Site-Specific Labeling of Transcription Factors Using Non-Canonical Amino Acids and Ring Strain-Promoted [2+3] Cycloaddition Via Copper-Free Click Chemistry

In this work, we demonstrate labeling of transcription factor proteins by incorporation of non-natural amino acids containing bioorthogonal chemical functionalites (azide or alkyne) followed by copper-free click chemistry. The ring strain promoted [3 + 2] dipolar cycloaddition of dibenzocyclooctyne with azides, known as “copper-free click chemistry”, provides rapid and site-specific labeling of zinc finger proteins (ZFPs) as transcription factors (TFs). Following expression, purification and labeling, TFs retain their sequence-specific DNA-binding activity under native conditions, whereas the activity of TFs labeled by copper-mediated click chemistry was ablated. Copper-free click chemistry exhibited high reaction yields, even at low temperatures or in the presence of high salt concentrations, which are generally unfavorable reaction conditions for common methods involving hydrolysis-mediated protein modification. Residue-specific replacement of methionine by azidohomoalanine was used to achieve mono- or multi-functionalization of engineered or “synthetic” transcription factor proteins. As a model transcription factor, we engineered a zinc-finger protein to contain a single methionine residue at the N-terminus. In addition, we constructed a chimeric TF protein by fusing a protein tag containing multiple methionine residues at the N-terminus and a zinc-finger domain without methionine sites at the C-terminus. In this way, the engineered protein contained multiple sites for chemical labeling, and the chimeric protein was assembled and assayed for functionality after the protein modification. For all engineered TF proteins, the copper-free click reaction afforded good yields as determined by spectroscopic and fluorescence measurements, which was independent of the protein purification buffer. Finally, using mass spectrometry analysis, we determined the number of solvent exposed methionine sites on TF proteins. Overall, we anticipate that copper-free click chemistry mediated by non-natural amino acid incorporation will provide a robust and viable route for site-specific labeling of metal-ion containing proteins, which comprise a universal class of transcription factor proteins.