Importance of single molecular determinants in the fidelity of expanded genetic codes

Abstract

The site-selective encoding of noncanonical amino acids (NAAs) is a powerful technique for the installation of novel chemical functional groups in proteins. This is often achieved by recoding a stop codon and requires two additional components: an evolved aminoacyl tRNA synthetase (AARS) and a cognate tRNA. Analysis of the most successful AARSs reveals common characteristics. The highest fidelity NAA systems derived from the Methanocaldococcus jannaschii tyrosyl AARS feature specific mutations to two residues reported to interact with the hydroxyl group of the substrate tyrosine. We demonstrate that the restoration of just one of these determinants for amino acid specificity results in the loss of fidelity as the evolved AARSs become noticeably promiscuous. These results offer a partial explanation of a recently retracted strategy for the synthesis of glycoproteins. Similarly, we reinvestigated a tryptophanyl AARS reported to allow the site-selective incorporation of 5-hydroxy tryptophan within mammalian cells. In multiple experiments, the enzyme displayed elements of promiscuity despite its previous characterization as a high fidelity enzyme. Given the many similarities of the TyrRSs and TrpRSs reevaluated here, our findings can be largely combined, and in doing so they reinforce the long-established central dogma regarding the molecular basis by which these enzymes contribute to the fidelity of translation. Thus, our view is that the central claims of fidelity reported in several NAA systems remain unproven and unprecedented. directed evolution | protein engineering

A platform for constructing, evaluating, and screening bioconjugates on the yeast surface

“…Display levels observed here are somewhat lower than the display levels of a reporter construct containing only canonical amino acids, but are similar among all aaRSs and 'cognate' ncAAs tested (Supplementary Figure S1, Supplementary Table SI). When the induction media is supplemented with a 'noncognate' ncAA, display levels remain elevated, indicating that the aaRSs used here are somewhat promiscuous (see also Supplementary Figure S1 and Supplementary Table SI) (Antonczak et al, 2011). Induction of cells in media lacking ncAAs results in reduced display levels, suggesting that ncAAs are present in the majority of constructs displayed on the yeast surface.…”

Efficient Synthesis and In Vivo Incorporation of Acridon-2-ylalanine, a Fluorescent Amino Acid for Lifetime and Förster Resonance Energy Transfer/Luminescence Resonance Energy Transfer Studies

“…S8, Supporting Information) Similar results have been observed by Tipmann for other UaaRSs. 56 This is probably generally true of TyrRS variants evolved for the incorporation of large amino acids. The pocket must be large enough to accommodate the Uaa and yet not permit Tyr to bind with hydrating water molecules.…”

Photoactivatable Mussel‐Based Underwater Adhesive Proteins by an Expanded Genetic Code

“…We aimed to develop an aaRS for ribosomal incorporationo f our newly developed photocaged DOPAd erivative o-nitrobenzyl DOPA( ONB-DOPA; [25] Scheme 1). This strategy circumvents the recently encountered problemo fh aving to evolve an aaRS that discriminates between DOPAa nd similar Tyr, [26,27] and to avoid DOPAa uto-oxidation,w hich has been shown to impair adhesive properties. [13,28] Additionally,t he ONB group, which is cleaved upon irradiation with l = 365 nm light and frequently used to produce caged proteins, [29][30][31] allows for spatiotemporal control of adhesive catechols ide chain of DOPA.…”