SELF-ASSEMBLY OF BACTERIOPHAGE-LAMBDA CI-REPRESSOR - EFFECTS OF SINGLE-SITE MUTATIONS ON THE MONOMER-DIMER EQUILIBRIUM

Dimerization of lambda cI repressor monomers is required for high-affi nity binding to bacteriophage lambda operator DNA and is known to invo lve protein-protein contacts between C-terminal domains of the repress or monomers. In order to address the importance of the C-terminal doma in in mediating the oligomeric properties of dimerization and cooperat ive binding to operator DNA, eight single-site mutant repressors were screened for possible deficiencies in cooperative interactions; all bu t one of the amino acid substitutions are located within the C-termina l domain. As a prelude to binding studies and the complete characteriz ation of cooperativity mutants of lambda cI repressor (Burz, D. S., an d Ackers, G. K. (1994) Biochemistry 33, 8406-8416), the thermodynamics of self-assembly of seven of these mutants was examined from 10(-11) to 10(-5) M total repressor using analytical gel chromatography. Resul ts show that the structural perturbation accompanying single amino aci d replacement does not significantly affect the monomer-dimer equilibr ium with the exception of that accompanying replacements of serine 228 ; mutations at that site weaken, by 2-4 kcal/mol, the protein-protein interactions responsible for self-association. An additional mutant re pressor, Pro158-->Thr, was also examined and found to associate revers ibly from monomers to a species with stoichiometry greater than 2. All mutations increase the apparent Stokes radius of the monomeric form b y 2-4.5 Angstrom and that of dimers by 1 or 3 Angstrom.