Improving target amino acid selectivity in a permissive aminoacyl tRNA synthetase through counter-selection
| Autor: | Lee C. Speight, Zachary M Hostetler, Camden M. Driggers, Jeffery G. Saven, Lea Z. Mbengi, Rahul M. Kohli, José A. Villegas, John J. Perona, Robert J. Blizzard, Itthipol Sungwienwong, E. James Petersson, Ryan A. Mehl, Joseph J. Porter |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
Models
Molecular 0301 basic medicine 010402 general chemistry 01 natural sciences Biochemistry Article Amino Acyl-tRNA Synthetases 03 medical and health sciences chemistry.chemical_compound Electron transfer In vivo Catalytic Domain Fluorescence Resonance Energy Transfer Amino Acids Physical and Theoretical Chemistry Permissive chemistry.chemical_classification 030102 biochemistry & molecular biology Aminoacyl tRNA synthetase Protein dynamics Organic Chemistry 0104 chemical sciences Amino acid Kinetics Förster resonance energy transfer chemistry Selectivity Protein Binding |
| Zdroj: | Organic & Biomolecular Chemistry. 15:3603-3610 |
| ISSN: | 1477-0539 1477-0520 |
| Popis: | The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein dynamics, either alone or as part of a Förster resonance energy transfer (FRET) or photo-induced electron transfer (eT) probe pair. We have previously reported the genetic incorporation of Acd by an aminoacyl tRNA synthetase (RS). However, this RS, developed from a library of permissive RSs, also incorporates N-phenyl-amino-phenylalanine (Npf), a trace byproduct of one Acd synthetic route. We have performed negative selections in the presence of Npf and analyzed the selectivity of the resulting AcdRSs by in vivo protein expression and detailed kinetic analyses of the purified RSs. We find that selection conferred a ~50-fold increase in selectivity for Acd over Npf, eliminating incorporation of Npf contaminants, and allowing one to use a high yielding Acd synthetic route for improved overall expression of Acd-containing proteins. More generally, our report also provides a cautionary tale on the use of permissive RSs, as well as a strategy for improving selectivity for the target amino acid. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|