Most theories about macromolecular crowding focus on two ideas: the macromolecular nature of the crowder and entropy. For proteins, the volume excluded by the crowder favors compact native states over expanded denatured states, enhancing protein stability by decreasing the entropy of unfolding. We tested these ideas with the widely used crowding agent Ficoll-70 and its monomer, sucrose. Contrary to expectations, Ficoll and sucrose have approximately the same stabilizing effect on chymotrypsin inhibitor 2. Furthermore, the stabilization is driven by enthalpy, not entropy. These results point to the need for carefully controlled studies and more sophisticated theories for understanding crowding effects.

Intrinsically disordered proteins are important in signaling, regulation, and translocation. Understanding their diffusion under physiologically relevant conditions will yield insight into their functions. We used NMR to quantify the translational diffusion of a globular and a disordered protein in dilute solution and under crowded conditions. In dilute solution, the globular protein chymotrypsin inhibitor 2 (CI2, 7.4 kDa) diffuses faster than the disordered protein α-synuclein (14 kDa). Surprisingly, the opposite occurs under crowded conditions; α-synuclein diffuses faster than CI2, even though α-synuclein is larger than CI2. These data show that shape is a key parameter determining protein diffusion under crowded conditions, adding to the properties known to be affected by macromolecular crowding. The results also offer a clue about why many signaling proteins are disordered.

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