Helix-forming ability of proteins in alkylammonium nitrate

Understanding the helical formation of proteins in concentrated aqueous solutions of alkylammonium-based ionic liquids (RAN-ILs) forming the glassy state is necessary to devise the guidelines for the design of ILs for use in protein cryopreservation. To reveal the effect of the alkyl-chain length of cation on the helix forming ability of RAN-ILs, helix formation in five model proteins (ribonuclease A,beta-lactoglobulin, ubiquitin, a-lactalbumin, and cytochrome c) with different secondary structure contents in the solutions of three RAN-ILs, namely methylammonium nitrate (MAN), ethylammonium nitrate (EAN), and propylammonium nitrate (PAN) were investigated over a wide concentration range X (mol%IL) using optical spectroscopy. Overall, although the addition of RAN-ILs to proteins caused protein unfolding, condensed solutions with EAN and PAN (X> 10) induced helical formation, which was disrupted by the tertiary structure of proteins, except for ubiquitin and a-lactalbumin. In contrast, aqueous MAN solutions did not induce the non-native helical structure of the five model proteins throughout the studied concentration range, X. The difference in the helix-forming ability between MAN and the other two RAN-ILs was dependent on the low polarity of the solutions. Moreover, although EAN and PAN have helix-forming abilities, these abilities were dependent on protein species. The present finding is that the consideration of solution polarity and dependence of protein species is related to the guidelines for the design of RAN-ILs for the cryopreservation of proteins such as recombinant proteins. (C) 2017 Published by Elsevier B.V.