Solutes preferentially excluded from macromolecules can drive depletion attractions in important biological association processes. The established Asakura-Oosawa theory relates depletion forces to the excluded volume reduction and the ensuing entropy gain upon macromolecular compaction. Accordingly, cosolute-induced protein stabilization is often described in terms of entropically driven “crowding”. In agreement, our experiments of peptide folding suggest that depletion forces are predominantly entropic for some cosolutes, such as polyethylene glycol polymers. However, for other cosolutes, such as polyol osmolytes, the effect is enthalpically dominated, while the entropic change can even be unfavorable. We show these properties persist for other proteins and macromolecular processes. To elucidate the molecular basis of these depletion interactions, we use simulations and analytic theory. Monte-Carlo simulations follow the association of two rod “macromolecules” in binary Lennard-Jones solutions. By dissecting the association free energy change into the respective thermodynamic components, we find different cosolutes induce stabilization through different thermodynamic driving mechanisms. Even for these simple liquids, considering intermolecular interactions beyond hard-cores can result in depletion forces that are completely enthalpic. We discuss how this newly resolved mechanism originates from intermolecular interactions and solvent restructuring. Finally, a mean-field theoretical model based on the Flory-Huggins solution theory complements the simulation analysis.
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