Controlling the DNA binding specificity of bHLH proteins through intramolecular interactions
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Reversible control of the conformation of proteins was employed to probe the relationship between flexibility and specificity of the basic helix-loop-helix protein MyoD. A fusion protein (apaMyoD) was designed where the basic DNA binding helix of MyoD was stablized by an amino-terminal extension with a sequence derived from the bee venom peptide apamin. The disulfide-stabilized helix from apamin served as a nucleus for a helix that extended for a further ten residues, thereby holding apaMyoD's DNA recognition helix in a predominantly alpha-helical conformation. The thermal stability of the DNA complexes of apaMyoD was increased by 13 degrees C relative to MyoD-bHLH. Measurements of the fluorescence anisotropy change on DNA binding indicated that apaMyoD bound to E-box-containing DNA sequences with enhanced affinity relative to MyoD-bHLH. Consequently, the DNA binding specificity of apaMyoD was increased 10-fold.
|Number of pages||9|
|Journal||Chemistry & Biology|
|Publication status||Published - Jan 2004|
- Amino Acid Sequence, Anisotropy, Basic Helix-Loop-Helix Transcription Factors, Binding Sites, Circular Dichroism, DNA, DNA-Binding Proteins, Magnetic Resonance Spectroscopy, Microscopy, Fluorescence, Models, Molecular, Molecular Sequence Data, Protein Binding, Structure-Activity Relationship, Substrate Specificity, Temperature, Transcription Factors