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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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Related in: MedlinePlus

Fluorescence anisotropy titration of insulin. Anisotropy of 10 µM FITC tagged NK9 is plotted as a function of increasing insulin concentration at 25 and 37°C temperature.The error bars represents the standard deviation of 3 measurements.
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pone-0072318-g007: Fluorescence anisotropy titration of insulin. Anisotropy of 10 µM FITC tagged NK9 is plotted as a function of increasing insulin concentration at 25 and 37°C temperature.The error bars represents the standard deviation of 3 measurements.

Mentions: Fluorescence anisotropy (FA) is a useful technique to study the binding interaction of a fluorescently labeled ligand with proteins. Theoretically, as the interaction between the ligand and the protein increases, rotational diffusion of the ligand decreases, that results in increase in anisotropy [57]. In Figure 7 as the concentration of insulin increases, the anisotropy also increases rapidly in the initial part of the curve. Thus, in the beginning, the added protein was immediately bound to NK9. The increase in anisotropy became smaller and finally reached a plateau. The plateau reflects anisotropy of the saturated insulin NK9 complex. This experiment was carried out at two different temperatures (25°C and 37°C) to validate the binding of NK9 with insulin at higher temperature. Anisotropy experiment at 25°C shows the plateau beyond an insulin concentration of 4 µM, which implies almost 2∶1 binding of NK9 to insulin. Whereas, anisotropy experiment at 37°C showed plateau beyond 2 μM concentration of insulin implying almost 4∶1 binding of NK9 to insulin. Binding curves were obtained with apparent Kd values of 2.2 μM and 1.2 μM for binding to insulin at 25°C and 37°C, respectively. Gibbs free energy change for this association process was found to be −32.2 and −35.1 KJ.mol−1 at 25°C and 37°C respectively. This observation clearly indicates that the rate of insulin-NK9 complex formation increases at elevated temperatures.


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)

Fluorescence anisotropy titration of insulin. Anisotropy of 10 µM FITC tagged NK9 is plotted as a function of increasing insulin concentration at 25 and 37°C temperature.The error bars represents the standard deviation of 3 measurements.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0072318-g007: Fluorescence anisotropy titration of insulin. Anisotropy of 10 µM FITC tagged NK9 is plotted as a function of increasing insulin concentration at 25 and 37°C temperature.The error bars represents the standard deviation of 3 measurements.
Mentions: Fluorescence anisotropy (FA) is a useful technique to study the binding interaction of a fluorescently labeled ligand with proteins. Theoretically, as the interaction between the ligand and the protein increases, rotational diffusion of the ligand decreases, that results in increase in anisotropy [57]. In Figure 7 as the concentration of insulin increases, the anisotropy also increases rapidly in the initial part of the curve. Thus, in the beginning, the added protein was immediately bound to NK9. The increase in anisotropy became smaller and finally reached a plateau. The plateau reflects anisotropy of the saturated insulin NK9 complex. This experiment was carried out at two different temperatures (25°C and 37°C) to validate the binding of NK9 with insulin at higher temperature. Anisotropy experiment at 25°C shows the plateau beyond an insulin concentration of 4 µM, which implies almost 2∶1 binding of NK9 to insulin. Whereas, anisotropy experiment at 37°C showed plateau beyond 2 μM concentration of insulin implying almost 4∶1 binding of NK9 to insulin. Binding curves were obtained with apparent Kd values of 2.2 μM and 1.2 μM for binding to insulin at 25°C and 37°C, respectively. Gibbs free energy change for this association process was found to be −32.2 and −35.1 KJ.mol−1 at 25°C and 37°C respectively. This observation clearly indicates that the rate of insulin-NK9 complex formation increases at elevated temperatures.

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