Tuning aromatic contributions by site-specific encoding of fluorinated phenylalanine residues in bacterial and mammalian cells

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 fluorinat...

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Published inbioRxiv
Main Authors Galles, Grace D, Infield, Daniel T, Clark, Colin J, Hemshorn, Marcus L, Manikandan, Shivani, Frederico Sassoli Fazan, Rasouli, Ali, Tajkhorshid, Emad, Galpin, Jason D, Cooley, Richard B, Mehl, Ryan A, Ahern, Christopher A
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 03.10.2022
Subjects
Amino acids
Aminoacyl-tRNA ligase
Electrostatic properties
Hydrophobicity
Mammalian cells
Mutagenesis
Phenylalanine
Transfer RNA
tRNA
Tryptophan
Tyrosine
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Summary: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 new group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA 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 will enable deconstruction of biological mechanisms which utilize aromatic-pi interactions in structural and cellular contexts. Competing Interest Statement The authors have declared no competing interest. Footnotes * We now include a new figure 7 with patch clamp evidence of macroscopic expression (7G through 7J) and MS/MS spectra for F1486(2,3,6F Phe)- Nav 1.5 (7F).
DOI:10.1101/2022.04.12.488046