Chain Entropy Modulates Cooperativity Selectively within Intermediate Subpopulations during Protein Unfolding

Area of Science:

• Biophysics
• Protein dynamics
• Structural biology

Background:

• Protein unfolding is often cooperative, but the molecular mechanisms driving coordinated structural changes are unclear.
• Understanding protein dynamics is crucial for deciphering biological function and disease mechanisms.

Purpose of the Study:

• To investigate the unfolding mechanism of the heterodimeric protein double-chain monellin (dcMN).
• To elucidate the role of interchain coupling in protein unfolding pathways.
• To characterize conformational heterogeneity during protein unfolding.

Main Methods:

• Site-specific time-resolved Förster Resonance Energy Transfer (FRET).
• Fluorescence anisotropy decay measurements under equilibrium conditions.
• Maximum entropy method for population-level analysis.

Main Results:

• Ensemble measurements suggested cooperative unfolding, but population analysis revealed conformational heterogeneity (N-like and U-like subpopulations).
• Time-resolved anisotropy showed gradual and asynchronous loss of local motional constraints.
• N-like subpopulations unfolded cooperatively, while U-like subpopulations showed chain-specific, noncooperative unfolding.
• Covalent linkage of chains suppressed heterogeneity and enforced coordinated unfolding.

Conclusions:

• Interchain coupling and covalent connectivity restrict chain entropy, influencing unfolding cooperativity.
• Conformational heterogeneity dictates whether protein unfolding intermediates transition cooperatively or in a chain-specific manner.
• The study provides molecular insights into the determinants of coordinated protein unfolding.