Pyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNA(Pyl). In this study, N(epsilon)-(tert-butyloxycarbonyl)-L-lysine (BocLys) and N(epsilon)-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNA(Pyl) by PylRS. Crystal structures of a PylRS catalytic fragment complexed with BocLys and an ATP analog and with AlocLys-AMP revealed that PylRS requires an N(epsilon)-carbonyl group bearing a substituent with a certain size. A PylRS(Y384F) mutant obtained by random screening exhibited higher in vitro aminoacylation and in vivo amber suppression activities with BocLys, AlocLys, and pyrrolysine than those of the wild-type PylRS. Furthermore, the structure-based Y306A mutation of PylRS drastically increased the in vitro aminoacylation activity for N(epsilon)-benzyloxycarbonyl-L-lysine (ZLys). A PylRS with both the Y306A and Y384F mutations enabled the large-scale preparation (>10 mg per liter medium) of proteins site-specifically containing N(epsilon)-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation.

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The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS-tRNA(Tyr)-L-tyrosine complex, solved at a resolution of 1.95 A, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNA(Tyr) identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.

We report a method of photo-cross-linking proteins in mammalian cells, which is based on site-specific incorporation of a photoreactive amino acid, p-benzoyl-L-phenylalanine (pBpa), through the use of an expanded genetic code. To analyze the cell signaling interactions involving the adaptor protein Grb2, pBpa was incorporated in its Src homology 2 (SH2) domain. The human GRB2 gene with an amber codon was introduced into Chinese hamster ovary (CHO) cells, together with the genes for the Bacillus stearothermophilus suppressor tRNA(Tyr) and a pBpa-specific variant of Escherichia coli tyrosyl-tRNA synthetase (TyrRS). The Grb2 variant with pBpa in the amber position was synthesized when pBpa was included in the growth medium. Upon exposure of cells to 365-nm light, protein variants containing pBpa in the positions proximal to the ligand-binding pocket were cross-linked with the transiently expressed epidermal growth factor (EGF) receptor in the presence of an EGF stimulus. Cross-linked complexes with endogenous proteins were also detected. In vivo photo-cross-linking with pBpa incorporated in proteins will be useful for studying protein-protein interactions in mammalian cells.

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25 at. % Rare-earth (RE)-doped ceria samples (RE=Sm, Dy, Y, and Yb) were examined using transmission electron microscopy and electron energy loss spectroscopy, from which the oxygen vacancy ordering in nanosized domains was confirmed. The relationships of the dopant type, oxygen vacancy ordering, and ionic conductivity of doped ceria were established. It is found that the ordering of oxygen vacancies depends strongly on the dopant type, and the development of nanosized domains with a higher degree of ordering can lead to a more dramatic decrease of ionic conductivity in doped ceria.

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