Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNA^Tyr Suppressor

Abstract

The Methanocaldcoccus jannaschii tyrosyl-tRNA synthetase (TyrRS):tRNATyr cognate pair has been used to incorporate a large number of noncanonical amino acids (ncAAs) into recombinant proteins in Escherichia coli. However, the structural elements of the suppressor tRNATyr used in these experiments have not been examined for optimal performance. Here, we evaluate the steady-state kinetic parameters of wild-type M. jannaschii TyrRS and an evolved 3-nitrotyrosyl-tRNA synthetase (nitroTyrRS) toward several engineered tRNATyr suppressors, and we correlate aminoacylation properties with the efficiency and fidelity of superfolder green fluorescent protein (sfGFP) synthesis in vivo. Optimal ncAA-sfGFP synthesis correlates with improved aminoacylation kinetics for a tRNATyr amber suppressor with two substitutions in the anticodon loop (G34C/G37A), while four additional mutations in the D and variable loops, present in the tRNATyr used in all directed evolution experiments to date, are deleterious to function both in vivo and in vitro. These findings extend to three of four other evolved TyrRS enzymes that incorporate distinct ncAAs. Suppressor tRNAs elicit decreases in amino acid Km values for both TyrRS and nitroTyrRS, suggesting that direct anticodon recognition by TyrRS need not be an impediment to superior performance of this orthogonal system and offering insight into novel approaches for directed evolution. The G34C/G37A tRNATyr may enhance future incorporation of many ncAAs by engineered TyrRS enzymes.

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

“…34 This is expected, since amino acid incorporation into protein in vivo can only occur upon aminoacylation. Thus, aminoacylation of an in vitro transcribed 32 P-labeled tRNA CUA was measured under single-turnover conditions at a variety of Acd or Npf concentrations for AcdRS1 and AcdRS2b.…”

The central role of tRNA in genetic code expansion

“…As illustrated by the M. jannaschii Tyr-OTS [112], the anticodon recognition impact on the active site's affinity for the ncAA might be underappreciated. Ideally, o-aaRSs should be mutagenized both in ncAA binding pocket and anticodon binding domain simultaneously.…”

“…In cases where the AA activation depends on tRNA binding, information on the wobble base recognition is transmitted to the distant aaRS active site [111]. In case of the M. jannaschii Tyr-OTS, the G34C mutation, needed to create a suppressor o-tRNA derivative, increases affinity for cognate tyrosine 30 times [112]. For M. maripaludis SepRS, cognate tRNA improves discrimination over glutamate and phosphothreonine in vitro [113].…”

Structural insights into a thermostable variant of human carbonic anhydrase II

“…A gene for recombinant C‐terminally His‐tagged HCAIIts, codon‐optimized for expression in E. coli , was put into a pBad expression vector; the protein was expressed and purified according to standard protocols with further purification by size exclusion chromatography using a HiLoad 16/600 Superdex 75 pg (GE) with isocratic elution in 10 m M HEPES pH 7.0. The protein was stored at 4°C in 10 m M HEPES pH 7.0 at 30 mg/mL (quantified using Bradford assay with BSA as a standard), and crystallized at 4°C in hanging drops (reservoir: 50 m M Tris pH 8.0, 2.5 M ammonium sulfate).…”