Fig. 1
GI N exhibits general molecular chaperone activity in vitro. a MBP-GIN decreases heat-mediated MDH aggregation with increasing stoichiometric parity. b MBP-GIC has no effect on heat-mediated MDH aggregation. Both GI polypeptides were tested using MDH (0.5 μM) as a model substrate under thermal denaturing conditions (45 °C) in various molar ratios. HSP70 and BSA used as positive and negative controls, respectively. c The mean MDH denaturation state at the treatment endpoint of a and b relative to thermal-denaturation of MDH alone. The holdase assay (a–c) measures the aggregation of the model substrate MDH (0.5 μM), by measuring the turbidity (light scattering) at 340 nm under thermal denaturing conditions for 15 min at 45 °C. The turbidity of MDH alone at 15 min was set to 100%, and that from each treatment expressed relative to it. d MBP-GIN refolds chemically denatured G6PDH. e MBP-GIC cannot refold chemically denatured G6PDH. f The mean G6PDH activity at the treatment endpoint of d and e relative to the activity of undenatured G6PDH. The foldase assay determines G6PDH activity by measuring absorbance at 340 nm (Abs340) from NADPH formation. G6PDH was denatured in 4 M guanidine-HCl for 2.5 h (−2.5 h), and the relative G6PDH activity (compared to native G6PDH activity, set to 100%) was monitored in the absence (Spon. Refolding, spontaneous refolding) or presence of MBP, GroEL, and MBP-GIN or MBP-GIC for 5 h in renaturation buffer. GroEL and MBP were used as a positive and negative control, respectively. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed Student’s t-test. Data are means ± s.e. (n = 3)
