Dimerisation mutants of Lac repressor. I. A monomeric mutant, L251A, that binds Lac operator DNA as a dimer.
Dimer formation between monomers of the Escherichia coli Lac repressor is substantially specificed by the interactions between three alpha-helices in each monomer which form a hydrophobic interface. As a first step in analysing the specificity of this interaction, we examined the mutant L251A. LacR bearing this mutation in a background lacking the C-terminal heptad repeats is completely incapable of forming dimers in solution, with a dimer-monomer equilibrium dissociation constant, or Kd, higher than 10(-5)M. This correlates with a 200-fold decrease in its ability to repress the lac operon in vivo compared to dimeric LacR. Surprisingly, the mutant is still capable of forming dimers upon binding to short operator DNA in vitro. Analysis of the kinetic parameters of binding of the mutant to operator DNA reveals a 2000 to 3000-fold increase in the equilibrium dissociation constant (Kd) of the mutant-DNA complex in comparison to dimeric LacR-operator complexes, with the change almost entirely due to a greater than 1000-fold decrease in association rate. The dissociation rate varies only by a factor of about two, in comparison to dimeric LacR. This change reflects a kinetic pathway in which dimer formation, in solution or on DNA, is the rate-limiting step. These findings have implications for the specificity and stability of the protein-protein interface in question.
Institut für Genetik der Universität zu Köln, Köln, Weyertal 121, 50931, Germany.
F
F
Dong
S
S
Spott
O
O
Zimmermann
B
B
Kisters-Woike
B
B
Müller-Hill
A
A
Barker
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D029968:Escherichia coli Proteins; D007700:Kinetics; D056968:Lac Repressors; D007930:Leucine / Q000378:metabolism; D009875:Operator Regions, Genetic; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D004247:DNA; D007930:Leucine