Repertoire selection of variant single-chain Cro: toward directed DNA-binding specificity of helix-turn-helix proteins.
A single-chain derivative of the lambda Cro repressor (scCro) has been randomly mutated in amino acid residues critical for specific DNA recognition to create libraries of protein variants. Utilizing phage display-afforded affinity selection, scCro variants have been isolated for binding to synthetic DNA ligands. Isolated scCro variants were analyzed functionally, both in fusion with phage particles and after expression of the corresponding free proteins. The binding properties with regard to specificity and affinity in binding to different DNA ligands were investigated by inhibition studies and determination of equilibrium dissociation constants for formed complexes. Variant proteins with altered DNA-sequence specificity were identified, which favored binding of targeted synthetic DNA sequences over a consensus operator sequence, bound with high affinity by wild-type Cro. The specificities were relatively modest (2-3-fold, as calculated from K(D) values), which can be attributed to the inherent properties in the design of the selection system; one half-site of the synthetic DNA sequences maintains the consensus operator sequence, and one "subunit" of the variant single-chain Cro dimers was conserved as wild-type sequence. The anticipated interaction between the wild-type subunit and the consensus DNA half-site of target DNA ligands is, hence, expected to contribute to the overlap in sequence discrimination. The binding affinity for the synthetic DNA sequences, however, was improved 10-30-fold in selected variant proteins as compared to "wild-type" scCro.
Nilsson
Mikael T I
MT
Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden.
Widersten
Mikael
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019077:Helix-Turn-Helix Motifs; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D017186:Titrimetry; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D008024:Ligands; D019151:Peptide Library; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA