ATP hydrolysis induces an intermediate conformational state in GroEL

The conformational properties of the molecular chaperone GroEL in the prese nce of ATP, its non-hydrolyzable analog 5'-adenylimidodiphosphate (AMP-PNP) , and ADP have been analyzed by differential scanning calorimetry (DSC), Fo urier-transform infra-red (FT-IR) and fluorescence spectroscopy. Nucleotide binding to one ring promotes a decrease in the T-m value of the GroEL ther mal transition that is reversed when both rings are filled with nucleotide, indicating that the sequential occupation of the two protein rings by thes e nucleotides has different effects on the GroEL thermal denaturation proce ss. In addition, ATP induces a conformational change in GroEL characterized by (a) the appearance of a reversible low temperature endotherm in the DSC profiles of the protein, and (b) an enhanced binding of the hydrophobic pr obe 8-anilino-naphthalene-1-sulfonate (ANS), which strictly depends on ATP hydrolysis. The similar sensitivity to K+ of the temperature range where ac tivation of the GroEL ATPase activity, the low temperature endotherm, and t he increase of the ANS fluorescence are observed strongly indicates the exi stence of a conformational state of GroEL during ATP hydrolysis, different from that generated on ADP or AMP-PNP binding. To achieve this intermediate conformation, GroEL mainly modifies its tertiary and quaternary structures , leading to an increased exposure of hydrophobic surfaces, with minor rear rangements of its secondary structure.