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Free keywords:
Protein-DNA interactions, protein-DNA binding, homeodomain, Bicoid, Ultrabithorax, Engrailed, DNA, fluorescence, FRET, stopped-flow, reaction kinetics, diffusion
Abstract:
In this work spectroscopic and transient-kinetic methods were applied to investigate homeodomain-DNA interactions. The analysis of intrinsic fluorescence and circular dichroism spectra of Bicoid (Bcd), Ultrabithorax (Ubx) and Engrailed (En) homeodomains revealed that the binding of either protein to 15 bp oligonucleotides induces changes in structure of these homeodomains, which are different for interaction with cognate and non-cognate sequences. Significant conformation changes were detected for DNA as well upon binding of Bcd or Ubx homeodomains to the 15 bp oligonucleotides containing respective cognate sequences using the technique of intramolecular fluorescence resonance energy transfer (FRET) between two fluorescent labels attached to the opposite ends of the DNA. The detection of FRET between the two fluorophores was found to be the most sensitive spectroscopic method for monitoring the homeodomain-DNA binding in both steady state and kinetic measurements. The fluorescence titration results obtained using this method indicate that Bcd and Ubx homeodomains bind to the specific oligonucleotides with a 2:1 stoichiometry. At protein concentrations exceeding this ratio, both complexes exhibited aggregation. The kinetic data of the Bcd and Ubx homeodomains specific binding to DNA are consistent with a two-step reaction mechanism. In the first step a specific complex between one protein molecule and the oligonucleotide is formed. In the next step the second protein molecule binds the specific complex interacting with both the first bound protein molecule and DNA. The binding affinities in these two steps are very different for Bcd and Ubx homeodomains. The analysis of the association kinetic data has shown that the process of Bcd and Ubx homeodomain binding to DNA is diffusion-controlled. Furthermore it was found that the strong electrostatic field of the alfa-helix III of Bcd homeodomain seems to play a significant role driving this molecule to a correct orientation relative to DNA thereby enhancing the association efficiency.
