Conformational plasticity of β-rich proteins: Molecular insights into stability, dynamics, and functional adaptation...

Proteins of the Bet v 1-like superfamily display remarkable functional versatility, supported by conformational flexibility. While conformational dynamics and ligand recognition in these proteins have been extensively investigated through NMR and MD approaches, the atomic-level features of transient high-energy states governing ligand binding and cavity gating remain incompletely understood. Here, we present one integrated experimental-computational characterization of such states in Bet v 1, a major birch pollen allergen and prototypical superfamily member. Using ¹⁵N NMR CPMG relaxation dispersion, urea-induced unfolding, and long-timescale molecular dynamics (MD) simulations, we show that Bet v 1 samples high-energy conformations on the microsecond-to-millisecond timescale. These transitions occur predominantly near the hydrophobic cavity entrance, involving mainly side-chain rearrangements with minimal changes to the overall fold. Urea titration reveals a three-state unfolding pathway involving a distinct intermediate, while MD combined with principal component analysis identifies flexible regions, gating motions, and provides structural models of gate-opening conformations. This atomic-level view of Bet v 1's dynamic landscape offers critical insight into its molecular recognition mechanisms and its behavior as an allergen. Our findings lay the groundwork for understanding how conformational dynamics influence function in this protein family and support future efforts to engineer proteins with tailored binding.