Inhibition of Insulin Fibrillation by a Non Toxic Peptide NK9

Abstract

Many proteins form ordered structural aggregates called amyloid fibrils, which are associated with numerous neurodegenerative diseases. Insulin, a largely α-helical protein, undergoes conformational changes under certain stress conditions leading to amyloid fibrils. Fibrillation of insulin poses a problem in its long-term storage, reducing its efficacy in treating type II diabetes. Oligomeric dissociation of insulin into its monomer is the key step for the onset of fibrillation. In the present study, we have used a non-toxic nine residue peptide, NK9 (NIVNVSLVK) constituting mostly with hydrophobic residues, which interferes fibrillation of insulin. The time course of insulin fibrillation at 62°C using Thioflavin T fluorescence shows an increase in the lag time from 120 min without peptide to 236 min with peptide. TEM micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. FTIR study shows, loss of α-helical structure of insulin upon incubation, but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. Size exclusion chromatography study confirms the resistance of the peptide towards the oligomeric dissociation of insulin into its monomer in the course of fibrillation. Saturation transfer difference (STD) NMR was used to confirm the binding epitope of NK9, where the hydrophobic residues give signal of resonance transfer being in close contact with insulin. MD simulation in conjunction with PCA analysis reveals how the binding of NK9 governs the interruption of insulin fibrillation. An estimation of probable aggregation site of insulin gives a clue for NK9 activity. The present study is considered to be helpful for architecting new peptide based leads for efficient stopping of aggregation of insulin.