Quantitative Protein Labeling in Live Cells by Controlling the Redox State of Encoded Tetrazines

Area of Science:

• Chemical Biology
• Biotechnology
• Molecular Imaging

Background:

• Site-specific protein modification is crucial for biological studies and drug development.
• 1,2,4,5-tetrazine (Tet) residues offer rapid kinetics for quantitative labeling in living systems.
• Intracellular redox environments can affect Tet reactivity, limiting labeling efficiency.

Purpose of the Study:

• To develop and validate methods for assessing and improving Tet-based protein labeling in living cells.
• To investigate the impact of intracellular redox states on Tet residue reactivity.
• To engineer novel Tet variants with tunable redox potentials for enhanced in-cell labeling.

Main Methods:

• Adaptation of a gel-shift assay into a "PEG Chaser assay" for evaluating labeling completeness.
• Utilizing photooxidation to convert reduced Tet forms to reactive oxidized forms.
• Employing genetic code expansion to encode new Tet non-canonical amino acids (ncAAs) with varying redox potentials.

Main Results:

• The PEG Chaser assay successfully distinguishes labeled from unlabeled proteins in living cells.
• Encoded Tet residues exist in an equilibrium between oxidized (Tz) and reduced (DHTz) forms intracellularly.
• A new Tet3H ncAA enables photoactivatable labeling with complete protein modification within 5 minutes.

Conclusions:

• Controlling the redox state of Tet residues is a key strategy for optimizing in-cell labeling.
• Engineered Tet ncAAs with tunable redox potentials significantly expand the utility of Tet chemistry in living cells.
• Photoactivatable Tet labeling offers a rapid and efficient method for quantitative protein modification in biological systems.