title: 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.
authors.affiliation: Department of Chemistry, University of Pennsylvania, 213 South 34th Street, Philadelphia, PA 19104, USA. ejpetersson@sas.upenn.edu.
authors.forename: Itthipol
authors.initials: I
authors.lastname: Sungwienwong
authors.forename: Zachary M
authors.initials: ZM
authors.lastname: Hostetler
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Blizzard
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Porter
authors.forename: Camden M
authors.initials: CM
authors.lastname: Driggers
authors.forename: Lea Z
authors.initials: LZ
authors.lastname: Mbengi
authors.forename: José A
authors.initials: JA
authors.lastname: Villegas
authors.forename: Lee C
authors.initials: LC
authors.lastname: Speight
authors.forename: Jeffery G
authors.initials: JG
authors.lastname: Saven
authors.forename: John J
authors.initials: JJ
authors.lastname: Perona
authors.forename: Rahul M
authors.initials: RM
authors.lastname: Kohli
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Mehl
authors.forename: E James
authors.initials: EJ
authors.lastname: Petersson
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding
chemical_list: D000596:Amino Acids; D000604:Amino Acyl-tRNA Synthetases