Tuning phenylalanine fluorination to assess aromatic contributions to protein function and stability in cells

Abstract

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.

Truncation-Free Genetic Code Expansion with Tetrazine Amino Acids for Quantitative Protein Ligations

“…The use of permissivity, the capacity of previously evolved aaRS/tRNA pairs to encode new ncAA variants, enables access to new functionality in proteins while circumventing the nontrivial process of selection new aaRS for a particular ncAA variant. 43,44 The improvement seen with Tet2-Et also underscores how the permissivity of aaRSs can be used to improve the encoding efficiency if minor structural changes do not impact the function. 13 An Improved Second-Generation Tet2RS.…”

“…We assessed the expression level using a small ubiquitin-like modifier SUMO 35 -sfGFP construct with the TAG site at position 35 in the SUMO domain. , We found that the suppression efficiency with the permissive Tet2-Et ncAA was 50% higher at 500 μM ncAA (Figure A), and the ncAA concentration required to reach half maximal expression (UP50) was 2-fold lower (Figure S4A). The use of permissivity, the capacity of previously evolved aaRS/tRNA pairs to encode new ncAA variants, enables access to new functionality in proteins while circumventing the nontrivial process of selection new aaRS for a particular ncAA variant. , The improvement seen with Tet2-Et also underscores how the permissivity of aaRSs can be used to improve the encoding efficiency if minor structural changes do not impact the function …”

Trimethyllysine Reader Proteins Exhibit Widespread Charge-Agnostic Binding via Different Mechanisms to Cationic and Neutral Ligands

“…To gain further insight into the mechanisms of binding of the unselective readers, we turned to tuning the electrostatics of aromatic residues, which is an established strategy to evaluate cation−π interactions. − Previously, we developed a genetic code expansion (GCE) methodology incorporating para -substituted Phe derivatives to systematically tune the electrostatic potentials (ESPs) of aromatic residues within cages of readers, which provides information on the electrostatic contribution to binding at the individual residue level (Figure ). This approach has elucidated the driving forces of various reader proteins binding to different PTMs, , including cation−π interactions with Kme3. , Using this GCE method, we investigated the DIDO1 PHD and the SGF29 TTD (Figure ).…”

Mechanism of Qiguiyin Decoction Treats Pulmonary Infection Caused by Pseudomonas aeruginosa Based on Gut Microbiota and Metabolomics

“…In addition, phenylalanine was one of the essential amino acids of human body, which belongs to the aromatic amino acid. 27 Most of them were catalyzed and oxidized to tyrosine by phenylalanine hydroxylase in the body. Together with tyrosine, they synthesized important neurotransmitters and hormones involved in the body’s metabolism of sugar and fat.…”