Exploring protein structure and dynamics under denaturing conditions by single-molecule FRET analysis

Area of Science:

• Biophysics
• Protein dynamics
• Chemical kinetics

Background:

• Proteins exhibit significant structural variability in native and denatured states.
• Understanding protein folding pathways requires exploring conformational energy landscapes.
• Single-molecule fluorescence spectroscopy offers atomic-scale resolution for real-time monitoring.

Purpose of the Study:

• To investigate the structure and dynamics of the enzyme RNase H.
• To explore protein folding pathways using single-molecule Förster resonance energy transfer (FRET).
• To analyze conformational changes in response to denaturant concentration.

Main Methods:

• Single-molecule fluorescence spectroscopy utilizing Förster resonance energy transfer (FRET).
• Immobilization of individual protein molecules on nanostructured surfaces.
• Real-time monitoring of protein conformational dynamics over extended periods.

Main Results:

• FRET analysis provided insights into structural changes of unfolded RNase H.
• Time traces revealed stepwise transitions in FRET levels, indicating conformational dynamics.
• Barriers in the protein's free energy landscape were estimated from transition kinetics.

Conclusions:

• Single-molecule FRET is a powerful tool for studying protein folding.
• The study elucidated conformational dynamics of RNase H in the presence of denaturants.
• This approach allows for the estimation of energy landscape features from kinetic data.