Controlling the DNA binding specificity of bHLH proteins through intramolecular interactions.
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.
Turner
Elizebeth C
EC
School of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
Cureton
Charlotte H
CH
Weston
Chris J
CJ
Smart
Oliver S
OS
Allemann
Rudolf K
RK
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*
D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D014157:Transcription Factors