OLIGOMERS OF THE CYTOPLASMIC FRAGMENT FROM THE ESCHERICHIA-COLI ASPARTATE RECEPTOR DISSOCIATE THROUGH AN UNFOLDED TRANSITION-STATE

The kinetic and equilibrium properties of a clustering process were st udied as a function of temperature for two point mutants of a 31 kDa f ragment derived from the cytoplasmic region of the Escherichia coli as partate receptor (C-fragment), which were shown previously to have a g reater tendency to form clusters relative to the wild-type C-fragment [Long, D.G., & Weis, R.M. (1992) Biochemistry 31, 9904-9911]. The clus tering equilibria were different for the two C-fragments. Monomers of a serine-461 to leucine (S461L) mutant C-fragment were in equilibrium with dimers. while monomers of a S325L C-fragment were in equilibrium with trimers. The positive values for Delta H degrees, Delta S degrees , and Delta C-p degrees of dissociation estimated from a van't Hoff an alysis, and the differences in the CD spectra of isolated monomers and oligomers, demonstrated that the monomers were less well-folded than the clustered forms, The oligomer dissociation rate exhibited a marked temperature dependence over the range from 4 to 30 degrees C and was remarkably slow at low temperatures; e.g. t(1/2) of dimer dissociation for the S461L C-fragment was 85 h at 4 degrees C. The value for Delta H degrees double dagger, Delta S degrees double dagger, and Delta C-p degrees double dagger derived from the temperature dependence of the dissociation rate were comparable to the corresponding parameters dete rmined in a DSC study of C-fragment denaturation [Wu, J., Long, D.G., & Weis, R.M. (1995) Biochemistry 34, 3056-3065], which indicated that the transition state resembled thermally denatured C-fragment. Octyl g lucoside accelerated the dissociation rate by 3-5-fold presumably by l owering the barrier to dissociation. This acceleration and the positiv e value of Delta C-p degrees double dagger were interpreted as evidenc e for an increase in solvent accessible hydrophobic groups in the tran sition state. The molecular basis for the low rate of dissociation is proposed to result from the conversion of intermolecular coiled coils in the oligomers to an intramolecular coiled coil in the monomer.