Concerted, rapid, quantitative, and site-specific dual labeling of proteins

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.

Scheme 1. Concerted, Rapid, One-Pot Quantitative Dual Labeling of Proteins in Aqueous Medium at Physiological pH and Temperature

(a) Unnatural amino acids and fluorophores used in this study.(b) Concerted labeling at an encoded terminal alkyne and an encoded cyclopropene via mutually orthogonal cycloadditions.

Figure 1

Specific protein labeling at genetically encoded unnatural amino acids 1 and 2. (a) Genetically encoded 1, but not 2, in calmodulin is specifically labeled with probe 3. Coomassie and fluorescence images demonstrate the specificity of labeling, and ESI MS before labeling (black, expected mass: 17875, found mass: 17874) and after labeling (red, expected mass: 18553, found mass: 18552) demonstrate the reaction is quantitative. (b) Genetically encoded 2, but not 1, in calmodulin is specifically labeled with probe 4. Coomassie and fluorescence images demonstrate the specificity of labeling, and ESI MS before labeling (black, expected mass: 17930, found mass: 17930) and after labeling (green, expected mass: 18484, found mass: 18485) demonstrate the reaction is quantitative. Raw (before deconvolution) ESI-MS spectra in Supplementary Figure 2.

Figure 2

Incorporating 1 and 2 at positions 1 and 40 of calmodulin and the kinetics of specific labeling. (a) Expression was performed in E. coli bearing ribo-Q1, O-gst-cam_1TAG-40AGTA, the PylRS/tRNA_UACU pair, and the MjPrpRS/tRNA_CUA pair. Amino acids 1 and 2 were used at 2 and 1 mM, respectively. (b) Labeling time course for reaction of CaM1₁2₄₀ with 3 and 4. Each reaction was followed for 2 h by in-gel fluorescence and mobility shift.

Figure 3

Concerted, quantitative, one-pot, dual labeling of calmodulin in 30 min. (a) Fluorophore-dependent labeling of CaM1₁2₄₀; sequential labeling with purification after first labeling in lane 4, sequential labeling without purification in lane 5, one-pot dual labeling in lane 6. (b) ESI-MS of one-pot protein labeling, before labeling (black, expected mass: 18000 found mass: 18000), after labeling (gold, expected mass: 19233 found mass: 19234). Raw (before deconvolution) ESI-MS spectra in Supplementary Figure 2.