Impact of imidazolium-based ionic liquids on the structure and stability of lysozyme

<p>Various types of water-miscible aprotic ionic liquids (ILs) with different cations (1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium, 1-octyl-3-methylimidazolium) and anions (ethylsulfate and chloride) were used as co-solvents to investigate the stability of lysozyme. Different techniques such as fluorescence, thermal absorption, and circular dichroism (CD) spectroscopy have been used for the study. Fluorescence results reveal that the addition of ILs (1-ethyl-3-methylimidazolium ethyl sulfate and 1-ethyl-3-methylimidazolium) increases the hydrophobicity around the tryptophan environment in lysozyme. CD analysis and temperature-dependent studies were done to investigate the stability of the protein. From the CD analysis, it was observed that the ILs keep the native structure of protein intact. Thermal denaturation studies depicted that the melting temperature of the protein increased in the presence of ILs (1-ethyl-3-methylimidazolium ethyl sulfate and 1-ethyl-3-methylimidazolium), which indicates the stabilization of the protein.</p

pH Dependence of Amylin Fibrillization

In type 2 diabetics, the hormone amylin misfolds into amyloid plaques implicated in the destruction of the pancreatic β-cells that make insulin and amylin. The aggregative misfolding of amylin is pH-dependent, and exposure of the hormone to acidic and basic environments could be physiologically important. Amylin has two ionizable residues between pH 3 and 9: the α-amino group and His18. Our approach to measuring the p<i>K</i>_a values for these sites has been to look at the pH dependence of fibrillization in amylin variants that have only one of the two groups. The α-amino group at the unstructured N-terminus of amylin has a p<i>K</i>_a near 8.0, similar to the value in random coil models. By contrast, His18, which is involved in the intermolecular β-sheet structure of the fibrils, has a p<i>K</i>_a that is lowered to 5.0 in the fibrils compared to the random coil value of 6.5. The lowered p<i>K</i>_a of His18 is due to the hydrophobic environment of the residue, and electrostatic repulsion between positively charged His18 residues on neighboring amylin molecules in the fibril. His18 acts as an electrostatic switch inhibiting fibrillization in its charged state. The presence of a charged side chain at position 18 also affects fibril morphology and lowers amylin cytotoxicity toward a MIN6 mouse model of pancreatic β-cells. In addition to the two expected p<i>K</i>_a values, we detected an apparent p<i>K</i>_a of ∼4.0 for the amylin-derived peptide NAc-SNN­F­G­A­ILSS-NH₂, which has no titratable groups. This p<i>K</i>_a is due to the pH-induced ionization of the dye thioflavin T. By using alternative methods to follow fibrillization such as the dye Nile Red or turbidimetry, we were able to distinguish between the titration of the dye and groups on the peptide. Large differences in reaction kinetics were observed between the different methods at acidic pH, because of charges on the ThT dye, which hinder fibril formation much like the charges on the protein