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Single-chain lambda Cro repressors confirm high intrinsic dimer-DNA affinity

R Jana¹, T R Hazbun, J D Fields

¹Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.

|June 13, 1998

Summary

Single-chain bacteriophage lambda Cro repressors, engineered with peptide linkers, exhibit significantly higher DNA binding affinity than their dimeric counterparts. This uncouples DNA recognition from subunit association, aiding structural and energetic studies.

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Area of Science:

Molecular Biology
Biophysics
Structural Biology

Background:

Bacteriophage lambda Cro repressor's DNA binding affinity is limited by dimer dissociation.
High affinity binding is expected for Cro dimers at low concentrations, yet experimental data shows otherwise.

Purpose of the Study:

To investigate the DNA binding affinity of single-chain Cro repressors.
To understand the structural and energetic factors governing DNA recognition by Cro repressor.

Main Methods:

Expression and purification of five single-chain Cro repressors with varying peptide linkers.
Circular dichroism (CD) spectroscopy and sedimentation equilibrium experiments.
Analysis of Cro-DNA complex dissociation rates.

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Main Results:

Single-chain Cro repressors bind operator DNA with picomolar affinities, over 100-fold higher than Cro V55C.
Engineered repressors show enhanced thermal stability and no self-association.
Dissociation rates of Cro-DNA complexes remain largely unchanged by covalent linkage.

Conclusions:

Covalent linkage of Cro subunits via peptide linkers dramatically enhances DNA binding affinity.
Single-chain constructs decouple DNA binding from subunit association, facilitating mechanistic studies.
This approach provides a model system for dissecting DNA recognition determinants.