Many Ways Out: Beyond the Two-State Model of Protein Unfolding

The conformational transition related to protein unfolding is often considered as the transition between the folded conformation and one unfolded form. However, there are many aspects that hint at a much richer and more complex unfolded state made of a conformational ensemble not only for the unfolded species but also during the unfolding process. Proteins often sample intermediate conformations that may retain elements of secondary structure, preserve part of the hydrophobic core, or display localized unfolding restricted to specific regions. During the unfolding pathway, there are often minor transitions involving local secondary-structure regions outside the hydrophobic core. Many researchers have also hypothesized the existence of one or more intermediates, albeit usually invisible because they are low populated. In the present Mini-Review, we re-examine critically crucial aspects of this fascinating problem starting from our own experience in protein stability and unfolding and revise the limitations and implications of the two-state model.