Oligomeric State and Holding Activity of Hsp60

Abstract

Similar to its bacterial homolog GroEL, Hsp60 in oligomeric conformation is known to work as a folding machine, with the assistance of co-chaperonin Hsp10 and ATP. However, recent results have evidenced that Hsp60 can stabilize aggregation-prone molecules in the absence of Hsp10 and ATP by a different, "holding-like" mechanism. Here, we investigated the relationship between the oligomeric conformation of Hsp60 and its ability to inhibit fibrillization of the Ab40 peptide. The monomeric or tetradecameric form of the protein was isolated, and its effect on beta-amyloid aggregation was separately tested. The structural stability of the two forms of Hsp60 was also investigated using differential scanning calorimetry (DSC), light scattering, and circular dichroism. The results showed that the protein in monomeric form is less stable, but more effective against amyloid fibrillization. This greater functionality is attributed to the disordered nature of the domains involved in subunit contacts.

Keywords: Hsp60; amyloid aggregation; monomer; non-canonical function; oligomer.

Figure 1

Time course of ThT fluorescence emission at 37 °C, for a sheared sample of 50 μM of Aβ₄₀ peptide alone (black line) and with the addition of 2 μM of Hsp60 in oligomeric (blue line) or monomeric (red line) form. The wavelengths of emission and excitation were 485 and 450 nm, respectively. ThT concentration was 12 μM.

Figure 2

Tapping mode AFM images of samples of 50 μM of Aβ₄₀ peptide alone (A) and with the addition of 2 μM of Hsp60 in oligomeric (B) or monomeric form (C). Note the different Z-scales. The images were taken at the end of the kinetics experiments of Figure 1.

Figure 3

Static light-scattered intensity (left panel) and average hydrodynamic radius (right panel) vs. temperature for 15 μM of Hsp60 in oligomeric (blue symbol) and monomeric (red symbol) form.

Figure 4

DSC thermograms of 15 μM of Hsp60 in oligomeric (blue line) or monomeric (red line) form. Calorimetric signals are given after subtraction of the instrumental baseline. The solid black lines show the best fit of the data to the simplest form of the Lumry–Eyring model. Additionally shown are linear extrapolations of the pre- and post-unfolding baseline (dotted lines) and the calculated chemical baseline (dashed line) for the signal of the protein in monomeric form. The scan rate was 30 °C/h. The thermal transition is not reversible for either form of the protein due to intermolecular aggregation. Notably, the monomeric fraction unfolds at a much lower temperature.

Figure 5

Fractions of secondary structure components vs. temperature for 15 μM of Hsp60 in monomeric (left panel) or oligomeric (right panel) form. Results in the figure were obtained by CDPro analysis of circular dichroism spectra at varying temperatures (see Figure S1).

Figure 6

Comparison of the temperature dependence of DSC, LS, and CD results for 15 μM of Hsp60 in monomeric (red line) or oligomeric (blue line) form. For data consistency, the temperature derivative of molecular ellipticity at 225 nm and the scattered light intensity were plotted with the DSC trace.