Protein instability in supercritical CO2 limits the application of this green solvent in enzyme-catalyzed reactions. CO2 molecules act as protein denaturant at high pressure in supercritical condition. Here, for the first time, we showed that natural osmolytes could stabilize protein conformation in supercritical CO2. Molecular dynamic simulation is used to monitor the effects of adding different natural osmolytes on Chymotrypsin Inhibitor 2 (CI2) conformation and dynamics in supercritical CO2. Simulations showed that CI2 is denatured at 200 bar in supercritical CO2, which is in agreement with experimental observations. Interestingly, protein conformation remains native after addition of about 1 M of amino acid and sugar-based osmolyte models. These molecules stabilize protein through formation of supramolecular self-assemblies resulting from macromolecule-osmolyte hydrogen bonds. Nevertheless, trimethylamine N-oxide (TMAO) which is known as a potent osmolyte for protein stabilization in aqueous solutions, amplifies protein denaturation in supercritical CO2. Based on our structural analysis, we introduce a new mechanism for osmolyte effect in supercritical CO2, as “inclusion mechanism”. To the best of our knowledge, this is the first osmolytes in a supercritical fluid and proposes the mechanistic insights of osmolyte action in non-aqueous media.

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