Udgaonkar, J. | Search bioRxiv

bioRxiv2026

Chain entropy modulates cooperativity selectively within intermediate sub-populations during protein unfolding

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) sub-populations 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 sub-populations underwent cooperative expansion across both intra- and inter-chain segments, indicating coordinated responses when inter-chain coupling is maintained. In contrast, the U-like sub-populations displayed pronounced chain-specific, non-cooperative 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 inter-chain coupling and covalent connectivity as a molecular determinant that governs whether heterogeneous intermediate sub-populations unfold cooperatively or in a chain-specific manner.

biophysics↗

bioRxiv2025

Intermediate heterogeneity modulates coupling between chain compaction and structure formation during protein folding

Polypeptide chains undergo both compaction and structure formation during folding, but the extent to which these processes are mechanistically coupled remains unclear. Although initial chain collapse can precede structure formation, the two processes invariably appear coupled at later stages of folding. This raises the question of whether the fraction of molecules that undergo initial collapse, as well as the degree of coupling between compaction and structure formation later during folding, are regulated by sequence-encoded structural constraints. To examine this, the folding of the small protein monellin was investigated using time-resolved fluorescence resonance energy transfer (trFRET) analyzed with the maximum entropy method to resolve sub-populations of molecules with native-like and unfolded-like dimensions. Mutation of Pro41 to Ala, or Pro93 to Ala, which relieve local backbone rigidity, selectively stabilized hidden minor conformations within the initial and later intermediate ensembles, respectively. In each case, the minor conformation had a segment that was more compact than in the major one, and its stabilization increased the number of molecules undergoing specific contraction to form the intermediate ensemble, without altering the extent of structure formation. Consequently, sub-populations within these intermediate ensembles could undergo chain contraction independently of structure formation. These findings identify intermediate-state heterogeneity, modifiable by backbone rigidity, as the basis for tunable coupling between chain compaction and structure formation during protein folding. Significance StatementChain compaction and structure formation are central features of protein folding, yet how these two processes are coupled remains unclear. Although early chain collapse can precede structure formation, the two often appear coupled at later stages, raising the question of whether this coupling is obligatory or tunable. Here, the coupling is shown to be tunable. Using a quantitative framework that resolves coexisting compact intermediates, this study demonstrates that proline-imposed backbone rigidity governs intermediate-state heterogeneity, which in turn determines the separability of these processes.

biophysics↗