Amyloid fibrillation of protein is associated with a great variety of pathologic conditions. The aggregation of protein is a complicated process with multisteps, whereas most of the inhibitors with elaborately designed structures can show an inhibition effect only on the nucleation stages of protein fibrillation. Herein, oxidized carbon dots (CDs) were achieved to study the relationship between the surface properties of CDs and their inhibition effect on human insulin (HI) fibrillation. More oxygen-containing function groups can be obtained after oxidation reaction of CDs, such as -OH and -COOH. The results show that 10-1 CDs (the mass ratios of CD/KMnO4 is 10:1), with the highest carboxyl group content, possess the best inhibition ability. All the nucleation, growth, and final phases can be retarded by 10-1 CDs, which have been studied in detail by fluorescence spectra. However, CDs without oxidation can show only a weak inhibition effect on the nucleation stage. The 10-1 CDs is demonstrated to binding with HI monomers much stronger than that of CDs by isothermal titration calorimetry (ITC). Moreover, molecular dynamics simulations (MD) studies indicate that CDs with more carboxyl groups can show stronger affinities with native or unfolded HI monomers, which may be mainly derived from the active binding sites of histidine residues (His5 and His10) on B-chain through electrostatic interaction. Because the unfolding of B-chain in HI is prior to that of A-chain in our MD simulations, the later aggregation of HI can be inhibited effectively by the stronger binding forces between 10 and 1 CDs and the B-chain of HI.
Keywords: amyloid fibrillation; carbon dots; electrostatic interaction.; human insulin; isothermal titration calorimetry.