Single-molecule fluorescence experiments determine protein folding transition path times.

Area of Science:

• Biophysics
• Protein dynamics
• Single-molecule biophysics

Background:

• The transition path represents the critical molecular trajectory during free-energy barrier crossing.
• Understanding transition paths is crucial for elucidating protein folding mechanisms.
• Single-molecule Förster resonance energy transfer (smFRET) experiments allow observation of these dynamic events.

Purpose of the Study:

• To investigate and compare the average transition-path times for fast- and slow-folding proteins.
• To determine if folding rate differences correlate with transition-path duration.
• To provide insights into the energy landscape theory of protein folding.

Main Methods:

• Photon-by-photon analysis of fluorescence trajectories.
• Single-molecule Förster resonance energy transfer (smFRET) experiments.
• Measurement of average transition-path times for distinct protein folding behaviors.

Main Results:

• While folding rate coefficients varied by 10,000-fold between proteins, their average transition-path times differed by less than 5-fold.
• This indicates that the actual time taken for folding to occur is remarkably similar, irrespective of overall folding speed.
• The results align with predictions from a simplified energy landscape model.

Conclusions:

• The duration of the transition path, the core folding event, is largely conserved across proteins with different folding rates.
• Protein folding speed is primarily determined by factors outside the transition path itself.
• Energy landscape theory provides a viable framework for explaining these observed similarities in transition-path times.