Unraveling the folding mechanism of the smallest knotted protein， MJ0366.

Understanding the mechanism by which polypeptide chains thread themselves into topologically knotted structures has emerged to be a challenging subject not least because of the additional complexity associated with the spontaneous and efficient knotting and folding events. While recent theoretical calculations have made significant progress in establishing the atomistic folding pathways for a number of knotted proteins， experimental data on the folding stabilities and kinetic pathways of knotted proteins has been sparse. Using MJ0366 from Methanocaldococcus jannaschii， the smallest knotted protein known to date， as a model system， we set out to systematically investigate its folding equilibrium， kinetics， and internal dynamics under native and chemically denatured states. NMR hydrogen-deuterium exchange analysis indicates that the knotted region is the most stable structural element within the novel fold. Additionally， N spin relaxation analysis reveals the presence of residual structures in urea-denatured MJ0366. Despite the apparent two-state equilibrium unfolding behavior during chemical denaturation， the kinetic unfolding pathway of MJ0366 involves the dissociation of the homodimeric native state into a native-like monomeric intermediate followed by unfolding into a denatured state. Our results provide comprehensive structural information regarding the folding dynamics and kinetic pathways of MJ0366， whose small size is ideal for converging experimental and theoretical findings to better understand the underlying principles of the folding of knotted proteins.