary pair. Wild-type tyrosyl-tRNA synthetase is a symmetrical dimer in which the side chains of the 2 Phe-164 residues interact at the subunit interface. Phe-164 was mutated to Asp in tyrosyl-tRNA synthetase and to Lys in a truncated enzyme (des-(321-419)tyrosyl-tRNA synthetase) which lacks the two tRNA-binding sites, but which can catalyze pyrophosphate exchange. The size difference allows subunit association to be studied by gel filtration chromatography. These changes induce reversible dissociation from active dimers into inactive monomers at pH values which favor ionization at position 164. A mixture of the two mutants near neutral pH is apparently fully active in pyrophosphate exchange and consists of a heterodimer of [Asp164]tyrosyl-tRNA synthetase and [Lys164]des-(321-419)tyrosyl-tRNA synthetase. Despite having only one binding site for tRNA, heterodimer has full aminoacylation activity at high concentrations of tyrosine. We have therefore produced a family of dimers that differ in stability near neutral pH. This novel approach using protein engineering allows specific dimerization of subunits of the same size that have different defined mutations, each subunit being tagged by the charge. Such hybrid proteins can be used to study subunit interaction.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009154:Mutation; D011108:Polymers / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*
D011108:Polymers; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase