The effects of high concentrations of ionic liquid on GB1 protein structure and dynamics probed by high-resolution magic-angle-spinning NMR spectroscopy

Abstract

Ionic liquids have great potential in biological applications and biocatalysis, as some ionic liquids can stabilize proteins and enhance enzyme activity, while others have the opposite effect. However, on the molecular level, probing ionic liquid interactions with proteins, especially in solutions containing high concentration of ionic liquids, has been challenging. In the present work the ¹³C, ¹⁵N-enriched GB1 model protein was used to demonstrate applicability of high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy to investigate ionic liquid - protein interactions. Effect of an ionic liquid (1-butyl-3-methylimidazolium bromide, [C₄-mim]Br) on GB1was studied over a wide range of the ionic liquid concentrations (0.6 to 3.5 M, which corresponds to 10%-60% v/v). Interactions between GB1 and [C₄-mim]Br were observed from changes in the chemical shifts of the protein backbone as well as the changes in ¹⁵N ps-ns dynamics and rotational correlation times. Site-specific interactions between the protein and [C₄-mim]Br were assigned using 3D methods under HR-MAS conditions. Thus, HR-MAS NMR is a viable tool that could aid in elucidation of the molecular mechanism of ionic liquid - protein interactions.

Keywords: GB1; HR-MAS NMR; imidazolium ionic liquid; ionic liquid – protein interaction.

Graphical abstract

Fig. 1

2D ¹H-¹⁵N HSQC spectra of GB1 in the presence of [C₄-mim]Br. (a) Overlay of spectra with 0% (blue), 10% (cyan), 25% (green), 40% (orange), and 50% (red) v/v [C₄-mim]Br. Aliased peaks are shown in dashed ovals.

Fig. 2

Chemical shift perturbations for each residue in the presence of 10–50% v/v [C₄-mim]Br. (a) Combined and weighted ¹H and ¹⁵N (ΔHN) chemical shift perturbations (CSP) of all residues in the presence of 10% (cyan), 25% (green), 40% (orange), 50% (red) v/v [C₄-mim]Br. (b) Cartoon representation of the crystal structure of GB1 (PDB: 2QMT) color coded by amide chemical shifts perturbation (CSP) in 10–50% v/v [C₄-mim]Br. Chemical shift perturbation is indicated by colors ranging from blue (least, 0.00 ppm) to red (most, 0.55 ppm), and the coloring gradient is scaled to the maximum CSP in the 50% [C₄-mim]Br sample. GB1 residues which have more 0.40 ppm CSP in the 50% v/v [C₄-mim]Br sample are identified. The maximum radius of the putty representation is scaled to maximum CSP in the shown condition (10% v/v [C₄-mim]Br, 25% v/v [C₄-mim]Br, 40% v/v [C₄-mim]Br or 50% v/v [C₄-mim]Br). The figure was generated by PYMOL.

Fig. 3

Rotational correlation times (τ_c) for GB1 in 10%, 25% and 50% v/v [C₄-mim]Br. Rotational correlation times were calculated from an average of T₁ and T₂ times of non-flexible residues for GB1 in the presence of 0% (blue), 25% (green), and 50% (red) v/v [C₄-mim]Br. The theoretical T₁ and T₂ curves were calculated using a home written Mathematica script. IL stands for ionic liquid.