Unknown

Dataset Information

0

Improving target amino acid selectivity in a permissive aminoacyl tRNA synthetase through counter-selection.


ABSTRACT: 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-aminophenylalanine (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.

SUBMITTER: Sungwienwong I 

PROVIDER: S-EPMC5507695 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

altmetric image

Publications

Improving target amino acid selectivity in a permissive aminoacyl tRNA synthetase through counter-selection.

Sungwienwong Itthipol I   Hostetler Zachary M ZM   Blizzard Robert J RJ   Porter Joseph J JJ   Driggers Camden M CM   Mbengi Lea Z LZ   Villegas José A JA   Speight Lee C LC   Saven Jeffery G JG   Perona John J JJ   Kohli Rahul M RM   Mehl Ryan A RA   Petersson E James EJ  

Organic & biomolecular chemistry 20170501 17


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-aminophenylalanine (Npf), a trace byproduct of one Acd synthetic route. We have performed negative selec  ...[more]

Similar Datasets

| S-EPMC6642615 | biostudies-literature
| S-EPMC5321558 | biostudies-literature
| S-EPMC6295713 | biostudies-literature
| S-EPMC3060492 | biostudies-literature
| S-EPMC7694997 | biostudies-literature
| S-EPMC5959800 | biostudies-literature
| S-EPMC2330213 | biostudies-literature
| S-EPMC2396676 | biostudies-literature
| S-EPMC5993425 | biostudies-literature
| S-EPMC1502431 | biostudies-literature