Conformational Changes of the Knotted Protein 1O6D during Denaturation.

Knotted proteins possess complex topologies that impose unique constraints on folding and unfolding. Whether the knot persists during denaturation and how its conformation reorganizes, however, remain unresolved. Here, we investigated the knotted protein 1O6D using site-specific fluorescence resonance energy transfer (FRET) and time-resolved fluorescence anisotropy (TRFA). The combined data support a staged denaturation process. At low denaturant concentrations, the knotted architecture undergoes progressive loosening accompanied by spatially nonuniform local rearrangement and C-terminal-directed reorganization. TRFA further reveals distinct site-dependent dynamical responses, including nonmonotonic anisotropy changes at W120 and W127, consistent with transient confinement within a motion-restricted intermediate microenvironment. At higher denaturant concentrations, the protein expands substantially, yet the FRET-derived distances and apparent thermodynamic parameters remain more consistent with a topologically constrained denatured ensemble than with a fully extended random coil. These results provide site-resolved evidence for asymmetric relaxation and persistence of a loosened but still tied topological state.