Figure 4.

Assignment of absorbance changes due to interdomain interaction. (A) (Upper panel) ATP-binding spectrum of wild-type DnaK, DnaK(1–507), and the ATPase domain of the protein, recorded in H₂O medium at 25°C. Average of 10 (wild-type DnaK), 7 (DnaK1–507), and 13 (ATPase) independent experiments. Protein concentration was 1.4 mM and the nucleotide:protein molar ratio was 1. Spectra were recorded during the first 10 sec after nucleotide release. (Lower panel) ADP-binding spectra of the same samples recorded in H₂O-based buffer under the same experimental conditions. Average of seven (wild-type DnaK), six (Dnak1–507), and eight (ATPase) different experiments. (B) (Upper panel) ATP-induced absorbance changes in the IR difference spectrum of wild-type DnaK, DnaK1–507, and the ATPase domain of the protein. Protein concentration was 1 mM and equimolecular amounts of nucleotide were released to the medium. Experiments were performed in D₂O-based buffer at 25°C and are the average of five (wild-type DnaK), four (DnaK1–507), and seven (ATPase) different experiments. (Lower panel) ADP-binding spectra of the same samples using the same experimental conditions. Average of 12, 5, and 4 independent experiments for wild-type DnaK, DnaK1–507, and ATPase domain, respectively. The photolysis spectra are shown in dotted lines. Other details are as in Figure 2​2.