Blue Native Polyacrylamide Gel Electrophoresis: Hsp60 at various concentrations.

<p>Blue Native PAGE (4–16%) image of naïve Hsp60 in the concentration range 1.9–4.8 µM. The pattern reveals that the protein exists in two oligomeric forms independently of the concentration.</p

Static light scattering characterization.

<p>Scattered light intensities from solutions of naïve Hsp60 protein at different concentrations, expressed in terms of the Rayleigh ratio <i>R_90°</i> at 18.7 µm⁻¹ normalized by the concentration values <i>c</i>. Together with the total scattered intensity (empty circles), the intensity contribution by species with diameter size lower than 70 nm (filled circles) is also reported. The lines represent the dependence of <i>R_90°/c</i> on concentration predicted for Hsp60 protein totally assembled in tetradecamers (solid) or heptamers (dashed). The experimental values of intensity scattered by Hsp60 species always fall between those predicted for totally tetradecameric or heptameric populations.</p

Size exclusion chromatography results.

<p>Size exclusion chromatography experiments on the naïve Hsp60 at different concentrations (0.8 µM: red, 1.6 µM: black, 2.2 µM: magenta, 6.4 µM: green, 16.0 µM: turquoise) compared with GroEL at 7.0 µM (blue) and BSA (dotted line). The vertical line drawn across the Hsp60 peak helps to highlight the independency of the retention time from protein concentration. The value of the retention time is consistent with that expected for heptameric and tetradecameric species in equilibrium.</p

SAXS profiles of GroEL and Hsp60.

<p>SAXS profiles of GroEL (red) and naïve Hsp60 (green). GroEL is at c = 52.5 µM, while Hsp60 is at c = 79.5 µM. Experimental curves are scaled for the sake of clarity. SAXS spectrum for Hsp60 is quite different from that of GroEL, which corresponds to a more well-defined structure.</p

Fluorescence correlation spectroscopy results.

<p>Diffusion times measured by FCS as a function of monomer concentration: GroEL (red), naïve Hsp60 (blue). Continuous lines correspond to interpolations of the experimental data to sigmoid functions and are drawn as a guide for the eye. Diffusion times, both for Hsp60 and GroEL remain constant with lowering the concentration down to 10 nM, thus indicating a high stability of the protein oligomeric complexes.</p

SAXS for GroEL and Hsp60: Guinier approach.

<p>SAXS profiles of naïve Hsp60 at 23.8 (green) and 79.5 µM (blue). Experimental curves are scaled for the sake of clarity. Continuous lines correspond to the theoretical fitting obtained by the Guinier approach, as reported in Material and Methods, SAXS. The gyration radius extrapolated by fitting procedure suggests, at both concentrations, the simultaneous presence of oligomeric structures in equilibrium.</p

Dynamic light scattering characterization.

<p>Dynamic light scattering characterization of the naïve Hsp60 at different concentrations (0.8 µM: black, 6.4 µM: green, 16.0 µM: turquoise, 27.0 µM: orange) compared with GroEL at 7.0 µM (blue). (A) Normalized intensity autocorrelation functions g⁽²⁾(t). (B) Number-weighted distribution functions P_N of the <i>z</i>-average hydrodynamic diameter D_H obtained by the analysis of the autocorrelation functions. At each concentration, the hydrodynamic diameter of Hsp60 is always compatible with that of heptamer/tetradecamer species.</p

Blue Native Polyacrylamide Gel Electrophoresis: Hsp60 at various concentrations.

<p>Blue Native PAGE (4–16%) image of naïve Hsp60 in the concentration range 1.9–4.8 µM. The pattern reveals that the protein exists in two oligomeric forms independently of the concentration.</p