The Methanococcus jannaschii tRNA(Tyr)/tyrosyl-tRNA synthetase pair has been engineered to incorporate unnatural amino acids into proteins in Escherichia coli site-specifically In older to acid other unnatural amino acids into proteins by this approach. the amino acid binding site of M jannaschn tyrosyl-tRNA synthetase need to be mutated The crystal structures of M.jannaschii tyrosyl-tRNA synthetase and Its maturation were determined, which provided all opportunity to design aminoacyl-tRNA synthetases specific for Unnatural amino acids In our Study. we attempted to design aminoacyl-tRNA synthetases being able to deliver p-acetyl-L-phenylalanine into proteins p-Acetyl-L-phenylalanine was Superimposed oil tyrosyl in M jannaschn tyrosyl-tRNA synthetase-tyrosine complex Tyr32 needed to be changed to non-polar amino acid with shorter side chain, Val, Leu, Ile, Gly or Ala, in order to reduce steric clash and provide hydrophobic environment to acetyl oil p-acetyl-L-phenylalanine Asp158 and Ile 159 Would be changed to specific amino acids for the same reason So we designed 60 aminoacyl-rRNA synthetases. Binding of these aminoacyl-tRNA synthetases with p-acetyl-L-phenylalanine indicated that only 15 of them turned Out to be able to bind p-acetyl-L-phenylalanine with reasonable poses Binding affinity computation proved that the mutation of Tyr32Leu and Asp158Gly benefited p-acetyl-L-phenylalanine binding And two of the designed aminoacyl-tRNA synthetases had considerable binding affinities. They seemed to be very promising to be able to incorporate p-acetyl-L-phenylalanine into proteins in E coli The results show that the combination of homology modeling and molecular docking is a feasible method to filter inappropriate mutations in molecular design and Point out beneficial Mutations (C) 2009 Elsevier Inc All rights reserved