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Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions
Published on: October 21, 2016
Long-range electrostatic interactions influence the orientation of Fos-Jun binding at AP-1 sites
V R Ramirez-Carrozzi1, T K Kerppola
1Howard Hughes Medical Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0650, USA.
Journal of Molecular Biology
|January 12, 2001
Summary
Transcription factor orientation, like Fos-Jun binding to DNA, is controlled by electrostatic interactions. Amino acid and DNA sequence changes outside the direct binding site dictate binding direction, influencing gene regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Heterodimeric transcription factors binding palindromic DNA can adopt two orientations.
- Binding orientation influences interactions with other regulatory proteins and transcriptional activity.
- Fos-Jun heterodimers exhibit orientation-dependent binding to AP-1 sites with varying flanking sequences.
Purpose of the Study:
- To elucidate the mechanism determining transcription factor binding orientation.
- To investigate the roles of amino acid residues and flanking DNA sequences in orientation control.
- To understand how electrostatic interactions govern nucleoprotein complex architecture.
Main Methods:
- Utilized a gel-based fluorescence resonance energy transfer (gelFRET) assay.
- Performed amino acid residue exchanges between Fos and Jun subunits.
- Introduced single amino acid and base-pair substitutions in flanking DNA sequences.
Main Results:
- Exchanging three amino acid residues reversed Fos-Jun heterodimer binding orientation.
- Binding orientation was dependent on flanking base-pairs and DNA bending propensities.
- Long-range electrostatic interactions, particularly DNA charge modifications, significantly affected binding orientation.
Conclusions:
- Heterodimer binding orientation is primarily determined by minimizing electrostatic free energy.
- Amino acid residues and flanking DNA sequences outside the core binding site dictate orientation.
- Electrostatic interactions play a crucial role in shaping nucleoprotein complex architecture and function.
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