Unveiling the Three-Step Model for the Interaction of Imidazolium-Based Ionic Liquids on Albumin

Abstract

The effect of the ionic liquids (ILs) 1-methyl-3-tetradecylimidazolium chloride ([C₁₄MIM][Cl]), 1-dodecyl-3-methylimidazolium chloride ([C₁₂MIM][Cl]), and 1-decyl-methylimidazolium chloride ([C₁₀MIM][Cl]) on the structure of bovine serum albumin (BSA) was investigated by fluorescence spectroscopy, ultraviolet-visible (UV-vis) spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations. Concerning the fluorescence measurements, we observed a blue shift and a fluorescence quenching as the IL concentration increased in the solution. Such behavior was observed for all three studied imidazolium-based ILs, being larger as the number of methylene groups in the alkyl chain increased. UV-vis absorbance measurements indicate that even at relatively small IL/protein ratios, like 1:1 or 1:2, ([C₁₄MIM][Cl]) is able to change, at least partially, the sample turbidity. SAXS results agree with the spectroscopic techniques and suggest that the proteins underwent partial unfolding, evidenced by an increase in the radius of gyration (R_g) of the scattering particle. In the absence and presence of ([C₁₄MIM][Cl]) = 3 mM BSA R_g increases from 29.1 to 45.1 Å, respectively. Together, these results indicate that the interaction of BSA with ILs is divided into three stages: the first stage is characterized by the protein in its native form. It takes place for protein/IL ≤ 1:2, and the interaction is predominantly due to the electrostatic forces provided by the negative charges on the surface of BSA and the cationic polar head of the ILs. In the second stage, higher IL concentrations induce the unfolding of the protein, most likely inducing the unfolding of domains I and III, in such a way that the protein's secondary structure is kept almost unaltered. In the last stage, IL micelles start to form, and therefore, the interaction with protein reaches a saturation point and free micelles may be formed. We believe that this work provides new information about the interaction of ILs with BSA.

Figure 1

(A) Relative fluorescence emission spectra of 30 μM BSA (λ_ex = 295 nm) in the absence and presence of the gradual addition of [C₁₄MIM][Cl]. Molar ratios [IL]/[BSA] are in the legend (IL concentration increases along the arrow). (B) Ratio between the total fluorescence intensity (the integral under each fluorescence spectra) in the absence and presence of an increasing IL concentration as a function of molar ratio [IL]/[BSA] on a linear-log scale. (C) Stern–Volmer plot as a function of IL concentration on a linear-log scale. (D) The wavelength at the maximum emission of fluorescence as a function of molar ratio [IL]/[BSA] on a linear-log scale.

Figure 2

(A) SAXS data of 60 μM BSA in the absence and presence of the gradual addition of [C₁₄MIM][Cl]. Molar ratios [IL]/[BSA] are on the left. Curves are displaced vertically for clarity. (B) Radii of gyration in the absence and presence of an increasing IL concentration as a function of molar ratio [IL]/[BSA] on a linear-log scale. The solid curve is used to guide the eyes. (C) Circular dichroism spectra of BSA in the absence and presence of the gradual addition of [C₁₄MIM][Cl]. Insert graph shows %α-helix content as a function of the molar ratio [C₁₄MIM][Cl]. The IL concentration increases along the arrow.

Figure 3

Radius of gyration of BSA in pure water (in black) and in IL/water solutions at different concentrations (1 BSA/10 IL in blue, 1 BSA/100 IL in red, and 1 BSA/200 IL in green). All simulations were performed using [C₁₄MIM][Cl] ionic liquid since it was the one with a larger effect on the protein structure according to our experimental data.

Figure 4

(A) Number of intramolecular and (B) intermolecular hydrogen bonds for simulations of BSA in pure water (in black) and in ionic liquid (IL)/water solutions in different concentrations (1 BSA/10 IL in blue, 1 BSA/100 IL in red, and 1 BSA/200 IL in green). (C, D) Snapshots extracted from the simulation of BSA/200 IL showing the protein (in yellow) and the C₁₄MIM molecules (in red), for simulation times t = 50 ns (C) and t = 500 ns (D); water molecules and counterions were excluded in the images for better visualization.

Figure 5

(A) Minimum distance between the tryptophan residues (TRP1 and TRP2) and the C14 IL molecules for BSA in ionic liquid/water solutions of different concentrations (1 BSA/100 and 1 BSA/200 IL). (B) Center of mass distances between the benzene and pyrrole rings of TRP1 and the imidazole ring of C14 for BSA in ionic liquid/water solution at 1 BSA/200 IL concentration.

Figure 6

Normalized data from 0 to 1 of the fluorescence shift, quenching, and radius of gyration.

Figure 7

Schematic representation of bovine serum albumin primary and secondary structure, indicating the main structural features, like domain I (from residue 1–185), domain II (from residue 186–378), and domain III (from residue 379–583). It is also indicated in the figure the regions with α-helix structures (red rectangular boxes) and all 17 disulfide bonds, involving the 34 cysteines (round circles connected with solid lines). These data represent the PDB entry 4F5S. This figure was created at uriwww.pdb.org.