Structure and function of proteins in hydrated choline dihydrogen phosphate ionic liquid

Abstract

Ionic liquids are being intensely studied as promising media for the stabilization of proteins and other biomolecules. Choline dihydrogen phosphate (CDHP) has been identified as one of the most promising candidates for this application. In this work we have probed in more detail the effects that CDHP may have on the thermodynamics, structure, and stability of proteins, including one of therapeutic interest. Microcalorimetry and circular dichroism spectropolarimetry (CD) were used to assess the thermal stability of protein solutions in CDHP/water mixtures at various concentrations. Increasing thermal stability of lysozyme and interleukin-2 in proportion to CDHP concentration was observed. Isothermal titration calorimetry (ITC) was used to quantify binding interactions, and indicate that the mechanism for stability does not appear to be dependent upon CDHP binding to protein. CD and small angle X-ray scattering (SAXS) analyses were used to probe for structural changes due to the presence of CDHP. SAXS indicates charge effects on the surface of the protein play a role in protein stability in ionic liquids, and no significant alteration of the overall tertiary conformation of lysozyme was observed at 25 °C. However, after incubation at 37 °C or at higher concentrations of CDHP, small changes in protein structure were seen. Effects on protein activity were monitored using turbidity assays, and CDHP decreases protein activity but does not eliminate it. Protein solubility was also monitored using a turbidity assay and was found to be inversely proportional to the concentration of CDHP in solution.

Extraction and stability of bovine serum albumin (BSA) using cholinium-based Good's buffers ionic liquids

“…Their application as media for maintaining the protein stability has been extensively studied, either employed as co-solvents with water, in biphasic systems, or as "neat" solvents [8][9][10][11]. Some reports also demonstrated that some ILs are able to maintain or even increase the proteins stability [11][12][13].…”

In Vitro Assessment of Choline Dihydrogen Phosphate (CDHP) as a Vehicle for Recombinant Human Interleukin-2 (rhIL-2)

“…9 Using measurements of far-UV circular dichroism spectropolarimetry at 222nm, we observed an increase in thermal midpoint of unfolding temperature (T m ) of recombinant human IL-2 (rhIL-2) in the presence of CDHP. However, the effects on rhIL-2 bioactivity were not explored.…”

“…The reported T m values are included here as follows: (1) 30mM NaH 2 PO 4 : T m = 61.7°C, (2) 30mM CDHP pH 7.4: T m =60.2°C, (3) 185mM CDHP pH 7.4: T m =63.0°C, and (4) 680mM CDHP pH 7.4: T m = 74.2°C. 9 …”

“…2,4,7 Stabilization effects have also been observed in model proteins when using lower concentrations (<40 wt %) of this salt. 8,9 …”

Small-Angle X-Ray Scattering of Ionic Liquids

“…Indirect Fourier Transformation (IFT) Analysis is a method that has been applied in studies of protein stability in a hydrated choline di-hydrogen phosphate ionic liquid [32]. The IFT analysis can also be useful in identifying unknown phases, as well as understanding the link between real space and reciprocal space.…”