Fig. 3.
cHSP27 monomers are potent chaperones in vitro. a The central ACD exists as a stable dimer in which residue C137 at the dimer interface can access both reduced (blue, PDB 4mjh) and oxidised (red, PDB 2n3j) states. b The cHSP27 variants used in this study together with their native mass spectra at 5 μM. For reduced cHSP27, 250 μM DTT was added. c Aggregation of 300 μM α-lactalbumin (αLac; black) at 37 °C and d 80 μM insulin at 40 °C following the addition of 2 mM DTT, both monitored by light scattering at 340 nm. The values of Tf are 10 (c) and 0.3 (d). Aggregation of both proteins is inhibited by C137S (green), reduced cHSP27 (blue), H124K/C137S (purple), and full length HSP27 (empty). For αLac and insulin, the sHSP concentrations were 70 and 40 μM, respectively. The traces represent the average of three experiments with errors bars indicating ± 1 SD. Under these conditions, C137S and reduced cHSP27 are predominantly dimeric whereas H124K/C137S is monomeric e Relative chaperone activity of the cHSP27 variants from panels c and d, with activity defined as one minus the ratio of the average signal with and without chaperone. f The self-aggregation of 800 μM C137S and H124K/C137S monitored by light scattering at 340 nm at 37 °C. The traces represent the average of six (C137S) or three (H124K/C137S) experiments with error bars indicating ± 1 SD. Inset: A representative transmission electron microscopy (TEM) micrograph at 40 h reveals large, non-fibrillar aggregates of H124K/C137S