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Engineered disulfide linking the hinge regions within lactose repressor dimer increases operator affinity, decreases
1Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251, USA.
Biochemistry
|December 19, 2001
Summary
The lactose repressor hinge region is crucial for DNA binding and allosteric regulation. A disulfide bond mutation enhances DNA binding but disrupts normal inducer response, revealing the hinge
Area of Science:
- Molecular Biology
- Protein-DNA Interactions
- Biochemistry
Background:
- The lactose repressor (LacI) hinge domain (amino acids 51-60) is vital for DNA binding and allosteric regulation.
- This region mediates critical hinge-DNA and hinge-hinge contacts and communicates inducer binding signals.
Purpose of the Study:
- To investigate the role of the LacI hinge region in DNA binding affinity and allosteric response.
- To characterize the effects of introducing a disulfide bond (V52C mutation) on LacI function.
Main Methods:
- Site-directed mutagenesis to introduce a V52C mutation in the LacI hinge domain.
- Assays to measure protein-DNA binding affinity and allosteric response to inducers.
- Thermodynamic analysis using temperature studies (DeltaCp).
Main Results:
- The V52C mutation, forming a disulfide bond, significantly increased LacI affinity for operator DNA.
- This disulfide-linked protein maintained high affinity despite altered operator symmetry, unlike wild-type LacI.
- Allosteric response to inducer was abolished in the disulfide-linked protein but remained for some operator variants.
- Thermodynamic studies showed the disulfide bond altered the protein-DNA interaction thermodynamics (smaller DeltaCp).
Conclusions:
- The hinge region is essential for both high-affinity LacI operator binding and allosteric communication.
- Disulfide linkage in the hinge region decouples DNA binding from allosteric signal transduction.
- DNA sequence and conformation critically influence allosteric regulation in LacI.
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