A synthetic peptide mimic of λ-Cro shows sequence-specific binding in vitro and in vivo.
ACS chemical biology 7:6 (2012) 1084-1094
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.Peptide based Molecules as Protein-Protein Interaction Inhibitors: Tools for Chemical Genetics and Therapy
Current Chemical Biology Bentham Science Publishers 6:2 (2012) 145-163
A genetic network that balances two outcomes utilizes asymmetric recognition of operator sites.
Biophysical journal 102:7 (2012) 1580-1589
Abstract:
Stability and induction of the lysogenic state of bacteriophage λ are balanced by a complex regulatory network. A key feature of this network is the mutually exclusive cooperative binding of a repressor dimer (CI) to one of two pairs of binding sites, O(R)1-O(R)2 or O(R)2-O(R)3. The structural features that underpin the mutually exclusive binding mode are not well understood. Recent studies have demonstrated that CI is an asymmetric dimer. The functional importance of the asymmetry is not fully clear. Due to the asymmetric nature of the CI dimer as well as its binding sites, there are two possible bound orientations. By fluorescence resonance energy transfer measurements we showed that CI prefers one bound orientation. We also demonstrated that the relative configuration of the binding sites is important for CI dimer-dimer interactions and consequent cooperative binding. We proposed that the operator configuration dictates the orientations of the bound CI molecules, which in turn dictates CI cooperative interaction between the O(R)1-O(R)2 or O(R)2-O(R)3, but not both. Modeling suggests that the relative orientation of the C- and N-terminal domains may play an important role in the mutually exclusive nature of the cooperative binding. This work correlates unique structural features of a transcription regulatory protein with the functional properties of a gene regulatory network.Recognition of different DNA sequences by a DNA-binding protein alters protein dynamics differentially.
FEBS letters 586:3 (2012) 258-262