title: A synthetic peptide mimic of λ-Cro shows sequence-specific binding in vitro and in vivo.
abstract: Development of small synthetic transcription factors is important for future cellular engineering and therapeutics. This article describes the chemical synthesis of α-amino-isobutyric acid (Aib) substituted, conformationally constrained, helical peptide mimics of Cro protein from bacteriophage λ that encompasses the DNA recognition elements. The Aib substituted constrained helical peptide monomer shows a moderately reduced dissociation constant compared to the corresponding unsubstituted wild type peptide. A suitably cross-linked dimeric version of the peptide, mimicking the dimeric protein, recapitulates some of the important features of Cro. It binds to the operator site O(R)3, a high affinity Cro binding site in the λ genome, with good affinity and single base-pair discrimination specificity. A dimeric version of an even shorter peptide mimic spanning only the recognition helix of the helix-turn-helix motif of the Cro protein was created following the same design principles. This dimeric peptide binds to O(R)3 with affinity greater than that of the longer version. Chemical shift perturbation experiments show that the binding mode of this peptide dimer to the cognate operator site sequence is similar to the wild type Cro protein. A Green Fluorescent Protein based reporter assay in vivo reveals that the peptide dimer binds the operator site sequences with considerable selectivity and inhibits gene expression. Peptide mimics designed in this way may provide a future framework for creating effective synthetic transcription factors.
authors.affiliation: Divisions of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Mazumder
authors.forename: Atanu
authors.initials: A
authors.lastname: Maiti
authors.forename: Koushik
authors.initials: K
authors.lastname: Roy
authors.forename: Siddhartha
authors.initials: S
authors.lastname: Roy
mesh_terms: D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000737:chemistry*; D020029:Base Pairing; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D012097:Repressor Proteins / Q000737:chemistry*; D014158:Transcription, Genetic / Q000187:drug effects*; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry*
chemical_list: D010455:Peptides; D012097:Repressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins