Lilie, Hauke

ABSTRACTThe ionic liquid 1‐ethyl‐3‐methyl imidazolium chloride (EMIM Cl) and the amino acid l‐arginine hydrochloride (l‐ArgHCl) have been successfully used to improve the yield of oxidative refolding for various proteins. However, the molecular mechanisms behind the actions of such solvent additives—especially of ionic liquids—are still not well understood. To analyze these mechanisms, we have determined the transfer free energies from water into ionic liquid solutions of proteinogenic amino acids and of diketopiperazine as peptide bond analogue. For EMIM Cl and 1‐ethyl‐3‐methyl imidazolium diethyl phosphate, which had a suppressive effect on protein refolding, as well as for l‐ArgHCl favorable interactions with amino acid side chains, but no favorable interactions with the peptide backbone could be observed. A quantitative analysis of other ionic liquids together with their already published effects on protein refolding showed that only solvent additives within a certain range of hydrophobicity, chaotropicity and kosmotropicity were effective for the refolding of recombinant plasminogen activator. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1129–1140, 2014.

The ionic liquid N‐ethyl‐N′‐methyl imidazolium chloride (EMIMCl) has been described as being very efficient in promoting refolding of the recombinant plasminogen activator rPA. Our study reveals that molar concentrations of EMIMCl increase the solubility of native and unfolded proteins due to favorable interactions with amino acid side chains rather than favorably interacting with the peptide backbone. This delicate balance of favorable interactions with side chains and unfavorable interactions with the peptide backbone provides a molecular explanation of how EMIMCl suppresses protein aggregation and simultaneously promotes refolding. By contrast, high concentrations of EMIMCl denature proteins because of a reduced water content and strong favorable interactions with amino acid side chains. This denatured species is not soluble and aggregates because, in contrast to the classical denaturants, guanidine hydrochloride and urea, EMIMCl does not solubilize the peptide backbone.Structured digital abstract PNP and PNP bind by molecular sieving (1, 2, 3, 4)