Insight into the co-solvent induced conformational changes and aggregation of bovine β-lactoglobulin

• Bovine β-lactoglobulin (β-lg) was incubated separately with two commonly used organic solvents MeCN and BA at room temperature at pH 7.0.
• Partial unfolding and increase of α-helical structure of β-lg at moderate concentrations of MeCN (5–50% v/v) and at very low concentrations of BA (0.1–1.5%).
• At higher concentrations of MeCN (50–80%) and at low concentrations of BA (1.5–3.0%), structural transitions from α-helical to non-native β-sheet structure leading to the self-assembly of β-lg.
• TEM study revealed the formation of amyloid fibrillar aggregates of β-lg in organic co-solvents.

Many proteins form ordered irreversible aggregates called amyloid fibrils which are responsible for several neurodegenerative diseases. β-lactoglobulin (β-lg), an important globular milk protein, self-assembles to form amyloid-like fibrils on heating at low pH. The present study investigated the effects of two commonly used organic solvents acetonitrile (MeCN) and antimicrobial preservative benzyl alcohol (BA) on the conformation and self-assembly of β-lg at ambient condition. Both MeCN and BA induced a concentration-dependent conformational change showing exposure of hydrophobic patches, loss of tertiary structure and higher α-helical structure at moderate concentrations. In the presence of 50–80% (v/v) MeCN and 1.5–3% (v/v) BA further structural transitions from α-helical to non-native β-sheet structure were observed with a molten globule-like intermediate at 70% MeCN. These non-native β-sheet structures have high tendency to form aggregates. The formation of β-lg self-assembly was confirmed by Thioflavin T studies, Congo red assay, Rayleigh scattering and dynamic light scattering analysis. Transmission electron microscopy studies showed amyloid fibril formation in both MeCN and BA. Our results showed that BA enhances the unfolding and self-assembly of β-lg at much lower concentration than MeCN. Thus solvent composition forces the protein to achieve the non-native structures which are responsible for protein aggregation.

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