Cation effects and charge inversion contribute to the electrostatic stabilisation of protein bioconjugates in neat ionic liquids

Phys Chem Chem Phys. 2024 Nov 7;26(43):27648-27659. doi: 10.1039/d4cp01811g.

Abstract

Dispersing and stabilising proteins in ionic liquids (ILs) provides significant opportunities for green solvent-based biocatalysis, especially in industrial processes at elevated temperatures. While unmodified proteins undergo denaturation, their polymer-conjugated counterparts have been stabilised in neat ILs. However, the nature of interactions and the generality of protein-bioconjugate stabilisation in neat ILs require further understanding. Using a combination of different physio-chemical experimental tools and molecular dynamics (MD) simulations, here we investigate the dispersion and driving force for the stabilisation of bioconjugates in neat ILs. Solvent-free bioconjugates of different proteins, viz. myoglobin, α-chymotrypsin, and regenerated silk fibroin having predominant α-helical, β-sheet, and random coiled secondary structures, respectively, were prepared by electrostatic coupling with polyethylene-glycol (PEG)-based polymer-surfactant (PS). Protic IL (PIL, N-methyl-2-pyrolidonium trifluoromethane sulfonate; [NMP][OTF]) and aprotic ionic liquid (AIL, 1-methyl-3-(4-sulfobutyl)-1H-imidazol-3-ium trifluoromethane sulfonate; [MEZ][OTF]) were synthesized to study the bioconjugate dispersion. Interestingly, time-dependent polarised optical microscopy combined with transmittance measurements showed complete dispersion of all bioconjugates only in AIL. MD simulations of the PS-cCT bioconjugate were carried out in the same ILs as the experiments. The surface electrostatic potential of PS-cCT reversed from positive in PIL to negative in AIL due to overcharging by the AIL anion and lower mobility of the AIL cation. Strong screening of electrostatic potentials between two PS-cCT complexes in PIL resulted in reduced dispersion stability. Lower diffusivity of long alkyl chain [MEZ] cations of AIL leads to a depletion zone of IL ions between the two PS-cCT complexes (separation <70 Å), thus resulting in a significant negative potential between the complexes. Hence, protein bioconjugates in AIL were stabilised by a combination of surface overcharging and steric exclusion of [MEZ] cations from the space between the approaching bioconjugate complexes.

MeSH terms

  • Cations* / chemistry
  • Chymotrypsin
  • Fibroins / chemistry
  • Ionic Liquids* / chemistry
  • Molecular Dynamics Simulation*
  • Myoglobin / chemistry
  • Protein Stability
  • Static Electricity*

Substances

  • Ionic Liquids
  • Cations
  • Myoglobin
  • alpha-chymotrypsin
  • Fibroins
  • Chymotrypsin