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.
Original language | English |
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Pages (from-to) | 69-77 |
Number of pages | 9 |
Journal | Chemistry & Biology |
Volume | 11 |
Issue number | 1 |
DOIs | |
Publication status | Published - 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