کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1391650 | 983611 | 2008 | 11 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nɛ-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nɛ-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification](/preview/png/1391650.png)
SummaryPyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNAPyl. In this study, Nɛ-(tert-butyloxycarbonyl)-L-lysine (BocLys) and Nɛ-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNAPyl 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 Nɛ-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 Nɛ-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 Nɛ-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation.
Journal: - Volume 15, Issue 11, 24 November 2008, Pages 1187–1197