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Use of a small peptide fragment as an inhibitor of insulin fibrillation process: a study by high and low resolution spectroscopy.

Banerjee V, Kar RK, Datta A, Parthasarathi K, Chatterjee S, Das KP, Bhunia A - PLoS ONE (2013)

Bottom Line: In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells.The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies.Further new peptide based leads may be developed from this nine residue peptide.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Bose Institute, Kolkata, India.

ABSTRACT
A non-toxic, nine residue peptide, NIVNVSLVK is shown to interfere with insulin fibrillation by various biophysical methods. Insulin 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. The dissociation of insulin oligomer to monomer is the key step for the onset of fibrillation. 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. Transmission electron micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. Upon incubation at 62°C and pH 2.6, insulin lost some α-helical structure as seen by Fourier transformed infra-red spectroscopy (FT-IR), but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. FT-IR spectroscopy also shows that insulin forms the cross beta structure indicative of fibrils beyond 2 h, but in the presence of the peptide, α-helix retention is seen till 4 h. Both size exclusion chromatography and dynamic light scattering show that insulin primarily exists as trimer, whose conversion to a monomer is resisted by the peptide. Saturation transfer difference nuclear magnetic resonance confirms that the hydrophobic residues in the peptide are in close contact with an insulin hydrophobic groove. Molecular dynamics simulations in conjunction with principal component analyses reveal how the peptide interrupts insulin fibrillation. In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells. The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies. Further new peptide based leads may be developed from this nine residue peptide.

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STD NMR Spectrum.(A) Reference spectrum of NK9 in presence of insulin at a molar ratio of insulin:NK9 = 1∶100. (B) STD NMR spectrum of same sample showing the resonance transfer within the binding epitope of insulin. The experiments were performed at 298 K with 2 sec of saturation time (on resonance  = 7.3 ppm; off resonance  = 40 ppm). (C) STD spectrum of NK9 alone (on resonance  = 7.3 ppm; off resonance  = 40 ppm). (D) The docking model of insulin-NK9 complex correlated of the STD NMR data.
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pone-0072318-g008: STD NMR Spectrum.(A) Reference spectrum of NK9 in presence of insulin at a molar ratio of insulin:NK9 = 1∶100. (B) STD NMR spectrum of same sample showing the resonance transfer within the binding epitope of insulin. The experiments were performed at 298 K with 2 sec of saturation time (on resonance  = 7.3 ppm; off resonance  = 40 ppm). (C) STD spectrum of NK9 alone (on resonance  = 7.3 ppm; off resonance  = 40 ppm). (D) The docking model of insulin-NK9 complex correlated of the STD NMR data.

Mentions: To obtain the structural insight of NK9-insulin complex, transferred NOESY experiment was performed (Figure S1). The peptide in absence and presence of insulin was predominantly characterized by intra residue and sequential NOEs between backbone proton and side chain proton resonances (Figure S1), clearly indicating that the peptide is highly flexible in solution state and does not adopt any folded conformation in presence of insulin. This result is in very good agreement with our CD data of NK9 in the context of insulin (Figure 3C). To understand the localization of NK9 in insulin we employed one dimensional proton STD NMR which is a powerful and very specific technique to identify the epitope of a ligand is in close proximity with the receptor [37]. Briefly, selective saturation to the receptor is transferred to the bound ligand via spin diffusion, without affecting the ligand signal alone. Therefore, from the difference spectrum (off resonance – on resonance) we obtain the distance information or the epitope of the ligand bound to the macromolecule. Excess of ligand compared to its receptor is used in the STD experiments to achieve effective magnetization transfer from the receptor to the ligand at its bound state (Figure 8). The strong STD effect was observed for the methyl groups of NK9 in presence of insulin (Figure 8B) but there was no STD effect in the absence of insulin (Figure 8C). Although it is difficult to pin point the aliphatic hydrophobic amino acid residue due to significant overlap of the methyl group signals, it could be from any or all of the residues from I2, V3, V5, L7 and V8 closely interacting with insulin. The I2γ and CβHs of V3/V5/V8 of NK9 show moderate STD effects (Figure 8B). The CαHs of NK9 are very close in proximity to the water signal. Interestingly, the CαH of S6 and L7 showed very weak STD signals (Figure 8B). Taken together, STD data qualitatively suggest a close association of hydrophobic amino acid residues with insulin (Figure 8B).


