FIGURE 3.

Conformational changes in cluster 1 mutants as analyzed by turbidimetric assay. Aggregation measured at 350 nm (A) and 600 nm (B) showed that cluster 1 mutants R33Q, K31A-R33A, and D29A-D32A are Ca²⁺-insensitive at low calcium concentrations. Shown is the percentage of protein precipitated due to Ca²⁺-induced aggregation (C) and Ca²⁺ chelation mediated by EGTA (D) as described under “Experimental Procedures.” Cluster 1 mutants required higher EGTA concentrations to become resolubilized. For EGTA-mediated Ca²⁺ chelation, the WT protein and cluster 1 mutants were first aggregated with 8 mm CaCl₂. The replot of aggregation-disaggregation of each of the cluster 1 mutants is shown in supplemental Fig. S5.