Thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL. II. GroEL recognizes thermally...

By thermal equilibrium measurements we found a three-state folding behavior of mature Escherichia coli beta-lactamase TEM2. The thermodynamically stable intermediate H had no enzymatic activity, but a native-like secondary structure. State H was 9 kcal mol-1 less stable than the native state N and 4 kcal mol-1 more stable than the totally unfolded state U, which is consistent with urea equilibrium measurements of mature beta-lactamase measured under similar conditions. Between 38 degrees C and 50 degrees C there was a decrease in the apparent equilibrium constant for dissociation K'D of the complex between GroEL and mature beta-lactamase, at least partially caused by a decrease in the thermodynamic stability of the native form of mature beta-lactamase. GroEL-bound beta-lactamase was released either after addition of ATP, or in the presence of a competing substrate (i.e. a single-chain antibody), or after lowering the temperature. Whereas at 10 degrees C the folding reaction of mature beta-lactamase was rate limiting, at 37 degrees C the release reaction was the rate-determining step for the regain of beta-lactamase activity, consistent with a decrease of the equilibrium constant for dissociation KD of the complex with temperature. A temperature dependent behavior of GroEL was also observed, when measuring the anilinonaphthalene sulfonic acid (ANS) fluorescence of the chaperone. Similar to all other substrate proteins studied so far, the maximal tryptohan fluorescence of GroEL-bound beta-lactamase was observed at 342 nm. Our results are compatible with a hydrophobic binding pocket of GroEL and confirm the suggested thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL.