Fig. 2: Oligomers of phosphorylation-mimicking mutant dissociate at lower temperatures and tend to be smaller.

A Determination of the oligomer size by AUC. The proteins were measured at a concentration of 23 µM and 35,000 rpm (98,500 × g) and 20 °C in PBS. Sedimentation was followed by UV absorption at 280 nm. Left panel: mutants of the NTR: WT: red, T42E: blue, S47E: black, T48E: purple, S47E/T48E: beige, S90E: brown. Middle panel: mutants of the ACD: S144E: brown, T163E: orange. Right panel: mutants of the CTR: S207E: blue, S208E: purple, S211E: black, S208E/S211E: gray. Runs with different chaperone concentrations are shown in Fig. S2A. HPLC runs are shown in Fig. S2B. B Thermal transitions of the proteins were measured in a Chirascan CD spectrometer at 218 nm in PBS. The protein samples (12.5 µM) were heated from 20 to 90 °C. Mean values and the standard deviation are plotted. N = 3 independent experiments. The color code is the same as in A and is also shown in the graph panels. C Formation of Hsp26 FRET pairs. In all, 5 µM AIAS and 5 µM LYI-labeled Hsp26S5C were mixed and incubated at 40 °C. At the indicated time points, spectra were measured at an excitation wavelength of 330 nm. D Subunit exchange was measured by the addition of unlabeled Hsp26 to the Hsp26 FRET complex in 39-fold molar excess. The dissociation of the FRET complex was followed at 520 nm (excitation at 330 nm). Subunit exchange was measured at 40 °C for 1500 s. An exemplary dissociation curve obtained for the WT protein is shown. The small inset shows the spectrum after the subunit exchange. E Dissociation constants were determined by exponentially fitting the dissociation curves. Mean values and the standard deviation (N = 3 independent experiments) are shown. Two-sided Student’s t tests were performed to check whether the changes were statistically significant. Only the changes observed for the S47E/T48E mutant were significantly different from the WT (p = 0.048).