Protein disorder prevails under crowded conditions

Crowding caused by the high concentrations of macromolecules in the living cell changes chemical equilibria, thus promoting aggregation and folding reactions of proteins. The possible magnitude of this effect is particularly important with respect to the physiological structure of intrinsically disordered proteins (IDPs), which are devoid of well-defined three-dimensional structures in vitro. To probe this effect, we have studied the structural state of three IDPs, α-casein, MAP2c, and p21(Cip1), in the presence of the crowding agents Dextran and Ficoll 70 at concentrations up to 40%, and also the small-molecule osmolyte, trimethylamine N-oxide (TMAO), at concentrations up to 3.6 M. The structures of IDPs under highly diluted and crowded conditions were compared by a variety of techniques, fluorescence spectroscopy, acrylamide quenching, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, fluorescence correlation spectroscopy (FCS), and far-UV and near-UV circular dichroism (CD) spectroscopy, which allow us to visualize various levels of structural organization within these proteins. We observed that crowding causes limited structural changes, which seem to reflect the functional requirements of these IDPs. α-Casein, a protein of nutrient function in milk, changes least under crowded conditions. On the other hand, MAP2c and, to a lesser degree, p21(Cip1), which carry out their functions by partner binding and accompanying partially induced folding, show signs of local structuring and also some global compaction upon crowded conditions, in particular in the presence of TMAO. The observations are compatible with the possible preformation of binding-competent conformations in these proteins. The magnitude of these changes, however, is far from that of the cooperative folding transitions elicited by crowding in denatured globular proteins; i.e., these IDPs do remain in a state of rapidly interconverting structural ensemble. Altogether, our results underline that structural disorder is the physiological state of these proteins.