Thermodynamic destabilization of azurin by four different tetramethylguanidinium amino acid ionic liquids

The thermal unfolding of the copper redox protein azurin was studied in the presence of four different amino acid-based ionic liquids (ILs), all of which have tetramethylguanidium as cation. The anionic amino acid includes two with alcohol side chains, serine and threonine, and two with carboxylic acids, aspartate and glutamate. Control experiments showed that amino acids alone do not significantly change protein stability and pH changes anticipated by the amino acid nature have only minor effects on the protein. With the ILs, the protein is destabilized and the melting temperature is decreased. The two ILs with alcohol side chains strongly destabilize the protein while the two ILs with acid side chains have weaker effects. Unfolding enthalpy (ΔH_unf^°) and entropy (ΔS_unf^°) values, derived from fits of the unfolding data, show that some ILs increase ΔH_unf^°while others do not significantly change this value. All ILs, however, increase ΔS_unf^°. MD simulations of both the folded and unfolded protein conformations in the presence of the ILs provide insight into the different IL-protein interactions and how they affect the ΔH_unf^° values. The simulations also confirm that the ILs increase the unfolded state entropies which can explain the increased ΔS_unf^° values.