Critical Phenomena in the Temperature-pressure-crowding Phase Diagram of a Large Protein*

In the cell, large proteins fold and perform complex functions through global structural rearrangements. Such large-scale dynamics are influenced by solvent fluctuations and the excluded volume from surrounding macromolecules. This requires a protein to be susceptible to small environmental agitations, yet stable to maintain structural integrity. These apparently competing behaviors are commonly exhibited by physical systems near a critical point, where distinct phases merge; a concept that is contrary to previous studies indicating proteins fold/unfold into well-defined phases on the pressure-temperature plane. Here, by characterizing the behavior of a large (~45 kDa) two-domain protein, phosphoglycerate kinase (PGK), on the temperature (T), pressure (P), and crowding volume-fraction (φ_c) phase diagram, we demonstrate a critical transition where several phases coexist. Above a critical point at a certain T and P, intermediate conformations between folded and unfolded phases disappear. When φ_c increases, this point moves along the T - P plane. Remarkably, crowding places PGK near a critical surface in its natural parameter space, where large conformational changes can occur without thermodynamic penalties.