Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Single-molecule Förster resonance energy transfer (smFRET) is crucial for real-time biomolecular dynamics.
• Existing smFRET methods face limitations due to dye availability, quantum yield, and spectral overlap, hindering ligand interaction studies.
• Correlating biomolecular conformation with ligand binding dynamics requires advanced smFRET techniques.

Purpose of the Study:

• To develop and validate a novel smFRET approach using nonfluorescent quenchers as acceptors.
• To investigate the conformational dynamics of the ribosome during elongation in real-time.
• To correlate ribosome conformational changes with transfer RNA (tRNA) dynamics.

Main Methods:

• Utilized a nonfluorescent quencher, Black Hole Quencher (BHQ), as a FRET acceptor.
• Employed a Cy3/BHQ dye pair for smFRET measurements.
• Applied single-color FRET to monitor ribosome conformational transitions and tRNA binding events.

Main Results:

• Successfully tracked ribosome conformational changes during elongation in real-time using the Cy3/BHQ pair.
• Demonstrated the correlation between ribosome conformational dynamics and tRNA compositional dynamics.
• Observed ribosome conformational transitions (locked to unlocked) upon fluorescently labeled tRNA arrival using a Cy5 acceptor.

Conclusions:

• Nonfluorescent quenchers are effective acceptors for smFRET, overcoming limitations of traditional fluorescent dyes.
• This method enables robust single-molecule correlation studies of biomolecular dynamics and ligand interactions.
• The developed approach holds significant potential for advancing the understanding of complex molecular mechanisms.