The effect of heparan sulfate on promoting amyloid fibril formation by β-casein and their binding research with multi-spectroscopic approaches.

As a molecular chaperone, β-casein is difficult to form amyloid fibrils under physiological conditions due to its chaperone activity. Heparan sulfate (HS) has drawn attention of technologists all over the word because of its relation to amyloid deposits in some amyloidosis diseases. For better understanding the relationship between the β-casein and HS, the multi-spectroscopic studies were employed. The data of thioflavin T (ThT) binding assay, transmission electron microscopy (TEM) and circular dichroism (CD) demonstrated that HS promoted fibril formation by β-casein in the amount and the growth speed. The results of steady-state UV-vis absorption spectra, fluorescence spectroscopy and fluorescence lifetime revealed that the β-casein-HS complexes were formed and HS quenched the fluorescence of β-casein by a static quenching mechanism. On the basis of fluorescence analysis, the value of binding constant was equal to 1.17 × 10 ⁷ L mol ^-1 at 338.15 K and there was about one binding site between them. According to thermodynamic parameters obtained, it was deduced that a spontaneous reaction happened, and protein-ligand complex was stabilized by hydrogen bonds and hydrophobic interaction. Furthermore, using fluorescence resonance energy transfer (FRET) assay, the value of binding distance between HS and Trp143 of β-casein was calculated to be 0.93 nm. Finally, on the basis of synchronous fluorescence experiment, the polarity increasing and hydrophobicity decreasing around Trp143 occurred during the period of fibril formation by β-casein.
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