Combinatorial mutations of lac repressor. Stability of monomer-monomer interface is increased by apolar substitution at position 84.
To examine the monomer-monomer subunit interface in the lac repressor, a mutation that generates dimeric protein (deletion of C-terminal amino acids to disrupt the dimer-dimer interface) has been combined with amino acid substitutions that alter the monomer-monomer interface (substitution at Lys84 or Tyr282). Dimeric proteins with significantly increased stability to urea denaturation were formed by the introduction of the apolar amino acids Ala or Leu in lieu of Lys84 in concert with the deletion of 11 C-terminal amino acids. K84A/-11 deletion protein retained wild-type affinity for operator DNA, while K84L/-11 deletion protein displayed operator affinity similar to its parent tetramer. To assess further the influence of monomer-monomer interface stability on assembly and DNA binding, triple mutants were generated with Y282D, an alteration that disrupts assembly completely in the wild-type background. The triple mutants were dimeric, but they exhibited diminished dimer stability to urea denaturation and decreased operator affinity compared with the double mutations. These results demonstrate directly the stabilizing influence of apolar substitution at position 84 on the monomer-monomer interface.
Nichols
J C
JC
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251, USA.
Matthews
K S
KS
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D007700:Kinetics; D056968:Lac Repressors; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics*; D014357:Trypsin / Q000378:metabolism; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside; D004247:DNA; D014357:Trypsin