Quantitative Measurement of Stability and Heterogeneity of Protein Amyloids using Disaggregation by Chemical Denaturants

Abstract

Amyloids are protein aggregates known to be involved in several neurodegenerative diseases e.g., Alzheimer's and Parkinson's disease. However, biological activities of the amyloids still remain unclear due to highly heterogeneous and polymorphic nature of the amyloid aggregates. We hypothesize that kinetics of dissolution of the amyloids can be dependent on the molecular structure of the amyloids. Here, we use amyloids of tetramethylrhodamine labelled Amyloid-β(1-42) (TMR-Aβ42) which are fluorescently dark as shown by Garai and Frieden previously. Our results using fluorescence correlation spectroscopy reveals that fluorescence of TMR increases upon dissolution of TMR-Aβ amyloids in 4M GdnCl. Increase of fluorescence is correlated with the increase in the concentration of monomeric TMR-Aβ42. We find that TMR-Aβ42 amyloid dissolved minimally in native buffer but the rate of dissolution increases exponentially with increase in the concentrations of Urea or GdnCl. However, dissolution of the amyloids doesn’t seem to follow thermodynamic solubility. In case of the amyloids we find that soluble concentration increases with the increase in total concentration of the fibrils. Furthermore, fibrils incubated at a particular concentration of denaturant do not dissolve any further in the same solvent even upon 100-fold dilution. Taken together, our data indicate that amyloids are a mixture of different species and each type of species requires a certain minimum concentration of denaturant for dissolution. Our data of apparent solubility as a function of concentration of denaturants can be fit assuming Gaussian distribution where the peak of the distribution indicates mean stability and width of the distribution indicates heterogeneity in a particular preparation of the amyloids. Methodologies used here are general; hence, they can be employed to characterize stability and heterogeneity of amyloids of other proteins as well.