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Molecular characterization of helix-loop-helix peptides
S J Anthony-Cahill1, P A Benfield, R Fairman
1Biotechnology Department, DuPont Merck Pharmaceutical Co., Wilmington, DE 19880-0328.
Science (New York, N.Y.)
|February 21, 1992
Summary
This study reveals how DNA-binding proteins, crucial for eukaryotic gene regulation, form specific structures. Findings support a parallel four-helix model for DNA-bound dimers, enhancing our understanding of gene expression control.
Area of Science:
- Molecular Biology
- Genetics
- Protein Structure and Function
Background:
- Eukaryotic gene expression is regulated by proteins containing conserved domains for DNA binding and oligomerization.
- A common structural motif involves a basic region rich in arginine and lysine, followed by a helix-loop-helix (HLH) domain, mediating sequence-specific DNA interaction.
Purpose of the Study:
- To investigate the oligomeric state and DNA-binding mechanism of the HLH motif in the MyoD protein.
- To elucidate the specific quaternary structure of the DNA-bound dimer and validate structural models.
Main Methods:
- Synthesis and characterization of peptides spanning the MyoD HLH motif.
- Analysis of peptide oligomerization in solution using biophysical techniques.
- DNA-binding assays to assess the affinity and specificity of peptide-DNA interactions.
- Structural modeling (parallel and antiparallel four-helix models) and experimental validation using disulfide bond engineering.
- Electron paramagnetic resonance (EPR) spectroscopy to measure inter-subunit distances.
Main Results:
- Peptides formed alpha-helical dimers and tetramers in solution.
- DNA binding occurred as dimers, with an observed increase in alpha-helical content upon binding.
- Disulfide bond engineering demonstrated that a configuration compatible with a parallel four-helix model enhanced specific DNA binding, while an antiparallel configuration abolished it.
- EPR measurements supported the parallel model by providing inter-subunit distance data consistent with this arrangement.
Conclusions:
- The helix-loop-helix motif of MyoD functions as a dimer to bind DNA.
- The DNA-bound dimer adopts a parallel four-helix quaternary structure.
- This structural insight clarifies the mechanism of DNA recognition by a key class of gene regulatory proteins.
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