The how’s and why’s of protein folding intermediates

The nature and role of intermediates have been the subject of much heated debate in the field of protein folding. Historically, intermediates were viewed as essential stepping-stones that guide a protein through the folding process to the native state. However, with the experimental identification of numerous small proteins that fold rapidly without intermediates, and the emergence from computational studies of new conceptual frameworks, came the thinking that intermediates can act as energy sinks, kinetic traps that result in less efficient folding. Whether ‘good’ or ‘bad’, it is without doubt that folding intermediates provide valuable information to protein chemists: at equilibrium they help to delineate the subdomain architecture of a protein and the hierarchy of subdomain stabilities; under kinetic conditions they provide experimentalists with additional snapshots of the folding reaction and, thereby, fundamental mechanistic details that are often lacking in the case of two-state folders. Intermediates give us valuable insights into the fluctuations from the native structure that may be important in regulating biological function. Lastly, intermediates are often the critical species in misfolding processes that lead to aggregation and disease. Here we review what we have learnt after almost half a century of protein-folding research, and we question two fundamental tests of our understanding: do we know enough about how proteins fold to design folding mechanisms de novo and can we exploit our knowledge to modulate protein-folding mechanisms in the cell for therapeutic benefit?