Molecular Determinants of Site-Specific Conformational Heterogeneity in Protein Folding and Unfolding

Abstract

Protein folding is a heterogeneous process in which multiple conformational sub-populations coexist within unfolded, intermediate, and native ensembles. However, the molecular determinants that give rise to this heterogeneity and that control the relative populations of coexisting conformations during folding and unfolding remain poorly understood. This thesis identifies the molecular origins of site-specific conformational heterogeneity and elucidates how sequence-encoded local backbone constraints and global chain topology govern folding pathways, cooperativity, and structural evolution. Intramolecular distance distributions were determined using site-specific time-resolved fluorescence resonance energy transfer analyzed by the maximum entropy method (trFRET–MEM). Monellin was employed as a model system in its single-chain variant (MNEI) to perturb local backbone rigidity and in its heterodimeric form (dcMN) to probe the effects of chain connectivity and inter-chain coupling while preserving native structure. Substitution of native cis-prolines to alanine in MNEI showed that although proline isomerization contributes to slow kinetic phases and unfolded-state heterogeneity, folding pathway heterogeneity persists even without cis-prolines, indicating that additional sequence-encoded factors give rise to heterogeneity. Importantly, proline substitutions abolished not only slow isomerization-limited phases but also a very fast folding phase through their effects on the unfolded ensemble. Site-specific measurements further revealed that folding intermediates comprise coexisting compact and expanded sub-populations, and that stabilization of minor compact intermediates through reduced local backbone rigidity (Pro→Ala substitution) enables chain compaction to proceed independently of structure formation, establishing that these processes are modular and decoupled during later folding stages. Equilibrium unfolding of heterodimeric monellin uncovered pronounced conformational heterogeneity masked by apparent two-state behavior in ensemble-averaged measurements. Chain connectivity was found to govern unfolding cooperativity, with covalent linkage suppressing heterogeneity and promoting cooperative structural responses. Refolding of dcMN further revealed concurrent barrier-limited conversion between expanded and compact sub-populations together with continuous structural contraction within compact intermediates, demonstrating the coexistence of activated and continuous processes during folding. Together, this thesis establishes conformational heterogeneity as an encoded and tunable feature of protein folding landscapes specified by local backbone constraints and global chain topology. The results define a unified mechanistic framework in which folding pathways, cooperativity, and structural evolution arise from redistribution of heterogeneous conformational ensembles, advancing beyond two-state and single-pathway descriptions of protein folding.