Chaperone Activity of Hsp33 on Denatured Citrate Synthase

Preparation of reduced, inactive Hsp33 (Hsp33_red) or HOCl-oxidized active Hsp33 (Hsp33_ox), and chaperone-activity measurements using either chemically or thermally denatured citrate synthase (CS) as client protein16 (link). To test the effects of Hsp33 on chemically denatured clients, CS from porcine heart (Sigma-Aldrich) was denatured to a final concentration of 12 μM in 6.0 M guanidinium-hydrochloride (GdmCl), 40 mM HEPES (pH 7.5) overnight at room temperature. To initiate aggregation of CS, the unfolded enzyme was diluted 1:160 into 1,600 μl 40 mM HEPES (pH 7.5) at either 20 or 30 °C in the absence or presence of Hsp33. To analyse Hsp33's effects on thermally unfolding CS, 0.15 μM CS were incubated in 1,600 μl 40 mM HEPES (pH 7.5) at the indicated temperatures in the absence or presence of Hsp33. For both assays, light scattering was monitored using a Hitachi F4500 fluorimeter equipped with a thermostated cell holder and stirrer. Excitation and emission wavelengths were set to 360 nm, and the excitation and emission slit widths were set to 2.5 nm. For competition studies between CS and NPY/NPY D4C IAM-TEMPO, a 10-fold molar excess of peptide to CS was used and the assay performed as described.