ATP hydrolysis induces an intermediate conformational state in GroEL
1999
The conformational properties of the molecular chaperone GroEL in the presence of ATP, its non-hydiolyzable analog 5'-adenylimidodiphosphate (AMP- PNP), and ADP have been analyzed by differential scanning calorimetry (DSC), Fourier-transform infia-red (FT-IR) and fluorescence spectroscopy. Nucleotide binding to one ring promotes a decrease in the T(m) value of the GroEL thermal transition that is reversed when both rings are filled with nucleotide, indicating that the sequential occupation of the two protein rings by these nucleotides has different effects on the GroEL thermal denaturation process. 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 probe 8-anilino-naphthalene-l-sulfonate (ANS), which strictly depends on ATP hydrolysis. The similar sensitivity to K(+) of the temperature range where activation of the GroEL ATPase activity, the low temperature endotherm, and the increase of the ANS fluorescence are observed strongly indicates the existence of a conformational state of GroEL during ATP hydrolysis, different from that generated on ADP or AMP-PNP binding. To achieve this intermediate conformation, G10EL mainly modifies its tertiary and quaternary structures, leading to an increased exposure of hydrophobic surfaces, with minor rearrangements of its secondary structure.
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