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Fluorescence quenching studies of Trp repressor-operator interaction.

Z Blicharska1, Z Wasylewski

  • 1Department of Physical Biochemistry, Institute of Molecular Biology, Jagiellonian University, Krakow, Poland.

Journal of Protein Chemistry
|June 6, 2000
PubMed
Summary

This study reveals how L-tryptophan binding alters the tryptophan repressor environment in Escherichia coli. DNA binding further changes the corepressor

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • The tryptophan repressor (Trp repressor) in Escherichia coli regulates gene expression by binding to L-tryptophan and subsequently to DNA operator sequences.
  • Understanding the microenvironment changes of the corepressor upon ligand binding is crucial for elucidating the regulatory mechanism.

Purpose of the Study:

  • To investigate the microenvironment changes of L-tryptophan within the tryptophan repressor mutant W19/99F upon binding to L-tryptophan.
  • To examine the effect of DNA operator binding on the corepressor's microenvironment.

Main Methods:

  • Steady-state quenching and time-resolved fluorescence spectroscopy were employed.
  • Iodide and acrylamide were used as quenchers to probe the accessibility of L-tryptophan.
  • Fluorescence emission spectra and lifetimes of L-tryptophan were analyzed.

Main Results:

  • L-tryptophan fluorescence emission in the holorepressor resolved into two components: a buried component (emission max 336 nm) and a solvent-exposed component.
  • The buried component exhibited a Stern-Volmer quenching constant of 2.0-2.3 M⁻¹, while the exposed component showed characteristics of free L-tryptophan.
  • Upon DNA binding, the Stern-Volmer quenching constant for the buried component significantly decreased to 0.56 M⁻¹, and fluorescence lifetimes decreased, suggesting a dynamic quenching mechanism.

Conclusions:

  • L-tryptophan binding induces distinct microenvironment changes within the Trp repressor, with a portion becoming buried.
  • DNA binding to the Trp holorepressor further alters the corepressor's microenvironment, impacting L-tryptophan accessibility and dynamics.
  • These findings provide insights into the allosteric regulation mechanism of the Trp repressor in Escherichia coli.

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