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The cell is highly crowded with biomacromolecules, and the excluded volume influences processes such as diffusion, folding, conformation, and aggregation or association of proteins and polynucleic acids. In Escherichia coli, the values reported for the total macromolecular content range from 200 to 400 mg/mL. Knowledge of the macromolecular crowding is needed to understand behavior and especially interactions of biomolecules in vivo, be it for drug development, fundamental knowledge, or to support computational efforts to model the living cell. Direct spatiotemporal read-out of the crowding would be a powerful asset to unravel the structure of the cytoplasm and the impact of excluded volume on protein function in living cells. Here, we introduce a Förster resonance energy transfer (FRET) sensor for quantification of the macromolecular crowding and apply the sensor in living cells.