Single-chain lambda Cro repressors confirm high intrinsic dimer-DNA affinity.
The overall affinity of the bacteriophage lambda Cro repressor for its operator DNA site is limited by dimer dissociation at submicromolar concentrations. Since Cro dimer-operator complexes form at nanomolar concentrations of Cro subunits where free dimers are rare, these dimers must bind with compensating high affinities. Previous studies of the covalent dimer Cro V55C suggest little change in DNA binding affinity even though the dimeric species is quantitatively populated; this is an apparent contradiction to the expectation of high intrinsic dimer-DNA affinity. In contrast to the disulfide linkage at the center of the dimer interface in Cro V55C, polypeptide linkers that join the two subunits allow single-chain Cro repressors to bind operator DNA with picomolar affinities. A series of five single-chain Cro repressors have been expressed from fused tandem cro genes. Each contains a peptide linker of 8-16 hydrophilic residues that connects the C-terminus of one subunit to the N-terminus of the next. All bind to operator DNA with at least 100-fold higher affinity than Cro V55C. Proteins containing the longest and shortest linkers have been purified and characterized in detail. Both exhibit similar CD spectra to wild-type Cro and enhanced thermal stability. Sedimentation equilibrium experiments show that single-chain Cro repressors do not associate at concentrations up to 30 microM. The rate of dissociation of Cro-DNA complexes is almost unchanged by covalent linkage. Biophysical characterization of Cro variants such as these, where DNA binding is uncoupled from subunit assembly, is necessary for a quantitative understanding of the structural and energetic determinants of DNA recognition in this simple model system.
Jana
R
R
Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Hazbun
T R
TR
Fields
J D
JD
Mossing
M C
MC
D010582:Bacteriophage lambda / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000138:chemical synthesis / Q000378:metabolism*; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000138:chemical synthesis / Q000378:metabolism*; D014764:Viral Proteins / Q000138:chemical synthesis / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA