Polyol and sugar osmolytes stabilize the molten globule state of α-lactalbumin and inhibit amyloid fibril formation.

Protein misfolding and aggregation are associated with several human diseases such as Alzheimer's, Parkinson's, prion related disorders, type-II diabetes, etc. Different strategies using molecular chaperones, synthetic and naturally occurring small molecules, osmolytes, etc. have been used to prevent protein aggregation and amyloid fibril formation. In this study, we have used bovine α-lactalbumin at pH 1.6, 37 °C, and shaking conditions to promote amyloid fibril formation. Polyol and sugar osmolytes like glycerol, sorbitol, and trehalose have been used to inhibit the fibrillation of a number of proteins. In the present work, amyloid fibril formation of α-lactalbumin has been shown by ThT assay and AFM, while changes in the secondary structure during fibrillation has been followed by circular dichroism spectroscopy. Our results show that glycerol, sorbitol, and trehalose affect amyloid fibril formation of α-lactalbumin in a concentration-dependent manner. There is a delay in the lag phase of amyloid fibril formation in sorbitol and trehalose and complete inhibition in 6 M glycerol. Our results indicate that delay in the lag phase and inhibition of amyloid fibril formation are due to the stabilization of molten globule state by these osmolytes. At pH 1.6, the molten globule as well as the amyloid fibrils bind to ANS. However, when pH was shifted from 1.6 to 7, only the oligomeric and the fibrillar species bind to ANS due to refolding of molten globule state. The outcome of this study might be useful in designing small molecules which may stabilize the intermediate states, thus preventing amyloid fibril formation.