Proteins are inherently unstable, which limits their use as therapeutic agents. However, the use of biocompatible cosolvents or surfactants can help to circumvent this problem through the stabilization of intramolecular and solvent-mediated interactions. Ionic liquids (ILs) have been known to act as cosolvents or surface-active compounds. In the presence of proteins, ILs can have a beneficial effect on their refolding, shelf life, stability, and enzymatic activities. In the work described herein, we used small-angle X-ray scattering (SAXS) to monitor the aggregation of different concentrations of ILs with protein models, lysozyme (Lys) and bovine serum albumin (BSA), and fluorescence microscopy to assess micelle formation of fluorinated ILs (FILs) with Lys. Furthermore, coarse-grained molecular dynamics (CG-MD) simulations provided a better understanding of Lys–FIL interactions. The results showed that the proteins maintain their globular structures in the presence of FILs, with signs of partial unfolding for Lys and compaction for BSA with increased flexibility at higher FIL concentrations. Lys was encapsulated by FIL, thus reinforcing the potential of ILs to be used in the formulation of protein-based pharmaceuticals.

Phenylketonuria (PKU) is an autosomal recessive disease caused by deficient activity of human phenylalanine hydroxylase (hPAH), which can lead to neurologic impairments in untreated patients. Although some therapies are already available for PKU, these are not without drawbacks. Enzyme-replacement therapy through the delivery of functional hPAH could be a promising strategy. In this work, biophysical methods were used to evaluate the potential of [N_1112(OH)][C₄F₉SO₃], a biocompatible fluorinated ionic liquid (FIL), as a delivery system of hPAH. The results herein presented show that [N_1112(OH)][C₄F₉SO₃] spontaneously forms micelles in a solution that can encapsulate hPAH. This FIL has no significant effect on the secondary structure of hPAH and is able to increase its enzymatic activity, despite the negative impact on protein thermostability. The influence of [N_1112(OH)][C₄F₉SO₃] on the complex oligomerization equilibrium of hPAH was also assessed.

Human carboxylesterase 2 (hCES2) is a glycoprotein involved in the metabolism of drugs and several environmental xenobiotics, whose crystallization has been proved to be a challenging task. This limitation could partly be due to glycosylation heterogeneity and has delayed the disclosure of the 3D structure of hCES2 which would be of upmost relevance for the development of new substrates and inhibitors. The present work evaluated the involvement of glycans in hCES2 activity and thermo stability in an attempt to find alternative active forms of the enzyme that might be adequate for structure elucidation. Partial or non-glycosylated forms of a secreted form of hCES2 have been obtained by three approaches: (i) enzymatic deglycosylation with peptide N-glycosidase F; (ii) incubation with the inhibitor tunicamycin; ii) site directed mutagenesis of each or both N-glycosylation sites. Deglycosylated protein did not show a detectable decrease in enzyme activity. On the other hand, tunicamycin led to decreased levels of secreted hCES2 but the enzyme was still active. In agreement, mutation of each and both N-glycosylation sites led to decreased levels of secreted active hCES2. However, the thermostability of the glycosylation mutants was decreased. The results indicated that glycans are involved, to some extent in protein folding in vivo, however, removal of glycans does not abrogate the activity of secreted hCES2.