title: 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.
authors.affiliation: Department of Mechanical Engineering and Engineering Science, The University of North Carolina at Charlotte, Charlotte, North Carolina 28223, USA.
authors.forename: Katherine D
authors.initials: KD
authors.lastname: Weaver
authors.forename: Regina M
authors.initials: RM
authors.lastname: Vrikkis
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Van Vorst
authors.forename: Jonathan
authors.initials: J
authors.lastname: Trullinger
authors.forename: Ranganathan
authors.initials: R
authors.lastname: Vijayaraghavan
authors.forename: David M
authors.initials: DM
authors.lastname: Foureau
authors.forename: Iain H
authors.initials: IH
authors.lastname: McKillop
authors.forename: Douglas R
authors.initials: DR
authors.lastname: MacFarlane
authors.forename: Joanna K
authors.initials: JK
authors.lastname: Krueger
authors.forename: Gloria D
authors.initials: GD
authors.lastname: Elliott
mesh_terms: D002151:Calorimetry; D002942:Circular Dichroism; D007376:Interleukin-2 / Q000737:chemistry*; D052578:Ionic Liquids / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D053838:Scattering, Small Angle; D012995:Solubility; D013696:Temperature; D013816:Thermodynamics; D014961:X-Ray Diffraction
chemical_list: D007376:Interleukin-2; D052578:Ionic Liquids; D009113:Muramidase