Open AccessGenetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells
Author(s) -
Riley M. Bednar,
Subhashis Jana,
Sahiti Kuppa,
Rachel Franklin,
Joseph S. Beckman,
Edwin Antony,
Richard B. Cooley,
Ryan A. Mehl
Publication year - 2021
Publication title -
acs chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.899
H-Index - 111
eISSN - 1554-8937
pISSN - 1554-8929
DOI - 10.1021/acschembio.1c00649
Subject(s) - bioorthogonal chemistry , tetrazine , protein engineering , protein tag , genetic code , amino acid , synthetic biology , biochemistry , encode , function (biology) , fusion protein , computational biology , biology , chemistry , microbiology and biotechnology , combinatorial chemistry , click chemistry , gene , recombinant dna , organic chemistry , enzyme
The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call d ual e ncoding a nd l abeling (DEAL), allowed us to efficiently encode tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically defined protein-protein cross-linking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro . By enabling the efficient dual modification of proteins in vivo , this DEAL approach provides a tool for the characterization and engineering of proteins in vivo .