Complex processes in nature often arise at an order-disorder transition [1][2][3][4]. In proteins, this complexity arises from an almost perfect compensation of entropy by enthalpy: molecular interactions that create structural integrity are on the same scale as thermal fluctuations from the environment. The resulting marginal stability of proteins suggests that they could behave like fluids near a critical point [5] -their structures fluctuate considerably subject to small perturbations without overcoming a large activation barrier.The concept of first-order and critical phase transitions does not rigorously apply to nanoobjects such as proteins; nevertheless, it is a useful one to classify folding transitions. For example, folding of small model proteins has been described as an abrupt, cooperative transition between the folded and unfolded phase for some proteins (the below-critical point scenario), or as a gradual barrier-less 'downhill' transition for other proteins (the above-critical point scenario) [6]. Even though critical behavior of proteins has been previously hinted [7-9], there has not been a direct observation of a critical point where one of these abrupt transitions simply disappears at Tc and Pc. In larger proteins, such as phosphoglycerate kinase (PGK) ( [10] in section S1), the situation can get even more complex: different parts or 'domains' of a large protein are more likely to be able to undergo separate order-disorder events [11], delicately poised between folded and partially unfolded structures to carry out their functions [12].Proteins must fold and function while crowded by surrounding macromolecules [13], which perturb the structure of the proteins at physiological conditions in the cell. The volume exclusion from macromolecules [14], which places shape and size (or co-volume) [15,16] constraints on the conformational space [ Fig. 1(a)], complicates protein folding and dynamics in living cells [17]. How the competing properties of a protein arise -being both stable yet dynamically sensitive to its environment -is mostly unknown; however, we show that the crowded environment provides a unique solution by placing PGK near a critical regime.We use pressure P, temperature T, and crowder-excluded volume fraction ϕ, to map PGK's folding energy landscape [12,18] and its critical regime on the T-P-ϕ phase diagram. Temperature can induce heat unfolding by favoring states of high conformational entropy, or cold denaturing by favoring reduced solvent entropy when hydrating core amino acids in the protein [19,20]. Since folded proteins contain heterogeneously distributed small, dry cavities due to imperfect packing of their quasi-fractal topology [21][22][23], high pressure also induces unfolding by introducing water molecules (as small granular particles) into the cavities in protein structures, leading to a reduced overall solvent-accessible volume of the unfolded protein [24]. Finally, in the presence of high crowding (large excluded volume fraction ϕ), compact desolvated (crystal) states ar...
