Site-selective introduction of unnatural amino acid into proteins and its use for analyses of structure-function relationship of proteins.

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1. E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA^<Tyr> (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA^<Tyr>/ tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering.2. Yeast mitochondrial tRNA^<Tyr> was shown to be possibly used as an Opal-suppressor and a mutant (F38A) of yeast mitochondrial tryptophanyl-tRNA synthetase having an altered amino acid specificity was created by genetic engineering.3. An efficient method for site-selective modification of proteins using an unnatural amino acid, 3-azido-tyrosine has been developed. Using rat calmodulin as a model protein, we prepared several unnatural calmodulin molecules, each carrying an azido-tyrosine at predetermined positions. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated calmodulin molecules. This method is intrinsically versatile in that it should be easily applicable to introducing any other desirable compounds (e.g. probes and cross-linkers) into selected sites of proteins as far as appropriate derivative compounds of triarylphosphine could be chemically synthesized.4. As an important supporting technique for X-ray crystallographic analyses (SAD method), we have developed an effective method for incorporating 3-iodo-tyrosine into site-selective positions in a target protein. We have also determined the crystal structure of yeast TyrRS complexed with a Tyr-AMP analogue and the native tRNA^<Tyr> at 2.4 Å resolution.