Figure 7.

The V181A mutational variant of Hsp21 is non-dodecameric and has decreased chaperone activity. A, non-denaturing agarose gel electrophoresis. Panels show aliquots withdrawn from cells and medium before harvesting the cells; in each panel pair there is Hsp21 (left) and Hsp21^V181A (right). Labels indicate the positions of Hsp21 (WT) and Hsp21^V181A (V181A) and the entry point for loading (E). The image shows the overexpressed proteins as they migrate toward the anode (+) and that, already before purification, the mutational variant Hsp21^V181A is of smaller size compared with Hsp21 and that Hsp21^V181A leaks out into the medium. C, denaturing SDS-PAGE of Hsp21 WT and Hsp21^V181A mutational variant cross-linked with lysine-specific cross-linker at various protein concentrations (25–250 μm) at protein/cross-linker ratios of 1:10 and 1:60. C, control samples without cross-linker. A protein concentration of 250 μm Hsp21 corresponds to ∼5 mg/ml (calculated based on monomeric mass 21 kDa). D, chaperone activity of Hsp21, determined as its suppression of heat-induced aggregation of the model substrate protein MDH at molar ratios of 1:1 and 2:1. E, chaperone activity of Hsp21^V181A mutational variant, determined as its suppression of heat-induced aggregation of the model substrate protein MDH at molar ratios of 1:1, 2:1, and 5:1. F, chaperone activity of Hsp21, determined as its suppression of heat-induced aggregation of the model substrate protein CS at molar ratios of 1:1, 2:1, and 5:1. G, chaperone activity of Hsp21^V181A mutational variant, determined as its suppression of heat-induced aggregation of the model substrate protein CS at molar ratios of 1:1, 2:1, 5:1, 10:1, and 20:1.