Extended Data Fig. 5: Cataract-associated crystallin mutants are thermodynamically destabilized.

(a) Far-UV CD spectroscopy analyses of secondary structure analysis of recombinant wild-type (black) and mutant (red) crystallins. Due to low solubility, no spectra were recorded for βA2-S47P. (b) NanoDSF measurements, recording the optical density to detect protein aggregation. The V124E mutation did not change the aggregation propensity of αA-crystallin, but decreased the chaperone activity towards the model substrate L-MDH (inset). Data shown as mean and s.d. from n = 3 independent samples, obtained with recombinant protein from the same batch. Wild-type crystallin (black) and crystallin mutant (red). Experiments were carried out in PBS, but for the βA2 mutant. Due to the low stability of βA2 S47P, Tris buffer was used and L-arginine was added for the measurements. The measurement with wild-type βA2 in PBS are shown in black and in grey for the Tris/Arginine buffer as a reference (c) SEC-MALS/ -HPLC and SV-AUC (inset) were employed to characterize the quaternary structure of wild-type (black) and mutant (red) crystallins. Note: Due to the low stability of βA2-S47P, quantitative unfolding of the protein occurs during SV-AUC runs. Data shown resembles a representative distribution from three independent samples, obtained with recombinant protein from the same batch. Error bars result from averaging consecutive scans during SV-AUC experiments. Apparent maxima of the g(s*) distribution show the most populated particle species.