The Complex Energy Landscape of miRFP709, a 4₁-Knotted Protein, Results in Its Irreversible Denaturation

Knotted proteins have a unique topological feature with an open knot, and the physiological significance of these knots remains elusive. In addition, these proteins challenge our understanding of the protein folding process, and whether they retain their native state during unfolding/refolding cycles like other proteins is debated. Most folding studies on knotted proteins have been performed on 3₁ and 5₂ knots, monitoring the tryptophan fluorescence. In this study, we probe the unfolding/refolding of a 4₁-knotted protein, miRFP709, which can be monitored through near-infrared fluorescence in addition to the intrinsic tryptophan emission. miRFP709, upon chemical unfolding and refolding, folds back to a compact, non-native, stable structure that loses its ability to bind to the biliverdin ligand and fluoresce. The refolded protein retains its secondary structure but behaves like a molten-globule state with an exposed hydrophobic surface. The complex folding landscape of these proteins results in hysteresis between the folding and refolding curves. We propose that upon refolding, either an altered knot or an unknotted structure prevents the formation of the native knotted structure.