Quantification of Macromolecular Crowding in Living Cells

Cells are highly crowded with proteins and polynucleotides, with concentrations ranging from 80 to 400 mg/mL. Knowledge of crowding is critical to understand cell physiology and to assess its relevance for medical science and biotechnology: Any reaction that depends on the available volume can be affected by crowding, including biopolymer diffusion, conformation, association, folding, phase separation, and aggregation. However, the effects of crowding in cells are complex and have remained difficult to tract. Here, we introduce a FRET-based sensor to quantify the effects of macromolecular crowding in living cells. This sensor is an excellent platform to determine the consequences of macromolecular crowding in living cells: We show that crowding effects are dependent on the structure of a protein, and that these effects are not constant over time.