Probing Transient Partial Unfolding in Proteins by Native-State Proteolysis

Even under native conditions, proteins can have various non-native conformations, which are in equilibrium with the native functional conformations. Though the populations of the non-native conformation are usually small under native conditions, knowledge of the energy and the structure of the non-native forms is critical in understanding how proteins acquire and lose their structures. Native-state proteolysis is an experimental approach to selectively investigate a dominant partially unfolded form using proteolysis as a structural probe. From analyzing the kinetics of proteolysis of a protein under native conditions, we determine the energetics of unfolding to the partially unfolded form. The effect of urea on the energetics of partial unfolding reports the degree of unfolding in the partially unfolded form. Also, by assessing the effect of a point mutation on the energetics of partial unfolding, we elucidate the structure of the partially unfolded form. Partially unfolded forms probed by native-state proteolysis provide valuable information on the mechanisms of protein folding and protein degradation. In this review, we survey the principle and the applications of native-state proteolysis and also examine the pros and cons of the method in comparison with other experimental approaches.