Cholinium-Based Ionic Liquids Modulate Protein Stability: A Comparative Study of Enzymes and Albumins

Abstract

This work aims to assess the intermolecular interaction of choline ionic liquids (ILs) (choline malonate ([Ch][Mal]), choline succinate ([Ch][Suc]), and choline valinate ([Ch][Val]) with two enzymes (lysozyme and α-chymotrypsin). We evaluated the state of the tertiary protein structure using circular dichroism (CD) spectrometry and quantified the binding parameters of the binding of the ionic liquids to the enzymes by fluorescence spectroscopy. The binding energies of the enzymes and the localization of ions on them were estimated using the molecular docking. We then analyzed the relationship between the enzymes' thermostability and their tendency towards aggregation in the enzyme/ionic liquid systems. The obtained results were compared with previous data on albumins to identify similarities and differences between the behavior of enzymes and albumins in ionic liquid solutions. Despite the comparable values of the binding constants, the effect of ionic liquids on the thermostability of enzymes was the opposite of their effect on albumins. In addition, although these ionic liquids promoted aggregation in both enzymes and albumins, this effect was much more pronounced for albumins.

Keywords: binding; enzyme; ionic liquid; protein; structure.

Figure A1

The CD spectra of lysosyme with ionic liquids at different temperatures, Black—30°, red—35°, blue—40°, green—45°, purple—50°, brown—55°, light blue—60°, dark brown—65°, dark yellow—70°, light red—75°, light blue—80°, light green—85°.

Figure A2

The CD spectra of a-chymotrypsim with ionic liquids at different temperatures, Black—30°, red—35°, blue—40°, green—45°, purple—50°, brown—55°, light blue—60°, dark brown—65°, dark yellow—70°, light red—75°, light blue—80°, light green—85°.

Figure A2

The CD spectra of a-chymotrypsim with ionic liquids at different temperatures, Black—30°, red—35°, blue—40°, green—45°, purple—50°, brown—55°, light blue—60°, dark brown—65°, dark yellow—70°, light red—75°, light blue—80°, light green—85°.

Figure 1

The structures of the choline-based ionic liquids that were studied.

Figure 2

CD spectra of lysozyme with ionic liquids at different concentrations.

Figure 3

CD spectra of a-chymotrypsin with ionic liquids at different concentrations.

Figure 4

The CD signal at 260 nm as a function of temperature for enzyme/IL system.

Figure 5

Fluorescence emission spectra of lysozyme in the presence of different concentrations of ionic liquids.

Figure 6

Fluorescence emission spectra of a-chymotrypsin in the presence of different concentrations of ionic liquids.

Figure 7

Stern–Volmer plots of lysozyme and a-chymotrypsin with ionic liquids.

Figure 8

Localization of ionic liquids’ ions on the surface of the lysozyme. Top row: left: [Ch][Mal], right: [Ch][Suc], bottom row: [Ch][Val]. The surface of the active site of the enzyme is highlighted in red, the enzyme backbone is represented by the blue line, its surface is shown in grey, and the ions of the ionic liquid are shown in light blue ([Ch][Mal]), green ([Ch][Suc]), and yellow ([Ch][Val]).

Figure 9

Localization of ionic liquids ions on the surface of the a-chymotrypsin. Top row: left: [Ch][Mal], right: [Ch][Suc], bottom row: [Ch][Val]. The surface of the active site of the enzyme is highlighted in red, the enzyme backbone is represented by the green line, its surface is shown in grey, and the ions of the ionic liquid are shown in light blue ([Ch][Mal]), green ([Ch][Suc]), and yellow ([Ch][Val]).