Abstract:
Protein unfolding invariably appears to be a cooperative transition; yet, the molecular basis by which structural elements could unfold in a coordinated manner remains unresolved. Here, the unfolding mechanism of the naturally occurring heterodimeric protein double-chain monellin (dcMN) was characterized using site-specific time-resolved FRET and fluorescence anisotropy decay measurements made under equilibrium conditions. Although ensemble-averaged measurements suggested an apparently cooperative transition, population-level analysis using the maximum entropy method coupled to time-resolved FRET revealed pronounced conformational heterogeneity, with partially contracted (N-like) coexisting with partially expanded (U-like) subpopulations during unfolding. Time-resolved fluorescence anisotropy decay measurements independently demonstrated that local motional constraints are lost gradually and asynchronously across different regions of the protein. The N-like subpopulations underwent cooperative expansion across both intra- and interchain segments, indicating coordinated responses when interchain coupling is maintained. In contrast, the U-like subpopulations displayed pronounced chain-specific, noncooperative behavior, consistent with independent unfolding of the two chains following loss of coupling. Comparison with a covalently linked single-chain variant demonstrates that chain connectivity suppresses heterogeneity and enforces coordinated unfolding. These results identify restriction of chain entropy arising from interchain coupling and covalent connectivity as a molecular determinant that governs whether heterogeneous intermediate subpopulations unfold cooperatively or in a chain-specific manner.