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.