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, 14 (12), 2793-2799

Addition of Isocyanide-Containing Amino Acids to the Genetic Code for Protein Labeling and Activation

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Addition of Isocyanide-Containing Amino Acids to the Genetic Code for Protein Labeling and Activation

Yuda Chen et al. ACS Chem Biol.

Abstract

Site-specific introduction of bioorthogonal handles into biomolecules provides powerful tools for studying and manipulating the structures and functions of proteins. Recent advances in bioorthogonal chemistry demonstrate that tetrazine-based bioorthogonal cycloaddition is a particularly useful methodology due to its high reactivity, biological selectivity, and turn-on property for fluorescence imaging. Despite its broad applications in protein labeling and imaging, utilization of tetrazine-based bioorthogonal cycloaddition has been limited to date by the requirement of a hydrophobic strained alkene reactive moiety. Circumventing this structural requirement, we report the site-specific incorporation of noncanonical amino acids (ncAAs) with a small isocyanide (or isonitrile) group into proteins in both bacterial and mammalian cells. We showed that under physiological conditions and in the absence of a catalyst these isocyanide-containing ncAAs could react selectively with tetrazine molecules via [4 + 1]-cycloaddition, thus providing a versatile bioorthogonal handle for site-specific protein labeling and protein decaging. Significantly, these bioorthogonal reactions between isocyanides and tetrazines also provide a unique mechanism for the activation of tetrazine-quenched fluorophores. The addition of these isocyanide-containing ncAAs to the list of 20 commonly used, naturally occurring amino acids expands our repertoire of reagents for bioorthogonal chemistry, therefore enabling new biological applications ranging from protein labeling and imaging studies to the chemical activation of proteins.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Genetic incorporation of ε-N-2-isocyanoisobutyryl-lysine (NCibK, 1) and ε-N-isocyano-lysine (NCK, 2) for protein labeling and decaging, respectively.
Figure 2.
Figure 2.
Characterization of reactions between NCibK and tetrazines. (A) Structures of tetrazine derivatives used in this study.(B) Kinetics of NCibK with dpTz resulted in a rate constant of k =0.29 ± 0.03 M−1 s−1. (C) Fluorogenic turn-on reaction between NCibK and BODIPY-m-Tz. (D) Fluorogenic turn-on reaction between NCibK and BODIPY-p-Tz.
Figure 3.
Figure 3.
Incorporation of NCK and NCibK into sfGFP proteins in E. coli. (A) Expression of sfGFP mutants in the presence or absence (−) of 1 mM NCK or NCibK, analyzed by SDS-PAGE. (B) ESI-MS analysis of sfGFP mutants containing NCibK or NCK. Two desired peaks were observed for each sample. One is the full-length sfGFP mutant, and the other one is the full-length sfGFP mutant without N-terminal Met. The 27577 Da peak corresponds to the misincorporation of Phe at 151 position of sfGFP. (C) ESI-MS analysis of NCibK-containing sfGFP mutant reacted with dpTz. The 27995 and 27864 Da peaks correspond to the conjugated products with and without N-terminal Met, respectively.
Figure 4.
Figure 4.
Incorporation of BocK, NCK, and NCibK into EGFPY40TAG in mammalian cells analyzed by fluorescence microscopy in the presence or absence (−) of ncAAs. Scale bar = 50 μm.
Figure 5.
Figure 5.
Tetrazine-mediated activation of isocyano-caged lipoic acid ligase-acceptor peptide (LAP) tag in living cells. (A) The reaction scheme for tetrazine-mediated activation of isocyano-caged proteins. NCK was site-specifically incorporated into the LAP-tagged neurexin1β (NRX-LAP) to block the critical lysine residue. The NCK-modified LAP tag was then decaged by tetrazine treatment, followed by reaction with coumarin fluorophore catalyzed by lipoic acid ligase (LplA). (B) HEK293T cells expressing NRX-LAP or NRXLAP-TAG with or without NCK were mixed with 1 mM dpTz in PBS for 20 min, followed by the incubation with 1 mM pyrrolidine for 8 h. After PRIME labeling (blue) and cell fixation, 0.1 μg/mL anti-Myc antibody and 7.5 μg/mL FITC-conjugated secondary antibody (green) were added to check the expression level of NRX-LAP. Nucleus staining was carried out with 5 μM DRAQ5 (red) before confocal imaging. Scale bars = 25 μm.

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