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-affinity binding to bacteriophage lambda operator DNA and is known to involve protein-protein contacts between C-terminal domains of the repressor monomers. In order to address the importance of the C-terminal domain in mediating the oligomeric properties of dimerization and cooperative binding to operator DNA, eight single-site mutant repressors were screened for possible deficiencies in cooperative interactions; all but one of the amino acid substitutions are located within the C-terminal domain. As a prelude to binding studies and the complete characterization of cooperativity mutants of lambda cI repressor (Burz, D. S., & 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. Results show that the structural perturbation accompanying single amino acid replacement does not significantly affect the monomer-dimer equilibrium 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 repressor, Pro158-->Thr, was also examined and found to associate reversibly from monomers to a species with stoichiometry greater than 2. All mutations increase the apparent Stokes radius of the monomeric form by 2-4.5 A and that of dimers by 1 or 3 A.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
D S
DS
Burz
D
D
Beckett
N
N
Benson
G K
GK
Ackers
D010582:Bacteriophage lambda / Q000235:genetics*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D008970:Molecular Weight; D009875:Operator Regions, Genetic; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins