Click strategies for single-molecule protein fluorescence

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Single-molecule methods are crucial for biological studies.
• Förster resonance energy transfer (FRET) is widely used for biomolecular structure and dynamics.
• Site-specific labeling of proteins with fluorescent dyes is a key challenge.

Purpose of the Study:

• To develop an optimized strategy for site-specific protein labeling using click chemistry and unnatural amino acids (UAAs).
• To evaluate various clickable UAAs for high-resolution single-molecule FRET (smFRET) applications.
• To enable structural studies of challenging proteins like RanBP3.

Main Methods:

• Systematic study of clickable UAAs for protein labeling.
• Optimization of parameters for single-molecule studies (probe accessibility, expression yield, labeling efficiency).
• Development of a biocompatible one-step dual-labeling strategy for proteins.

Main Results:

• Genetically encoding propargyllysine was identified as advantageous for smFRET measurements.
• Multiparameter fluorescence analysis provided insights into dye-UAA photophysical properties.
• The regulatory protein RanBP3 was successfully dual-labeled, revealing a disordered, collapsed state of its FxFG repeat region.

Conclusions:

• The presented click chemistry and UAA strategy overcomes limitations in protein labeling for smFRET.
• Propargyllysine is a highly effective UAA for advanced smFRET applications.
• This method enables structural characterization of proteins not amenable to traditional techniques.