Abstract
Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small molecules at physiological pH, temperature, and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-containing amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quantitative, site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, respectively. Protein double labeling in aqueous buffer at physiological pH, temperature, and pressure is quantitative in 30 min.
Highlights
The ability to attach two distinct molecules to programmed sites in proteins will facilitate a variety of applications including FRET1,2 to study protein structure, conformation, and dynamics
One approach relies on the installation of one unnatural amino acid that is labeled in combination with cysteine thiol labeling, but this approach is generally limited to proteins that do not contain more than one free thiol.[3,4]
We demonstrated that the PyrrolysyltRNA synthetase (PylRS)/tRNA pair and synthetically evolved derivatives of the Methanococcus janaschii Tyrosyl-tRNA synthetase (MjTyrRS)/MjtRNA pair are mutually orthogonal in their aminoacylation specificity and can be used to direct the incorporation of pairs of unnatural amino acids in response to amber and quadruplet codons.[6]
Summary
The ability to attach two distinct molecules to programmed sites in proteins will facilitate a variety of applications including FRET1,2 to study protein structure, conformation, and dynamics. An ideal strategy for dual labeling requires (i) the efficient, cellular incorporation of two distinct unnatural amino acids bearing bioorthogonal functional groups that do not react together, into a protein and (ii) the quantitative, rapid, sitespecific labeling of each encoded functional group at physiological temperature, pressure, and pH upon the simultaneous addition of both labeling reagents.
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