Controlling the DNA binding specificity of bHLH proteins through intramolecular interactions

Research output: Contribution to journalArticlepeer-review

Authors

Colleges, School and Institutes

Abstract

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.

Details

Original languageEnglish
Pages (from-to)69-77
Number of pages9
JournalChemistry & Biology
Volume11
Issue number1
Publication statusPublished - Jan 2004

Keywords

  • 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