Steinberg X et al. (2020), A rationally designed orthogonal synthetase for...

XB-ART-57461

Heliyon 2020 Oct 01;610:e05140. doi: 10.1016/j.heliyon.2020.e05140.

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A rationally designed orthogonal synthetase for genetically encoded fluorescent amino acids.

Steinberg X , Galpin J , Nasir G , Sepúlveda RV , Ladron de Guevara E , Gonzalez-Nilo F , Islas LD , Ahern CA , Brauchi SE .

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The incorporation of non-canonical amino acids into proteins has emerged as a promising strategy to manipulate and study protein structure-function relationships with superior precision in vitro and in vivo. To date, fluorescent non-canonical amino acids (f-ncAA) have been successfully incorporated in proteins expressed in bacterial systems, Xenopus oocytes, and HEK-293T cells. Here, we describe the rational generation of a novel orthogonal aminoacyl-tRNA synthetase based on the E. coli tyrosine synthetase that is capable of encoding the f-ncAA tyr-coumarin in HEK-293T cells.

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Genes referenced: mt-tr trna

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Figure 1. Homology modeling of the CoumRS enzyme and molecular docking studies. (a) Structure of the EcTyrRS enzyme (PDB: 1X8X) used to compute the CoumRS homology model. The Tyr ligand is shown in red. Color code depicts the different regions of the enzyme. C- and N- terminal regions in green, catalytic domains in blue, the linker between them in brown, and the KMSKS region in red. (b) Linear scheme of TyrRS highlighting the nine engineered mutations present in the CoumRS. Color code is the same as in panel a. (c) Tyr ligand (red) in the binding pocket of TyrRS. (d) Network of amino acids stabilizing Try within the binding region. (e) Molecular docking showing that TyrRS binds Tyr leaving Phe, Trp, and Tyr-coumarin (yellow, green, and pink respectively) out of the principal binding region. (f) Tyr-coumarin ligand (pink) docked in the molecular model for CoumRS. The new enzyme presents a wider ligand binding region when compared to the wildtype enzyme in c. (g) The network of amino acids stabilizing Tyr-coumarin is not well conserved when compared to the wildtype enzyme. Nevertheless, the lowest energy configuration suggests that the orientation of both ligands is similar. (h) View of the lowest energy configuration of Tyr/TyrCoum molecular docking shows that CoumRS binds Tyr-coumarin leaving the natural aromatic amino acids (Phe, yellow; Tyr, red; Trp, green) out of the principal binding region.

Figure 2. Amber codon suppression and incorporation of a coumarinyl aminoacid in HEK-293T cells. (a) Western blots for the detection of amber suppression in HEK-293T cells expressing the GFP40TAG gene in the absence or presence of Tyr-coumarin and Lys-NBD (lower band at 30 kDa, green box). 2nd lane is transfected with GFP40TAG alone and the 3rd lane represent the effect of expressing a TyrRS and the correspondant tRNA. The rescue of GFP40TAG of the BpaRS (lane 4) is comparable to the rescue in the presence of CoumRS (lane 6). (b) Structures corresponding to the f-ncAAs used in this study and the natural amino acid tyrosine. (c) The suppression of the amber codon was estimated by analyzing the expression of GFP40TAG on cultured HEK-293T cells. Images correspond to a representative filed of view in which transmitted light and fluorescent signals are merged for both the condition with (left) or without (right) Tyr-coumarin in the culture media. Bar = 40um (d) Quantification of GFP40TAG expression in the absence or presence of Tyr-coumarin in the culture media or absence of CoumRS in the transfection mix. Bars correspond to SD. (e) Colocalization of coumarin and GFP signals. Images taken from transiently transfected HEK293T cells expressing GFP rescued with a coumarin side chain at position 40. The signal of coumarin fluorescence is absent in the cells not expressing GFP. Bar = 10 um.

References [+] :

Bae, Expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins. 2003, Pubmed