RESUMEN
The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.
Asunto(s)
Aminoacil-ARNt Sintetasas , Fenilalanina , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Halogenación , Fenilalanina/metabolismo , ARN de Transferencia/metabolismo , Humanos , Células HEK293RESUMEN
The development of bioorthogonal fluorogenic probes constitutes a vital force to advance life sciences. Tetrazine-encoded green fluorescent proteins (GFPs) show high bioorthogonal reaction rate and genetic encodability, but suffer from low fluorogenicity. Here, we unveil the real-time fluorescence mechanisms by investigating two site-specific tetrazine-modified superfolder GFPs via ultrafast spectroscopy and theoretical calculations. Förster resonance energy transfer (FRET) is quantitatively modeled and revealed to govern the fluorescence quenching; for GFP150-Tet with a fluorescence turn-on ratio of ~9, it contains trimodal subpopulations with good (P1), random (P2), and poor (P3) alignments between the transition dipole moments of protein chromophore (donor) and tetrazine tag (Tet-v2.0, acceptor). By rationally designing a more free/tight environment, we created new mutants Y200A/S202Y to introduce more P2/P1 populations and improve the turn-on ratios to ~14/31, making the fluorogenicity of GFP150-Tet-S202Y the highest among all up-to-date tetrazine-encoded GFPs. In live eukaryotic cells, the GFP150-Tet-v3.0-S202Y mutant demonstrates notably increased fluorogenicity, substantiating our generalizable design strategy.
RESUMEN
Genetic code expansion (GCE) technologies commonly use the pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei (Mm) and Methanosarcina barkeri (Mb) for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Recently a homologous PylRS/tRNAPyl pair from Candidatus Methanomethylophilus alvus Mx1201 (Ma) was developed that, lacking the N-terminal tRNA-recognition domain of most PylRSs, overcomes insolubility, instability, and proteolysis issues seen with Mb/Mm PylRSs. An open question is how to alter Ma PylRS specificity to encode specific ncAAs with high efficiency. Prior work focused on "transplanting" ncAA substrate specificity by reconstructing the same active site mutations found in functional Mm/Mb PylRSs in Ma PylRS. Here, we found that this strategy produced low-efficiency Ma PylRSs for encoding three structurally diverse ncAAs: acridonyl-alanine (Acd), 3-nitro-tyrosine, and m-methyl-tetrazinyl-phenylalanine (Tet3.0-Me). On the other hand, efficient Ma PylRS variants were generated by a conventional life/death selection process from a large library of active site mutants: for Acd encoding, one variant was highly functional in HEK293T cells at just 10 µM Acd; for nitroY encoding, two variants also encoded 3-chloro, 3-bromo-, and 3-iodo-tyrosine at high efficiency; and for Tet-3.0-Me, all variants were more functional at lower ncAA concentrations. All Ma PylRS variants identified through selection had at least two different active site residues when compared with their Mb PylRS counterparts. We conclude that Ma and Mm/Mb PylRSs are sufficiently different that "active site transplantation" yields suboptimal Ma GCE systems. This work establishes a paradigm for expanding the utility of the promising Ma PylRS/tRNAPyl GCE platform.