THERMODYNAMIC PARTITIONING MODEL FOR HYDROPHOBIC BINDING OF POLYPEPTIDES BY GROEL .1. GROEL RECOGNIZES THE SIGNAL SEQUENCES OF BETA-LACTAMASE PRECURSOR

From equilibrium measurements with urea we found a three-state thermod ynamic and kinetic folding behavior for the precursor and mature form of Escherichia coli beta-lactamase TEM2. The thermodynamic intermediat e H of Escherichia coli beta-lactamase and its precursor had no enzyma tic activity, and a quenched tryptophan fluorescence intensity, but a native-like wavelength of maximum intensity. State H of mature beta-la ctamase was 8.7 kcal mol(-1) less stable than the native state N and a bout 4.2 kcal mol(-1) more stable than the unfolded state U, extrapola ted to absence of urea. In contrast, state H of precursor beta-lactama se 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 s tabilized by lowering the pH value from 7.0 to 5.5, probably by proton ating a histidine residue leading to an improved solubility of the sig nal 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 inac tivation of mature beta-lactamase by p38, consistent with competition between GroEL and mature beta-lactamase for binding to p38. The equili brium constant for dissociation K-D of the complex between GroEL and p 23, a peptide containing exclusively the signal sequence of pre-beta-l actamase, was measured with the BIAcore(TM) instrument to be in the ra nge 10(-7) to 10(-8). Our results are consistent with co-operative bin ding of GroEL to the mature part and to the signal sequence of pre-bet a-lactamase. We suggest a thermodynamic partitioning model for hydroph obic binding of polypeptides by GroEL.