Specific Interactions and Environment Flexibility Tune Protein Stability under Extreme Crowding

Macromolecular crowding influences protein mobility and stability in vivo. A precise description of the crowding effect on protein thermal stability requires the estimate of the combined effects of excluded volume, specific protein–environment interactions, as well as the thermal response of the crowders. Here, we explore an ideal model system, the lysozyme protein in powder state, to dissect the factors controlling the melting of the protein under extreme crowding. By deploying state-of-the art molecular simulations, supported by calorimetric experiments, we assess the role of the environment flexibility and of intermolecular electrostatic interactions. In particular, we show that the temperature-dependent flexibility of the macromolecular crowders, along with specific interactions, significantly alleviates the stabilizing contributions of the static volume effect.