ATP HYDROLYSIS IS CRITICAL FOR INDUCTION OF CONFORMATIONAL-CHANGES INGROEL THAT EXPOSE HYDROPHOBIC SURFACES

The degree of hydrophobic exposure in the molecular chaperone GroEL du ring its cycle of ATP hydrolysis was analyzed using 1,1'-bis(4-anilino )naphthalene-5,5'disulfonic acid (bisANS), a hydrophobic probe, whose fluorescence is highly sensitive to the environment, In the presence o f 10 mM MgCl, and 10 mM KCI the addition of ATP, but not ADP or AMP-PN P, resulted in a time-dependent, linear increase in the bisANS fluores cence, The rate of the increase in the bisANS fluorescence depended on the concentrations of both GroEL and the probe, The effect could be s ubstantially inhibited by addition of excess ADP or by converting ATP to ADP using hexokinase, showing that the increase in the bisANS fluor escence was correlated with ATP hydrolysis. The rate of ATP hydrolysis catalyzed by GroEL was uncompetitively inhibited in the presence of b isANS, This uncompetitive inhibition suggests that the probe can inter act with the GroEL-ATP complex, The inability of the nonhydrolyzable A TP analog, AMP-PNP, to cause a similar effect is explained by the inte raction of bisANS with a transient conformational state of GroEL forme d consequent to ATP hydrolysis, It is suggested that this short lived hydrophobic exposure reflects a conformational shift in GroEL that res ults from electrostatic repulsion between the bound products of ATP hy drolysis, and it plays an important role in the mechanism of the chape ronin cycle.