Impact of in-cell and in-vitro crowding on the conformations and dynamics of an intrinsically disordered protein.

The conformations and dynamics of proteins can be influenced by crowding from the large concentrations of macromolecules within cells. We recently showed that intrinsically disordered proteins (IDPs) exhibit chain compaction in crowded solutions in vitro, but, surprisingly, no such effects were observed in cultured mammalian cells. Here, to increase intracellular crowding, we reduced the cell volume by hyperosmotic stress and used an IDP as a crowding sensor for in-cell single-molecule spectroscopy. In these more crowded cells, the IDP exhibits compaction, slower chain dynamics, and much slower translational diffusion, indicating a pronounced concentration and length-scale dependence of crowding. In vitro, these effects cannot be reproduced with small but only with large polymeric crowders. The observations can be explained with polymer theory and depletion interactions and indicate that IDPs can diffuse much more efficiently through a crowded cytosol than a globular protein of similar dimensions, which may facilitate the target search important for many IDP functions.