title: Controlling the DNA binding specificity of bHLH proteins through intramolecular interactions.
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.
authors.affiliation: School of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
authors.forename: Elizebeth C
authors.initials: EC
authors.lastname: Turner
authors.forename: Charlotte H
authors.initials: CH
authors.lastname: Cureton
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Weston
authors.forename: Oliver S
authors.initials: OS
authors.lastname: Smart
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
mesh_terms: D000595:Amino Acid Sequence; D016880:Anisotropy; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008856:Microscopy, Fluorescence / Q000295:instrumentation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D014157:Transcription Factors