Mapping Multiple Distances in a Multidomain Protein for the Identification of Folding Intermediates

Area of Science:

• Structural biology
• Biophysics
• Protein folding

Background:

• Multidomain protein folding mechanisms remain challenging to elucidate.
• Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for observing protein conformational changes.

Purpose of the Study:

• To investigate the denaturant-induced unfolding transitions of yeast phosphoglycerate kinase.
• To map inter- and intradomain distances to understand its folding pathway.

Main Methods:

• Utilized single-molecule Förster resonance energy transfer (smFRET).
• Monitored multiple inter- and intradomain distances during unfolding.
• Studied yeast phosphoglycerate kinase.

Main Results:

• Observed heterogeneous unfolding behavior in yeast phosphoglycerate kinase.
• Interdomain distance suggested two-state unfolding, while intradomain distances indicated a molten globule intermediate.
• Different dye attachment sites yielded distinct unfolding profiles.

Conclusions:

• Multidomain protein unfolding is complex and can be heterogeneous.
• Monitoring multiple distances is crucial for accurate characterization.
• Single reporters may lead to oversimplified interpretations of protein folding.