ABSTRACT
The search for biocompatible ionic liquids (ILs) with novel biochemical and biomedical applications has recently gained greater attention. In this report, we characterize the effects of two novel amino acid-based aqueous ILs composed of tetramethylguanidinium (TMG) and amino acids on the structure and stability of a widely used red fluorescent protein (mCherry). Our experimental data shows that while the aspartic acid-based IL (TMGAsp) has effects similar to previously studied conventional ILs (BMIBF4, EMIAc, and TMGAc), the alanine-based IL (TMGAla) has a much stronger destabilization effect on the protein structure. Addition of 0.30 M TMGAla to mCherry decreases the unfolding temperature from 83 to 60 °C. Even at 25 °C, TMGAla results in a blue shift of the mCherry absorbance and fluorescence peaks and an increased Stokes shift. Molecular dynamics simulations show that the chromophore conformation and its interaction with mCherry with TMGAla are changed relative to those with TMGAsp or in the absence of ILs. Protein-ILs contact analysis indicates that the mCherry-Asp interactions are hydrophilic but the (fewer) mCherry-Ala interactions are more hydrophobic and may modulate the TMG interaction with the protein. Hence, the anion hydrophobicity may explain the special TMGAla destabilization of mCherry.
