Figure 2.

Interaction with aggregated protein. (a) ClpC together with MecA and ATP associates with aggregated protein. Heat-aggregated MDH was incubated for 5 min at room temperature with ClpC in the presence or absence of MecA with or without ATP [with pyruvate kinase (PK) and phosphoenolpyruvate (PEP) as ATP regenerating system], as indicated below the gel. In addition, a control without aggregated MDH (agg MDH) is shown. A subsequent centrifugation for 30 min at 16,000 × g separated supernatant and pellet fractions. Supernatant (S) and pellet (P) fractions were analyzed by Coomassie-stained SDS/PAGE. Positions of the different proteins on the gel are marked. (b) ClpC together with MecA can disaggregate heat-aggregated MDH. Previously heat-aggregated MDH was incubated with the indicated proteins ATP and the PK/PEP regenerating system. The disaggregation reaction was subsequently followed in time by light-scattering measurements. In addition, curve fits of the data points are shown. (c) ClpC together with MecA can disaggregate and refold previously heat-aggregated MDH. Previously heat-aggregated MDH was incubated with the proteins as indicated, and MDH activity was determined. (d) ClpC together with MecA can disaggregate and refold previously heat-aggregated luciferase. Previously heat-aggregated luciferase was incubated with the indicated proteins, and luciferase activity was determined.