Cytochrome c folds through foldon-dependent native-like intermediates in an ordered pathway

Area of Science:

• Protein folding dynamics
• Biophysical chemistry
• Molecular biology

Background:

• Cytochrome c (Cyt c) is a protein with 104 residues.
• Previous studies indicated Cyt c unfolds via a series of cooperative folding units (foldons).
• An energy ladder of partially folded intermediates governs Cyt c's equilibrium unfolding pathway.

Purpose of the Study:

• To directly investigate Cyt c intermediates and folding pathways during kinetic folding.
• To compare kinetic folding pathways with equilibrium unfolding pathways.
• To validate the existence of defined protein folding pathways.

Main Methods:

• Utilized hydrogen exchange (HX) pulse labeling.
• Employed fragment separation coupled with mass spectrometry.
• Analyzed folding intermediates and pathways in real-time.

Main Results:

• Over 95% of Cyt c population follows a folding pathway consistent with equilibrium unfolding intermediates.
• The kinetic folding pathway involves sequential transitions through foldon-dependent intermediates.
• Identified a conserved set of intermediates for both kinetic and equilibrium processes.

Conclusions:

• Protein folding proceeds through a defined sequence of native-like intermediates.
• The findings support a classical pathway model of protein folding.
• Kinetic barriers are condition-dependent and can be misinterpreted with less sensitive methods.