Fig. 1: Phospho-mimetic mutants tend to be more active at room temperature. | Nature Communications

Fig. 1: Phospho-mimetic mutants tend to be more active at room temperature.

From: Phosphorylation activates the yeast small heat shock protein Hsp26 by weakening domain contacts in the oligomer ensemble

Fig. 1

A Schematic structure of Hsp26 with highlighted phosphorylation sites. B Endpoints of insulin assays performed at 25 °C with 40 µM insulin and 8 µM chaperone concentration are plotted. Data are presented as mean values +/− SD. Single measurement points are indicated in the plot. Aggregation of insulin was induced by the addition of 20 mM DTT. The assay was performed in PBS for 70 min. The light scattering signal (360 nm) was normalized on the saturation value of the model substrate without chaperone. N = 3 independent experiments for insulin alone, WT, T42E, S90E, S207E, S208E, and S211E; N = 4 for S47E, T48E, S144E, T163E, S208E/S211E; N = 5 for S47E/T48E. C Endpoints of insulin assays performed at 43 °C with 45 µM insulin and 2 µM chaperone concentration are plotted. Data are presented as mean values +/− SD. Single measurement points are indicated in the plot. The assay was performed in 40 mM HEPES/KOH pH 7.5 for 36 min. The light scattering signal (360 nm) was normalized on the saturation value of the model substrate without chaperone. N = 3 independent experiments for insulin alone, S207E, and S208E/S311E; n = 4 for all remaining samples. More detailed titrations are shown in Fig. S1. D The aggregation assay was performed as described in B. For the visualization of chaperone substrate complexes, samples were taken after 20 min and analyzed with TEM at a magnification of 60,000. The scale bar represents 50 nm. E The aggregation assay was performed as described in C. For the visualization of chaperone substrate complexes, samples were taken after 20 min and analyzed with TEM at a magnification of 60,000. The scale bar represents 50 nm.

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