In Cellulo Cysteine Umpolung for Protein Structure Probing

Area of Science:

• Chemical Biology
• Proteomics
• Structural Biology

Background:

• Intramolecular cross-linking of amino acids is crucial for probing protein structure.
• Existing methods like carbene insertion require two steps, while bifunctional electrophiles have limitations in capturing transient protein conformations.
• A single-step, in situ cross-linking strategy is needed for efficient protein structure analysis.

Purpose of the Study:

• To introduce a novel single-step cross-linking strategy using vinyl thianthrenium tetrafluoroborate (VTT).
• To demonstrate the efficiency and speed of VTT-mediated cross-linking within living cells.
• To highlight VTT's utility for proteome-wide protein structure determination.

Main Methods:

• Utilized vinyl thianthrenium tetrafluoroborate (VTT) for a single-step cross-linking reaction.
• Leveraged the umpolung of native cysteinyl thiols to generate electrophilic episulfonium ions.
• Performed cross-linking reactions in various cell types and compared with existing reagents (maleimide, iodoacetamide).

Main Results:

• VTT mediates rapid (within minutes) in situ cross-linking of amino acids in diverse cell types.
• VTT exhibits fast cellular uptake, enabling efficient intracellular labeling even in the presence of competing reagents.
• The reaction forms stable ethylene linkers between cysteine and nucleophilic amino acids.

Conclusions:

• VTT provides a facile and efficient single-step method for in cellulo protein cross-linking.
• The rapid generation of episulfonium ions without exogenous activation facilitates structure prediction.
• This approach offers a valuable tool for generating quantitative proteome-wide structural information.