Cosolvents Induced Unfolding and Aggregation of Keyhole Limpet Hemocyanin

Abstract

The objective of this study was to examine the effects of 2,2,2 trifluoroethanol (TFE) and acetonitrile (ACN) on the stability, behavior, and structural characteristics of giant multimeric protein Keyhole Limpet hemocyanin (KLH) by combining the circular dichroism (CD) and fluorescence measurements of KLH solution. In concentration range 20-50% (v/v) TFE, protein at pH 7.4 shows visible aggregation while no aggregation was observed in the entire concentration range of TFE at molten globule (MG) state (pH 2.8) and resulted in stable α-helix. Our result shows that in the presence of 80% (v/v) and 40% (v/v) TFE, at native (pH 7.4) and MG state (pH 2.8) occurred in a highly helical state referred to as TFE denatured state I and II, respectively. However, in case of ACN, aggregation starts above 40% (v/v) for pH 7.4 and at 80% (v/v) for acid-induced MG (pH 2.8) state, which was dominated by β-sheet structure and referred to as ACN denatured state III and IV. An important object of our investigation is to get more detail study of efficiency of cosolvents in inducing structural changes in KLH. The dependence of scattering intensity and the R h on alcohol concentrations was investigated at 25 °C.

Systematic Development of a Size Exclusion Chromatography Method for a Monoclonal Antibody with High Surface Aggregation Propensity (SAP) Index

“…Given that acetonitrile is a non-polar solvent, it can dissociate non-covalent aggregate species that are formed by hydrophobic interactions, in the same way that it minimizes the interaction between hydrophobic regions of the protein and the column resin. According to Varshney et al , 34 acetonitrile induced aggregation was observed at concentrations above 40% at pH 7.4 and above 70% at pH 2.8, confirming that high concentration of organic leads to significant loss of tertiary structure, breaking up presumed hydrophobically driven interactions and thus the formation of aggregates. We opted out using acetonitrile and moved forward with the evaluation of arginine containing mobile phase.…”

Hydrolases: The Most Diverse Class of Enzymes

“…A protein exists in equilibrium with unfolded conformational states in solution with the folded ensemble being favored at ambient conditions. This equilibrium between the folded and the unfolded states can be perturbed by changing the thermodynamic state of the system (temperature, pressure, and pH) or by changing the composition by the addition of co-solvents to the solution [16]. Interestingly, the effect of co-solvents on the protein can alter this equilibrium in any direction.…”

“…On the other hand, protective osmolytes/kosmotropes, such as trimethylamine N-oxide (TMAO), dimethyl sulphoxide (DMSO), glycine, betaine, glycerol, and sugars, induce stabilization of the folded proteins (Figure 3b). Studies on solvent-mediated structural and conformational transitions of proteins can provide insight into their stability, folding pathways, and intermolecular aggregation behavior [16,17].…”

Kanamycin-Mediated Conformational Dynamics of Escherichia coli Outer Membrane Protein TolC

“…It is also useful to understand the mode of interaction and determine the change in protein secondary structure (Rabbani and Choi, 2018;Rabbani et al, 2015). The intermolecular and intramolecular interactions involved in the protein secondary structure stability are affected upon binding with ligands or drug molecules (Varshney et al, 2014;Thakur et al, 2017). CD spectra is established with one positive spectra on 195 nm and two significant negative peaks centered on 209 nm and 222 nm in the far-UV region due to the n → π* transition that shifts the peptide bond, which is characteristic of α-helix structure (Varlan and Hillebrand, 2010;Rogozea, 2012;Suryawashi, 2016;Zhang et at., 2016).…”