Use of a small peptide fragment as an inhibitor of insulin fibrillation process: a study by high and low resolution spectroscopy.

Banerjee V, Kar RK, Datta A, Parthasarathi K, Chatterjee S, Das KP, Bhunia A - PLoS ONE (2013)

STD NMR Spectrum.(A) Reference spectrum of NK9 in presence of insulin at a molar ratio of insulin:NK9 = 1∶100. (B) STD NMR spectrum of same sample showing the resonance transfer within the binding epitope of insulin. The experiments were performed at 298 K with 2 sec of saturation time (on resonance  = 7.3 ppm; off resonance  = 40 ppm). (C) STD spectrum of NK9 alone (on resonance  = 7.3 ppm; off resonance  = 40 ppm). (D) The docking model of insulin-NK9 complex correlated of the STD NMR data.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756998&req=5

pone-0072318-g008: STD NMR Spectrum.(A) Reference spectrum of NK9 in presence of insulin at a molar ratio of insulin:NK9 = 1∶100. (B) STD NMR spectrum of same sample showing the resonance transfer within the binding epitope of insulin. The experiments were performed at 298 K with 2 sec of saturation time (on resonance  = 7.3 ppm; off resonance  = 40 ppm). (C) STD spectrum of NK9 alone (on resonance  = 7.3 ppm; off resonance  = 40 ppm). (D) The docking model of insulin-NK9 complex correlated of the STD NMR data.
Mentions: To obtain the structural insight of NK9-insulin complex, transferred NOESY experiment was performed (Figure S1). The peptide in absence and presence of insulin was predominantly characterized by intra residue and sequential NOEs between backbone proton and side chain proton resonances (Figure S1), clearly indicating that the peptide is highly flexible in solution state and does not adopt any folded conformation in presence of insulin. This result is in very good agreement with our CD data of NK9 in the context of insulin (Figure 3C). To understand the localization of NK9 in insulin we employed one dimensional proton STD NMR which is a powerful and very specific technique to identify the epitope of a ligand is in close proximity with the receptor [37]. Briefly, selective saturation to the receptor is transferred to the bound ligand via spin diffusion, without affecting the ligand signal alone. Therefore, from the difference spectrum (off resonance – on resonance) we obtain the distance information or the epitope of the ligand bound to the macromolecule. Excess of ligand compared to its receptor is used in the STD experiments to achieve effective magnetization transfer from the receptor to the ligand at its bound state (Figure 8). The strong STD effect was observed for the methyl groups of NK9 in presence of insulin (Figure 8B) but there was no STD effect in the absence of insulin (Figure 8C). Although it is difficult to pin point the aliphatic hydrophobic amino acid residue due to significant overlap of the methyl group signals, it could be from any or all of the residues from I2, V3, V5, L7 and V8 closely interacting with insulin. The I2γ and CβHs of V3/V5/V8 of NK9 show moderate STD effects (Figure 8B). The CαHs of NK9 are very close in proximity to the water signal. Interestingly, the CαH of S6 and L7 showed very weak STD signals (Figure 8B). Taken together, STD data qualitatively suggest a close association of hydrophobic amino acid residues with insulin (Figure 8B).

Bottom Line: In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells.The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies.Further new peptide based leads may be developed from this nine residue peptide.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Bose Institute, Kolkata, India.

ABSTRACT
A non-toxic, nine residue peptide, NIVNVSLVK is shown to interfere with insulin fibrillation by various biophysical methods. Insulin 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. The dissociation of insulin oligomer to monomer is the key step for the onset of fibrillation. 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. Transmission electron micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. Upon incubation at 62°C and pH 2.6, insulin lost some α-helical structure as seen by Fourier transformed infra-red spectroscopy (FT-IR), but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. FT-IR spectroscopy also shows that insulin forms the cross beta structure indicative of fibrils beyond 2 h, but in the presence of the peptide, α-helix retention is seen till 4 h. Both size exclusion chromatography and dynamic light scattering show that insulin primarily exists as trimer, whose conversion to a monomer is resisted by the peptide. Saturation transfer difference nuclear magnetic resonance confirms that the hydrophobic residues in the peptide are in close contact with an insulin hydrophobic groove. Molecular dynamics simulations in conjunction with principal component analyses reveal how the peptide interrupts insulin fibrillation. In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells. The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies. Further new peptide based leads may be developed from this nine residue peptide.

Show MeSH
Related in: MedlinePlus