Thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL. I. GroEL recognizes the signal sequences of beta-lactamase precursor.

From equilibrium measurements with urea we found a three-state thermodynamic and kinetic folding behavior for the precursor and mature form of Escherichia coli beta-lactamase TEM2. The thermodynamic intermediate H of Escherichia coli beta-lactamase and its precursor had no enzymatic activity, and a quenched tryptophan fluorescence intensity, but a native-like wavelength of maximum intensity. State H of mature beta-lactamase was 8.7 kcal mol-1 less stable than the native state N and about 4.2 kcal mol-1 more stable than the unfolded state U, extrapolated to absence of urea. In contrast, state H of precursor beta-lactamase was even more stable than N by about 0.5 kcal mol-1 and about 6.9 kcal mol-1 more stable than U. Native pre-beta-lactamase could be stabilized by lowering the pH value from 7.0 to 5.5, probably by protonating a histidine residue leading to an improved solubility of the signal sequence. Synthetic peptides, containing 23 or 38 N-terminal amino acid residues of pre-beta-lactamase, were unable to compete with pre-beta-lactamase for binding to GroEL. However, GroEL prevented the inactivation of mature beta-lactamase by p38, consistent with competition between GroEL and mature beta-lactamase for binding to p38. The equilibrium constant for dissociation KD of the complex between GroEL and p23, a peptide containing exclusively the signal sequence of pre-beta-lactamase, was measured with the BIAcore instrument to be in the range 10(-7) to 10(-8) M. Our results are consistent with co-operative binding of GroEL to the mature part and to the signal sequence of pre-beta-lactamase. We suggest a thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL.